WO2022103871A1 - Compositions thérapeutiques pour le traitement de la covid-19 - Google Patents

Compositions thérapeutiques pour le traitement de la covid-19 Download PDF

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WO2022103871A1
WO2022103871A1 PCT/US2021/058831 US2021058831W WO2022103871A1 WO 2022103871 A1 WO2022103871 A1 WO 2022103871A1 US 2021058831 W US2021058831 W US 2021058831W WO 2022103871 A1 WO2022103871 A1 WO 2022103871A1
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antibodies
composition
bovine
proteins
antibody
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PCT/US2021/058831
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Gary Robinson
Nada JAIN
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Wyomingv Immune, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • C07K2317/12—Immunoglobulins specific features characterized by their source of isolation or production isolated from milk
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • 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
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Coronavirus disease 2019 (COVID-19) is a global pandemic infectious disease caused by severe acute respiratory syndrome coronavirus-2 (SARS CoV-2).
  • SARS CoV-2 severe acute respiratory syndrome coronavirus-2
  • SARS CoV-2 is a new member of the Coronaviridae family, which is closely related (approximately 88%) to two bat-derived SARS- like coronaviruses.
  • SARS-CoV-2 is highly contagious, and the epidemic of SARS CoV-2 is posing a great threat for global public health.
  • COVID-19 being a multi-faceted disease attacking multiple organs, such as the gastrointestinal tract (GI), and further due to the vaccine hesitancy around the world, both general and organ-specific therapies are required.
  • This viral pandemic therefore remains a global medical need.
  • Applicants* focus on mucosal polyclonal antibody delivery (oral and nasal) offers advantages over the mainstream approaches.
  • SARS CoV-2 is composed of a single-stranded ribonucleic acid (RNA) structure, which belongs to the ⁇ -coronavirus (P-CoVs) and falls within the P-CoVs 2b lineage (Coutard et al.. 2020: Zhai et al.. 2020). Similar to other p-CoVs, SARS CoV-2 virus particles look like a solar corona under transmission electron micrographs imaging. The virus particles are spherical and polymorphic, with a diameter of about 60 to 140 nm and specific spines of 9 to 12 nm in length (Zhu N. et al.. 2020).
  • SARS-CoV-2 The viral genome of SARS-CoV-2 is about 29.8 kb, the G+C content is 38%, and there are six open reading frames common to coronaviruses and some other accessory genes. Moreover, SARS-CoV-2 has 5' and 3' terminal sequences, which are typical P-CoVs, 265 nt at 5' ends and 358 nt at 3' ends, respectively (Chan et al.. 2020). SARS CoV-2 contains four main structural proteins, namely spike (S), membrane (M), nucleocapsid (N), and envelope (E) proteins.
  • S spike
  • M membrane
  • N nucleocapsid
  • E envelope
  • S, M, and E proteins are all embedded in the viral envelope, while the N protein is the only protein that interacts with the viral RNA at the core of the viral particle to form the nucleocapsid (Fehr and Perlman. 2015).
  • the severely glycosylated S protein forms homotrimeric spikes on the surface of the virus particle, and mediates receptor binding to the host cell membrane through the receptor binding domain (RBD) of the SI domain.
  • RBD receptor binding domain
  • SARS-CoV-2 shares 89.8% sequence identity in S2 subunits with SARS-CoV, implying their similar mechanism of membrane fusion through S2 subunit (Xia et al. 2020).
  • the M and E protein play a critical role in coordinating virus assembly and forming mature viral envelopes, while the N protein binds to the viral RNA and is involved in the transcription and replication of viral RNA, as well as packaging of the encapsidated genome into virions (Ashour et al.. 2020; Nishiga et al.. 2020).
  • the amino acid sequence of the SARS CoV- 2 membrane protein is known, see GenBank Accession No YP009825055.1
  • the amino acid sequence of a SARS CoV-2 membrane protein is set forth in SEQ ID NO:2.
  • the amino acid sequence of the SARS CoV-2 envelope protein is known, see GenBank Accession No YP 009724392 and variant(s): Leu37His and/or Pro72Leu set forth in SEQ ID NO:3.
  • SARS CoV-2 spike protein is a glycoprotein, which mediates attachment of the virus to the host cell.
  • the structure of the spike (S) protein and amino acid sequence are known, see for example, SARS CoV-2 Genbank Accession No. BCA87361.1. See also Genbank YP_009724390.1 ; and is set forth in SEQ ID NO: 1.
  • the spike protein recognizes the human angiotensin-converting enzyme 2 (ACE2) protein on the host cell surface.
  • ACE2 human angiotensin-converting enzyme 2
  • bovine colostra! antibodies have been used experimentally to treat and prevent infection and inflammation in die digestive tract (Jones et al., Crit Rev Biotechnol 2015; July 15: 1-15). In contrast to other types of antibodies, which are readily digested, bovine colostral antibodies naturally resist intestinal digestion, making them uniquely suited for oral, topical and mucosal use in the digestive tract and airways. Studies of bovine colostral antibodies have consistently shown that they survive in the human digestive tract (Jaison et al. Nutrition Journal 2015;14:1-8).
  • Bovine colostrum contains a mixture of immunoglobulins including IgGl, lgG2, IgM, and IgA. In addition to proteins, colostrum contains large quantities of lipids, sugars, and small molecules. While bovine colostrum is a complex mixture of bioactive substances, the structural features of imparting digestion resistance have been recently elucidated. (Burton et al., J Bio! Chem. 2020 Aug 21;295(34):12317-12327,doi: 10.1074/jbc.RA120.014327. Epub 2020 Jul 14.)
  • Immunoglobulins in bovine colostrum are transferred from the serum to the udder shortly before parturition. Most transfer of IgG occurs by active transport via mammary epithelial cells, which secrete substances into the colostrum. During transport through these cells, IgG can be modified by glycosylation or other means.
  • Immunoglobulin from the colostrum (early milk) of immunized ruminants and notably cows is an ideal source of antibody. Cows secrete a bolus of immunoglobulin into colostrum to provide passive immunity to the newborn calf (antibody does not cross the placenta in ruminants). Colostrum contains 23 - 30 g/L immunoglobulin, approximately 50% of the total protein. In addition, modem dairy cows have been bred to produce large amounts of milk - ⁇ 7 gallons of milk per day. As a result of the large volume and high concentration, approximately 1 kg of antibody can be isolated from a single cow in the first week after calving.
  • Colostrum is expected to be an exceptionally safe source of polyclonal antibody.
  • bovine immunoglobulin - regular milk contains 1.5 g/L IgG and the average American will have consumed 6.5 kg of bovine immunoglobulin by the time they reach 18.
  • immunoglobulin is such an unusual reactivity that it is generally not detected.
  • the majority of patients allergic to milk proteins react to 6-lactoglobulin, caseins, lactalbumin or seroalbumin (Esteban, M. M. et al., (1998), Adverse reactions to cow's milk proteins. Allergol Immunopathol (Madr), 26, 171-194)).
  • Bovine spongiform encephalopathy is not transmitted through milk or colostrum (Castilla, J., et al., (2005). Vertical transmission of bovine spongiform encephalopathy prions evaluated in a transgenic mouse model. J Virol, 79( 13), 8665-68; and EMA. (2002). NOTE FOR GUIDANCE ON MINIMISING THE RISK OF TRANSMITTING ANIMAL SPONGIFORM ENCEPHALOPATHIES VIA HUMAN AND VETERINARY MEDICINAL PRODUCTS. EMEA/410/01 Rev.2,) and there are no international restrictions on the distribution of dairy products.
  • Bovine immunoglobulin concentrate-clostridium difficile retains C difficile toxin neutralising activity after passage through the human stomach and small intestine.
  • the present disclosure is directed to colostral polyclonal antibodies for induction of passive and active immunity against Covid- 19 (eliciting anti-idiotypic antibodies).
  • the colostral polyclonal antibodies are elicited after immunization of ruminants, notably cows with one or more of the foregoing SARS CoV-2 proteins (envelope, nucleocapsid, membrane and spike proteins or polypeptides), including without limitation the spike protein, or immunogenic fragments of such proteins and harvesting of the antibodies from the colostrum or early milk.
  • the present disclosure is also directed to a platform technology for generating such antibodies, not only against Covid- 19 but also against other pathogens and notably viruses.
  • polyclonal antibodies are provided which can be used as therapeutics (passive immunization) against Covid- 19.
  • the antibodies are derived from the early milk (colostrum) of immunized ruminants, e.g., cows, purified to remove milk proteins and other constituents and pathogens, incorporated into pharmaceutical compositions and used therapeutically for passive immunization of subjects afflicted with Covid- 19.
  • Colostrum of immunized ruminants may be utilized in compositions at various levels of purification, e.g. anti-SARS-CoV-2 antibodies need not be separated from other colostrum immunoglobulins.
  • Such compositions may be in a form of a medical food or dietary supplement.
  • compositions of the present disclosure include medical foods and dietary supplements in addition to pharmaceutical compositions and biologic medicaments.
  • These antibodies can also be used intranasally or by inhalation for more direct delivery to tissues afflicted by SARS-CoV-2, such as the nasal mucosa, the larynx, the bronchial passages and the lungs.
  • the present disclosure also encompasses colostrum-derived monoclonal antibodies, and combinations thereof (which encompass cocktails of two or more monoclonal antibodies having different specificities) and mixtures of monoclonal and polyclonal antibodies.
  • the polyclonal antibodies can be made by immunization of ruminants notably bovines with whole virus (attenuated or inactivated) or viral antigens and collecting colostra! polyclonal antibodies directed against one or more epitopes of one or more proteins of SARS CoV-2, for example against the spike polypeptide, or an immunologically active fragment thereof.
  • Such epitopic regions of the spike protein have been identified and can be further refined for this protein and other viral proteins. See for example Huang, Y., et al.
  • the monoclonal and polyclonal antibodies are, or are based on the structural features of, ruminant early colostrum antibodies that exhibit resistance to proteases and intestinal digestion.
  • pharmaceutical compositions comprising these antibodies as described herein are passive immunogens, i.e., provide an immune response directed against SARS CoV-2 virus, while they advantageously exhibit resistance to proteases and intestinal digestion and may be delivered in both the digestive tract (oral cavity, pharynx, esophagus, stomach and intestine) and the airways (nasal, laryngeal, bronchial and lung mucosa). The latter can be accomplished by nasal or by-inhalation administration. Site specific targeting of therapy or a dual approach can thus be implemented to manage Covid 19 infection and/or symptoms.
  • the airways delivery can be employed to generate active immunity as well (as described below).
  • compositions containing (e.g., comprising, consisting of or consisting essentially of) colostra! polyclonal antibodies from ruminants notably bovines directed against one or more SARS CoV-2 antigens such as a spike polypeptide, or an immunologically active fragment thereof, and optionally a carrier or diluent (e.g., a pharmaceutically acceptable carrier or diluent), formulated for digestive tract or airway administration.
  • the composition further comprises a preservative.
  • the composition is lyophilized.
  • the composition is a sprayable liquid (e.g., an aqeous suspension) or a gel or other physiologically acceptable film-forming material that adheres to the nasal mucosa; in other embodiments, it is an aerosolizable powder.
  • a sprayable liquid e.g., an aqeous suspension
  • a gel or other physiologically acceptable film-forming material that adheres to the nasal mucosa; in other embodiments, it is an aerosolizable powder.
  • kits for treating a viral disease comprising administering, directed against one or more SARS CoV-2 antigens such as a spike polypeptide, or an immunologically active fragment thereof, to a subject in need thereof, such as a human patient infected with or exposed to a viral disease.
  • the disease is COVID-19.
  • the present disclosure is directed to anti-idiotypic (anti-Id) antibodies produced in the colostrum of cows immunized with (i) human polyclonal antibodies to SARS-CoV-2 proteins harvested from human patients infected with COVID-19, or (ii) colostral polyclonal antibodies harvested from ruminants, such as cows, immunized with colostral polyclonal antibodies elicited by immunization of ruminants such as cows with SARS CoV-2 virus; and to monoclonal anti-idiotypic antibodies based on such colostral anti-idiotypic antibodies.
  • anti-Id anti-idiotypic antibodies produced in the colostrum of cows immunized with (i) human polyclonal antibodies to SARS-CoV-2 proteins harvested from human patients infected with COVID-19, or (ii) colostral polyclonal antibodies harvested from ruminants, such as cows, immunized with colostral polyclonal antibodies
  • the bovine anti-idiotypic antibodies can be used for active immunization of subjects and may be characterized further to select Anti-Ab2.
  • the anti-Id antibodies employed are of the Anti-Ab2 type and are elicited in response to immunization of cows with polyclonal colostral antibodies that bind to SARS CoV-2 antigens. It is preferred that a cocktail of monoclonal anti-Id be used for active immunization of subjects. Kohler, H. et al, Frontiers in Immunol. 10:808, April 12, 2019; doi: 103389/fimmu.2019.00808 incorporated by reference in its entirety.
  • the recombinant antibody retains at least the antigen binding CDRs or variable region (VR) from a parent (non-bovine) antibody, and includes all or a portion of a bovine IgG 1 constant region (e.g., a bovine IgG 1 CH 1 and/or hinge domain).
  • a bovine IgG 1 constant region e.g., a bovine IgG 1 CH 1 and/or hinge domain.
  • the antibody comprises the variable region of a parent (non-bovine) antibody, and the constant region (i.e., CH1 domain, hinge region, CH2 domain, and CH3 domain) of a bovine IgGl antibody.
  • a parent antibody non-bovine
  • the constant region i.e., CH1 domain, hinge region, CH2 domain, and CH3 domain
  • Bovine chimeric antibodies Bovinized antibodies and bovine chimeric antibodies are described in PCT/US 16/54304, published as WO2017/062253, the disclosure of which is herein incorporated by reference in its entirety.
  • isolated recombinant monoclonal antibodies comprising (a) heavy and light chain CDR regions from a non-bovine antibody and (b) a constant region comprising all or a portion of a bovine IgGl constant region (e.g., a bovine IgGl constant region selected from any of SEQ ID NOs: 4 -7).
  • the non-bovine antibody is a human antibody.
  • the non-bovine antibody is directed against a virus polypeptide.
  • the virus polypeptide is a structural polypeptide.
  • the structural polypeptide is a spike polypeptide, or an immunologically active fragment of the spike polypeptide, of SARS CoV-2.
  • isolated recombinant polyclonal antibodies comprising (a) heavy and light chain CDR regions from a non-bovine antibody and (b) a constant region comprising all or a portion of a bovine IgGl constant region (e.g., a bovine IgGl constant region selected from any of SEQ ID NOs: 4 -7).
  • the non-bovine antibody is a human antibody.
  • the non-bovine antibody, monoclonal or polyclonal is directed against a virus polypeptide.
  • the virus polypeptide is a structural polypeptide.
  • the structural polypeptide is a spike polypeptide, or an immunologically active fragment of the spike polypeptide, of SARS CoV-2.
  • the constant region comprises all or a portion of a bovine IgG 1 hinge region.
  • the constant region comprises all or a portion of a bovine IgGl CH1 domain.
  • the constant region comprises all or a portion of a bovine IgGl CH2 domain.
  • the constant region comprises all or a portion of a bovine IgGl CH3 domain.
  • the constant region further comprises a portion of a human constant region (e.g., a human IgGl or IgG2 constant region).
  • bovinized antibodies that incorporate one or more of the following structural features found in bovine IgGl that impart protease resistance to the antibody:
  • the parent (non-bovine) antibody comprises a human IgG hinge, wherein sites within the hinge which are susceptible to enzymatic or spontaneous proteolysis are replaced with the equivalent bovine hinge region sequences.
  • the parent (non-bovine) antibody comprises a bovinized hinge region, in which some or all of the amino acid residues of the hinge region are replaced with the equivalent portion of the bovine IgGl hinge region sequence.
  • isolated recombinant monoclonal human antibodies wherein the constant region of the antibodies comprise one or more of the following features found in bovine IgGl: a) a disulfide bond linking the N-terminus of the CH1 domain to the N-terminus of the hinge region, b) a disulfide bond linking the N-terminus of the CH1 domain to the C-terminus of the light chain, and c) a cluster of three disulfide bonds linking the hinge regions of two heavy chains together.
  • the antibody may have fully human sequences, except for one or more of these structural features that exist in bovine IgGl (or structurally equivalent features of IgG1 from other ruminants or rabbit) which confer protease resistance.
  • the antibodies described herein comprise a constant region comprising one or more of the following substitutions (Kabat numbering): threonine at position 252 is substituted with methionine; glycine at position 255 is substituted with arginine; glutamine at position 309 is substituted with leucine; threonine at position 314 is substituted with leucine; and glycine at position 315 is substituted with asparagine.
  • the bovinized or frilly bovine recombinant IgG 1 monoclonal antibodies or polyclonal antibodies described herein bind to a biological antigen (e.g., a spike polypeptide of SARS CoV-2).
  • a biological antigen e.g., a spike polypeptide of SARS CoV-2.
  • Bovinized antibodies described herein exhibit greater protease resistance relative to the parent antibody. In some embodiments, the bovinized antibodies also retain greater antigen- binding activity after protease digestion relative to the parent antibody. In some embodiments, the bovinized antibodies, upon being subjected to protease digestion in the digestive tract, retain antigen-binding activity.
  • frilly bovine recombinant IgG 1 monoclonal antibodies as well as host cells that secrete the antibodies.
  • bispecific bovinized antibodies directed against a spike polypeptide, or an immunologically active fragment, of SARS CoV-2 or bispecific fully bovine recombinant IgGl antibodies directed against a spike polypeptide, or an immunologically active fragment, of SARS CoV-2.
  • immunoconjugates comprising the bovinized polyclonal antibodies directed against a spike polypeptide, or an immunologically active fragment, of SARS CoV-2 or fully bovine recombinant IgGl monoclonal antibodies directed against a spike polypeptide, or an immunologically active fragment, of SARS CoV-2.
  • nucleic acid molecules that encode the recombinant antibodies described herein.
  • expression vectors that comprise the nucleic acids.
  • host cells comprising the nucleic acids or expression vectors.
  • compositions containing (e.g., comprising, consisting of or consisting essentially of) the bovinized antibody or fully bovine poolyclonal or recombinant IgGI monoclonal antibodies or bovinized antibodies directed against a spike polypeptide, or an immunologically active fragment, of SARS CoV-2, and optionally a carrier (e.g., a pharmaceutically acceptable carrier), formulated for oral administration.
  • a carrier e.g., a pharmaceutically acceptable carrier
  • the composition further comprises a preservative.
  • the composition is lyophilized.
  • kits for treating a viral infection comprising administering the bovinized antibodies directed against a spike polypeptide, or an immunologically active fragment, of SARS CoV-2 or fully bovine recombinant IgGl monoclonal antibodies directed against a spike polypeptide, or an immunologically active fragment, of SARS CoV-2 to a subject in need thereof.
  • the disease is COVID-19.
  • kits comprising the bovinized antibodies directed against a spike polypeptide, or an immunologically active fragment, of SARS CoV-2 or folly bovine recombinant IgG 1 monoclonal antibodies directed against a spike polypeptide, or an immunologically active fragment, of SARS CoV-2 described herein.
  • kits for producing the bovinized antibodies directed against a spike polypeptide, or an immunologically active fragment, of SARS CoV-2 or fully bovine recombinant IgG1 monoclonal antibodies directed against a spike polypeptide, or an immunologically active fragment, of SARS CoV-2 described herein comprising expressing the antibodies in a host cell and isolating the antibodies from the cells.
  • a composition for delivery via the digestive tract to a human patient comprising (i) polyclonal antibodies or antigen binding fragments thereof specific to one or more proteins of SARS CoV-2, including without limitation the spike (S) protein thereof, wherein the polyclonal antibodies are obtained from the colostrum of bovine or other ruminant that has been immunized with SARS CoV-2 virus or one or more proteins thereof or an antigenic fragment of the one or more proteins; or (ii) one or more monoclonal antibodies specific to one or more proteins of SARS CoV-2, wherein the antibodies are derived from antibodies obtained from the colostrum of a bovine or other ruminant that has been immunized with SARS CoV-2 virus or one or more proteins thereof or an antigenic fragment of the one or more proteins, and wherein upon administration to the digestive tract of the patient, the composition is therapeutically effective to treat COVID 19 in the patient, and wherein the antibodies are present in the composition at a dosage of about 0.01 mg per dose to about
  • composition of embodiment 1 formulated as a solid dosage form for oral administration.
  • composition of embodiment 2 wherein the solid dosage form is selected from capsules, tablets, pills, powders and granules.
  • composition of embodiment 1 which is formulated as a dietary supplement or medical food.
  • composition of embodiment 1 or 2 wherein the solid dosage form comprises a carrier or diluent.
  • composition of embodiment 1 wherein the ruminant is a bovine.
  • a method of treating COVID-19 in a human patient in need thereof comprising orally administering to a patient, a composition according to any one of embodiments 1 - 6.
  • a composition for delivery via the digestive tract to a human patient comprising (i) polyclonal antibodies or antigen binding fragments thereof specific to one or more proteins of SARS CoV-2, including without limitation the spike (S) protein thereof, wherein the polyclonal antibodies are obtained from the colostrum of bovine or other ruminant that has been immunized with SARS CoV-2 virus or one or more proteins thereof or an antigenic fragment of the one or more proteins; or (ii) one or more monoclonal antibodies specific to one or more proteins of SARS CoV-2, wherein the antibodies are derived from antibodies obtained from the colostrum of a bovine or other ruminant that has been immunized with SARS CoV-2 virus or one or more proteins thereof or an antigenic fragment of the one or more proteins, and wherein upon administration to the digestive tract of the patient, the composition is therapeutically effective to treat COVID 19 in the patient, and wherein the antibodies are present in the composition at a dosage of about 0.01 mg per dose to about
  • composition of embodiment 2 wherein the solid dosage form is selected from capsules, tablets, pills, powders and granules.
  • composition of embodiment 1 which is formulated as a dietary supplement or medical food.
  • composition of embodiment 1 or 2 wherein the solid dosage form comprises a carrier or diluent.
  • composition of embodiment 1 wherein the ruminant is a bovine.
  • a method of treating COVID-19 in a human patient in need thereof comprising orally administering to a patient, a composition according to any one of embodiments 1 - 6.
  • a composition for delivery via the airway to a human patient comprising (i) polyclonal antibodies or antigen binding fragments thereof specific to one or more proteins of SARS CoV-2, including without limitation the spike (S) protein thereof, wherein the polyclonal antibodies are obtained from the colostrum of bovine or other ruminant that has been immunized with SARS CoV-2 virus or one or more proteins thereof or an antigenic fragment of the one or more proteins; or (ii) one or more monoclonal antibodies specific to one or more proteins of SARS CoV-2, wherein the antibodies are derived from antibodies obtained from the colostrum of a bovine or other ruminant that has been immunized with SARS CoV-2 virus or one or more proteins thereof or an antigenic fragment of the one or more proteins, and wherein upon administration to the airways of the patient, the composition is therapeutically effective to treat COVID 19 in the patient, and wherein the antibodies are present in the composition at a dosage of about 0.01 mg per dose to about
  • composition of embodiment 8 formulated as a liquid dosage form for nasal administration.
  • composition of embodiment 9 formulated as a powder for by-inhalation administration into the upper and lower respiratory tract.
  • composition of embodiment 8 or 9 wherein the dosage form further comprises a carrier or diluent.
  • composition of embodiment 8 wherein the ruminant is a bovine.
  • a method of treating COVID-19 in a human patient in need thereof comprising administering to a patient, a composition according to any one of embodiments 8- 12 intranasally or by inhalation.
  • composition of embodiment 1 or 8 wherein the antibodies are specific to one or more protein of SARS CoV-2 selected from the group consisting of spike (S) protein/polypeptide, membrane (M) polypeptide/protein, envelope (E) polypeptide/protein, SARS CoV-2 capsid (C) structural protein and combinations of two or more of the foregoing.
  • a composition for delivery via intravenous route to a human patient comprising (i) polyclonal antibodies or antigen binding fragments thereof specific to one or more proteins of SARS CoV-2, including without limitation the spike (S) protein thereof, wherein the polyclonal antibodies are obtained from the colostrum of bovine or other ruminant that has been immunized with SARS CoV-2 virus or one or more proteins thereof or an antigenic fragment of the one or more proteins; or (ii) one or more monoclonal antibodies specific to one or more proteins of SARS CoV-2, wherein the antibodies are derived from antibodies obtained from the colostrum of a bovine or other ruminant that has been immunized with SARS CoV-2 virus or one or more proteins thereof or an antigenic fragment of the one or more proteins, and wherein upon administration to the patient, the composition is therapeutically effective to treat CO VID 19 in the patient, and wherein the antibodies are present in the composition at a dosage of about 0.01 mg per dose to about to about 1
  • composition of embodiment 22 formulated as an infusible solution or suspension for administration by infusion.
  • composition of embodiment 23 wherein the solution or suspension comprises one or more diluents and excipients suitable for administration by infusion.
  • a method of treating COVID-19 infection in a human patient in need thereof comprising administering to a patient by infusion, a composition according to any one of embodiments 22 - 24.
  • a composition for delivery via intravenous route to a human patient comprising (i) polyclonal antibodies or antigen binding fragments thereof specific to one or more proteins of SARS CoV-2, including without limitation the spike (S) protein thereof, wherein the polyclonal antibodies are obtained from the serum of bovine or other ruminant that has been immunized with SARS CoV-2 virus or one or more proteins thereof or an antigenic fragment of the one or more proteins; or (ii) one or more monoclonal antibodies specific to one or more proteins of SARS CoV-2, wherein the antibodies are derived from antibodies obtained from the serum of a bovine or other ruminant that has been immunized with SARS CoV- 2 virus or one or more proteins thereof or an antigenic fragment of the one or more proteins, and wherein upon administration to the patient, the composition is therapeutically effective to treat COVID 19 in the patient, and wherein the antibodies are present in the composition at a dosage of about 0.01 mg per dose to about to about 1 gram or higher up
  • composition of embodiment 29 wherein the solution or suspension comprises one or more diluents and excipients suitable for administration by infusion.
  • a method of treating COVID-19 infection in a human patient in need thereof comprising administering to a patient by infusion, a composition according to any one of embodiments 28 - 30.
  • Figure 1 Generation and characterization of bovine anti-SARS-CoV-2 antibodies. As seen in Figure 1, antibodies directed against SARS-CoV-2 Spike Protein Receptor Binding Domain from group A (calves #1-3) bind to the wildtype SARS-CoV-2 Spike Protein Receptor Binding Domain.
  • Figure 2 Generation and characterization of bovine anti-SARS-CoV-2 antibodies.
  • FIG. 3 Generation and characterization of bovine anti-SARS-CoV-2 antibodies.
  • the binding of antibodies directed against a wildtype SARS-CoV-2 Spike protein for example, a wildtype SARS-CoV-2 Spike protein in the form of a trimer
  • SARS-CoV-2 Spike protein variant N501Y was measured.
  • calves immunized with the wildtype SARS-CoV-2 Spike protein provide antibodies which bind to a variant SARS-CoV-2 Spike proteins.
  • Figure 4 Generation and characterization of bovine anti-SARS-CoV-2 antibodies.
  • the binding of antibodies directed against a wildtype SARS-CoV-2 Spike protein for example, a wildtype SARS-CoV-2 Spike protein in the form of a trimer
  • SARS-CoV-2 Spike protein variant E48K was measured.
  • calves immunized with the wildtype SARS-CoV-2 Spike protein provide antibodies which bind to a variant SARS-CoV-2 Spike protein.
  • Figure 5 Pseudovirus neutralization assay for SARS-CoV-2.
  • the pseudovirus neutralization inhibition rate of the tested serum is calculated based on the luciferase luminescence value.
  • the results of the neutralization assay for SARS-CoV-2 pseudovirus and a spike protein variant is shown in Figure 5.
  • the EC50 values for Sample 1 and Sample 2 are presented in the figure.
  • the example demonstrates that the serum antibodies of Sample 1 and Sample 2 are capable of binding the Spike Protein Receptor Binding Domain of SARS-CoV-2 and variants thereof and blocking the binding of the SARS-CoV-2 spike protein RBD to its receptor ACE2.
  • Figure 6 Pseudovirus neutralization assay for SARS-CoV-2.
  • the pseudovirus neutralization inhibition rate of the tested serum is calculated based on the luciferase luminescence value.
  • the results of the neutralization assay for SARS-CoV-2 pseudovirus spike protein variants is shown in Figure 6.
  • the EC50 values for Sample 1 and Sample 2 are presented in the figure.
  • the example demonstrates that the serum antibodies of Sample 1 and Sample 2 are capable of binding the Spike Protein Receptor Binding Domain of SARS-CoV-2 variants and blocking the binding of the SARS-CoV-2 spike protein RBD to its receptor ACE2.
  • Figure 7 Pseudovirus neutralization assay for SARS-CoV-2.
  • the pseudovirus neutralization inhibition rate of the tested serum is calculated based on the luciferase luminescence value.
  • the results of the neutralization assay for SARS-CoV-2 pseudovirus spike protein variants is shown in Figure 7.
  • the EC50 values for Sample 1 and Sample 2 are presented in the figure.
  • the example demonstrates that the serum antibodies of Sample 1 and Sample 2 are capable of binding the Spike Protein Receptor Binding Domain of SARS-CoV-2 variants and blocking the binding of the SARS-CoV-2 spike protein RBD to its receptor ACE2.
  • antibody is referred to in the broadest sense and encompasses monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments including antigen binding fragments, such as Fab and Fab’ fragments.
  • antibody fragments include, but are not limited to, (i) the Fab fragment consisting of VL, VH, CL and CH1 domains, (ii) the Fd fragment consisting of the VH and CH1 domains, (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment which consists of a single variable, (v) isolated CDR regions, (vi) F(ab’)2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site, (viii) bispecific single chain Fv dimers and (ix) "diabodies” or "triabodies", multivalent or multispecific fragments constructed by gene fusion.
  • the antibody fragments may be modified. For example, the molecules may be stabilized by the incorporation of
  • Antibodies thus can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins.
  • Antibodies are typically tetramers of immunoglobulin molecules.
  • the antibodies in the present disclosure may exist in a variety of forms including, for example, polyclonal antibodies and monoclonal antibodies.
  • the use of the singular terms “a” or “an” or “the” antibody are not meant to be limited to a single antibody when it is clear that more than one antibody is present in the composition or preparation.
  • the singular term for "antibody” may include a collection of antibodies that are not necessarily heterogenous in their structures or specificities unless indicated otherwise.
  • Antibodies are generally glycoproteins comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, i.e., covalent heterotetramers comprised of two identical Ig H chains and two identical L chains that are encoded by different genes.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region of an IgG subclass of immunoglobulins for example, is comprised of three domains, CH1 , CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • CL The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRI, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells). Formation of a mature functional antibody molecule can be accomplished when two proteins are expressed in stoichiometric quantities and self-assemble with the proper configuration.
  • the term "monoclonal antibody” refers to an antibody that displays a single binding specificity and affinity for a particular epitope or a composition of antibodies in which all antibodies display a single binding specificity and affinity for a particular epitope.
  • an “isolated antibody” refers to an antibody that is substantially free of colostrum proteins and non-antibody constituents or, in the case of monoclonal antibody free of other antibodies having different antigenic specificities.
  • isotype refers to the antibody class (e.g., IgG 1 , IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE antibody) that is encoded by the heavy chain constant region genes.
  • the recombinant antibody of the present disclosure antibody is an IgG antibody, e.g. IgGl, IgG2.
  • the recombinant antibody of the present disclosure is an IgGl antibody.
  • Fc or “Fc region”, as used herein is meant the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain.
  • Hinge regions are generally encoded by unique exons, and contain disulfide bonds that link the two heavy chain fragments of the antibody.
  • the amino acid sequence of a hinge region can be generally rich in proline, serine, and threonine residues.
  • the extended peptide sequences between the CH1 and CH2 domains of IgG, IgD, and IgA are rich in prolines.
  • IgM and IgE antibodies include a domain of about 110 amino acids that possesses hinge-like features and are included in the term "hinge region" as used herein.
  • region equivalent to the hinge region of a ruminant antibody is intended to include naturally occurring allelic variants of the hinge region of a ruminant (e.g., bovine) immunoglobulin of any isotype as well as variants having alterations which produce substitutions, additions, or deletions but which do not decrease substantially the ability of die immunoglobulin to resist enzyme digestion. Such variants can be selected according to general rules known in the art so as to have minimal effect on activity.
  • a CH1 domain is an immunoglobulin heavy chain constant region domain.
  • ruminant immunoglobulin CH 1 domains such as a bovine, ovine or caprine with the bovine CH1 domains being preferred.
  • the amino acid sequence of immunoglobulin CH11 domains of various species are known or are generally available to the skilled artisan (Kabat et al., Sequences of proteins of immunological interest Fifth Ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
  • the CH1 domain is a bovine IgGl CH1 domain.
  • the immunoglobulin CH1 domain is of the d allotype.
  • region equivalent to CH 1 domain of a ruminant antibody is intended to include naturally occurring allelic variants of the CH1 domain of a ruminant (e.g., bovine) immunoglobulin of any isotype as well as variants having alterations which produce substitutions, additions, or deletions but which do not decrease substantially the ability of the immunoglobulin to resist enzyme digestion. Such variants can be selected according to general rules known in the art so as to have minimal effect on activity.
  • a CH2 domain is an immunoglobulin heavy chain constant region domain.
  • the CH2 domain is preferably the CH2 domain of one of the five immunoglobulins subtypes indicated above.
  • Preferred are ruminant immunoglobulin CH2 domains such as a bovine, ovine or caprine with the bovine CH2 domains being preferred.
  • the amino acid sequence of immunoglobulin CH2 domains of various species are known or are generally available to the skilled artisan (Rabat et al., Sequences of proteins of immunological interest Fifth Ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
  • a preferred immunoglobulin CH2 domain within the context of the present disclosure is a bovine IgG and preferably from bovine IgG 1 , or IgG2, and more preferably a bovine IgGl.
  • ruminant antibody molecules reference is made to the IgG class in which an N-linked oligosaccharide is attached to the amide side chain of Asn of the inner face of the CH2 domain of the Fc region. This site is equivalent to Asn 297 of the human IgG I immunoglobulin molecule. It is characteristic of the recombinant antibody of the present disclosure that it contain or be modified to contain at least a CH2 domain.
  • region equivalent to CH2 domain of a ruminant antibody is intended to include naturally occurring allelic variants of the CH2 domain of a ruminant (e.g. bovine) immunoglobulin of any isotype as well as variants having alterations which produce substitutions, additions, or deletions but which do not decrease substantially the ability of the immunoglobulin to resist enzyme digestion. Such variants can be selected according to general rules known in the art so as to have minimal effect on activity.
  • a CH3 domain is an immunoglobulin heavy chain constant region domain.
  • the CH3 domain is preferably the CH3 domain of one of the five immunoglobulins subtypes indicated above.
  • Preferred are ruminant immunoglobulin CH3 domains such as a bovine, ovine or caprine with the bovine CH3 domains being preferred.
  • the amino acid sequence of immunoglobulin CH3 domains of various species are known or are generally available to the skilled artisan (Kabat et al., Sequences of proteins of immunological interest Fifth Ed., U.S. Department of Health and Human Services, NIH Publication No. 91- 3242).
  • a preferred immunoglobulin CH3 domain within the context of the present disclosure is from bovine IgG CH3 domain, and preferably a bovine IgGl CH3 domain or bovine IgG2 CHS domain, and more preferably a bovine IgGl CHS domain.
  • region equivalent to CHS domain of a ruminant antibody is intended to include naturally occurring allelic variants of the CHS domain of a ruminant (e.g., bovine) immunoglobulin of any isotype as well as variants having alterations which produce substitutions, additions, or deletions but which do not decrease substantially the ability of the immunoglobulin to resist enzyme digestion. Such variants can be selected according to general rules known in the art so as to have minimal effect on activity.
  • region equivalent to the Fc region of a ruminant antibody is intended to include naturally occurring allelic variants of the Fc region of a ruminant immunoglobulin of any isotype as well as variants having alterations which produce substitutions, additions, or deletions but which do not decrease substantially the ability of the immunoglobulin to resist enzyme digestion. Such variants can be selected according to general rules known in the art so as to have minimal effect on activity.
  • the term "host cell” covers any kind of cellular system which can be engineered to generate the antibodies disclosed herein
  • Host cells include cultured cells, e.g., mammalian cultured cells, such as CHO cells, HEK293T cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, fungal cells, and insect cells, to name only a few, but also cells comprised within a transgenic animal or cultured tissue.
  • mammalian cultured cells such as CHO cells, HEK293T cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, fungal cells, and insect cells, to name only a few, but also cells comprised within
  • host cells are ruminant mammary epithelial cells including but not limited to: ruminant mammary epithelial cell lines such as Bovine cell lines BMEC+H (Bovine Mammary Epithelial Cells of the Hormone-adapted), HH2A (spontaneously immortalized bovine mammary epithelial cell line), ET-C (epithelial and myoepithelial-like characteristics) and Mac-T (Mammary Alveolar Cells).
  • ruminant mammary epithelial cell lines such as Bovine cell lines BMEC+H (Bovine Mammary Epithelial Cells of the Hormone-adapted), HH2A (spontaneously immortalized bovine mammary epithelial cell line), ET-C (epithelial and myoepithelial-like characteristics) and Mac-T (Mammary Alveolar Cells).
  • an "isolated antibody” or “isolated antibodies”, as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities.
  • An isolated antibody may be substantially free of other cellular material and/or chemicals.
  • the term “substantially” in any of the definitions herein generally means at least about 60%, at least about 70%, at least about 80%, or more preferably at least about 90%, and still more preferably at least about 95%. Isolated also means separation from contaminant components of the antibodies' natural environment that would typically interfere with diagnostic or therapeutic uses of the antibody, such as enzymes, hormones, and other materials.
  • a non-naturally occurring antibody does not require “isolation” to distinguish it from its naturally occurring counterpart.
  • a “concentrated”, “separated” or “diluted” antibody is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is generally greater than that of its naturally occurring counterpart.
  • an antibody made by recombinant means and expressed in a host cell is considered to be “isolated.”
  • specific binding refers to antibody binding to a predetermined epitope, isoform or variant of an antigen.
  • the antibody binds with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen other than the predetermined antigen or a closely-related antigen. Therefore, the antibodies provided herein in some embodiments specifically bind a target antigen.
  • affinity or “binding affinity” as used herein is meant the strength of interaction between an antibody and its target antigen.
  • the strength of affinity is often reported with a equilibrium dissociation constant, KD, which is obtained from the ratio of kd to ka (i.e,. kd/ka) and is expressed as a molar concentration (M).
  • KD values for antibodies can be determined using methods well established in the art (e.g., Biacore surface Plasmon resonance, flow cytometry, Scatchard analysis). Lower values of KD correspond to tighter binding and higher affinity. Higher values of KD correspond to weaker binding and lower affinity.
  • target antigen refers to the molecule that is bound specifically by the variable region of a given antibody.
  • a target antigen may be a protein, carbohydrate, lipid, or other chemical compound.
  • bovine chimeric antibody refers to a recombinant antibody that retains at least the antigen binding CDRs or variable region ( VR) from a parent (non-bovine) antibody, and includes at least a portion of a bovine IgGl constant region (e.g., a bovine IgGl CH1 and/or hinge domain).
  • a bovinized antibody comprises the variable regions and CH2 and CH3 domains of a parent antibody (e.g., a human antibody), and the hinge region and CH1 domain of a bovine IgGl antibody.
  • a bovinized antibody comprises one or more amino acid residues in the parent antibody that are replaced with the equivalent residue(s) of a bovine constant region, e.g., a bovine IgGl constant region.
  • the bovinized antibody comprises the variable region of a parent antibody, and the CH1 domain, hinge region, CH2 domain, and CH3 domain of a bovine IgGl antibody (i.e., a "bovine chimeric antibody").
  • a "parent antibody” is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments including antigen binding fragments such as an Fc-fusion protein wherein the "parent antibody” is the basis for modification to generate a variant of the present disclosure also referred to herein as a "bovinized antibody".
  • the term parent antibody may refer to the parent antibody itself, a composition comprising the parent antibody or the amino acid sequence or nucleic acid sequence that encodes the parent antibody.
  • the term "fully bovine recombinant monoclonal antibody” is intended to include recombinant antibodies having variable and constant regions derived from bovine germline immunoglobulin sequences.
  • the fully bovine antibodies of the present disclosure may include amino acid residues not encoded by the bovine germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo),
  • bovine antibody is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse have been grafted onto bovine framework sequences.
  • the antibody is a full-length antibody.
  • the full-length antibody comprises a heavy chain and a light chain.
  • a "therapeutic recombinant antibody” as that term is used herein is a recombinant antibody that use useful in preventing or ameliorating a disease, disorder or condition when administered to a subject in need of treatment.
  • recombinant antibody is intended to include antibodies that are prepared, expressed, created or isolated by recombinant means including, but not limited to, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences.
  • recombinant antibodies may be single-chain antibodies.
  • N-linked oligosaccharides are those oligosaccharides that are linked to a peptide backbone through asparagine, by way of an asparagine-N-acetylglucosamine linkage. N linked oligosaccharides are also called “N-linked glycans.” All N-linked oligosaccharides have a common pentasaccharide core of Man3GlcNAc2. They differ in the presence of, and in the number of branches (also called antennae) of peripheral sugars such as N acetylglucosamine, galactose, N-acetylgalactosamine, fucose and sialic acid. Optionally, this structure may also contain a core fucose molecule and/or a xylose molecule.
  • O-linked oligosaccharides are those oligosaccharides also referred to herein as "O linked glycans" are linked to a peptide backbone through threonine, serine, hydroxyproline, tyrosine, or other hydroxy-containing amino acids.
  • sialic acid refers to any member of a family of nine-carbon carboxylated sugars.
  • the most common member of the sialic acid family is N-acetyl neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-l-onic acid (often abbreviated as Neu5Ac, NeuAc, or NANA).
  • a second member of the family is N-glycolyl- neuraminic acid (Neu5Gc or NeuGc), in which the N-acetyl group of NeuAc is hydroxylated.
  • a third sialic acid family member is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al, JBC 1986;261:11550-7; Kanamori et al., JBC 1990;65:21811-9). Also included are 9-substituted sialic acids such as a 9-0-C 1-C6 acyl-Neu5Ac like 9-0-lactyl Neu5Ac or 9-0-acetyl-Neu5Ac, 9-deoxy- 9-fluoro-Neu5 Ac and 9-azido-9-deoxy-Neu5Ac.
  • KDN 2-keto-3-deoxy-nonulosonic acid
  • 9-substituted sialic acids such as a 9-0-C 1-C6 acyl-Neu5Ac like 9-0-lactyl Neu5Ac or 9-0-acetyl-Neu5Ac, 9-deoxy- 9-fluoro-Neu5 Ac and 9-azid
  • sialylated or “sialylation” refers to the addition of sialyl acid groups to oligosaccharide groups present on a glycosylated peptide such as an antibody. Such addition may be by natural enzymatic processes taking place in, for example a cell or via chemical glycoengineering.
  • isolated nucleic acid refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs.
  • the term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid nucleic acid encoding additional peptide sequence.
  • a "polynucleotide” means a single strand or parallel and anti-parallel strands of a nucleic acid.
  • a polynucleotide may be either a single-stranded or a double-stranded nucleic acid.
  • nucleic acid typically refers to large polynucleotides.
  • oligonucleotide typically refers to short polynucleotides, generally no greater than about 50 nucleotides.
  • Conventional notation is used herein to describe polynucleotide sequences: the left- hand end of a single-stranded polynucleotide sequence is the 5 -end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction.
  • the direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • nucleic acid sequence encodes a protein if transcription and translation of mRNA corresponding to that nucleic acid produces the protein in a cell or other biological system.
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that nucleic acid or cDNA.
  • a "nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.
  • homologous refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two peptide molecules.
  • two nucleic acid molecules e.g., two DNA molecules or two RNA molecules
  • two peptide molecules e.g., two amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, or a sequence similarity between two polymeric molecules.
  • the homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology.
  • the DNA sequences 3'ATTGCC5' and 3TATGGC share 50% homology.
  • BLAST protein searches can be performed with the XBLAST program (designated "blastn” at the NCBI web site) or the NCBI “blastp” program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein.
  • Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res 1997;25:3389-402).
  • PSI-Blast or PHl-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.) and relationships between molecules which share a common pattern.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
  • a "heterologous nucleic acid expression unit" encoding a peptide is defined as a nucleic acid having a coding sequence for a peptide of interest operably linked to one or more expression control sequences such as promoters and/or repressor sequences wherein at least one of the sequences is heterologous, i.e., not normally found in the host cell.
  • two polynucleotides as "operably linked” is meant that a single stranded or double-stranded nucleic acid moiety comprises the two polynucleotides arranged within the nucleic acid moiety in such a manner that at least one of the two polynucleotides is able to exert a physiological effect by which it is characterized upon the other.
  • a promoter operably linked to the coding region of a nucleic acid is able to promote transcription of the coding region.
  • promoter/regulatory sequence means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulator sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • a "constitutive promoter is a promoter which drives expression of a gene to which it is operably linked, in a constant manner in a cell. By way of example, promoters which drive expression of cellular housekeeping genes are considered to be constitutive promoters.
  • An "inducible" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living cell substantially only when an inducer which corresponds to the promoter is present in the cell.
  • a "tissue-specific" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
  • a "vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term "vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • Expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant polynucleotide.
  • a “genetically engineered” or “recombinant” cell is a cell having one or more modifications to the genetic material of the cell. Such modifications are seen to include, but are not limited to, insertions of genetic material, deletions of genetic material and insertion of genetic material that is extrachromasomal whether such material is stably maintained or not.
  • “native form” means the form of the peptide when produced by the cells and/or organisms in which it is found in nature. When the peptide is produced by a plurality of cells and/or organisms, the peptide may have a variety of native forms.
  • Peptide refers to a polymer in which the monomers are amino acids and are joined together through amide bonds, alternatively referred to as a peptide. Additionally, unnatural amino acids, for example, p-alanine, phenylglycine and homoarginine are also included.
  • amino acids that are not nucleic acid-encoded may also be used in the present disclosure.
  • amino acids that have been modified to include reactive groups, glycosylation sites, polymers, therapeutic moieties, biomolecules and the like may also be used in the present disclosure. All of the amino acids used in the present disclosure may be either the D- or L isomer thereof. The L-isomer is generally preferred.
  • other peptidomimetics are also useful in the present disclosure.
  • “Peptides'* include, for example, oligopeptides, polypeptides, peptides, proteins, or glycoproteins.
  • polypeptide refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit on the length of the chain.
  • One or more amino acid residues in the protein may contain a modification such as, but not limited to, glycosylation, phosphorylation or a disulfide bond.
  • a “protein” may comprise one or more polypeptide chains.
  • protein and polypeptide can be and commonly are used interchangeably
  • Analogs may differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. For example, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function. Conservative amino acid substitutions typically include substitutions within the following groups:
  • valine isoleucine, leucine
  • Modifications include in vivo, or in vitro, chemical derivatization of peptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a peptide during its synthesis and processing or in further processing steps; e.g., by exposing the peptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
  • the peptides may incorporate amino acid residues which are modified without affecting activity.
  • the termini may be derivatized to include blocking groups, i.e. chemical substituents suitable to protect and/or stabilize the N- and C-termini from "undesirable degradation", a term meant to encompass any type of enzymatic, chemical or biochemical breakdown of the compound at its termini which is likely to affect the function of the compound, i.e. sequential degradation of the compound at a terminal end thereof.
  • MALDI Matrix Assisted Laser Desorption Ionization
  • SA-PEG sialic acid-poly(ethylene glycol)
  • a "ruminant” is any mammal of the suborder Ruminantia which include domestic animals such as camel, deer, cows (bovine), sheep (ovine) and goats (caprine).
  • "early colostrum” or “early colostra! includes colostrum produced
  • early colostrum is the colostrum produced within 1 to 3 days after birth. In a most preferred embodiment, early colostrum is the colostrum produced within 24 to 36 hours after birth.
  • the "digestive tract” consists of the mouth, pharynx, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum) and anus.
  • the digestive tract is a human digestive tract
  • airway includes the nasal cavity, the larynx, bronchial passages and lung, including the mucosal surfaces thereof.
  • the "oral cavity” includes the mouth, the pharynx and the esophagus.
  • oral degradation of an antibody is used herein to mean degradation of an antibody in the oral cavity by endogenous or exogenous enzymes present in the oral cavity.
  • GI tract includes the stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum) and anus.
  • intestinal digestion refers to digestion in the small intestine and/or the large intestine.
  • intestinal degradation of an antibody refers to degradation of an antibody in the small intestine and/or large intestine by endogenous or exogenous enzymes present in the small intestine and large intestine or due to exposure to acidic conditions during intestinal digestion.
  • “enhanced” or “increased” ability to resist cleavage by proteases and/or enhanced or increased resistance to digestion or degradation during intestinal digestion in a mammal is intended to refer to a recombinant antibody or composition thereof that exhibits greater resistance to protease cleavage or intestinal digestion produced by the methods of the present disclosure that impart the unique features of ruminant early colostral IgG antibodies that enable such antibodies to resist cleavage and digestion as compared to a recombinant antibody or composition thereof produced by a method that does not impart such features to the antibodies.
  • Proteases to which bovinized antibody variants of the present disclosure have increased resistance may include, but are not limited to, simulated intestinal fluid, papain, pepsin, a matrix metalloproteinase including MMP-7, neutrophil elastase (HNE), stromelysin (MMP 3), macrophage elastase (MMP- 12), trypsin, chymotrypsin, and other proteases as compared to antibodies that do not possess the features of the present disclosure.
  • antibody preparation as used herein is used to define a composition comprising antibodies of the present disclosure wherein contaminant components, such as materials which would interfere with diagnostic or therapeutic uses for the antibodies are substantially reduced.
  • the antibody preparations of the present disclosure will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • treatment or “treating,” or “palliating” or “ameliorating” is used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit.
  • therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated.
  • a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.
  • the compositions may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.
  • a "therapeutic effect”, as used herein, refers to a physiologic effect, including but not limited to the cure, mitigation, amelioration, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental well being of humans or animals, wherein the such therapeutic effect is facilitated by a recombinant antibody of the present disclosure.
  • terapéuticaally effective amount refers to an amount of a polyclonal or monoclonal antibody of the present disclosure, either alone, or in combination with another therapeutic, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject. Such effect need not be absolute to be beneficial.
  • terapéuticaally effective dose regimen refers to a schedule for consecutively administered doses of an antibody of the present disclosure, either alone or as part of a combination with another therapeutic, wherein the doses are given in therapeutically effective amounts to result in sustained beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition.
  • patient and “subject” refer to any human or non-human animal that receives either prophylactic or therapeutic treatment.
  • the methods and compositions described herein can be used to treat a subject having a gastrointestinal disorder associated with SARS-CoV-2 infection.
  • non-human animal includes all vertebrates, e.g., mammals and non mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.
  • bottle refer to members of the subfamily Bovinae, and include the species Bos indicus and Bos taurus. As used herein, “cattle” is intended to encompass calves, mature cows, steers, and bulls.
  • Described herein are recombinant antibodies having an increased ability to resist cleavage by proteases and resist degradation during digestion in the digestive tract of a mammal (e.g., during human digestion), as well as methods for preparing the same.
  • Bovine antibodies and particularly early colostral bovine IgG, are known to have a natural resistance to digestion in the human gastrointestinal tract (Wamy et al., Gut 1999;44:212-217).
  • colostrum The initial secretion present in the mammary gland at or near the time of parturition is termed colostrum, which arises during a distinct physiological and functional stage of mammary gland development that is completely different from the gland's primary role of milk production.
  • colostrogenesis the transfer of immunoglobulins from the maternal circulation into mammary secretions in domestic ruminants begins several weeks prior to parturition and ceases after parturition. During this time 500 g - 3 kg of IgG are transferred into mammary secretions and early studies have established that the source of colostral immunoglobulin is the maternal circulation.
  • Colostrum is unique in its composition and function.
  • the principal difference between colostrum and milk is the high concentration of colostral immunoglobulin, specifically IgGl .
  • IgGl is concentrated to levels 5-10 times that of serum where it accounts for greater than 90% of total colostral protein.
  • the process by which immunoglobulin is transferred from maternal circulation into the mammary gland is the result of transcytosis, a process by which various macromolecules including immunoglobulins are transported across the interior bovine mammary epithelial cells.
  • Immunoglobulins are captured in vesicles on the basal- lateral side of the mammary epithelium cell, drawn across the cell and ejected at the apical surface into colostrum.
  • Polyclonal antibodies of the invention derived from the ruminant, for example cow, which are found in the serum of immunized animals may be used for injection/infusion and by inhalation. While serum antibodies are not as stable in the gut as colostrum antibodies, the serum- derived antibodies would be suitable for other modes of administration known in the art.
  • spike polypeptide having structural features associated with bovine IgGl antibodies that impart enhanced protease resistance and which can be orally administered for the treatment of COVID-19.
  • bovine antibodies that retain at least the antigen binding CDRs or variable region (VR) from a parent (non-bovine) antibody, and includes all or a portion of a bovine IgGl constant region (e.g., a bovine IgG1 CH1 and/or hinge domain).
  • a bovine IgGl constant region e.g., a bovine IgG1 CH1 and/or hinge domain.
  • bovinized antibodies which comprises the variable region of a parent antibody, and the constant region (i.e., CH1 domain, hinge region, CH2 domain, and CH3 domain) of a bovine IgGl antibody.
  • bovine chimeric antibodies These antibodies are referred to as "bovine chimeric antibodies.”
  • bovinized antibodies that fully or partially replace one or more constant region domain(s) of a parent (i.e., non-bovine) antibody with the equivalent bovine IgG 1 constant region domain(s).
  • Exemplary bovine IgG 1 antibody constant regions are presented in Table 1.
  • bovinized antibodies that incorporate 1, 2, 3, or all of the following structural features found in bovine IgGl that impart protease resistance to the antibody:
  • the bovinized antibody comprises the variable domain, CH2 domain, and CH3 domain of the parent antibody, and at least a portion (i.e., one or more amino acid residues) of a bovine IgGl CH1 and/or hinge domain.
  • the CH1 domain of the antibody is at least 85% identical, for example, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% identical, to the bovine IgGl CH1 and/or hinge domain.
  • the bovinized antibodies described herein comprise a CH1 domain from a parent antibody (e.g., a human CH1 domain), wherein the amino acid residues equivalent to Cys 127 and Cys 128 (according to Rabat numbering) in bovine IgGl are replaced with cysteine in the parent antibody.
  • the antibody comprises a human CH1 domain with S127C and/or SI28C substitutions (numbering according to Rabat).
  • the antibody comprises a human CH1 domain with S127C and/or SI 28C amino acid substitutions, wherein 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids upstream (N-terminal) and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids downstream (C-terminal) of amino acid residues 127 and 128 are replaced with the equivalent bovine IgGl CH1 residues.
  • the bovinized antibody comprises a bovinized hinge region.
  • the antibody comprises a human IgG hinge, wherein sites within the hinge which are susceptible to enzymatic or spontaneous proteolysis are replaced with the equivalent bovine hinge region sequences.
  • the bovine IgGl hinge region sequence differs from the human IgG 1 hinge region sequence at sites known to be susceptible to protease digestion.
  • the bovinized antibody comprises a bovinized hinge region, in which some or all of the amino acid residues of the hinge region are replaced with the equivalent portion of the bovine IgGl hinge region sequence.
  • the bovinized antibody comprises a bovinized hinge region which comprises a bovine heavy chain-heavy chain linkage, wherein a cluster of three disulfide bonds link the hinge regions of two heavy chains together.
  • the antibody comprises a hinge of the parent antibody, wherein the amino acid residues equivalent to the underlined cysteine residues in the bovine hinge region below are replaced with cysteine (if not already cysteine) in the hinge of the parent antibody, for example, DRAVDPRCRPSPCDCCPPPELPGGP (SEQ ID NO:8)
  • the antibody comprises a human hinge region sequence, wherein Pro241 (Rabat numbering) is replaced with a cysteine residue (i.e., a P241C substitution).
  • the bovinized antibodies exhibit greater protease resistance relative to the parent antibody.
  • the bovinized antibodies retain greater antigen-binding activity after protease digestion relative to the parent antibody.
  • the bovinized antibodies upon being subjected to protease digestion in the digestive tract, retain antigen-binding activity. In certain embodiments, the bovinized antibodies remain bivalent upon digestion by proteases in the digestive tract. In one embodiment, the bovinized antibodies are digested into F(ab') 2 fragments by proteases in the digestive tract.
  • the protease resistance of bovinized antibodies comprising a bovine IgG1 hinge is increased by partially incorporating bovine IgG3 hinge region sequences into the IgGl hinge region.
  • pepsin cleavage can be prevented by adding a bovine IgG3 hinge domain having a putative O-glycosylation site to the bovine IgGl constant region.
  • the antigen-binding region of the bovinized antibodies lack protease-sensitive sequences. Such determinations can be made by comparing the primary sequence of the variable region (e.g., the CDR regions) with known protease consensus sequences, or tested experimentally using pancreatin digestion methods described in the Examples.
  • portions of the constant regions of other ruminants that share structural similarities with the bovine constant region that imparts protease stability can be incorporated into the recombinant antibodies described herein.
  • these structurally similar sequences are derived from the rabbit IgG1 constant region.
  • the bovinized antibody comprises one or more additional amino acid substitutions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions) in the hinge or Fc region that increase or decrease antibody effector activity and/or FcRn binding activity.
  • additional amino acid substitutions e.g., 1, 2, 3, 4, or 5 amino acid substitutions
  • Particular amino acid substitutions include those that generate an Fc variant that (a) has increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased or decreased complement mediated cytotoxicity (CDC), (c) has increased or decreased affinity for Cl q and/or (d) has increased or decreased affinity for a Fc receptor relative to the parent Fc.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement mediated cytotoxicity
  • c has increased or decreased affinity for Cl q
  • d has increased or decreased affinity for a Fc receptor relative to the parent Fc.
  • the Fc region is altered by species replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody.
  • amino acids selected from amino acid residues 234, 235,
  • the effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. See, e.g., U.S. Patent Nos. 5,624,821 and 5,648,260.
  • one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC). See, e.g., U.S. Patent No. 6,194,551.
  • one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement See, e.g., PCT Publication WO 94/29351.
  • the Fc region may be modified to increase antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity for an Fey receptor by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294.295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333,334,335, 337, 338, 340, 360, 73, 376, 378, 3823 88, 389, 398, 414, 416, 419, 430, 433,
  • Exemplary substitutions include 236A, 239D, 239E, 268D 267E, 268E, 268F, 324T, 332D. and 332E.
  • Exemplaiy variants include 239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F, 267E/324T, and 267E/268F/324T.
  • Fc modifications that increase binding to an Fey receptor include amino acid modifications at any one or more of amino acid positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285., 298, 289, 290, 292., 29, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 324, 327, 329, 330, 335, 337, 3338, 340, 360, 373, 376, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Rabat (WO00/42072).
  • Fc modifications that can be made to Fes are those for reducing or ablating binding to FeyR and/or complement proteins, thereby reducing or ablating Fc-mediated effector functions such as ADCC, ADCP, and CDC.
  • Exemplary modifications include but are not limited substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, and 328, wherein numbering is according to the EU index.
  • Exemplary substitutions include but are not limited to 2340, 235G, 236R, 237K, 267R, 269R, 325L, and 328R, wherein numbering is according to the EU index.
  • An Fc variant may comprise 236R/328R.
  • the Fc region may comprise a non-naturally occurring amino acid residue at additional and/or alternative positions known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; PCT Patent Publications WO 00/42072; WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752; WO 04/074455; WO 04/099249; WO 04/06335 1; WO 05/070963; WO 05/040217, WO 05/092925 and WO 06/020 114).
  • Fc variants that enhance affinity for an inhibitory receptor FcyRllb may also be used. Such variants may provide an Fc fusion protein with inmunomodulatory activities related to FcyRllb, cells, including for example B cells and monocytes. In one embodiment, the Fc variants provide selectively enhanced affinity to FcyRllb relative to one or more activating receptors. Modifications for altering binding to FcyRllb include one or more modifications at a position selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, and 332, according to the EU index.
  • Exemplary substitutions for enhancing FcyRllb affinity include but are not limited to 234D, 234E, 234F, 234W, 235D, 235F, 23R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E.
  • Exemplary substitutions include 235 Y, 236D, 2391 , 266M, 267E, 268D, 268E, 328F, 328W, and 328Y.
  • Fc variants for enhancing binding to FeyRulb include 235Y/267E, 236D/267E, 239D/268D, 239D/267E, 267E/268D, 267E/268E, and 267E/328F.
  • the hinge is a bovinized hinge, and comprises one or more of the amino acid substitutions described herein.
  • IgGl mutants containing L235V, F243L, R292P, Y300L and P396L mutations which exhibited enhanced binding to FcyRJIIa and concomitantly enhanced ADCC activity in transgenic mice expressing human FcyRlIIa in models of B cell malignancies and breast cancer have been identified (Stavenhagen et al., 2007; Nordstrom et al., 2011).
  • Other Fc mutants include: S298A/E333A/L334A, S239D/1332E, S239D/1332E/A330L,
  • Fc variants that enhance affinity for Protein A from Staphylococcus aureus may also be used.
  • Previous work (US Patent Application US20140154270A1) has shown that IgG from ruminant species binds to Protein A with poor affinity under conditions typically used for manufacturing.
  • Variants of the bovine sequence may provide an Fc fusion protein that can be purified by methods commonly used in the manufacture of biological therapeutics.
  • the antibody can be modified at positions 250-255, 288, 307-317, or 430-436 to amino-acids found in IgG molecules known to bind Protein A with high affinity.
  • substitutions may be chosen to enhance Protein A binding while retaining weak binding to human FcRn.
  • the Fc domain contains one or more (i.e., I, 2, 3, 4, or 5) of the following substitutions: T252M, G255R, Q309L, T314L, and G315N.
  • the Fc domain contains a T252M/G255R substitution.
  • the Fc domain contains a Q309L/T314L/G315N substitution.
  • the Fc domains contains a T252M/G255R/Q309L/T314L/G315N substitution.
  • the parent antibody comprises a hinge region, or is altered to comprise a hinge region, that is at least 85%, such as at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the equivalent portion of a hinge region comprising the amino acid sequence of bovine IgGl (S22080): DKAVDPRCKPSPCDCCPPPELPGGP (SEQ ID NO: 8).
  • the parent antibody comprises a hinge region, or is altered to comprise a hinge region, that is at least 85%, such as at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the equivalent portion of a hinge region disclosed in PCT/US2016/054304.
  • the parent antibody comprises or is altered to comprise a hinge region and a CH1 domain is at least 85%, such as at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the equivalent portion of a bovine, CH1 domain amino acid sequence (e.g., a bovine IgGl CH1 domain).
  • the parent antibody comprises or is altered to comprise a hinge region and a CH2 domain is at least 85%, such as at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the equivalent portion of a bovine, CH2 domain amino acid sequence (e.g., a bovine IgGl CH2 domain).
  • the parent antibody comprises or is altered to comprise a fusion between a VL domain with specific binding to a desired antigen and a bovine CL domain.
  • the parent antibody contains a CL domain corresponding to die encoded protein from the bovine IGLC 1, IGLC2, 1GLC3 or IGLC4 loci.
  • minimally bovinized human IgGl, 2, and 4 constant region sequences which exhibit protease resistance and be incorporated into parent antibodies (e.g., combined with the variable regions of the parent antibody) to create highly stable, digestion resistant antibodies which can be administered orally.
  • the constant regions of the parent antibody are replaced with the minimally bovinized sequences using routine recombinant techniques. Protease resistance can then be tested using the art-recognized methods described herein.
  • the minimally bovinized human IgGl construct was purified and tested by pancreatin digestion and shown to have comparable protease stability to bovine colostra! IgGl and significantly greater protease stability than the parent human IgGl molecule.
  • Table 2 Minimally bovinized human IgG1
  • the recombinant antibodies are capable of surviving digestion and reaching their target antigens.
  • the recombinant antibodies are therapeutic antibodies directed against SARS CoV-2 polypeptides that are bovinized as described herein or variants of existing therapeutic antibodies modified to increase resistance to cleavage by proteases and resist degradation during digestion in the digestive tract of a mammal, such as during human digestion.
  • the recombinant therapeutic antibody is a monoclonal antibody, a polyclonal antibody, or an anti-idiotype antibody which is specific for a target antigen.
  • the target antigen is derived from a virus.
  • the target antigen is derived from SARS CoV-2, for example, the target antigen is selected from a spike (S) protein/polypeptide, a membrane (M) polypeptide/protein, envelope (E) polypeptide/protein, SARS CoV-2 capsid (C) structural proteins and combinations of two or more of the foregoing.
  • S spike
  • M membrane
  • E envelope
  • C SARS CoV-2 capsid
  • the target antigen is a biological antigen (e.g., a human protein, a human peptide, or other antigenic molecule specific to humans), such as the antigens described further herein.
  • the bovinized antibody facilitates a therapeutic effect on a subject when administered to the subject.
  • Antibody fragments for example, which contain specific binding sites of the target antigen of interest may be generated by known techniques.
  • fragments include, but are not limited to, F(ab*) 2 fragments which can be produced by pepsin or other enzymic digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab*) 2 fragments.
  • Fab expression libraries may be constructed (Huse et al., Science 1989;246:1275-81) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity to the target protein of interest.
  • antibodies (bovinized antibodies and fully bovine recombinant monoclonal IgGl antibodies, polyclonal antibodies or anti-idiotype antibodies) which bind to the same epitope on a target of interest as a reference antibody (e.g., a therapeutic antibody), or competes with a reference antibody for binding to a target of interest. Whether a particular antibody binds to the same epitope on a target antigen as a reference antibody, or competes with binding to the same epitope on a target antigen as a reference antibody, can be readily determined by the skilled artisan using art-recognized epitope mapping and competition assays, respectively.
  • the antibodies described herein may be used to generate bispecific molecules.
  • the antibodies can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target
  • the antibodies described herein are administered systemically to a subject.
  • the antibody is not immunogenic in the subject being treated.
  • Such antibodies can be designed to avoid increasing the predicted risk for T cell epitopes, for example, as assessed using art-recognized algorithms and databases (see, e.g., Vita et al., Nucleic Acids Res 2015;43:D405-12; Vita et al, Nucleic Acids Res 38:D854-62, Nielsen et al., Protein Sci 2003;12:1007-17; Bui et al, Immunogenetics 2005;57:304-14; Lundegaard et al., J Immunol Methods 2011;374:226-34).
  • mutations are made in the Fc region of the antibodies described herein to increase resistance to host proteases, as described in, e.g., U.S. Patent No. 8,871,204, CA2.822.366, and US2013/0011386.
  • Fully bovine recombinant monoclonal IgGl antibodies also provided herein are fully bovine recombinant monoclonal IgGl antibodies and related oral compositions. Such antibodies can be generated by immunizing cattle (calves or mature cows, steers or bulls) with an antigen of interest, using the methods described in the Examples below. In certain embodiments, cattle are immunized with DNA encoding the antigen of interest. General methods for immunizing animals with genetic vaccines (e.g., DNA vaccines) are known in the art (see, e.g., U.S. Patent No. 8,927,508, W02003/012117, W02003/048371, W01997/040839).
  • cattle e.g., Holstein breed
  • an adjuvant Any an recognized adjuvant can be used. Suitable adjuvants include, but are not limited to, Emulsigen- D, Carbigen, Quil A, and Seppic ISA. In a preferred embodiment, the adjuvant is Quil A.
  • cattle are inoculated with antigen 1 , 2, 3, 4, 5, or 6 times, or more, to induce a specific immune response.
  • inoculations can be performed in 1, 2, 3, 4, or 5 week intervals.
  • cattle are inoculated 4 times at 2-3 week intervals.
  • the antigen for immunization is injected into the area drained by the target superficial lymph node (targeted for harvesting lymphocytes).
  • cattle are immunized 1, 2, 3, 4, or 5 days prior to removal of the target lymph nodes for subsequent hybridoma production.
  • the optimal concentration of target antigen used in the immunizations can be readily determined by the skilled artisan.
  • lymph nodes or the spleen
  • lymphocytes are isolated and fused with an immortalized cell line using a suitable fusing agent, e.g., polyethylene glycol (PEG), in order to form a hybridoma cell (see, e.g., Coding (1986) Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103).
  • PEG polyethylene glycol
  • Other suitable methods for generating hybridomas are described, for example, in U.S. Patent No. 5,026,646, U.S. Patent No. 5,087,693, Tucker et al.
  • Immortalized cell lines may be transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Rat or mouse myeloma cell lines may be used.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium”), which substances prevent the growth of HGPRT- deficient cells.
  • Immortalized cell lines suitable for use are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • Preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Manassas, Va.
  • the medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the target of interest (e.g., SARS CoV-2 spike polypeptide).
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells can be determined by inmunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are well known in the art.
  • the binding affinity of the monoclonal antibody can be determined by using art-recognized assays, such as Scatchard analysis and Biacore surface plasmon resonance assays.
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal. [0247] In certain embodiments, the hybridoma cells are screened for the secretion of target specific monoclonal IgGl antibodies. The antibody isotype can be determined using standard sequencing procedures known in the art.
  • the fully bovine recombinant monoclonal IgGl antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures, such as, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies purified from hybridoma culture or ascites fluid can also be made using standard recombinant DNA methods known in the art.
  • DNA encoding the bovine monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of urine antibodies).
  • the hybridoma cells of the present disclosure serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of mnonoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of mnonoclonal antibodies in the recombinant host cells.
  • host cells include fungal cells (aspergillus), insect cells, yeast cells, bacteria cells, and other art-recognized host cells.
  • fully bovine recombinant monoclonal IgG 1 antibodies are generated through art-recognized single B cell sequencing and cloning methods, as described in the examples herein. See also, e.g., US201 1/03 12505, US2012/030855, Murugan et al., Eur Immiunol 2015;45:2698;700, Liao et al., J Virol Methods 2009;158:171-9; Busse et al., Eur J Inmunol 2014;44:597-603, the contents of all of which are herein incorporated by reference.
  • the fully bovine recombinant monoclonal IgGl antibodies are formulated with a preservative. In another embodiment, the fully bovine recombinant monoclonal IgGl antibodies are lyophilized.
  • nucleic acid molecules Also provided herein are nucleic acid molecules that encode the bovinized and fully bovine recombinant monoclonal IgGl antibodies described herein. Once an antibody having increased resistance to proteases is identified, the coding nucleic acid sequence can be identified and isolated using art-recognized techniques. [0253] The nucleic acids may be present in whole cells (e.g., hybridomas, ascites fluid, stable cell lines), in a cell lysate, or in a partially purified or substantially pure form.
  • a nucleic acid is "isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other chromosomal DNA, e.g., the chromosomal DNA that is linked to the isolated DNA in nature) or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al, ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York.
  • a nucleic acid described herein can be, for example, DNA or RNA and may or may not contain intronic sequences.
  • the nucleic acid is a cDNA molecule.
  • Nucleic acids described herein can be obtained using standard molecular biology techniques.
  • cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques.
  • VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene.
  • a VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.
  • the term "operatively linked”, as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.
  • the isolated DNA encoding the VH region or VL chain can be converted to a full length heavy chain or light chain gene by operatively linking the VH-encoding DNA or VL encoding DNA to another DNA molecule encoding heavy chain or light chain constant regions. Nucleotide sequences for these regions are known in the art, and DNA fragments encompassing these regions can be obtained by standard PCR amplification. Also provided are nucleic acid molecules with conservative substitutions (i.e., substitutions that do not alter the resulting amino acid sequence upon translation of nucleic acid molecule), e.g., for codon optimization.
  • Methods of production also provided herein are host cell expression systems for producing the bovinized and fully bovine recombinant monoclonal antibodies described herein.
  • Such host cell expression systems may be engineered to comprise a recombinant nucleic acid molecule encoding the bovinized antibodies or bovine monoclonal antibodies described herein, operatively linked to an art-recognized regulatory sequences, which include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes.
  • promoters include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes.
  • Exemplary regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA ( 1990)).
  • regulatory sequences may depend on factors such as the choice of host ceil to be transformed, the level of expression of protein desired, etc.
  • Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • nonviral regulatory sequences may be used, such as the ubiquitin promoter or J-globin promoter.
  • regulatory elements composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).
  • recombinant expression vectors may include additional sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.), and allows for the establishment of stable cell lines (which can be stored for later use).
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • neo gene for G418 selection.
  • Transfection is intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
  • electroporation e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
  • expression of antibodies in eukaryotic cells, and most preferably mammalian host cells is the most preferred because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.
  • any type of cultured cell line can be used as a background to engineer the host cell lines of the present disclosure including but not limited to CHO cells (e.g., dhfr CHO cells used with a DHFR selectable marker), BHK cells, NSO myeloma cells, COS cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells (e.g., Pichia pastoris, S. cerevisiae), bacterial cells (e.g., E.
  • CHO cells e.g., dhfr CHO cells used with a DHFR selectable marker
  • BHK cells e.g., NSO myeloma cells, COS cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells
  • host cell systems capable of glycosylating the recombinantly produced antibody are used.
  • these host cells are used for large-scale production of antibodies. Methods for large-scale production of antibodies are also known in the art (and are described in the Examples below), e.g., in Aspergillus (Ward et al., Appl Environ Microb 2004;70:2567-576), yeast (e.g., Pichia pastoris, S.
  • stable expression is generally preferred to transient expression because it typically achieves more reproducible results and also is more amenable to large scale production.
  • host cells can be transformed with the respective coding nucleic acids controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows selection of cells which have stably integrated the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.
  • Antibodies can be recovered from the culture medium using standard protein purification methods.
  • compositions e.g., oral pharmaceutical compositions
  • Formulations of polyclonal immunoglobulin (Ig) purified from the colostrum of ruminants, namely cows, immunized with SARS-CoV-2 antigens, or with other desired viral antigens, are well suited for preparation of dietary supplements, nutraceuticals and medical foods but are also used as intermediary products for further purification (e.g. chromatography) of drug substance towards pharmaceutical product manufacturing.
  • the colostrum upon collection and pooling of colostrum, the colostrum is subjected to whey intermediate processing including the following steps: fat removal; de-fatted colostrum acidification (casein precipitation); and casein precipitate removal (e.g. by depth filtration).
  • Such intermediate processing may also include pasteurization (e.g. prior to fat removal); and casein precipitate centrifugation involving depth filtration and 0.2 micron filtration).
  • Such whey intermediate may be frozen or dried into bulk powder. Additional steps towards the manufacture of drug substance may then be taken such as serial chromatography (flow through Capto S and Capto Q), tangential flow filtration to condition and concentrate, and heat treatment (e.g. 10 hrs @ 60°) to produce bulk drug substance which is enriched in immunoglobulin fraction and may be stored frozen.
  • the drug substance may be further enriched and desired antibodies purified using affinity chromatography.
  • Any of the above-mentioned materials may be further processed into dosage forms (e.g. tablets, capsule, suspension powders) using processes well known in the art.
  • compositions described herein comprise a therapeutically effective amount of a bovinized and/or fully bovine recombinant monoclonal antibody, polyclonal antibody, anti-idiotype antibody, and mixtures of such antibodies, optionally formulated together with one or mote pharmaceutically acceptable carriers or excipients.
  • compositions comprising or consisting essentially of bovinized and/or fully bovine recombinant monoclonal antibodies, polyclonal antibodies, anti-idiotype antibodies, and mixtures of such antibodies, and optionally a carrier (e.g., a pharmaceutically acceptable carrier and/or preservative).
  • a carrier e.g., a pharmaceutically acceptable carrier and/or preservative.
  • the composition is substantially free, e.g., at least 90%, 95%, or 99% free, from other (i.e., non-bovine IgGl or bovinized) antibodies.
  • the pharmaceutical composition is formulated for oral administration.
  • the bovinized and/or fully bovine recombinant monoclonal antibodies, polyclonal antibodies, anti-idiotype antibodies, and mixtures of such antibodies may be systemically administered to a subject by intravenous route, which includes intravenous injection and intravenous infusion.
  • intravenous route which includes intravenous injection and intravenous infusion.
  • the diluents and other excipients, if any, for this mode of administration are known in the art.
  • the compositions can be delivered in a mouthwash, rinse, paste, gel, or other suitable formulation.
  • the antibodies described herein can be delivered using formulations designed to increase the contact between the active antibody and the mucosal surface, such as buccal patches, buccal tape, mucoadhesive films, sublingual tablets, lozenges, wafers, chewable tablets, quick or fast dissolving tablets, effervescent tablets, or a buccal or sublingual solid.
  • the antibody can be delivered by oral ingestion in the form of a capsule, tablet, liquid formulation or similar form designed to introduce drug to the subject.
  • antibody may be administered by suppository or enema for delivery to the lower digestive tract.
  • the term "pharmaceutically acceptable carrier or excipient” means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • Some examples of materials which can serve as pharmaceutically acceptable carriers are water, sterile water, sugars such as lactose, glucose and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and ahiminun hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible
  • composition may also include a pharmaceutically acceptable antioxidant.
  • pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), but
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and
  • compositions for rectal administration are preferably suppositories which can be prepared by mixing the compounds of this disclosure with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • compositions for rectal administration are in the form of an enema.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl
  • the dosage form may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • enteric coatings take advantage of the post-intestinal change in pH to dissolve a film coating and release the active ingredient Coatings and formulations have been developed to deliver protein therapeutics to the small intestine and these approaches could be adapted for the delivery of an antibody of the present disclosure.
  • enteric-coated form of insulin has been developed for oral delivery (Toorisaka et al., J Control Release 2005;107:91-6).
  • enteric coating is described in, e.g., U.S. Patent Nos. 5,225,202 and 6,306,900, and US2008/0020041.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with other coatings and shells well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredients) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.
  • the antibody is formulated in enterically-coated microparticles delivered as a liquid suspension.
  • fee antibody is formulated in enterically-coated microparticles delivered as a capsule.
  • compositions e.g., oral pharmaceutical compositions
  • fee pharmaceutical compositions comprising fee antibodies described herein also include a preservative.
  • compositions containing polyclonal antibodies for immunization of ruminants notably cows in order to generate anti-idiotypic antibodies an adjuvant is included.
  • Carriers, diluents and other excipients suitable for veterinary animal use may be included as described above. Any adjuvant safe for animal vaccination would be suitable, subject of course to optimization.
  • immunogenic compositions containing anti-idiotypic antibodies for human use may optionally include an adjuvant or other stimulator or enhancer of immune response, as well as a carrier, diluent other excipient as described above.
  • fee carrier or diluent is a carrier or diluent suitable for intranasal or by-inhalation use.
  • fee composition is formulated as an inhalable powder or solution or suspension feat can be aerosolized.
  • Devices for administration or delivery to fee respiratory tract or airway(s) are known and recognized in fee skilled art and in clinical or medical practice and are applicable in the methods, protocols and compositions of the present invention. Devices include metered dose inhalers, metered spray pumps, hand-bulb atomizers, small or large volume nebulizers, ultrasonic nebulizers and dry powder inhalers.
  • Effective doses will vary depending on route of administration, as well as the possibility of co-usage with other agents. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; die timing of delivery of the compound relative to food intake; the duration of the treatment; drags used in combination or contemporaneously with the specific compound employed; and like factors well known in the medical arts.
  • Particular embodiments of the present disclosure involve administering via the digestive tract a pharmaceutical composition comprising an antibody of the present disclosure at a dosage typically in the range from about 0.01 mg per dose to about to about 1 gram or higher per dose (not per antibody). Multiple doses per day may be administered and up to 30 grams daily have been generally recognized as safe. In some embodiments, it may be desirable to reach the maximum tolerated daily dose.
  • the frequency of administration will usually be a single dose but administration can be repeated as needed within a day and on subsequent days.
  • Oral administration can also be combined with intraperitoneal or intravenous administration of the same antibody-containing therapeutic composition or of a different antibody-containing composition.
  • Usual ranges for administration in the digestive tract thus may be from 1 mg per day to about 1 g/day, more preferably from about 10 mg/day to about 500 mg/day, and most preferably from about 20 mg/day to about 100 mg/day, to a subject.
  • a polyclonal antibody preparation is administered at a dosage of antibody from about 100 mg to about 50 g/day, more preferably from about 500 mg/day to about 10 g/day, and most preferably from about 1 g/day to about 5 g/day, to a subject, wherein the polyclonal antibody preparation has not been enriched for antibodies specific for the target antigen.
  • Treatment regimens include administering an antibody composition of the present disclosure one time per day, two times per day, or three or more times per day, to treat COVID- 19.
  • an antibody composition of the present disclosure is administered four times per day, 6 times per day or 8 times per day to treat a disease or disorder or condition disclosed herein.
  • an antibody composition of the present disclosure is administered one time per week, two times per week, or three or more times per week, to treat a medical disorder disclosed herein.
  • Similar treatment regimens are contemplated for the compositions administered via the respiratory route (intranasal or by-inhalation) subject to the limitations described herein.
  • compositions of the present disclosure include the use of bovinized and/or fully bovine recombinant monoclonal antibodies, polyclonal antibodies, anti- ideotype antibodies, and mixtures of such antibodies, directed against SARS CoV-2 antigens in combination with one or more additional therapeutic agents useful in treating the condition with which the patient is afflicted.
  • additional therapeutic agents include both proteinaceous and non- proteinaceous drugs.
  • dosages may be adjusted accordingly, as is recognized in the pertinent art.
  • “Co administration” and combination therapy are not limited to simultaneous administration, but also include treatment regimens in which an antibody of the present disclosure is administered at least once during a course of treatment that involves administering at least one other therapeutic agent to the patient.
  • the one or more additional therapeutic agent may be a bovinized antibody specific for target antigens such as cytokines and chemokines that promote inflammation, including but not limited to TNF, TNF-kappa, IFN-gamma, IL- 1 alpha, IL-I beta, IL-2, IL-6, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL27, IL 32, IL-33, and IL-35 or receptors thereof; and/or chemokines, including but not limited to CCL1/TCA3 CCL11 CCL12/MCP-5 CCL13/MCP-4 CCL14 CCL15 CCL16 CCL17/TARC CCL18 CCL19 CCL2/MCP-1 CCL20 CCL21 CCL22/MDC CCL23 CCL24 CCL25
  • target antigens such as cytokines and chemokines that promote inflammation, including but not limited to TNF, TNF-kappa
  • the anti-TNF antibody is a bovinized or fully bovine recombinant monoclonal antibody, as described in PCT/US2016/054304. In one embodiment, the anti-TNF antibody is an antibody with enhanced mucosal permeability.
  • the one or more additional therapeutic agent which is an antibody directed against cytokines that promote inflammation which is delivered intravenously, mucosally, orally and/or to the digestive tract reduces levels of those cytokines. Levels of cytokine can be determined by direct measurement of the cytokine or by analysis of a surrogate marker that responds to the cytokine. In one aspect, antibodies delivered to the digestive tract that are specific for soluble cytokines reduce levels of those cytokines in both the digestive tract and systemic circulation.
  • An antibody to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. The formulation ordinarily will be stored in lyophilized form, as spray dried particles or in solution.
  • Bovinized and/or fully bovine recombinant monoclonal antibodies, polyclonal antibodies, anti-idiotype antibodies, and mixtures of such antibodies, directed against SARS CoV-2 antigens may be used in the oral cavity and may be specific for receptors or other antigens expressed on the apical surface of the oral cavity, against receptors or other antigens expressed on the basolateral surface of the mucosal barrier of the oral cavity, or against receptors or other antigens expressed in the mucosa, submucosa, or any other region of the body accessible to topically applied antibody.
  • the bovinized and/or fully bovine recombinant monoclonal antibodies, polyclonal antibodies, anti-idiotype antibodies, and mixtures of such antibodies, directed against SARS CoV-2 antigens exhibit improved stability in the oral cavity and improved stability to gastric and intestinal digestion.
  • the therapeutic compositions comprise bovinized and/or fully bovine recombinant monoclonal antibodies, polyclonal antibodies, and mixtures of such antibodies, directed against SARS CoV-2 antigens as well as anti-idiotypic polyclonal antibodies elicited by immunization of ruminants with polyclonal antibodies against one or more SARS CoV-2 proteins which are delivered topically to the luminal face of the digestive tract.
  • the antibodies described herein may be administered topically to the digestive tract by, for example, oral administration, rectal administration, and all forms of administration to the oral cavity such as by buccal, mucoadhesive films and the like.
  • the antibodies may cross the mucosal barrier of the digestive tract to enter the submucosal space to interact with their targets.
  • the bovinized and/or fully bovine recombinant monoclonal antibodies, colostra! polyclonal antibodies, and mixtures of monoclonal and polyclonal antibodies are specific for a SARS CoV-2 spike polypeptide.
  • compositions comprising bovinized and/or fully bovine recombinant monoclonal antibodies, colostral polyclonal antibodies, anti-idiotype antibodies, and mixtures of such antibodies are suitable for the treatment of CO VID-19.
  • Example 1 Generation and characterization of bovine anti-SARS-CoV-2 antibodies.
  • Group A (calves #1-3) were immunized with a Spike Protein Receptor Binding Domain (RBD), (Recombinant SARS-CoV-2 Spike RBD His-tag (CHO Expressed), (R&D Systems, biotechne), Lot: DNZP0220091) and Group B (calves #4-6) with Spike Protein Trimer (Recombinant SARS-CoV-2 Spike (Active Trimer) His Protein (R&D Systems, biotechne). Lot: DOJBO 120091.). Immunizations and observations were under the direction of the PI and attending veterinarian. Calves were observed daily by veterinary staff for three days following immunization. No abnormal or unusual findings were observed. Daily observations were recorded by Animal Care Staff throughout this study.
  • RBD Spike Protein Receptor Binding Domain
  • Group B (calves #4-6) with Spike Protein Trimer (Recombinant SARS-CoV-2 Spike (Active Trimer) His Protein (R&D Systems, biotechne). Lot: DOJBO 120091.
  • the example demonstrates that ruminants, including bovines, can be immunized with SARS-CoV-2 recombinant proteins to produce potent antibodies against the wildtype S ARS- CoV-2 virus, and that these antibodies recognize variant SARS-CoV-2 viruses.
  • Example 2. Pseudovirus neutralization assay for SARS-CoV-2.
  • Controls included: a virus control (VC) without serum, which is the control for 100% infectivity; positive control having a SARS-CoV-2 positive control antibody with neutralization response to SARS-CoV-2 WT and variants; negative control with negative control antibody without neutralization response to SARS-CoV-2.
  • VC virus control
  • a total volume of the 2X diluted serum of 50 uL/well for a 96-well plate was prepared.
  • a Human ACE2 Stable Cell Line - HEK293T (hsACE2/293T cells; Creative Diagnostics Cat# CSC-ACE01) was grown to sub-confluency in Cell Culture media (DMEM + 10% FBS + 10 mM HEPES + lx Penicillin-Streptomycin) (Coming Cat# 10017CM, Summerlin Cat# SS-100, Coming Cat# 25-060-CI, Coming Cat# 30-002-CI).
  • the hsACE2/293T cells were trypsinized, pelleted and resuspended in Cell Culture medium. The cell density was determined using a hemocytometer or equivalent cell counter. For seeding a 96-well plate, cells were resuspended at a density of 4 x 10 s cells ZmL.
  • the cell suspension 100 ul (4 x 10 4 cells), was added to each well of the 96 well plate containing the SARS-CoV-2 Spike pseudovirus or variants thereof and mixed.
  • a cell only control (CC) without pseudovirus was also added to the 96 well plate.
  • the plate was incubated in a 5% CO2, 37°C humidified incubator for 48-72 hours.
  • the SARS-CoV-2 Spike pseudovirus or variants thereof may infect the 293T/ACE2 cells after incubation with the serum and luciferase may be produced in the infected cell. Antibodies present in the serum may prevent the SARS-CoV-2 Spike pseudovirus or variants thereof from infecting the 293T/ACE2 cells and producing luciferase.
  • the pseudovirus neutralization inhibition rate of the tested serum is calculated based on the luciferase luminescence value.
  • the results of the neutralization assay for SARS- CoV-2 pseudovirus and variants thereof is shown in Figures 5, 6 and 7.
  • the EC50 values for Sample 1 and Sample 2 are presented in the figures.
  • the example demonstrates that the serum antibodies of Sample I and Sample 2 are capable of binding the Spike Protein Receptor Binding Domain of SARS-CoV-2 and variants thereof and blocking the binding of the SARS-CoV-2 spike protein RBD to its receptor ACE2
  • ruminants including bovines
  • SARS-CoV-2 recombinant proteins for example Spike Protein Receptor Binding Domain
  • the binding and neutralizing potency across SARS- CoV-2 virus variants is caused by the polyclonal character of the immune response.
  • Polyclonal antibodies recognize a multitude of different epitopes on the SARS- CoV-2 spike protein of the wildtype SARS-CoV-2 virus. The majority of these epitopes appear to be preserved in variants and thus are targeted by the polyclonal antibody immune response. As these antibodies are being used in COVID-19 pandemic, it is remarkable that immunizing bovine with wildtype spike protein antigens induces the broadly binding and neutralizing response. Therefore, it would not be necessary to make customized antibodies against variant proteins in each infection offering significant advantages over monoclonal antibodies targeting a single epitope.
  • Polyclonal antibodies are used to rapidly confer passive immunity to infectious agents, neutralize viral binding to host cells, and block action of cytokines and toxins (Newcombe et al. 2007; Casadevall et al. 2004; Sawyer et al. 2000). Sequencing analyses showed that SARS- Cov2 shares 79.5% sequence identity with SARS-Cov (Zhou et al. 2020; Lu et al. 2020).
  • S surface spike glycoprotein
  • SARS-CoV-2 and SARS-CoV which are phylogenetically related, have an amino-acid sequence identity of around 77% (Zhou et al. 2020).
  • Analysis of the receptor binding domain (RBD) in the S protein showed that most of the amino acid residues essential for receptor binding were conserved between SARS-CoV and SARS-CoV-2, suggesting that the two strains use the same host receptor for cell entry (Shang et al. 2020).
  • the entry receptor utilized by SARS-CoV is Angiotensin-Converting Enzyme 2 ( ACE- 2)(Li et aI. 2003).
  • SARS-Cov2-specific polyclonal antibodies will be generated using selected inactivated virus particles and whole recombinant S protein. Antigens will be suspended in PBS and emulsified 1: 1 (vol/vol) with adjuvant. Pregnant, healthy, mastitis-free Holstein dairy cows are immunized subcutaneously in the rear thigh with 100 ⁇ g of the antigen mixture in a total volume.
  • Cows are immunized with an immunogen (detailed below) three times using two distinct adjuvants (that are approved for use in lactating dairy cows to maximize immunoglobulin titers (for example 1: 1 (vol/vol) with CARBIGEN® (MVP Laboratories, Omaha, Nebr.), a carbomer-based adjuvant).
  • the amounts of immunogen and immunization protocol are anticipated to be essentially the same as those used in Example 2 of US Patent 8268971. Briefly, the first immunization occurs 50 to 60 days before the expected parturition date for each animal. The second immunization is given 3 weeks after the first, and the third injection is administered 2 weeks later.
  • Serum samples are collected immediately before each immunization, then once a week until parturition. A final serum sample is collected from each cow at parturition. Colostrum samples are collected from all cows twice a day for 3 days (a total of 6 milkings) beginning on the day of parturition. Serum and colostrum aliquots are stored at -20°C. A fresh aliquot is thawed before each ELISA experiment. As control, both serum and colostrum samples will be collected from a non-immunized cow.
  • Colostrum is collected from day 1 to 3 post-parturition and the three samples from each cow are used to assess inclusion into a combined pooled colostrum sample. Colostrum from each animal is processed and assayed separately to compare the efficiency of immunization and efficacy of adjuvants. Colostrum is centrifuged at 4000 x g to remove fat. The pH is slowly adjusted to 4.6 using 1 N HC1, incubated for 30 min at 37°C to precipitate casein, and centrifuged. Whey is then stored at -20°C.
  • Titers are determined by ELISA in triplicate using a human SARS-Cov2 IgG ELISA kit (Euroimmun, Mountain Lakes, NJ, or Biovendor, Asheville, NC). The concentration of total immunoglobulin in each whey sample is determined by ELISA using sheep antibovine IgG (h+1) (Bethyl Laboratories). Purified bovine immunoglobulin is used as a control. The specific activity of each colostrum sample is calculated (titer per mg immunoglobulin).
  • Example 4 Purification of anti-SARS-Cov2 antibodies from bovine colostrum.
  • the levels of anti-SARS-Cov2 antibodies in colostrum samples from individual cows will be determined by ELISA. Given the multitude of active molecules present in the unpurified colostra! whey that may lead to false positives, we will purify immunoglobulin to be used in the characterization assays. Immunoglobulin will be purified on the thiophilic gel T-gel (Pierce, Rockford, IL). T-gel is preferred over Protein G affinity chromatography because it binds IgM and IgA as well as IgG, and thus will capture all major isotypes in bovine colostrum.
  • the protein concentration of the starting pool and the purified immunoglobulin is measured using the BCA assay (BCA1 kit, Sigma-Aldrich, St. Louis, MO). Standard curves are generated using reagent grade bovine immunoglobulin (Sigma-Aldrich). Purity is assessed using non-reducing SDS-PAGE. T-gel may not provide immunoglobulin that is sufficiently pure for use in the functional studies.
  • the resin has been used previously to purify immunoglobulin from colostrum but not in the context of SARS-Cov2. In the event that non-specific binding occurs, anti-SARS- Cov2 antibodies will be purified with Protein G Sepharose, which is highly specific for immunoglobulins.
  • IgG 600 mg of purified Ig will be passed over a 40 ml Protein G Sepharose column using a protocol specifically developed for bovine IgG (Akita et al. 1998).
  • IgG is eluted with 0.1 M glycine-HCl, pH 2.8 into a neutralizing buffer (0.2 M Tris, pH 8.3) to give a final pH of approximately 7.
  • IgA and IgM should pass through the column while IgG should be retained. The flow through is collected and concentrated to a volume of 6 mL.
  • IgM and IgA is separated on a Sephacryl S-200 gel filtration column.
  • IgGl and IgG2 is separated by ion exchange chromatography (Fang et al. 1976).
  • the purified IgG is loaded onto a 100 ml TEAE cellulose column in 0.32 NaCl/0.01 M Tris HC1, pH 8.0.
  • the column is washed and eluted in a stepwise fashion using a higher pH buffer.
  • IgG2 is eluted with 100 ml 0.001 M Tris HC1 pH 8.6 followed by a 200 mL linear gradient of the same buffer containing 0 - 0.32M NaCI to elute IgG 1.
  • Fractions are collected and pooled based on absorbance at 280 nm. Samples are analyzed using ELISA.
  • Example 5 Production of bovine anti-idiotype SARS-Cov2 antibodies.
  • a single injection is used for immunization of cows with either purified polyclonal antibodies from convalescent COVID-19 patients or with the previously identified antibody from a recovered SARS-CoV-2 patient, CR3022, that recently has been shown to recognize the receptor-binding domains of SARS-CoV-2.
  • a single injection is used to avoid severe side effects encountered when cows receive human antibody. Two groups of 3 cows each will be employed, with group #1 injected with purified antibodies from convalescent patients and group #2 injected with CR022 antibody. It is anticipated that the injections will generate anti-idiotype polyclonal antibodies against human anti-SARS-Cov2 antibodies. The titration and purification processes will be the same as used for the anti-SARS-CoV-2 polyclonal antibodies described in Examples 1 and 2.
  • Standard ELISA methods can be used to determine the binding specificities and approximate affinities of Ab-antigen interactions. However, more accurate binding affinities and information on the kinetics of binding are determined by surface plasmon resonance (SPR) analyses.
  • SPR surface plasmon resonance
  • the antibody or antigen binding proteins is immobilized on a surface. The other binding partner is then added at different known concentrations. The binding of the added partner increases the mass of the bound material on the surface, and this mass difference can be detected and used to calculate the binding affinity (KD) derived from dissociation and association constants (KofiTKon) (Wittenberg, N. J., et al, (2014).
  • KD binding affinity
  • KofiTKon dissociation and association constants
  • Example 7 Antibody activity in cell-based assays.
  • bovine colostrum-derived antibodies to neutralize SARS-Cov2 will be assessed by measuring infection and viral replication in two independent cell-based assays: a SARS-Cov2 plaque assay and a neutralization assay using lentiviral vector pseudotyped with SARS-Cov2 spike protein.
  • HEK293T human embryonic kidney cells
  • ACE-2XCatalog# NR-52511, BE1 resources the main entry point of SARS-Cov2 in host cells
  • L-132 lung epithelial cell line ATCC, ATCC® CCL-5TM
  • Both cell lines are grown in MEM (G1BCO-BRL Cat # 41600-016) in the presence of L-glutamine, antibiotics, and 10% FBS in 75 cm2 flasks at 37°C.
  • Confluent monolayers of cells are trypsinized and dispensed into 12-well plates. Cells are plated at a density that allows for the formation of confluent monolayers within 48-72 hours. Different concentrations of (pseudotyped) SARS-Cov2 are added to the cells and incubated for 2 hours at 33°C to allow the virus absorption to the cell monolayer. Each dilution is titrated in triplicate.
  • Each monolayer is overlaid with 2 mL of an overlay medium containing supplemented MEM, 2% FBS, DEAE- Dextran, 26 mM MgCI2 and purified agar (Oxoid L28, ThermoFisher Scientific). The ratio of the agar and the supplemented medium is 1 : 1.
  • the overlay solidifies, the plates are held for 6 days in a 5% CO2 atmosphere at 33°C. Cells are then fixed and stained with 0.5% crystal violet solution in 25% methanol.
  • bovine colostrum-derived antibody to neutralize SARS-Cov2 is determined by counting the number of viral plaques generated. Control will be HEK293T and L- 132 cells infected with irrelevant viruses. Different anti-SARS-Cov2 antibody titers are tested to evaluate the neutralizing effects on viral infection and replication. Effective cell entry inhibition results in no formation of viral plaques.
  • a higher throughput assay can be used which does not require enhanced safety measures (BSL-3 facilities).
  • BSL-3 facilities This is a neutralization assay using S-protein pseudotyped viruses.
  • Crawford et al reported a lentiviral system pseudotyped with the spike protein and have made all the key experimental reagents publicly available in the BEI Resources repository of ATCC and the NIH (Crawford et al. 2020). Briefly, 293T cells are transfected with a HIV backbone plasmid encoding a luciferase reporter protein, a plasmid expressing Spike protein, and plasmids expressing the minimal set of HIV proteins necessary to assemble viral particles.
  • the transfected cells then produce Spike-pseudotyped HIV1 particles that can be used to infect 293T cells that express the SARS-CoV-2 receptor protein, ACE2.
  • Serial dilutions of anti-SARS-Cov2 antibody are tested to evaluate the neutralizing effects on viral entry.
  • Anti-SARS-Cov 2 antibodies are added either prior to or following incubation of the cells with pseudovirus. Results then demonstrate whether antibodies can block viral binding to the cell receptor and/or any other early step of viral entry.
  • the level of pseudovirus entry will be measured by the expression of luciferase reporter at 60 h post-infection. Effective cell entry inhibition results in reduction of the luciferase signal.
  • Non-human antibodies are frequently administered as F(ab’)2 or Fab antibody fragments to reduce immunogenicity and the development of serum sickness. Often, the full intact Fc is not required for neutralizing activity and Fab or F(ab’)2 fragments are enough even if their activity is reduced compared to intact antibody.
  • Fab and F(ab’)2 fragments are generated by enzymatic cleavage of intact purified Ig. Papain digestion of purified antibody is a common way to generate the Fab fragments and the optimal conditions for digestion are routinely determined by a pilot study. Ig: papain ratios of 10:1 and 50:1 are incubated for times ranging from 1 - 24 hr and samples analyzed by SDS-PAGE.
  • F(ab’)2 is commonly generated by pepsin digestion of purified Ig.
  • a pilot study is performed to determine the optimal conditions. Digestions at pH 4.0 or 4.5 are incubated for times ranging from 1 - 24 hr and samples analyzed by SDS-PAGE. When the conditions have been defined, 200 mg of purified Ig is dialyzed into acetate buffer at the appropriate pH and incubated at 2 mg/ml with 1 mg pepsin (Sigma- Aldrich) at 37°C. The reaction are stopped by the additionof 20 ml 2M Tris base.
  • Both Fab and F(ab’)2 fragments are dialyzed into PBS and residual intact Ig is removed on a T-Gel column.
  • the proteins are concentrated and the fragments separated from contaminating papain / pepsin and other cleavage fragments on a Sephacryl S-200 superfine column.
  • the purity of the final cleavage fragments is assessed using non-reducing SDS-PAGE.
  • the activity of the fragments is assessed in a functional cell-based assay (see Example 7) and compared with the activity of Fc antibodies. Comparative activity assessment may include antibodies purified from sera of recovering CO VID- 19 patients.
  • Example 9 Nebulization of antibody products for inhalation delivery.
  • a lyophilized formulation of colostrum-derived anti-SARS CoV-2 polyclonal antibodies is used.
  • the nebulizer technology of Carbon dioxide Assisted Nebulization with a Bubble Dryer® (CAN-BD) technology (available as a fee-for-service or license from Activ-Dry LLC, Boulder, CO through Robert Sievers, University of Colorado) is used to produce free- flowing, dry, inhalable powders of antibody products (Cape SP et al., Pharm Res. 2008 Sep;25(9): 1967-90. doi: 10.1007/sl 1095-008-9575-6. Epub 2008 Jun 26. PMID: 185812122008, incorporated by reference).
  • CAN-BD is a patented technique (see US Patents 10,206,873; 9,895,321 ; 6,095, 134 and 5,639,441; and US Patent Publications 20190038552; 20180271826; 20120045479; 20020018815, all incorporated by reference) for producing fine, dry powders from solutions or suspensions using either aqueous or non-aqueous solvents.
  • the particles produced have high specific surface area, and aerodynamic diameters of 0.5 to 5 pm amenable for sterilization.
  • the process does not introduce product stresses like traditional freezing from lyophilization, shear from jet milling, or thermal stresses from spray drying.
  • CAN-BD produces powders with moisture content of 2% or less, a value desirable for stability of dry protein formulations produced by freeze-drying.
  • Solutions containing the antibody products are pumped through 1/16” outer diameter (OD) stainless steel tubing into one inlet of a stainless T at room temperature and 80 bar pressure.
  • the nebulization fluid either liquid carbon dioxide or liquid nitrogen, is pumped into the other inlet at the same temperature and pressure.
  • Either Isco piston for CO2 or conventional HPLC pumps are preferably used.
  • the two-phase flow of product solution and nebulizing fluid exits the outlet connection, flowing into a 75 pm inner diameter (ID), 10 cm long fused silica restrictor connected to a glass drying chamber maintained at atmospheric pressure, into which is also pumped heated dry nitrogen gas.
  • Rapid decompression from the restrictor to the drying chamber from 80 to 1 bar induces vaporization of liquid and dissolved nebulizing fluid (CO2 or N2), forming a fine aerosol plume of the product fluid, which is then dried into a powder by the heated nitrogen gas.
  • the resulting powder is collected on a 0.45 um pore size filter downstream of the drying chamber. After collection and placement in sample tubes with loose caps, the powder is dried further with at least 24 hours in a vacuum dessicator at room temperature.
  • nebulizers are commercially available and inhalable powders can be made by known techniques, subject to adaptation to the particular materials and circumstances involved.
  • Example 10 Safety and efficacy of antibody products in an animal model.
  • Nebulized antibodies are then further tested for safety and efficacy in appropriate animal models.
  • One such animal model has been described by Chan et al. at the University of Hong-Hong. The authors report infection of eight golden Syrian hamsters with SARS CoV-2 (Chan JF, at al,. Simulation of the clinical and pathological manifestations of Coronavirus Disease 2019 (COVID-19) in golden Syrian hamster model: implications for disease pathogenesis and transmissibility. Clin Infect Dis. 2020 Mar 26:ciaa325. doi: 10.1093/cid/ciaa325, incorporated by reference in its entirety). All animals were consistently infected and displayed weight loss, lethargy, hunched posture and rapid and/or difficult breathing.
  • SARS-CoV-2 High levels of SARS-CoV-2 were found in the lungs, intestines, and other tissues where ACE2 is highly expressed. Clinical signs and viral loads closely resemble the human manifestations of upper and lower respiratory tract infection. Antibody efficacy and safety can be assessed in this model of COVID- 19. Efficacy, safety and mucosal targeting efficiency can be assessed following oral or inhalation administration of anti-SARS-Cov2 antibodies, both monoclonal and polyclonal, and anti-idiotype antibodies administered by oral mucosal or by-inhalation administration.
  • Example 11 DNA vaccination for generation of bovine antibodies.
  • a strong antibody response to a protein antigen can be induced by vaccination of cattle with a DNA vector containing a gene encoding the desired antigen (see, e.g., van Drunen Little-van den Hurk et al., Clin Vaccine Immunol 2013;20: 166-73).
  • Individual animals are injected in the right gluteus maximus muscle with 1.5 mg of plasmid DNA in 1 mL, followed by application of a 250-V/cm electrical field for 400 ms at a 10% duty cycle, using a TriGrid electrode array (Ichor Medical Systems).
  • the immunizations are repeated two or three times, 21 days apart, and are expected to induce a significant antibody response.
  • Example 12 Generation of hybridomas producing monoclonal bovine antibodies following immunization
  • Hybridomas can be generated from immunized cows to establish bovine antibody secreting cell lines, essentially as described in U.S. Patent No. 5,026,646. Also see Examples 14- 16 of PCT/US2016/054304.
  • Immunization of cows with SARS CoV-2 antigen e.g., spike polypeptide, nucleocapsid polypeptide, membrane polypeptide and/or envelope polypeptide
  • adjuvants consists of a series of three subcutaneous injections of SARS CoV-2 antigen plus adjuvant at 2- 3 week intervals in pregnant Holstein dairy cows prior to calving.
  • Fusions are performed with modified version of the PEG protocol (Van Deusen et al., Am Assoc Vet Lab Diagnost 24’ Annual Proc 1981:211-228) using myeloma celklymph node cell ratios of approximately 1, and seeded onto 96-well plates. Cells are cultured at a 1: 1 ratio of DMEM and high glucose with 10% horse serum to conditioned media from fusion partner cultures. Fusion partner selection for methotrexate sensitivity is achieved by passage in media containing 6- thioguanine and 8-azaguanine.
  • SP2/0 murine myeloma cells are fused with right prescapular calf lymph nodes cells in a 3: 1 mixture to generate bovine Ig secreting primary cell lines. After secretion ceases, the lines are selected for methotrexate sensitivity by passage in media containing 6-thioguanine and 8- azaguanine.
  • Methotrexate-sensitive bovine:murine heterohybridomas are combined in a 1:1 ratio and mixed with lymph node cells from the immunized and extracted left prescapular lymph node from the same calf used previously at a 1:1 ratio. This fusion results in transiently bovine Ig secreting cell lines, which are then selected for methotrexate sensitivity once secretion ceases.
  • Cells from the methotrexate-sensitive bovine x murine heterohybridomas are combined in a 1:1 ratio and fused with fresh lymph node cells from an immunized, right prefemoral lymph node from the same calf as before at a 1 :2 mixture, respectively.
  • the fusion results in bovine x murine primary heterohybridomas that secrete bovine antibodies.
  • Stable heterohybridomas secreting antibodies having the function of interest e.g., binding to SARS CoV-2 antigen
  • heterohybridomas can be further screened for those which produce IgGl antibodies by sequencing using routine methods.
  • Example 13 Single B cell sequencing and recombinant antibody production
  • Single B cells are sequenced essentially as described by Tiller et al. (J Immunol Methods 2008;329: 122-4). Mononuclear cells are isolated from peripheral venous blood or serum of cows immunized as described in the example above and purified by Ficoll-Paque density gradient centrifugation, and optionally followed by enrichment of B cells using anti-CD19 magnetic beads. Single mononuclear cells are sorted by flow cytometry into 96-well PCR plates. cDNA is synthesized directly in each well.
  • RNA from single cells are reverse transcribed with random hexamers using the Superscript III reverse transcriptase kit, and IgG, IgX, and IgK V transcripts are amplified with gene-specific primers using the isolated cDNA as template. Aliquots of the VH, V and V chain PCR products are purified and sequenced. Sequences are analyzed by IgBLAST comparison with GenBank to identify germline V(D)J gene segments with highest identity.
  • PCR products are purified using the Qia-Quick 96 PCR Purification Kit (Qiagen). PCR samples are digested and ligated into the multiple cloning site of human Igyl, IgK and IgX expression vectors. Ligation products are transformed into competent DH10B bacteria cells, and colonies are PCR screened for the presence of bands of the expected size. Plasmid DNA is isolated from bacterial cultures using QIAprep Spin columns (Qiagen) and purified.
  • Antibodies are produced by transiently transfecting the purified plasmids into HEK293 cells or 293T cells cultured in 150 mm plates. Six days after transfection, culture supernatants are harvested and antibodies are purified using Protein G beads. Recombinant antibody concentrations are then determined by ELISA.
  • Example 14 Selection of recombinant bovine monoclonal IgGl antibodies, polyclonal antibodies and anti-idiotype antibodies.
  • Antibodies produced are sequenced to determine whether they have the structural properties that confer resistance to proteases and are tested for functional activity (e.g., binding to target SARS CoV-2 antigen) and stability. Further, as discussed supra, potential protease cut sites in the antigen-binding region are also considered when generating stable bovine IgGl antibodies. Thus, the antibodies are tested for both SARS CoV-2 antigen binding and pancreatin stability.
  • the heavy and light chains of antibodies found to have the desired properties are cloned into expression vectors, expressed, harvested, and purified using standard recombinant methods.
  • Example 15 Construct design for folly bovine recombinant monoclonal antibodies and bovinized antibodies.
  • DNA constructs may be made which encode bovinized and/or fully bovine recombinant monoclonal antibodies, polyclonal antibodies, anti-idiotype antibodies, and mixtures of such antibodies, directed against SARS CoV-2 antigens.
  • Recombinant expression vectors comprising DNA encoding SARS CoV-2 antigen -bovinized antibodies are transformed/transfected into host cells (e.g. any mammalian host cell or a ruminant mammary epithelial cell line such as MAC-T) and cultured for expression of the bovinized and/or folly bovine recombinant monoclonal antibodies, polyclonal antibodies, anti-idiotype antibodies.
  • Example 16 Large-scale production of bovine monoclonal and bovinized antibodies.
  • Bovinized and/or folly bovine recombinant monoclonal antibodies, polyclonal antibodies, anti-idiotype antibodies, and mixtures of such antibodies, directed against SARS CoV-2 antigens can be recombinantly produced in large-scale in various host organisms for use in research and clinical settings, as described below. Production in yeast, Aspergillus, mammalian and insect cells can be utilized using methods known in the art. By way of example, production may be as described below.
  • plasmid DNA two plasmids: one encoding the heavy chain and the other encoding the light chain, or alternatively one plasmid comprising sequences encoding both heavy and light chains
  • plasmid DNA are added to the cells.
  • the cells and plasmid DNA are incubated on ice for 5 min and transferred to 0.2 cm electroporation cuvettes.
  • a Bio-Rad Gene Pulser (Richmond, Calif.) is used to perform the electroporation. Transformants are selected on YNB media lacking uracil.
  • a preculture is grown in 100 ml YNB media (ura-, leu*) + 2% glucose for 24 hrs at 30°C, followed by inoculation in 200 ml fermentation media, (ura-, leu-) + 2% glucose, at 0.5 OD.
  • Cultures are grown and harvested and samples taken at various time points and induced with 2% galactose.
  • Cells are separated from the culture media by centrifugation, and culture supernatants are concentrated with Centricon 30 filters by ultrafiltration and used for SDS-PAGE analysis. Antibody purity and yield can be detected by running concentrated supernatant on SDS- PAGE.
  • Nucleic acids encoding the heavy and light chain of an antibody of interest are cloned into separate expression vectors with selectable markers known in the art (e.g., pGAMpR, or those described for filamentous fungi in Sambrook et al., 1989 and Ausubel FM et al., 1989).
  • Aspergillus niger or Aspergillus niger var. awamori strain (which has a deletion in the gene encoding a major secreted aspartyl protease) are used as host cells. Transformations and large-scale fermentations are carried out as described in Ward et al. (Appl Environ Microb 2004;70:2567-576). Yields using the protocol described in Ward et al. can reach 0.20.9 g/L.
  • Antibodies are purified from the culture supernatant by removal of the fungal cells by filtration through a cellulose pad and filtration of the supernatant. Hydrophobic charge induction chromatography (HCIC) and high-performance liquid chromatography (HPLC) are used to purify the supernatant. Antibody purity and yield are detected by running concentrated supernatant on SDS-PAGE.
  • HCIC Hydrophobic charge induction chromatography
  • HPLC high-performance liquid chromatography
  • Mammalian cells e.g., Chinese hamster ovary (CHO) cells, and NSO murine myeloma cells
  • Nucleic acids encoding the heavy and light chain of an antibody of interest e.g., anti-SARS-Cov_2 antigen antibody
  • adherent cell lines are adapted to suspension culture formats for large-scale production (see, e.g., Sinacore et al., Biotechnol Bioeng 1996;52: 518-28).
  • Stable cell line clones are selected using methods known in the art, such as through metabolic markers, including methotrexate (MTX). To identify high-producing clones, cells are separated from the culture media by centrifugation, and culture supernatants are concentrated by ultrafiltration and used for SDS-PAGE analysis. Antibody purity and yield can be detected by running concentrated supernatant on SDS-PAGE. The top yielding clones are selected for further evaluation in large- scale bioreactors (e.g., 2,000 liters) to determine the final production clone.
  • large- scale bioreactors e.g., 2,000 liters
  • Insect cells can be used in the large-scale production of antibodies of interest.
  • Nucleic acids encoding the heavy and light chain of an antibody of interest e.g., SARS CoV-2 spike polypeptide antibody
  • Cells are cultured at 25°C-30°C in Grace's Supplemented (TNM-FH) medium and grown overnight.
  • Celis are infected with die baculovirus and harvested at various time points to measure antibody production.
  • Example 17 Antibody purification.
  • the antibodies described herein may be purified by several procedures commonly used in the production of recombinant antibodies, including precipitation by ammonium sulfete, antigen affinity chromatography, thioaffinity chromatography, or binding to bacterial proteins that have high affinity for mammalian immunoglobulins. Beads coupled to recombinant Streptococcus Protein G have been used to purify the IgG molecules described herein. A 5 mL Protein G Sepharose HiTrap column (GE Healthcare cat #17-0405-01) was equilibrated with 1x PBS (10 mM sodium phosphate, 150 mM sodium chloride, pH 7.4).
  • 1x PBS 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.4
  • the sample was loaded onto the column, washed with PBS, and the antibody was eluted with 5 column volumes of 100 mM glycine, pH 2.7.
  • the fraction collection tubes contained 100 pL 1 M TrisCi, pH 9 to neutralize the eluate fractions.
  • the elution fractions were pooled and concentrated with an Amicon 10k MWCO spin filter to purify the antibodies.
  • Example 18 Treatment of patients infected with COVID- 19.
  • Subjects having been diagnosed with COVID-19 are passively immunized with bovinized and folly bovine recombinant monoclonal antibodies combinations thereof having different specificities, polyclonal antibodies, and/or mixtures of the foregoing directed against SARS CoV-2 antigens for the treatment and/or alleviation of the symptoms of COVID- 19.
  • the antibodies, and/or mixtures of antibodies, directed against SARS CoV-2 antigens are purified or derived from the colostrum of cows having been immunized with SARS CoV-2 antigens (e.g., spike polypeptides, membrane polypeptides, envelope polypeptides, nucleocapsid polypeptides).
  • Subjects diagnosed with COVID-19 are administered a therapeutically effective amount of a pharmaceutical composition comprising one or a combination of recombinant monoclonal antibodies, or polyclonal antibodies and/or mixtures of such antibodies, directed against SARS CoV-2 antigens, for example, administration by systemic (e.g., intraperitoneal or intravenous), oral, and/or mucosal routes.
  • a pharmaceutical composition comprising one or a combination of recombinant monoclonal antibodies, or polyclonal antibodies and/or mixtures of such antibodies, directed against SARS CoV-2 antigens, for example, administration by systemic (e.g., intraperitoneal or intravenous), oral, and/or mucosal routes.
  • the effective amount of polyclonal or monoclonal antibody or monoclonal antibody combination or mixture of monoclonal and polyclonal antibody contained in a therapeutic composition effective for treatment or prophylaxis of a viral infection, particularly, COVID-19, for oral cavity or GI tract administration according to the present disclosure will vary depending on such factors as affinity for die target, specificity for the target, and number of target epitopes recognized by the antibody or antibodies administered.
  • the quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of inhibition or neutralization of virus desired. Broadly speaking, the effective amount will typically be in the range from about 0.01 mg per dose to about to about 1 gram or higher per dose (not per antibody).
  • Multiple doses per day may be administered and up to 30 grams daily have been generally recognized as safe. In some embodiments, it may be desirable to reach the maximum tolerated daily dose.
  • the frequency of administration will usually be a single dose but administration can be repeated as needed within a day and on subsequent days. Oral administration can also be combined with intraperitoneal or intravenous administration of the same antibody-containing therapeutic composition or of a different antibody-containing composition.
  • the effective unit dose of polyclonal or monoclonal antibody or monoclonal antibody combination or mixture of monoclonal and polyclonal antibody contained in a therapeutic composition for intranasal or by-inhalation administration effective and useful for treatment or prophylaxis of virus, particularly COVID-19, is comparatively reduced versus that indicated or required for digestive tract administration, and much reduced compared to that that required for IP or IV administration.
  • an antibody composition for airway administration, particularly intranasal or by-inhalation administration wherein the unit dose is further and substantially reduced in order to be applied to the target tissue and to avoid eliciting a reflex reaction.
  • intranasal and by-inhalation administration will typically be in an amount within the range from 0.01 mg per dose to up to about 3 mg per dose.
  • the amount can be higher up to the highest amount that can be applied to the target tissue and/or tolerated. Multiple daily doses are contemplated if needed and tolerated up to the maximum tolerated daily amount.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount for intranasal or by-inhalation administration.
  • quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of inhibition or neutralization of virus desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. Suitable regimes for initial administration and follow-on administration are also variable. In one regime, there is an initial administration followed by repeated subsequent dose(s), a single or multiple subsequent doses, at one or more hour intervals by a subsequent injection or other administration of the same or a different anti-SARS CoV-2 antibody.
  • Initial administration intranasally may be followed by administration of higher doses of antibody intraperitoneally or intravenously or by other suitable, including the oral, route.
  • a novel dosing approach or parameter is provided wherein a patient or subject is administered antibody intranasally, and either concomitantly, subsequently or after the lapse of a few hours administered a neutralizing or non-neutralizing antibody intraperitoneally or intravenously.

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Abstract

La présente invention concerne des compositions de Tor et des procédés de traitement d'une infection par le SARS-CoV-2 chez un patient qui en a besoin, en particulier, l'invention concerne des compositions pharmaceutiques comprenant des anticorps monoclonaux, des anticorps polyclonaux, des anticorps anti-idéotype ou des mélanges de tels anticorps, dirigés contre des antigènes de la SARS-CoV-2. Ainsi, les compositions pharmaceutiques et les formulations de l'invention peuvent être utilisées pour administrer des agents thérapeutiques à base d'anticorps au patient pour le traitement d'une infection par le SARS-CoV-2 .
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