WO2022157774A1 - Antibodies, peptides and combinations of same for the treatment or prevention of coronavirus infection - Google Patents

Antibodies, peptides and combinations of same for the treatment or prevention of coronavirus infection Download PDF

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Publication number
WO2022157774A1
WO2022157774A1 PCT/IL2022/050088 IL2022050088W WO2022157774A1 WO 2022157774 A1 WO2022157774 A1 WO 2022157774A1 IL 2022050088 W IL2022050088 W IL 2022050088W WO 2022157774 A1 WO2022157774 A1 WO 2022157774A1
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Prior art keywords
antibody
coronavirus
antigen binding
binding domain
peptide
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PCT/IL2022/050088
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French (fr)
Inventor
Ziv SHULMAN
Roei David MAZOR
Liat STOLER-BARAK
Lihee ASAF
Nachum NATHAN
Sharon KAGAN BEN TIKVA
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Yeda Research And Development Co. Ltd.
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Publication of WO2022157774A1 publication Critical patent/WO2022157774A1/en

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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
    • 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
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • 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

  • the present invention in some embodiments thereof, relates to antibodies, peptides and combinations of same for the treatment or prevention of Coronavirus infection.
  • SARS-CoV-2 Worldwide infections by SARS-CoV-2 is the cause of the COVID-19 pandemic.
  • the development of vaccination that endows long-lasting immunity at the population level is critical for harnessing the rapid global spread of the virus. So far, several vaccine candidates are enrolled in clinical trials and show promising induction of high titers of virus-specific antibodies as well as cellular-mediated responses.
  • RBD receptor-binding domain
  • Phagocytic cells contribute to the antibody-mediated elimination of pulmonary-infected SARS coronavirus. Virology. (2014) 454-455:157-68. doi: 10.1016/j.virol.2014.02.005
  • composition comprising at least one antigenic determinant of Coronavirus and at least one antibody comprising an antigen binding domain which binds the at least one antigenic determinant of Coronavirus.
  • composition for use in preventing or treating Coronavirus infection there is provided the composition for use in preventing or treating Coronavirus infection.
  • a method of preventing or treating Coronavirus infection in a subject in need thereof comprising administering to the subject an effective amount of the composition of claim 1, thereby treating or preventing the Coronavirus infection in the subject.
  • composition comprising a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 2 or analog or a fragment thereof, the peptide being shorter than 50 amino acids in length and capable of specifically binding an A7 antibody.
  • composition comprising a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 1 or analog or a fragment thereof, the peptide being shorter than 50 amino acids in length and capable of specifically binding a C1 or C3 antibody.
  • composition for use in preventing or treating Coronavirus infection in a subject in need thereof there is provided the composition for use in preventing or treating Coronavirus infection in a subject in need thereof.
  • the at least one antigenic determinant is a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 1 or analog or a fragment thereof, the peptide being shorter than 50 amino acids in length and capable of specifically binding a C1 or C3 antibody.
  • the at least one antigenic determinant is a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 2 or analog or a fragment thereof, the peptide being shorter than 50 amino acids in length and capable of specifically binding an A7 antibody.
  • the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9.
  • CDRs complementarity determining regions
  • a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus for use in preventing or treating Coronavirus infection in a subject in need thereof, wherein the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9.
  • CDRs complementarity determining regions
  • a method of preventing or treating Coronavirus infection in a subject in need thereof comprising administering to the subject an effective amount of a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus, wherein the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9, thereby preventing or treating Coronavirus in the subject.
  • CDRs complementarity determining regions
  • a method of producing an antibody capable of binding an antigenic determinant of Coronavirus comprising:
  • expressing in a host cell a heterologous polynucleotide encoding a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus, wherein the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9; and optionally
  • CDRs complementarity determining regions
  • a vaccine comprising an effective amount of a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus and an excipient, wherein the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9.
  • CDRs complementarity determining regions
  • a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus attached to a heterologous effector moiety or carrier, wherein the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9.
  • CDRs complementarity determining regions
  • the antibody is a recombinant antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is capable of activating antibody-dependent cellular phagocytosis.
  • the antibody is of an IgGl serotype.
  • the antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of A7, C1 or C3.
  • the antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of A7.
  • the antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of C1 or C3.
  • the human antibody comprising an antigen binding domain which binds the Coronavirus comprises a plurality of different human antibodies each comprising an antigen binding domain which binds a Coronavirus.
  • a method of detecting a Coronavirus infection comprising contacting a biological sample suspected of being infected with Coronavirus with a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus under conditions which allow a specific immunocomplex formation between the antibody and the Spike, wherein a presence of the immunocomplex is indicative of Coronavirus infection, wherein an antigen binding domain of the antibody comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9.
  • CDRs complementarity determining regions
  • the antibody is labeled.
  • the contacting is effected in-vivo. According to some embodiments of the invention, the contacting is effected ex-vivo. According to an aspect of some embodiments of the present invention there is provided a diagnostic kit for detecting a Coronavirus infection, the kit comprising a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus which allow a specific immunocomplex formation between the antibody and the Spike, wherein an antigen binding domain of the antibody comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9.
  • CDRs complementarity determining regions
  • the antibody is labeled.
  • the Coronavirus is SAR-CoV-2, Middle East respiratory syndrome Coronavirus (MERS-CoV) or severe acute respiratory syndrome Coronavirus (SARS-CoV).
  • MERS-CoV Middle East respiratory syndrome Coronavirus
  • SARS-CoV severe acute respiratory syndrome Coronavirus
  • the Coronavirus is SAR-CoV-2.
  • Figs. 1A-B show that patient-derived serum can form immune complexes that mediate antibody-dependent phagocytosis.
  • A. Spike-coated beads were incubated with patient or healthy control sera and the amount of bound IgG was measured by flow cytometry.
  • B. Coated beads from A, are able to induce phagocytosis by TCP-1 macrophages.
  • Figs. 2A-C show monoclonal antibodies that bind the spike protein.
  • Fig. 3 shows peptide targets of WIS-C1 and WIS-A7-germline. Peptides derived from the spike protein were used for detection of the antibody binding sites. The specific targets are shown in red. The sequences of the overlapping target peptides are shown below.
  • Figs. 4A-D show phagocytic and neutralization activity of the cloned antibodies.
  • a and B Binding of the cloned antibodies to spike-coated beads. Present bead binding is shown in A and the amount of binding is shown in B.
  • C Phagocytic activity of the cloned antibodies.
  • D Neutralization activity of the clones antibodies.
  • FIGs. 5A-B show that vaccination of mice with immune complexes composed of spike and WES-A7 induce a rapid antibody response.
  • Figures 5 A and B Mice were either immunized with immune complex, isotype control + spike or spike adjuvanted in alum. The amount of IgG1 antibodies are shown for each mouse (A) or as an average of 3 mice (B).
  • the present invention in some embodiments thereof, relates to antibodies, peptides and combinations of same for the treatment or prevention of Coronavirus infection.
  • SARS-CoV-2 invasion triggers the activation of the adaptive immune response and the rapid generation of antibodies.
  • SARS-CoV-2-specific monoclonal antibodies are tested for virus-neutralizing activity, less is known about their effector mechanisms.
  • polyclonal antibodies derived from recovered patient sera formed immune complexes and supported antibody-dependent cellular phagocytosis (ADCP).
  • ADCP antibody-dependent cellular phagocytosis
  • the present inventors generated monoclonal antibodies (mAbs) of different affinities to the SPIKE protein and defined their specific binding domains.
  • the present inventors revealed a high-affinity mAb that carried only 2 affinity enhancing mutations and was highly potent in supporting ADCP through the generation of ICs.
  • Mouse vaccination with ICs induced a spike-specific antibody response without adjuvant at a faster rate than traditional adjuvanted immunization such as Alum.
  • composition comprising at least one antigenic determinant of Coronavirus and at least one antibody comprising an antigen binding domain which binds said at least one antigenic determinant of Coronavirus.
  • antigenic determinant refers to a peptide (i.e., essentially a peptide) which comprises an epitope that is recognized by an antigen binding domain of an antibody. Hence the antigenic determinant may comprise one or more epitopes. According to a specific embodiment, the antigenic determinant forms a portion of a viral protein with or without amino acid alterations with respect to the wild-type viral sequence.
  • the antigenic determinant is of a Coronavirus.
  • Coronavirus refers to enveloped positive-stranded RNA viruses that belong to the family Coronaviridae and the order Nidovirales.
  • Corona viruses which are contemplated herein include, but are not limited to, 229E, NL63, OC43, and HKU1 with the first two classified as antigenic group 1 and the latter two belonging to group 2, typically leading to an upper respiratory tract infection manifested by common cold symptoms.
  • Coronaviruses which are zoonotic in origin, can evolve into a strain that can infect human beings leading to fatal illness.
  • SARS-CoV Middle East respiratory syndrome Coronavirus
  • MERS- CoV Middle East respiratory syndrome Coronavirus
  • SARS-CoV-2 2019-nCoV
  • the Corona virus is SARS-CoV-2.
  • binding refers to a mode of binding that reflects an antibody and an antigen binding.
  • the affinity is between 0.05-100 nM, as determined by ELISA assay which is described in the Examples section which follows.
  • the composition comprises at least one antibody.
  • a composition may be prepared under conditions (e.g., buffers) which allow for a structural lattice to form (see Figure 1 of Wang et al. supra).
  • the formation of ICs is expected to augment ADCP, a well as antigen presentation that results in an anti Coronavirus-specific T cell response enhancement and antibody response enhancement.
  • ADCP antibody-dependent cellular phagocytosis
  • An Fc receptor-dependent function of antibody-dependent cellular phagocytosis provides mechanisms for clearance of virus and virus-infected cells, as well as for stimulation of downstream adaptive immune responses by facilitating antigen presentation, or by stimulating the secretion of inflammatory mediators.
  • the activity necessitates specific antibody-antigen binding and myeloid cells such as macrophages or neutrophils.
  • the assay involves incubation of THP-1 cell line with fluorescentiy-labeled spike coated beads and monoclonal antibodies.
  • the ADCP is measured by analysis of fluorescent signals in the THP-1 cells by flow cytometer.
  • the composition comprises 1 or more antibodies e.g., 2, 3, 4, 5 or up to 10 antibodies, each binding different epitopes of the at least one antigenic determinant, to form complex ICs.
  • immune complexes also termed “an antigen-antibody complex” or “antigen-bound antibody”, a molecule formed from the binding of multiple antigens to antibodies.
  • the bound antigen and antibody act as a unitary object, effectively an antigen of its own with a specific epitope.
  • the immune complexes can be subject to any of a number of responses, including complement deposition, opsonization, phagocytosis, or processing by proteases.
  • Red blood cells carrying CRl-receptors on their surface may bind C3b-coated immune complexes and transport them to phagocytes, mostly in liver and spleen, and return to the general circulation.
  • the complex can be formed in vitro by mixing of the antibody with the spike antigen or a peptide.
  • peptide(s) and antibody(s) will depend of the desired use.
  • the virus is SARS-CoV-2 and the antigenic determinant is derived from the SPIKE protein of the virus.
  • the composition is produced and maintained to form immunocomplexes (ICs) in vitro.
  • ICs immunocomplexes
  • peptide and “antigenic determinant” which are interchangeably used herein encompass native peptides backbone (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body, more capable of penetrating into cells improving clearance, biodistribution and/or pharmacokinetics. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, backbone modifications, and residue modification.
  • Natural aromatic amino acids, Tyr and Phe may be substituted by non-natural aromatic amino acids such as l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), naphthylalanine, ring-methylated derivatives of Phe, halogenated derivatives of Phe or O-methyl-Tyr.
  • Tic l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid
  • naphthylalanine naphthylalanine
  • ring-methylated derivatives of Phe halogenated derivatives of Phe or O-methyl-Tyr.
  • the peptide comprises naturally occurring Trp residues.
  • the peptides of some embodiments of the invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
  • modified amino acids e.g. fatty acids, complex carbohydrates etc.
  • amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.
  • amino acid includes both D- and Lramino acids (stereoisomers).
  • Tables 1 and 2 below list naturally occurring amino acids (Table 1), and non-conventional or modified amino acids (e.g., synthetic, Table 2) which can be used with some embodiments of the invention.
  • amino acids of the peptides of some embodiments of the present invention may be substituted either conservatively or non-conservatively.
  • conservative substitution refers to the replacement of an amino acid present in the native sequence in the peptide with a naturally or non-naturally occurring amino or a peptidomimetics having similar steric properties.
  • side-chain of the native amino acid to be replaced is either polar or hydrophobic
  • the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side-chain of the replaced amino acid).
  • amino acid analogs synthetic amino acids
  • a peptidomimetic of the naturally occurring amino acid is well documented in the literature known to the skilled practitioner.
  • the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.
  • non-conservative substitutions refers to replacement of the amino acid as present in the parent sequence by another naturally or non-naturally occurring amino acid, having different electrochemical and/or steric properties.
  • the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the native amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted.
  • non-conservative substitutions of this type include the substitution of phenylalanine or cycohexylmethyl glycine for alanine, isoleucine for glycine, or -NH-CH[(-CH 2 ) 5 -COOH]-CO- for aspartic acid.
  • Those non- conservative substitutions which fall under the scope of the present invention are those which still constitute a peptide capable of forming ICs.
  • peptides of some embodiments of the invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.
  • the peptides of some embodiments of the invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.
  • a composition comprising a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 1 or analog or a fragment thereof, the peptide being shorter than 50 amino acids in length and capable of specifically binding a C1 or C3 antibody.
  • the composition comprises a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 2 or analog or a fragment thereof, the peptide being shorter than 50 amino acids in length and capable of specifically binding an A7 antibody.
  • an A7 antibody refers to a germline antibody or a mutated version thereof such as set forth in A7, A7-sm1, A7-sm2, A7-sm3 a7-dm1, A7-dm2 and A7-dm3 (see sequences of Table 2).
  • SEQ ID NO: 2 is bound by all A7 family members, namely, WIS-A7 (the fully mutated version, WIS-A7sm1, WIS-A7sm2, WIS-A7sm3 (single mutant synthetic variants), WIS-A7dm1, WIS- A7dm2, WIS-A7dm3 (double mutant synthetic variants) and WIS-A7gl (germline, none mutated variant).
  • WIS-A7 the fully mutated version, WIS-A7sm1, WIS-A7sm2, WIS-A7sm3 (single mutant synthetic variants), WIS-A7dm1, WIS- A7dm2, WIS-A7dm3 (double mutant synthetic variants) and WIS-A7gl (germline, none mutated variant).
  • the A7 antibody is WIS-A7, carrying 3 mutations compared to the germline version (gl).
  • the peptide is shorter than the full length SPIKE.
  • the peptide does not include a receptor binding domain (RBD) or SPIKE.
  • RBD receptor binding domain
  • SPIKE receptor binding domain
  • the peptide is 4-50 amino acids long.
  • the peptide is 4-45 amino acids long.
  • the peptide is 4-40 amino acids long.
  • the peptide is 4-35 amino acids long.
  • the peptide is 4-30 amino acids long.
  • the peptide is 4-25 amino acids long.
  • the peptide is 4-20 amino acids long.
  • the peptide is 4-15 amino acids long.
  • the peptide is 4-10 amino acids long.
  • the peptide is 5-50 amino acids long.
  • the peptide is 6-50 amino acids long.
  • the peptide is 7-50 amino acids long.
  • the peptide is 8-50 amino acids long.
  • the peptide is 10-50 amino acids long.
  • the peptide is 12-50 amino acids long.
  • the peptide is 14-50 amino acids long.
  • the peptide is 16-50 amino acids long.
  • the peptide is 20-50 amino acids long.
  • the peptide is 25-50 amino acids long.
  • the peptide is 30-50 amino acids long.
  • the peptide is 35-50 amino acids long.
  • the peptide is 40-50 amino acids long.
  • the peptide When comprising substitutions (e.g., 1-10 amino acids substitution) or deletions (e.g., 1-5 amino acids deletions) relative to the wild-type sequence, the peptide is considered as an analog or derivative, though it is still to maintain the function of forming ICs with the cognate antibodies and ultimately activate ADCP or regardless of ICs or activation of ADCP simply used for vaccination in vivo.
  • substitutions e.g., 1-10 amino acids substitution
  • deletions e.g., 1-5 amino acids deletions
  • the peptide comprises a protecting moiety or a stabilizing moiety.
  • protecting moiety refers to any moiety (e.g. chemical moiety) capable of protecting the peptide from adverse effects such as proteolysis, degradation or clearance, or alleviating such adverse effects.
  • stabilizing moiety refers to any moiety (e.g. chemical moiety) that inhibits or prevents a peptide from degradation.
  • a protecting moiety or a stabilizing moiety typically results in masking the charge of the peptide terminus, and/or altering chemical features thereof, such as, hydrophobicity, hydrophilicty, reactivity, solubility and the like.
  • suitable protecting moieties can be found, for example, in Green et al., "Protective Groups in Organic Chemistry", (Wiley, 2.sup.nd ed. 1991) and Harrison et al., “Compendium of Synthetic Organic Methods", Vols. 1-8 (John Wiley and Sons, 1971-1996).
  • the protecting moiety (or group) or stabilizing moiety (or group) may be added to the N- (amine) terminus and/or the C- (carboxyl) terminus of the peptide.
  • N-terminus protecting/stabilizing moieties include, but are not limited to, formyl, acetyl (also denoted herein as “Ac”), trifluoroacetyl, benzyl, benzyloxycarbonyl (also denoted herein as “CBZ”), tert-butoxycarbonyl (also denoted herein as “BOC”), trimethylsilyl (also denoted “TMS”), 2-trimethylsilyl-ethanesulfonyl (also denoted “SES”), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (also denoted herein as "FMOC”), nitro-veratryloxycarbonyl (also denoted herein as "NVOC”), t- amyloxycarbonyl, adamantyl-oxycarbonyl, and p-methoxybenzyloxycarbonyl, 2- chloro
  • the protecting/stabilizing moiety is an amine protecting moiety.
  • the protecting/stabilizing moiety is a terminal cysteine residue.
  • C-terminus protecting/stabilizing moieties are typically moieties that lead to acylation of the carboxy group at the C-terminus and include, but are not limited to, benzyl and trityl ethers as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers, allyl ethers, monomethoxytrityl and dimethoxytrityl.
  • the -COOH group of the C-terminus may be modified to an amide group.
  • peptides include replacement of the amine and/or carboxyl with a different moiety, such as hydroxyl, thiol, halide, alkyl, aryl, alkoxy, aryloxy and the like.
  • chemical derivative of a peptide or analog.
  • Such chemical derivates contain additional chemical moieties not normally a part of the peptide.
  • Covalent modifications of the peptide are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
  • organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
  • salts of the peptides and analogs of the invention are also included in the scope of the invention.
  • the term “salts” refers to both salts of carboxyl groups and to acid addition salts of amino groups of the peptide molecule.
  • Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases such as those formed for example, with amines, such as triethanolamine, arginine, or lysine, piperidine, procaine, and the like.
  • Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids, such as, for example, acetic acid or oxalic acid.
  • mineral acids such as, for example, hydrochloric acid or sulfuric acid
  • organic acids such as, for example, acetic acid or oxalic acid.
  • Such chemical derivatives and salts are preferably used to modify the pharmaceutical properties of the peptide insofar as stability, solubility, etc., are concerned.
  • the isolated peptide capable of binding the antibody is attached to a heterologous moiety proteinaceous or non-proteinaceous moiety.
  • heterologous moiety refers to an amino acid sequence which does not endogenously form a part of the peptide’s amino essentially viral acid sequence.
  • the heterologous moiety does not affect the biological activity of the isolated peptide (e.g. capability of binding the antibody or eliciting an immune response against Coronavirus).
  • the heterologous moiety may thus serve to ensure stability of the isolated peptide of the present invention without compromising its activity.
  • the heterologous moiety may increase the half-life of the isolated peptide in the serum.
  • the heterologous moiety does not induce an immune response.
  • it may contain human sequences that do not produce an immune response in a subject administered therewith.
  • heterologous amino acid sequences examples include, but are not limited to, immunoglobulin, galactosidase, glucuronidase, glutathione-S-transferase (GST), carboxy terminal peptide (CTP) from chorionic gonadotrophin (CGb) and chloramphenicol acetyltransferase (CAT) [see for example U.S. Publication No. 20030171551].
  • the heterologous amino acid sequence is an immunoglobulin.
  • heterologous amino acid sequence is localized at the amino- or carboxyl- terminus (N-ter or C-ter, respectively) of the isolated peptide of the present invention.
  • the heterologous amino acid sequence may be attached to the isolated peptide amino acid sequence by any of peptide or non-peptide bond. Attachment of the isolated peptide amino acid sequence to the heterologous amino acid sequence may be effected by direct covalent bonding (peptide bond or a substituted peptide bond) or indirect binding such as by the use of a linker having functional groups.
  • the molecules (i.e., antibodies or peptides) of the present invention can be generated using recombinant techniques such as described by Bitter et al. (1987) Methods in Enzymol. 153:516-544; Studier et al. (1990) Methods in Enzymol. 185:60-89; Brisson et al. (1984) Nature 310:511-514; Takamatsu et al. (1987) EMBO J. 3:1671-1680; Brogli et al. (1984) Science 224:838-843; Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic
  • a method of producing an antibody capable of binding an antigenic determinant of Coronavirus comprising:
  • expressing in a host cell a heterologous polynucleotide encoding a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9; and optionally
  • CDRs complementarity determining regions
  • the heterologous moiety may also be chemically linked to the isolated peptide following the independent generation of each.
  • the two peptides may be covalently or non-covalently linked using any linking or binding method and/or any suitable chemical linker known in the art.
  • Such linkage can be direct or indirect, as by means of a peptide bond or via covalent bonding to an intervening linker element, such as a linker peptide or other chemical moiety, such as an organic polymer.
  • Such chimeric peptides may be linked via bonding at the carboxy (C) or amino (N) termini of the peptides, or via bonding to internal chemical groups such as straight, branched, or cyclic side chains, internal carbon or nitrogen atoms, and the like.
  • cross-linkers and cross linking methods are preferably adapted to the type and nature of the peptides used.
  • this variant is referred to a fusion protein or a chimeric protein.
  • fused means that at least a protein or peptide is physically associated with another protein or peptide, which naturally don’t form a complex.
  • the fused molecule is a “fusion peptide” or “fusion protein”, a protein created by joining two or more heterologously related peptide sequences together.
  • fusion protein “chimera”, “chimeric molecule”, or “chimeric protein” are used interchangeably.
  • non-proteinaceous moiety refers to a molecule, not including peptide bonded amino acids, that is attached to the above-described isolated peptide’s amino acid sequence.
  • the non-proteinaceous moiety is non-toxic.
  • non-proteinaceous moieties which may be used according to the present teachings include, but are not limited to, polyethylene glycol (PEG), Polyvinyl pyrrolidone (PVP), poly(styrene comaleic anhydride) (SMA), and divinyl ether and maleic anhydride copolymer (DIVEMA).
  • PEG polyethylene glycol
  • PVP Polyvinyl pyrrolidone
  • SMA poly(styrene comaleic anhydride)
  • DIVEMA divinyl ether and maleic anhydride copolymer
  • antibody as used in this invention includes intact molecules as well as functional fragments thereof (such as Fab, F(ab')2, Fv, scFv, dsFv, or single domain molecules such as VH and VL) that are capable of binding to the antigenic determinant such as of SEQ ID NO: 1 or 2.
  • the antibody is a whole or intact antibody.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP immunecomplexes
  • the antibody is an antibody fragment.
  • Suitable antibody fragments for practicing some embodiments of the invention include a complementarity-determining region (CDR) of an immunoglobulin light chain (referred to herein as “light chain”), a complementarity-determining region of an immunoglobulin heavy chain (referred to herein as “heavy chain”), a variable region of a light chain, a variable region of a heavy chain, a light chain, a heavy chain, an Fd fragment, and antibody fragments comprising essentially whole variable regions of both light and heavy chains such as an Fv, a single chain Fv (scFv), a disulfide-stabilized Fv (dsFv), an Fab, an Fab’, and an F(ab’) 2 .
  • CDR complementarity-determining region
  • light chain referred to herein as “light chain”
  • heavy chain a complementarity-determining region of an immunoglobulin heavy chain
  • variable region of a light chain a variable region of a heavy chain
  • a light chain a variable region of
  • CDR complementarity-determining region
  • VH VH1; CDRH2 or H2; and CDRH3 or H3
  • the identity of the amino acid residues in a particular antibody that make up a variable region or a CDR can be determined using methods well known in the art and include methods such as sequence variability as defined by Kabat et al. (See, e.g., Rabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.), location of the structural loop regions as defined by Chothia et al. (see, e.g., Chothia et al., Nature 342:877-883, 1989.), a compromise between Rabat and Chothia using Oxford Molecular's AbM antibody modeling software (now Accelrys®, see, Martin et al., 1989, Proc.
  • variable regions and CDRs may refer to variable regions and CDRs defined by any approach known in the art, including combinations of approaches.
  • Fv defined as a genetically engineered fragment consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) expressed as two chains;
  • scFv single chain Fv
  • dsFv disulfide-stabilized Fv
  • Fab a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme papain to yield the intact light chain and the Fd fragment of the heavy chain which consists of the variable and CH1 domains thereof;
  • Fab’ a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin, followed by reduction (two Fab’ fragments are obtained per antibody molecule);
  • F(ab’)2 a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin (i.e., a dimer of Fab’ fragments held together by two disulfide bonds); and
  • Single domain antibodies or nanobodies are composed of a single VH or VL domains which exhibit sufficient affinity to the antigen.
  • the antibody heavy chain constant region is chosen from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE.
  • the antibody is an IgG antibody.
  • the antibody isotype is IgG1 which is capable of eliciting ADCP.
  • antibody type will depend on the immune effector function that the antibody is designed to elicit.
  • the antibody comprises an Fc domain.
  • the Fc domain may be a wild-type Fc domain or a domain which elicits the same effector functions such as that of IgGl, but is can also be modified such as to increase immune effector functions e.g., ADCP.
  • ADCP recruitment depends on the following activating receptors: 5 activating Fc ⁇ Rs: the high affinity Fc ⁇ RI that can bind to monovalent antibody, and the lower affinity Fc ⁇ RIIa and IIe and Fc ⁇ RIIIa (HGNC:3619) and nib that require avidity-based interactions.
  • an engineered Fc variant with G236A (and optionally S239D/I332E) that provides selectively enhanced binding to FcgammaRIIa relative to FcgammaRIIb is specifically contemplated.
  • Variants containing this substitution have up to 70-fold greater FcgammaRIIa affinity and 15-fold improvement in FcgammaRIIa/FcgammaRIIb ratio and mediate enhanced phagocytosis of antibody-coated target cells by macrophages.
  • the Fc portion is a-fucosylated (N297A on IgG1) which also exhibits enhanced binding to FcRl.
  • the antibody is a naked antibody.
  • naked antibody refers to an antibody which does not comprise a heterologous effector moiety e.g. therapeutic moiety, detectable moiety.
  • heterologous means not occurring in nature in conjunction with the antibody.
  • the antibody comprises a heterologous effector moiety e.g. e.g. therapeutic moiety, detectable moiety.
  • the effector moiety can be proteinaceous or non- proteinaceous; the latter generally being generated using functional groups on the antibody and on the conjugate partner.
  • the effector moiety may be any molecule, including small molecule chemical compounds and polypeptides.
  • the effector moiety can be a known drag to Coronavirus infection.
  • the antibody is a recombinant antibody.
  • the antibody is a monoclonal antibody.
  • Antibody fragments according to some embodiments of the invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • cleaving antibodies such as separation of heavy chains to form monovalent light- heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross- linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker.
  • These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
  • humanized or human antibodies are preferably used.
  • the antibody is a human antibody.
  • the human antibody carries human Vh,Dh, Jh, VI, J, gene segments such as in germ line antibodies or natural variants thereof.
  • Table 2 below lists antibodies, heavy chains, light chains, CDRs or antibodies which are specifically contemplated according to some embodiments of the invention.
  • the Table describes germ-line antibodies and natural variants and synthetic variants thereof.
  • the subject can be treated with a plurality of antibodies to achieve maximal inhibition of the virus either as a treatment or as a vaccine. Also diagnosis may be benefited by the use of a plurality of antibodies.
  • plurality refers to at least 2 antibodies having different antigen binding domains (at least one different CDR), e.g., 2-3, 2-4.
  • the plurality of antibodies bind different epitopes on the virus.
  • the plurality of antibodies bind identical epitopes on the virus, but may be different in their effector (Fc-mediate) functions.
  • the human antibody comprising an antigen binding domain which binds said antigenic determinant comprises a plurality of different human antibodies each comprising an antigen binding domain which binds a Coronavirus.
  • the antibody binds the SPIKE protein of a
  • receptor binding domain refers to the receptor (ACE2) binding domain of SARS-CoV-2 of SPIKE, residues Arg319-Phe541 of SPIKE.
  • Binding can be qualified using various methods known in the art, such as ELISA (exemplified in the section which follows) and surface plasmon resonance (SPR).
  • ELISA exemplified in the section which follows
  • SPR surface plasmon resonance
  • the present invention envisages immunization against-, and prevention or treatment of Coronavirus infection with any of the immunocomplexes, peptides, antibodies described herein.
  • treating refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology.
  • pathology disease, disorder or condition
  • Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology (i.e., Coronavirus infection, e.g., COVID19 or related complications).
  • the term “preventing” refers to keeping a disease, disorder or condition (i.e., Coronavirus infection, e.g., COVID19 or related complications) from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.
  • a disease, disorder or condition i.e., Coronavirus infection, e.g., COVID19 or related complications
  • Prevention can be done by means of immunization, in an embodiment passive immunization, where the antibody is administered, or active where the peptide is administered.
  • the term “subject” includes mammals, preferably human beings, male or female, at any age or gender, which suffer from the pathology. Preferably, this term encompasses individuals who are at risk to develop the pathology (e.g., above 65 of age) or exposed to the virus, e.g., healthcare personnel, education personnel etc.
  • composition of matter comprising any of the immunocomplexes, peptides, and antibodies (active ingredient(s)) of the present invention can be administered to the subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
  • a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the immunocomplexes, peptides or antibodies accountable for the biological effect.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intrapulmonary or intraocular injections.
  • Conventional approaches for drag delivery to the central nervous system include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport polypeptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin polypeptide).
  • neurosurgical strategies e.g., intracerebral injection or intracerebroventricular infusion
  • molecular manipulation of the agent e
  • each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
  • compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • compositions of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (composition of matter comprising the antibodies) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., Coronaviral infection) or prolong the survival of the subject being treated.
  • a disorder e.g., Coronaviral infection
  • an effective amount of the composition of matter comprising the antibodies of some embodiments of the present invention is an amount selected to eliminate infected cells e.g. by initiating ADCC or ADCP and optionally neutralize Coronaviruses.
  • any in vivo or in vitro method of evaluating Coronavirus viral load may be employed.
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 P-1) ⁇
  • Dosage amount and interval may be adjusted individually to provide the active ingredient at a sufficient amount to induce or suppress the biological effect (minimal effective concentration, MFC).
  • MFC minimum effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drags or of an approved product insert.
  • Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
  • the present teachings further envisage treating with other anti-viral drags or anti- inflammatory drags or anti-coagulants as separate treatments or in a co-formulation.
  • the antiviral drag is selected from the group consisting of remdesivir, an interferon, ribavirin, adefovir, tenofovir, acyclovir, brivudin, cidofovir, fomivirsen, foscamet, ganciclovir, penciclovir, amantadine, rimantadine and zanamivir.
  • plasma treatments from infected persons who survived and/or anti- HIV drags such as lopinavir and ritonavir, as well as chloroquine.
  • Drags that are routinely used for the treatment of COVID-19 include, but are not limited to, Lopinavir /Ritonavir, Nucleoside analogues, Neuraminidase inhibitors, Remdesivir, polypeptide (EK1), abidol, RNA synthesis inhibitors (such as TDF, 3TC), anti- inflammatory drags (such as hormones and other molecules), Chinese traditional medicine, such ShuFengJieDu Capsules and Lianhuaqingwen Capsule, could be the drag treatment options for COVID19.
  • a method of diagnosing a Coronavirus infection in a subject in need thereof comprising:
  • a biological sample which may comprise a SPIKE protein of a Coronavirus with antibodies as described herein under conditions which allow complex formation between the composition and the SPIKE;
  • diagnosis refers to classifying a disease, determining a severity of a disease (grade or stage), monitoring progression, forecasting an outcome of the disease and/or prospects of recovery.
  • the subject may be a healthy subject (e.g., human) undergoing a routine well-being check-up.
  • the subject may be at risk of the disease or infection.
  • the method may be used to monitor treatment efficacy.
  • biological sample refers to a sample of tissue or fluid isolated from a subject, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, sputum, and also samples of in vivo cell culture constituents. It should be noted that a “biological sample obtained from the subject” may also optionally comprise a sample that has not been physically removed from the subject (in vivo as opposed to in vitro).
  • tissue or fluid collection methods can be utilized to collect the biological sample from the subject in order to determine the level of Coronaviruses or infected cells in the sample. Regardless of the procedure employed, once a biopsy/sample is obtained the level of the variant can be determined and a diagnosis can thus be made.
  • the method of the present invention is effected under conditions sufficient to form protein-protein interactions i.e., complex (e.g. a complex between).
  • complex e.g. a complex between
  • Such conditions e.g., appropriate concentrations, buffers, temperatures, reaction times
  • methods to optimize such conditions are known to those skilled in the art, and examples are disclosed herein below.
  • the antibody-SPIKE complex may comprise e.g., be attached, to an identifiable moiety.
  • the complex may be identified indirectly such as by using a secondary antibody.
  • diagnosis is corroborated using any diagnostic method known in the art, such as by measuring the viral load or titer, by antigen level measurement, antibody level measurement, virus isolation and/or genomic detection by reverse transcriptase- polymerase chain reaction (RT-PCR), etc.
  • RT-PCR reverse transcriptase- polymerase chain reaction
  • a higher viral load or titre often correlates with the severity of an active viral infection.
  • the quantity of virus per mL can be calculated for example by estimating the live amount of virus in an involved body fluid (e.g. serum sample or whole blood).
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
  • PBMCs Peripheral blood mononuclear cells
  • Plasma samples were obtained from 30ml blood extracted from recruited individuals by a certified physician and processed in an enhanced biosafety level 2 facility. Samples underwent a gradient separation protocol, in which 12ml of Ficoll-Paque (GE Healthcare) were added to the bottom of a 50-ml falcon tube containing 30ml of peripheral blood.
  • mice C57BL/6 mice were provided by Harlan. All experiments using mice were approved by the Weizmann Institute Animal Care and Use Committee.
  • mice were injected intraperitoneally with 200 ⁇ 1 PBS containing either 10 ⁇ g spike protein in alum (2:1), or immune complex containing 10 ⁇ g spike protein with 60 ⁇ g A7 or isotype control after 2h incubation at room temperature. Blood samples were collected prior to the immunization, followed by blood collection on day 3 and every 7 days up to 21 days after the injection. Blood samples were centrifuged at 3000 RPM, 4°C, for 10 minutes and plasma was aliquoted and frozen. Flow cytometry
  • Antibody-dependent cellular phagocytosis was assessed by the measurement of the uptake of antibody-opsonized, antigen-coated fluorescent beads by a monocytic cell line. Briefly, 2 ⁇ g of Biotinylated Spike protein was used to saturate the binding sites of 0.5mg 1 ⁇ m fluorescent NeutrAvidin beads (Invitrogen). Excess antigen was removed by washing the beads, which were then blocked with 1% BSA. Next, the beads were washed and incubated with antibodies at a final concentration of 0.5 ⁇ or plasma diluted 1:100 for 2h at 37°C. Following opsonization, beads were washed, and unbound antibodies were removed.
  • the beads were then either stained for IgG to determine the IgG coating or incubated with phagocytotic cells.
  • phagocytosis assay THP-1 cells (ATCC) were added, and the cells were incubated for lh at 37°C to allow phagocytosis. The cells were then fixed, and the extent of phagocytosis was measured via flow cytometry (CytoFLEX). The data are reported as a phagocytic score, which takes into account the proportion of effector cells that phagocytosed and the degree of phagocytosis (integrated MFI: frequency x MFI) as previously described (Darrah, Patricia A., et al.
  • Multifunctional TH 1 cells define a correlate of vaccine-mediated protection against Leishmania major.” Nature medicine 13.7 (2007): 843-850).
  • the beads were incubated with anti-human IgG secondary antibody (Jackson Immuno Research) in blocking buffer at a 1:100 dilution for 30 min on ice. The beads were then washed, and the IgG was measured using the CytoFLEX flow cytometer.
  • Lentiviruses expressing S-Covidl9 spikes were produced by transfecting HEK293T cells with Luciferase-pLenti6, ⁇ 19 S_covid-pCMV3 and ⁇ R89 ⁇ vectors at 1:1:1 ratio, using Lipofectamine 2000 (Thermo Fisher). Media containing Lentiviruses was collected at 48h post- transfection, centrifuged at 600g for 5 min for clarifying from cells, and aliquots were frozen at - 80°C. For neutralization assays, HEK293T were transiently transfected with hACE2-pCDNA using Lipofectamine 2000.
  • ELISA reactions to evaluate antibodies binding to SARS-CoV-2 RBD and trimeric spike proteins were carried out using flat-bottom MaxiSorpTM 96- well plates (Invitrogen). The plates were coated with 5 ⁇ g/ml protein solution in PBS at 100 ⁇ per well and left overnight at 4°C. The Plates were washed 5 times with washing buffer (lx PBS with 0.05% Tween-20 (Sigma- Aldrich)) and incubated with 100 ⁇ blocking buffer (lx PBS with 1% BSA) for lh at room temperature. The blocking solution was subsequently replaced by a serial dilutions of either monoclonal antibodies or plasma samples for 2.5h at RT.
  • Plasma samples were assayed at a 1:10 starting dilution and 3 additional tenfold serial dilutions. Monoclonal antibodies were tested at 10 ⁇ g/ml starting concentration and 8 additional fourfold serial dilutions. Plates were washed 6 times with washing buffer and then incubated with anti-human IgG or mouse IgG secondary antibody conjugated to horseradish peroxidase (HRP) (Jackson Immuno Research) in blocking buffer at a 1:5,000 dilution. Plates were developed by addition of the HRP substrate, TMB (Thermo Fisher) and absorbance was measured at 630nm with an ELISA microplate reader (Synergy HT, Biotek).
  • HRP substrate Thermo Fisher
  • Spike reactive CD19 + , CD27 + , IgGl + , IgK* peripheral blood memory B cells were single cell sorted into 96 well plates. These in turn underwent nested PCR amplification and Sanger sequencing of their heavy and light chain transcripts, as previously described (PMID: 17996249). Upon collection of all immunoglobulin transcripts, data analysis was performed as detailed below. Determination of clonality and reconstruction of lineage trees
  • Ig Fasta sequences were aligned against the IMGT human heavy chain gene database (downloaded at Dec. 2019) and light chain gene database (downloaded at Feb. 2017) using NCBI IgBlast (version 1.14.0) (Ye et al., 2013).
  • Post processing of IgBlast output, and clonal clustering were performed using Change-0 vO.4.6 (Gupta et al., 2015), Alakazam v0.3.0, SHazaM vO.2.3, and custom scripts within the R statistical computing environment, as follows.
  • V(D)J sequences were assigned to clonal groups by partitioning sequences based on identity of IGHV gene annotations, IGHJ gene annotations, and junction region lengths.
  • sequences differing from one another by a distance of more than 15 nucleotides between the V genes were defined as separate clones.
  • the clonal distance threshold was determined by manual inspection using heatmaps of V genes hamming distance.
  • Full-length germline sequences were reconstructed for each clonal cluster with D segment and N/P regions masked (replaced with Ns), with any ambiguous gene assignments within clonal groups resolved by the majority rule.
  • Lineage trees were constructed for each clone having at least two unique sequences using PHYLIP (v3.697) (Felsenstein, 2005) and Alakazam. Selection quantification was calculated using BASELINe’s local test (Yaari et al., 2012).
  • Antibody transcripts were chosen for cloning and expression on the basis of several criteria. These included: relation of the candidate to an expanded B cell clone, a high degree of somatic hypermutation or homology to the CDR3 consensus sequence motif as generated by aggregating all sequences whose V gene appeared in more than 5% of the sample size.
  • Candidate heavy and light chain transcripts including 5’ & 3’ vector homologous, 30 nucleotide long arms were ordered as gene blocks from IDT. These were cloned into IgGl/K expression vectors via the restriction free method using Phusion High-Fidelity DNA polymerase (NEB), according to the manufacturer’s protocol.
  • the previous expression vector templates were degraded by incubating the products with the DPN1 restriction enzyme (NEB) for 16 hours at 37C.
  • the products were then transformed into DH5a competent bacteria via the heat shock method (42°C, 90s). Selection of plasmid bearing bacteria was performed on the basis of Ampicillin resistance inherent to the vectors. 24 hours following transformation, single bacterial colonies were isolated. These underwent colony PCR and were re-plated on an index plate.
  • the colony PCR products were sequenced using the Sanger method to confirm that their corresponding plasmids indeed contain the desired constructs in-frame and absent of any de- novo mutations.
  • Colonies harboring successfully cloned plasmids were used to form glycerol stocks as well as a larger biomass of the desired vectors using a Maxiprep kit (Qiagen). The Maxiprep products were then sequenced again to confirm their accuracy.
  • Antibody expression was performed in 293T HEK cells. Cells were grown to 90% confluence in 15cm plates, in complete growth medium (DMEM, 10% foetal bovine serum, IX MEM-Eagle non essential amino acids, 2mM glutamine, 1:100 Pen-Strep solution). Prior to transfection, the 293T HEK monolayers were carefully washed with PBS and complemented with serum free complete growth medium.
  • DMEM 10% foetal bovine serum
  • IX MEM-Eagle non essential amino acids 2mM glutamine, 1:100 Pen-Strep solution
  • the transfection mix was formed in 1ml of serum free medium per plate - and included 12.5 ⁇ g of each vector (IgGl & IgK) and 50 ⁇ g of linear, 25kDa linear polyethylenimine as a transfection reagent.
  • Antibody purification was performed five days following introduction of the transfection mix to the plates. Supernatants were collected, filtered, and reacted overnight at 4°C with protein G sepharose beads (GE Healthcare). The beads were washed with PBS and eluted using IgG elution buffer (Thermo-scientific) into 1M Tris buffer after which they were dialyzed to PBS overnight. Measurement of the antibody concentration was performed using Nanodrop.
  • SARS-CoV2 SPIKE Ectodomain Construct SEQ ID Nos: 113-114)
  • ADCP Antibody-Dependent Cellular Phagocytosis
  • ADCP is one of the major functions of antibodies that promote the internalization of antibody-coated pathogens or antigens by cells of the innate immune system.
  • the present inventors examined the capacity of antibodies in serum derived from convalescent COVID-19 patients to mediate antigen capture. For this purpose, 10 recovered individuals that donated blood samples 6-10 weeks after recovery were recruited. Whereas SARS-CoV-2-neutralizing antibodies usually interact with the receptor-binding domain (RBD) of the spike, antibodies with ADCP potential are not necessarily restricted to this domain. Therefore, the ADCP potential of antibodies that bind spike triplex protein and not necessarily the RBD domain was examined. For this purpose, the SARS-CoV-2 spike protein complex was generated as well as the single- molecule RBD domain.
  • RBD receptor-binding domain
  • ELISA ELISA was performed using patient-derived sera. It was found that all of the recovered patients have anti-spike IgGl antibodies in their blood, however, the quantity of the antibodies varied significantly among the examined individuals. Thus, the study cohort of recovered patients has detectable IgGl antibodies that bind the spike protein in their sera. In order for an antibody to mediate ADCP, it must effectively bind its target in multiple sites to form a high-density surface coating. To test whether the amount of spike-specific antibodies in the recovered patient blood are sufficient to induce ADCP, serum IgG was first examined for the ability to form soluble immune complexes (ICs).
  • ICs soluble immune complexes
  • spike complexes were biotinylated and conjugated to streptavidin-coated beads.
  • the beads were incubated with recovered patient sera or with samples that were taken from healthy controls, for 2 hours followed by fluorescent anti-IgG staining.
  • This assay revealed that IgG antibodies in the recovered patients can form ICs in various efficiencies whereas beads from healthy donors showed low background level of antibody coating (Figure 1A).
  • immune complexes were generated with serum antibodies and incubated with THP-1 macrophage cell line.
  • the left side is a quantitative measure of the amount of beads phagocytosed and the right side, a measure of how many macrophages participate in phagocytosis in general.
  • the ADCP analysis of recovered patients was performed with polyclonal antibodies that most likely bind multiple sites on the spike trimeric complex.
  • the aim is to expose and characterize monoclonal antibodies that effectively mediate ADCP in COVID-9 convalescent patients.
  • immunoglobulins of memory B cells from two patients were sequenced, one of which showed the highest ADCP activity in the IC-uptake assay.
  • Memory B cells were defined as class-switched (IgGl) CD 19+ CD27+ cells that bind fluorescently labeled spike trimeric complexes.
  • IgGl class-switched CD 19+ CD27+ cells that bind fluorescently labeled spike trimeric complexes.
  • the present inventors focused only on IgK expressing memory B cells. Spike-specific single MBCs were sorted and subjected to PCR amplification of their immunoglobulin genes followed by gene sequencing.
  • mAb WIS-A7 (or A7) which carried only 3 mutations (2 and 1 in the heavy and light chains respectively) compared to the germline version (also referred to herein as “gl”) in IMGT was the most effective in binding the spike protein, whereas the mutated antibodies interacted modestly with the spike (A9, C1 and C3) ( Figure 2A).
  • mAb A7 was reverted to its germline configuration and its binding capacity to the spike protein was tested.
  • ELISA revealed that the germline version of WIS-A7 was 100-fold less effective in binding the spike (Figure 2B).
  • Antibodies can bind either a linear sequence or a 3D structure of a protein.
  • an array of linear peptides that cover all of the spike sequences was used [CelluSpots HullB (Intavis)].
  • C1 showed a clear target binding of two overlapping peptides
  • A7 and C3 showed binding patterns that were similar to a control nonspecific antibody.
  • A9 did not show any binding activity in this assay (not shown).
  • the germline version of A7 bound two specific overlapping peptides that were not detected in the mutated version of the antibody ( Figure 3).
  • Patient-derived monoclonal antibodies support antibody-dependent cellular phagocytosis and virus neutralization
  • Example 1 above showed that patient-derived serum IgG can mediate ADCP.
  • the present inventors first examined if the spike-specific immunoglobulins can form ICs.
  • spike-coated beads were mixed with the same amount of different monoclonal antibodies.
  • Flow cytometric analysis revealed that besides A9, all of the cloned antibodies, including the germline version of A7, bound all of the spike coated beads and formed IC ( Figure 4A).
  • Additional mean fluorescent intensity (MET) measurements revealed that A7 and its germline version were the most effective in coating the beads (Figue 4B). This assay indicates that many A7 antibodies bind one bead and possibly several antibodies bind one spike complex.
  • MET mean fluorescent intensity
  • the A7 monoclonal antibody was very effective in the generation of ICs that promoted efficient antigen uptake by innate immune cells through ADCP. Immune complexes are very potent inducers of antibody generation and were previously used as vaccines. Whereas IgGl binds to activating FC receptors in humans, in mice this function is mediated by IgG2a/b. Thus, a mouse version of A7 was produced that has an IgG2a FC domain. To examine whether A7 can be used as IC for vaccination the present inventors generated immune complexes in vitro and used them for i.p. immunization of mice.
  • mice were immunized with traditional antigen in alum vaccine.
  • Spike in alum immunization triggered robust IgGl antibody formation only at day 14 after administration of the vaccine whereas immunization with IC triggered endogenous antibody generation very rapidly that was already detected at day 7 of the response ( Figures 5A-B).
  • IC immunization induced a stronger IgGl antibody response than the traditional vaccination at day 14 after administration ( Figures 5A-B).

Abstract

A composition comprising at least one antigenic determinant of Coronavirus and at least one antibody comprising an antigen binding domain which binds said at least one antigenic determinant of Coronavirus is provided. Also provides uses of such compositions.

Description

ANTIBODIES, PEPTIDES AND COMBINATIONS OF SAME FOR THE TREATMENT OR PREVENTION OF CORONAVIRUS INFECTION
RELATED APPLICATION
This application claims priority from Israeli Application No. 280340 filed on January 21, 2021 and is hereby incorporated by reference in its entirety.
SEQUENCE LISTING STATEMENT
The ASCII file, entitled 90393 Sequence Listing.txt, created on 18 January 2022, comprising 69,632 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.
FIELD AND BACKGROUND OF THE INVENTION
The present invention, in some embodiments thereof, relates to antibodies, peptides and combinations of same for the treatment or prevention of Coronavirus infection.
Worldwide infections by SARS-CoV-2 is the cause of the COVID-19 pandemic. The development of vaccination that endows long-lasting immunity at the population level is critical for harnessing the rapid global spread of the virus. So far, several vaccine candidates are enrolled in clinical trials and show promising induction of high titers of virus-specific antibodies as well as cellular-mediated responses. In addition, many studies examined the antibody immune response against SARS-CoV-2 and identified antibodies with very potent neutralization activity by blocking the interaction between the virus envelope spike protein and its receptor in the host. Most neutralizing antibodies prevent the receptor-binding domain (RBD) within the spike from binding the ACE2 receptor that mediates virus docking on host cells and internalization. Passive transfer of these antibodies can induce a reduction of viral load and this type of therapy is under evaluation for the use in the clinic. Despite the critical role of neutralizing antibodies in the protection and clearance of the virus, there is a major gap in understanding other antibody- mediated effector functions and their potential contribution to effective protection. In addition, less is known whether non-neutralizing antibody activity can be harnessed for therapeutic applications.
Hence, to date it is not clear if antibody effector functions are sufficient or to what extent a necessity in combating a Coronavirus infection.
Additional background art includes: Yasui F, Kohara M, Kitabatake M, Nishiwaki T, Fujii H, Tateno C, et al. Phagocytic cells contribute to the antibody-mediated elimination of pulmonary-infected SARS coronavirus. Virology. (2014) 454-455:157-68. doi: 10.1016/j.virol.2014.02.005
Zirui Tay et al. Front. Immunol., 28 February 2019 www(dot)doi(dot)orgZ10.3389/fimmu(dot)2019.00332
Wang et al. npj Vaccines (2019) 4:2 ; www(dot)doi(dot)org/10.1038/s41541-018-0095-z.
SUMMARY OF THE INVENTION
According to an aspect of some embodiments of the present invention there is provided a composition comprising at least one antigenic determinant of Coronavirus and at least one antibody comprising an antigen binding domain which binds the at least one antigenic determinant of Coronavirus.
According to an aspect of some embodiments of the present invention there is provided the composition for use in preventing or treating Coronavirus infection.
According to an aspect of some embodiments of the present invention there is provided a method of preventing or treating Coronavirus infection in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of claim 1, thereby treating or preventing the Coronavirus infection in the subject.
According to an aspect of some embodiments of the present invention there is provided a composition comprising a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 2 or analog or a fragment thereof, the peptide being shorter than 50 amino acids in length and capable of specifically binding an A7 antibody.
According to an aspect of some embodiments of the present invention there is provided a composition comprising a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 1 or analog or a fragment thereof, the peptide being shorter than 50 amino acids in length and capable of specifically binding a C1 or C3 antibody.
According to an aspect of some embodiments of the present invention there is provided the composition for use in preventing or treating Coronavirus infection in a subject in need thereof.
According to an aspect of some embodiments of the present invention there is provided a method of treating or preventing Coronavirus infection in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition, thereby treating or preventing Coronavirus infection in the subject. According to some embodiments of the invention, the at least one antigenic determinant is a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 1 or analog or a fragment thereof, the peptide being shorter than 50 amino acids in length and capable of specifically binding a C1 or C3 antibody.
According to some embodiments of the invention, the at least one antigenic determinant is a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 2 or analog or a fragment thereof, the peptide being shorter than 50 amino acids in length and capable of specifically binding an A7 antibody.
According to some embodiments of the invention, the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9.
According to an aspect of some embodiments of the present invention there is provided a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus for use in preventing or treating Coronavirus infection in a subject in need thereof, wherein the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9.
According to an aspect of some embodiments of the present invention there is provided a method of preventing or treating Coronavirus infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus, wherein the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9, thereby preventing or treating Coronavirus in the subject.
According to an aspect of some embodiments of the present invention there is provided a method of producing an antibody capable of binding an antigenic determinant of Coronavirus, the method comprising:
(a) expressing in a host cell a heterologous polynucleotide encoding a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus, wherein the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9; and optionally
(b) recovering the antibody from the host cell.
According to an aspect of some embodiments of the present invention there is provided a vaccine comprising an effective amount of a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus and an excipient, wherein the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9.
According to an aspect of some embodiments of the present invention there is provided a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus attached to a heterologous effector moiety or carrier, wherein the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9.
According to some embodiments of the invention, the antibody is a recombinant antibody.
According to some embodiments of the invention, the antibody is a monoclonal antibody.
According to some embodiments of the invention, the antibody is capable of activating antibody-dependent cellular phagocytosis.
According to some embodiments of the invention, the antibody is of an IgGl serotype. According to some embodiments of the invention, the antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of A7, C1 or C3.
According to some embodiments of the invention, the antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of A7.
According to some embodiments of the invention, the antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of C1 or C3.
According to some embodiments of the invention, the human antibody comprising an antigen binding domain which binds the Coronavirus comprises a plurality of different human antibodies each comprising an antigen binding domain which binds a Coronavirus.
According to an aspect of some embodiments of the present invention there is provided a method of detecting a Coronavirus infection, the method comprising contacting a biological sample suspected of being infected with Coronavirus with a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus under conditions which allow a specific immunocomplex formation between the antibody and the Spike, wherein a presence of the immunocomplex is indicative of Coronavirus infection, wherein an antigen binding domain of the antibody comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9.
According to some embodiments of the invention, the antibody is labeled.
According to some embodiments of the invention, the contacting is effected in-vivo. According to some embodiments of the invention, the contacting is effected ex-vivo. According to an aspect of some embodiments of the present invention there is provided a diagnostic kit for detecting a Coronavirus infection, the kit comprising a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus which allow a specific immunocomplex formation between the antibody and the Spike, wherein an antigen binding domain of the antibody comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9.
According to some embodiments of the invention, the antibody is labeled.
According to some embodiments of the invention, the Coronavirus is SAR-CoV-2, Middle East respiratory syndrome Coronavirus (MERS-CoV) or severe acute respiratory syndrome Coronavirus (SARS-CoV).
According to some embodiments of the invention, the Coronavirus is SAR-CoV-2.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
Figs. 1A-B show that patient-derived serum can form immune complexes that mediate antibody-dependent phagocytosis. A. Spike-coated beads were incubated with patient or healthy control sera and the amount of bound IgG was measured by flow cytometry. B. Coated beads from A, are able to induce phagocytosis by TCP-1 macrophages.
Figs. 2A-C show monoclonal antibodies that bind the spike protein. A. Spike binding activity of the cloned antibodies was tested by ELISA. B. Comparison of spike binding by ELISA of mutated A7 antibody and its germline version. C. Binding activity of A7 antibody that carries different mutations. Lines represent binding to the spike complex, dashed lines represent binding to BSA.;
Fig. 3 shows peptide targets of WIS-C1 and WIS-A7-germline. Peptides derived from the spike protein were used for detection of the antibody binding sites. The specific targets are shown in red. The sequences of the overlapping target peptides are shown below.
Figs. 4A-D show phagocytic and neutralization activity of the cloned antibodies. A and B. Binding of the cloned antibodies to spike-coated beads. Present bead binding is shown in A and the amount of binding is shown in B. C. Phagocytic activity of the cloned antibodies. D. Neutralization activity of the clones antibodies.
Figs. 5A-B show that vaccination of mice with immune complexes composed of spike and WES-A7 induce a rapid antibody response. Figures 5 A and B. Mice were either immunized with immune complex, isotype control + spike or spike adjuvanted in alum. The amount of IgG1 antibodies are shown for each mouse (A) or as an average of 3 mice (B).
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The present invention, in some embodiments thereof, relates to antibodies, peptides and combinations of same for the treatment or prevention of Coronavirus infection.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. SARS-CoV-2 invasion triggers the activation of the adaptive immune response and the rapid generation of antibodies. Whereas SARS-CoV-2-specific monoclonal antibodies are tested for virus-neutralizing activity, less is known about their effector mechanisms.
Whilst conceiving embodiments of the invention, the present inventors envisaged that efficient treatment or immunization against Coronavirus infection necessitates the formation of immune complexes (ICs) which is independent of neutralization.
As is shown in the Examples section which follows, polyclonal antibodies derived from recovered patient sera formed immune complexes and supported antibody-dependent cellular phagocytosis (ADCP). Using single-cell immunoglobulin sequencing, the present inventors generated monoclonal antibodies (mAbs) of different affinities to the SPIKE protein and defined their specific binding domains. The present inventors revealed a high-affinity mAb that carried only 2 affinity enhancing mutations and was highly potent in supporting ADCP through the generation of ICs. Mouse vaccination with ICs induced a spike-specific antibody response without adjuvant at a faster rate than traditional adjuvanted immunization such as Alum.
These findings suggest that the uncovered peptides, antibodies and in particular combinations thereof in the form of ICs are highly potent in triggering rapid antibody response and may provide immediate treatment for infected patients.
Thus, according to an aspect of the invention there is provided a composition comprising at least one antigenic determinant of Coronavirus and at least one antibody comprising an antigen binding domain which binds said at least one antigenic determinant of Coronavirus.
As used herein “antigenic determinant” refers to a peptide (i.e., essentially a peptide) which comprises an epitope that is recognized by an antigen binding domain of an antibody. Hence the antigenic determinant may comprise one or more epitopes. According to a specific embodiment, the antigenic determinant forms a portion of a viral protein with or without amino acid alterations with respect to the wild-type viral sequence.
The antigenic determinant is of a Coronavirus.
As used herein, “Coronavirus” refers to enveloped positive-stranded RNA viruses that belong to the family Coronaviridae and the order Nidovirales.
Examples of Corona viruses which are contemplated herein include, but are not limited to, 229E, NL63, OC43, and HKU1 with the first two classified as antigenic group 1 and the latter two belonging to group 2, typically leading to an upper respiratory tract infection manifested by common cold symptoms. However, Coronaviruses, which are zoonotic in origin, can evolve into a strain that can infect human beings leading to fatal illness. Thus particular examples of Coronaviruses contemplated herein are SARS-CoV, Middle East respiratory syndrome Coronavirus (MERS- CoV), and the recently identified SARS-CoV-2 [causing 2019-nCoV (also referred to as “COVID-19”)].
It would be appreciated that any Coronavirus strain is contemplated herein even though SARS-CoV-2 is emphasized in a detailed manner.
According to specific embodiments, the Corona virus is SARS-CoV-2.
As used herein “binds” refers to a mode of binding that reflects an antibody and an antigen binding.
Typically the affinity is between 0.05-100 nM, as determined by ELISA assay which is described in the Examples section which follows.
According to a specific embodiment, the composition comprises at least one antibody. Such a composition may be prepared under conditions (e.g., buffers) which allow for a structural lattice to form (see Figure 1 of Wang et al. supra). The formation of ICs is expected to augment ADCP, a well as antigen presentation that results in an anti Coronavirus-specific T cell response enhancement and antibody response enhancement.
An Fc receptor-dependent function of antibody-dependent cellular phagocytosis (ADCP) provides mechanisms for clearance of virus and virus-infected cells, as well as for stimulation of downstream adaptive immune responses by facilitating antigen presentation, or by stimulating the secretion of inflammatory mediators. The activity necessitates specific antibody-antigen binding and myeloid cells such as macrophages or neutrophils. The assay involves incubation of THP-1 cell line with fluorescentiy-labeled spike coated beads and monoclonal antibodies. The ADCP is measured by analysis of fluorescent signals in the THP-1 cells by flow cytometer.
According to a specific embodiment, the composition comprises 1 or more antibodies e.g., 2, 3, 4, 5 or up to 10 antibodies, each binding different epitopes of the at least one antigenic determinant, to form complex ICs.
As used herein “immune complexes (ICs)” also termed “an antigen-antibody complex” or “antigen-bound antibody”, a molecule formed from the binding of multiple antigens to antibodies. The bound antigen and antibody act as a unitary object, effectively an antigen of its own with a specific epitope. After an antigen-antibody reaction, the immune complexes can be subject to any of a number of responses, including complement deposition, opsonization, phagocytosis, or processing by proteases. Red blood cells carrying CRl-receptors on their surface may bind C3b-coated immune complexes and transport them to phagocytes, mostly in liver and spleen, and return to the general circulation. The complex can be formed in vitro by mixing of the antibody with the spike antigen or a peptide.
The choice of peptide(s) and antibody(s) will depend of the desired use.
According to a specific embodiment, the virus is SARS-CoV-2 and the antigenic determinant is derived from the SPIKE protein of the virus.
Hence, according to a specific embodiment, the composition is produced and maintained to form immunocomplexes (ICs) in vitro.
The term "peptide" and “antigenic determinant” which are interchangeably used herein encompass native peptides backbone (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body, more capable of penetrating into cells improving clearance, biodistribution and/or pharmacokinetics. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drag Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.
Peptide bonds (-CO-NH-) within the peptide may be substituted, for example, by N- methylated amide bonds (-N(CH3)-CO-), ester bonds (-C(=O)-O-), ketomethylene bonds (-CO- CH2-), sulfinylmethylene bonds (-S(=O)-CH2-), α-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl (e.g., methyl), amine bonds (-CH2-NH-), sulfide bonds (-CH2-S-), ethylene bonds (-CH2- CH2-), hydroxyethylene bonds (-CH(OH)-CH2-), thioamide bonds (-CS-NH-), olefinic double bonds (-CH=CH-), fluorinated olefinic double bonds (-CF=CH-), retro amide bonds (-NH-CO-), peptide derivatives (-N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally present on the carbon atom.
These modifications can occur at any of the bonds along the peptide chain and even at several (e.g. 2-3) bonds at the same time. Natural aromatic amino acids, Tyr and Phe, may be substituted by non-natural aromatic amino acids such as l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), naphthylalanine, ring-methylated derivatives of Phe, halogenated derivatives of Phe or O-methyl-Tyr.
According to a specific embodiment, the peptide comprises naturally occurring Trp residues.
The peptides of some embodiments of the invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
The term "amino acid" or "amino acids" is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term "amino acid" includes both D- and Lramino acids (stereoisomers).
Tables 1 and 2 below list naturally occurring amino acids (Table 1), and non-conventional or modified amino acids (e.g., synthetic, Table 2) which can be used with some embodiments of the invention.
Table la
Figure imgf000011_0001
Table lb
Figure imgf000012_0001
Figure imgf000013_0001
The amino acids of the peptides of some embodiments of the present invention may be substituted either conservatively or non-conservatively.
The term “conservative substitution” as used herein, refers to the replacement of an amino acid present in the native sequence in the peptide with a naturally or non-naturally occurring amino or a peptidomimetics having similar steric properties. Where the side-chain of the native amino acid to be replaced is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side-chain of the replaced amino acid).
As naturally occurring amino acids are typically grouped according to their properties, conservative substitutions by naturally occurring amino acids can be easily determined bearing in mind the fact that in accordance with the invention replacement of charged amino acids by sterically similar non-charged amino acids are considered as conservative substitutions.
For producing conservative substitutions by non-naturally occurring amino acids it is also possible to use amino acid analogs (synthetic amino acids) well known in the art.A peptidomimetic of the naturally occurring amino acid is well documented in the literature known to the skilled practitioner.
When affecting conservative substitutions the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.
The phrase "non-conservative substitutions" as used herein refers to replacement of the amino acid as present in the parent sequence by another naturally or non-naturally occurring amino acid, having different electrochemical and/or steric properties. Thus, the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the native amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted. Examples of non-conservative substitutions of this type include the substitution of phenylalanine or cycohexylmethyl glycine for alanine, isoleucine for glycine, or -NH-CH[(-CH2)5-COOH]-CO- for aspartic acid. Those non- conservative substitutions which fall under the scope of the present invention are those which still constitute a peptide capable of forming ICs.
The peptides of some embodiments of the invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.
Since the present peptides are preferably utilized in therapeutics which requires the peptides to be in soluble form, the peptides of some embodiments of the invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain. Thus, according to an embodiment of the invention there is provided a composition comprising a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 1 or analog or a fragment thereof, the peptide being shorter than 50 amino acids in length and capable of specifically binding a C1 or C3 antibody.
According to an additional or an alternative embodiment, the composition comprises a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 2 or analog or a fragment thereof, the peptide being shorter than 50 amino acids in length and capable of specifically binding an A7 antibody.
As used herein “an A7 antibody” refers to a germline antibody or a mutated version thereof such as set forth in A7, A7-sm1, A7-sm2, A7-sm3 a7-dm1, A7-dm2 and A7-dm3 (see sequences of Table 2).
SEQ ID NO: 2 is bound by all A7 family members, namely, WIS-A7 (the fully mutated version, WIS-A7sm1, WIS-A7sm2, WIS-A7sm3 (single mutant synthetic variants), WIS-A7dm1, WIS- A7dm2, WIS-A7dm3 (double mutant synthetic variants) and WIS-A7gl (germline, none mutated variant).
According to a specific embodiment, the A7 antibody is WIS-A7, carrying 3 mutations compared to the germline version (gl).
The peptide is shorter than the full length SPIKE. According to a specific embodiment, the peptide does not include a receptor binding domain (RBD) or SPIKE. According to some embodiments, by referring to the length, the skilled artisan would appreciate that the reference is made to the viral (e.g., SPIKE fragment) portion of the peptide and not to the length of the peptide when attached to a proteineceous heterologous moiety.
Thus, according to a specific embodiment the peptide is 4-50 amino acids long.
Thus, according to a specific embodiment the peptide is 4-45 amino acids long.
Thus, according to a specific embodiment the peptide is 4-40 amino acids long.
Thus, according to a specific embodiment the peptide is 4-35 amino acids long.
Thus, according to a specific embodiment the peptide is 4-30 amino acids long.
Thus, according to a specific embodiment the peptide is 4-25 amino acids long.
Thus, according to a specific embodiment the peptide is 4-20 amino acids long.
Thus, according to a specific embodiment the peptide is 4-15 amino acids long.
Thus, according to a specific embodiment the peptide is 4-10 amino acids long.
Thus, according to a specific embodiment the peptide is 5-50 amino acids long.
Thus, according to a specific embodiment the peptide is 6-50 amino acids long. Thus, according to a specific embodiment the peptide is 7-50 amino acids long.
Thus, according to a specific embodiment the peptide is 8-50 amino acids long.
Thus, according to a specific embodiment the peptide is 10-50 amino acids long.
Thus, according to a specific embodiment the peptide is 12-50 amino acids long.
Thus, according to a specific embodiment the peptide is 14-50 amino acids long.
Thus, according to a specific embodiment the peptide is 16-50 amino acids long.
Thus, according to a specific embodiment the peptide is 20-50 amino acids long.
Thus, according to a specific embodiment the peptide is 25-50 amino acids long.
Thus, according to a specific embodiment the peptide is 30-50 amino acids long.
Thus, according to a specific embodiment the peptide is 35-50 amino acids long.
Thus, according to a specific embodiment the peptide is 40-50 amino acids long.
When comprising substitutions (e.g., 1-10 amino acids substitution) or deletions (e.g., 1-5 amino acids deletions) relative to the wild-type sequence, the peptide is considered as an analog or derivative, though it is still to maintain the function of forming ICs with the cognate antibodies and ultimately activate ADCP or regardless of ICs or activation of ADCP simply used for vaccination in vivo.
According to another embodiment, the peptide comprises a protecting moiety or a stabilizing moiety.
The term "protecting moiety" refers to any moiety (e.g. chemical moiety) capable of protecting the peptide from adverse effects such as proteolysis, degradation or clearance, or alleviating such adverse effects.
The term “stabilizing moiety” refers to any moiety (e.g. chemical moiety) that inhibits or prevents a peptide from degradation.
The addition of a protecting moiety or a stabilizing moiety to the peptide typically results in masking the charge of the peptide terminus, and/or altering chemical features thereof, such as, hydrophobicity, hydrophilicty, reactivity, solubility and the like. Examples of suitable protecting moieties can be found, for example, in Green et al., "Protective Groups in Organic Chemistry", (Wiley, 2.sup.nd ed. 1991) and Harrison et al., "Compendium of Synthetic Organic Methods", Vols. 1-8 (John Wiley and Sons, 1971-1996).
The protecting moiety (or group) or stabilizing moiety (or group) may be added to the N- (amine) terminus and/or the C- (carboxyl) terminus of the peptide.
Representative examples of N-terminus protecting/stabilizing moieties include, but are not limited to, formyl, acetyl (also denoted herein as “Ac”), trifluoroacetyl, benzyl, benzyloxycarbonyl (also denoted herein as "CBZ"), tert-butoxycarbonyl (also denoted herein as "BOC"), trimethylsilyl (also denoted "TMS"), 2-trimethylsilyl-ethanesulfonyl (also denoted "SES"), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (also denoted herein as "FMOC"), nitro-veratryloxycarbonyl (also denoted herein as "NVOC"), t- amyloxycarbonyl, adamantyl-oxycarbonyl, and p-methoxybenzyloxycarbonyl, 2- chlorobenzyloxycarbonyl and the like, nitro, tosyl (CH3C6H4S02-), adamantyloxycarbonyl, 2, 2, 5, 7, 8-pentamethylchroman-6-sulfonyl, 2,3,6-trimethyl-4-methoxyphenylsulfonyl, t-butyl benzyl (also denoted herein as “BZL”) or substituted BZL, such as, p-methoxybenzyl, p- nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl, 2,6-dichlorobenzyl, t-butyl, cyclohexyl, cyclopentyl, benzyloxymethyl (also denoted herein as “BOM”), tetrahydropyranyl, chlorobenzyl, 4-bromobenzyl, and 2,6-dichlorobenzyl.
According to one embodiment of the invention, the protecting/stabilizing moiety is an amine protecting moiety.
According to a specific embodiment, the protecting/stabilizing moiety is a terminal cysteine residue.
Representative examples of C-terminus protecting/stabilizing moieties are typically moieties that lead to acylation of the carboxy group at the C-terminus and include, but are not limited to, benzyl and trityl ethers as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers, allyl ethers, monomethoxytrityl and dimethoxytrityl. Alternatively the -COOH group of the C-terminus may be modified to an amide group.
Other modifications of peptides include replacement of the amine and/or carboxyl with a different moiety, such as hydroxyl, thiol, halide, alkyl, aryl, alkoxy, aryloxy and the like.
Also included in the scope of the present invention are "chemical derivative" of a peptide or analog. Such chemical derivates contain additional chemical moieties not normally a part of the peptide. Covalent modifications of the peptide are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Many such chemical derivatives and methods for making them are well known in the art, some are discussed hereinbelow.
Also included in the scope of the invention are salts of the peptides and analogs of the invention. As used herein, the term “salts” refers to both salts of carboxyl groups and to acid addition salts of amino groups of the peptide molecule. Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases such as those formed for example, with amines, such as triethanolamine, arginine, or lysine, piperidine, procaine, and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids, such as, for example, acetic acid or oxalic acid. Such chemical derivatives and salts are preferably used to modify the pharmaceutical properties of the peptide insofar as stability, solubility, etc., are concerned.
According to one embodiment of the invention, the isolated peptide capable of binding the antibody is attached to a heterologous moiety proteinaceous or non-proteinaceous moiety.
As used herein the phrase "heterologous moiety" refers to an amino acid sequence which does not endogenously form a part of the peptide’s amino essentially viral acid sequence. Preferably, the heterologous moiety does not affect the biological activity of the isolated peptide (e.g. capability of binding the antibody or eliciting an immune response against Coronavirus).
The heterologous moiety may thus serve to ensure stability of the isolated peptide of the present invention without compromising its activity. For example, the heterologous moiety may increase the half-life of the isolated peptide in the serum.
According to one embodiment, the heterologous moiety does not induce an immune response. Thus, for instance, in the case of Ig, it may contain human sequences that do not produce an immune response in a subject administered therewith.
Examples of heterologous amino acid sequences that may be used in accordance with the teachings of the present invention include, but are not limited to, immunoglobulin, galactosidase, glucuronidase, glutathione-S-transferase (GST), carboxy terminal peptide (CTP) from chorionic gonadotrophin (CGb) and chloramphenicol acetyltransferase (CAT) [see for example U.S. Publication No. 20030171551].
According to a specific embodiment, the heterologous amino acid sequence is an immunoglobulin.
Generally the heterologous amino acid sequence is localized at the amino- or carboxyl- terminus (N-ter or C-ter, respectively) of the isolated peptide of the present invention. The heterologous amino acid sequence may be attached to the isolated peptide amino acid sequence by any of peptide or non-peptide bond. Attachment of the isolated peptide amino acid sequence to the heterologous amino acid sequence may be effected by direct covalent bonding (peptide bond or a substituted peptide bond) or indirect binding such as by the use of a linker having functional groups. Functional groups include, without limitation, a free carboxylic acid (C(=O)OH), a free amino group (ΝH2), an ester group (C(=O)OR, where R is alkyl, cycloalkyl or aryl), an acyl halide group (C(=O)A, where A is fluoride, chloride, bromide or iodide), a halide (fluoride, chloride, bromide or iodide), a hydroxyl group (OH), a thiol group (SH), a nitrile group (C≡N), a free C-carbamic group (NR”-C(=O)-0R\ where each of R’ and R” is independently hydrogen, alkyl, cycloalkyl or aryl).
The molecules (i.e., antibodies or peptides) of the present invention can be generated using recombinant techniques such as described by Bitter et al. (1987) Methods in Enzymol. 153:516-544; Studier et al. (1990) Methods in Enzymol. 185:60-89; Brisson et al. (1984) Nature 310:511-514; Takamatsu et al. (1987) EMBO J. 6:307-311; Coruzzi et al. (1984) EMBO J. 3:1671-1680; Brogli et al. (1984) Science 224:838-843; Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic
Press, NY, Section VIII, pp 421-463.
Thus, according to an aspect of the invention there is provided a method of producing an antibody capable of binding an antigenic determinant of Coronavirus, the method comprising:
(a) expressing in a host cell a heterologous polynucleotide encoding a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9; and optionally
(b) recovering the antibody from the host cell (e.g., such as to 90 % protein purity). Similar methods are contemplated for peptide production e.g., such as the peptides specifically described herein and exemplified by SEQ ID NO: 1 or 2.
The heterologous moiety may also be chemically linked to the isolated peptide following the independent generation of each. Thus, the two peptides may be covalently or non-covalently linked using any linking or binding method and/or any suitable chemical linker known in the art. Such linkage can be direct or indirect, as by means of a peptide bond or via covalent bonding to an intervening linker element, such as a linker peptide or other chemical moiety, such as an organic polymer. Such chimeric peptides may be linked via bonding at the carboxy (C) or amino (N) termini of the peptides, or via bonding to internal chemical groups such as straight, branched, or cyclic side chains, internal carbon or nitrogen atoms, and the like. The exact type and chemical nature of such cross-linkers and cross linking methods is preferably adapted to the type and nature of the peptides used. Where a heterologous amino acid sequence is fused to the viral peptide, this variant is referred to a fusion protein or a chimeric protein.
As used herein, the term “fused” means that at least a protein or peptide is physically associated with another protein or peptide, which naturally don’t form a complex. According to a specific embodiment the fused molecule is a “fusion peptide” or “fusion protein”, a protein created by joining two or more heterologously related peptide sequences together. The terms “fusion protein”, “chimera”, “chimeric molecule”, or “chimeric protein” are used interchangeably.
The phrase “non-proteinaceous moiety” as used herein refers to a molecule, not including peptide bonded amino acids, that is attached to the above-described isolated peptide’s amino acid sequence.
According to one embodiment, the non-proteinaceous moiety is non-toxic.
Exemplary non-proteinaceous moieties which may be used according to the present teachings include, but are not limited to, polyethylene glycol (PEG), Polyvinyl pyrrolidone (PVP), poly(styrene comaleic anhydride) (SMA), and divinyl ether and maleic anhydride copolymer (DIVEMA).
The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof (such as Fab, F(ab')2, Fv, scFv, dsFv, or single domain molecules such as VH and VL) that are capable of binding to the antigenic determinant such as of SEQ ID NO: 1 or 2.
According to specific embodiments, the antibody is a whole or intact antibody.
This is of specific value in order to activate effector functions such as antibody-dependent cellular cytotoxicity (ADCC), but particularly ADCP. Of note is the generation of immunecomplexes (ICs) with the antigenic determinant for the activation of ADCP.
According to another embodiment, the antibody is an antibody fragment.
Suitable antibody fragments for practicing some embodiments of the invention include a complementarity-determining region (CDR) of an immunoglobulin light chain (referred to herein as “light chain”), a complementarity-determining region of an immunoglobulin heavy chain (referred to herein as “heavy chain”), a variable region of a light chain, a variable region of a heavy chain, a light chain, a heavy chain, an Fd fragment, and antibody fragments comprising essentially whole variable regions of both light and heavy chains such as an Fv, a single chain Fv (scFv), a disulfide-stabilized Fv (dsFv), an Fab, an Fab’, and an F(ab’)2. As used herein, the terms "complementarity-determining region" or "CDR" are used interchangeably to refer to the antigen binding regions found within the variable region of the heavy and light chain polypeptides. Generally, antibodies comprise three CDRs in each of the VH (CDRH1 or H1; CDRH2 or H2; and CDRH3 or H3) and three in each of the VL (CDRL1 or L1; CDRL2 or L2; and CDR L3 or L3).
The identity of the amino acid residues in a particular antibody that make up a variable region or a CDR can be determined using methods well known in the art and include methods such as sequence variability as defined by Kabat et al. (See, e.g., Rabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.), location of the structural loop regions as defined by Chothia et al. (see, e.g., Chothia et al., Nature 342:877-883, 1989.), a compromise between Rabat and Chothia using Oxford Molecular's AbM antibody modeling software (now Accelrys®, see, Martin et al., 1989, Proc. Natl Acad Sci USA. 86:9268; and world wide web site www(dot)bioinf-org(dot)uk/abs), available complex crystal structures as defined by the contact definition (see MacCallum et al., J. Mol. Biol. 262:732-745, 1996) and the "conformational definition" (see, e.g., Makabe et al., Journal of Biological
Chemistry, 283:1156-1166, 2008).
As used herein, the “variable regions” and "CDRs" may refer to variable regions and CDRs defined by any approach known in the art, including combinations of approaches.
Functional antibody fragments comprising whole or essentially whole variable regions of both light and heavy chains are defined as follows:
(i) Fv, defined as a genetically engineered fragment consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) expressed as two chains;
(ii) single chain Fv (“scFv”), a genetically engineered single chain molecule including the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
(iii) disulfide-stabilized Fv (“dsFv”), a genetically engineered antibody including the variable region of the light chain and the variable region of the heavy chain, linked by a genetically engineered disulfide bond.
(iv) Fab, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme papain to yield the intact light chain and the Fd fragment of the heavy chain which consists of the variable and CH1 domains thereof; (v) Fab’, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin, followed by reduction (two Fab’ fragments are obtained per antibody molecule);
(vi) F(ab’)2, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin (i.e., a dimer of Fab’ fragments held together by two disulfide bonds); and
(vii) Single domain antibodies or nanobodies are composed of a single VH or VL domains which exhibit sufficient affinity to the antigen.
According to specific embodiments the antibody heavy chain constant region is chosen from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE.
According to specific embodiments, the antibody is an IgG antibody.
According to a specific embodiment the antibody isotype is IgG1 which is capable of eliciting ADCP.
The choice of antibody type will depend on the immune effector function that the antibody is designed to elicit.
According to specific embodiments, the antibody comprises an Fc domain.
The Fc domain may be a wild-type Fc domain or a domain which elicits the same effector functions such as that of IgGl, but is can also be modified such as to increase immune effector functions e.g., ADCP.
Typically, ADCP recruitment depends on the following activating receptors: 5 activating FcγRs: the high affinity FcγRI that can bind to monovalent antibody, and the lower affinity FcγRIIa and IIe and FcγRIIIa (HGNC:3619) and nib that require avidity-based interactions.
At least one of the following modifications on an FC scaffold are contemplated herein G236A/S239D/I332E where the reference sequence is human Ighyl gene.
For instance, it has been reported that optimization of antibody binding to FcgammaRIIa enhances macrophage phagocytosis of tumor cells (John O Richards 1, Sher Karki, Greg A
Lazar, Hsing Chen, Wei Dang, John R Desjarlais Affiliations expand, PMID: 18723496).
Thus, for example, an engineered Fc variant with G236A (and optionally S239D/I332E) that provides selectively enhanced binding to FcgammaRIIa relative to FcgammaRIIb is specifically contemplated. Variants containing this substitution have up to 70-fold greater FcgammaRIIa affinity and 15-fold improvement in FcgammaRIIa/FcgammaRIIb ratio and mediate enhanced phagocytosis of antibody-coated target cells by macrophages. According to a specific embodiment, the Fc portion is a-fucosylated (N297A on IgG1) which also exhibits enhanced binding to FcRl.
According to specific embodiments, the antibody is a naked antibody.
As used herein, the tern "naked antibody" refers to an antibody which does not comprise a heterologous effector moiety e.g. therapeutic moiety, detectable moiety.
As used herein “heterologous” means not occurring in nature in conjunction with the antibody.
According to specific embodiments, the antibody comprises a heterologous effector moiety e.g. e.g. therapeutic moiety, detectable moiety. The effector moiety can be proteinaceous or non- proteinaceous; the latter generally being generated using functional groups on the antibody and on the conjugate partner. The effector moiety may be any molecule, including small molecule chemical compounds and polypeptides. For example the effector moiety can be a known drag to Coronavirus infection.
According to specific embodiments, the antibody is a recombinant antibody.
According to specific embodiments, the antibody is a monoclonal antibody.
Antibody fragments according to some embodiments of the invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light- heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross- linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97- 105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
Another form of an antibody fragment is a peptide coding for a single complementarity- determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
It will be appreciated that for human therapy, humanized or human antibodies are preferably used.
According to preferred embodiments, the antibody is a human antibody.
According to a specific embodiment, the human antibody carries human Vh,Dh, Jh, VI, J, gene segments such as in germ line antibodies or natural variants thereof.
Table 2 below lists antibodies, heavy chains, light chains, CDRs or antibodies which are specifically contemplated according to some embodiments of the invention. The Table describes germ-line antibodies and natural variants and synthetic variants thereof.
Table 2 C1one A7 (8 mAbs)
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000028_0002
C1one A9 -
Figure imgf000028_0003
C1one C1/3 (2 mAbs)
Figure imgf000029_0001
Notes:
• Nucleotide sequences were identified using IgBlast, based on the human IMGT database
• Amino acid sequences were obtained using the Expasy Translate tool • CDR3 sequences appear underline & are derived based on IgBlast
• For the A7 clone, mutations in the nucleotide sequences appear in bold
It will be appreciated that the subject can be treated with a plurality of antibodies to achieve maximal inhibition of the virus either as a treatment or as a vaccine. Also diagnosis may be benefited by the use of a plurality of antibodies.
As used herein “plurality” refers to at least 2 antibodies having different antigen binding domains (at least one different CDR), e.g., 2-3, 2-4.
According to a specific embodiment, the plurality of antibodies bind different epitopes on the virus.
According to a specific embodiment, the plurality of antibodies bind identical epitopes on the virus, but may be different in their effector (Fc-mediate) functions.
According to a specific embodiment, the human antibody comprising an antigen binding domain which binds said antigenic determinant comprises a plurality of different human antibodies each comprising an antigen binding domain which binds a Coronavirus.
According to a specific embodiment, the antibody binds the SPIKE protein of a
Coronavirus.
As used herein “receptor binding domain (RBD)” refers to the receptor (ACE2) binding domain of SARS-CoV-2 of SPIKE, residues Arg319-Phe541 of SPIKE.
Binding can be qualified using various methods known in the art, such as ELISA (exemplified in the section which follows) and surface plasmon resonance (SPR).
The present invention envisages immunization against-, and prevention or treatment of Coronavirus infection with any of the immunocomplexes, peptides, antibodies described herein.
The term “treating” refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology (i.e., Coronavirus infection, e.g., COVID19 or related complications).
As used herein, the term “preventing” refers to keeping a disease, disorder or condition (i.e., Coronavirus infection, e.g., COVID19 or related complications) from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.
Prevention can be done by means of immunization, in an embodiment passive immunization, where the antibody is administered, or active where the peptide is administered. As used herein, the term “subject” includes mammals, preferably human beings, male or female, at any age or gender, which suffer from the pathology. Preferably, this term encompasses individuals who are at risk to develop the pathology (e.g., above 65 of age) or exposed to the virus, e.g., healthcare personnel, education personnel etc.
A composition of matter comprising any of the immunocomplexes, peptides, and antibodies (active ingredient(s)) of the present invention can be administered to the subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
Herein the term "active ingredient" refers to the immunocomplexes, peptides or antibodies accountable for the biological effect.
Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.
Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
Techniques for formulation and administration of drags may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.
Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intrapulmonary or intraocular injections.
Conventional approaches for drag delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport polypeptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin polypeptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.
Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (composition of matter comprising the antibodies) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., Coronaviral infection) or prolong the survival of the subject being treated.
According to an embodiment of the present invention, an effective amount of the composition of matter comprising the antibodies of some embodiments of the present invention is an amount selected to eliminate infected cells e.g. by initiating ADCC or ADCP and optionally neutralize Coronaviruses.
Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
For example, any in vivo or in vitro method of evaluating Coronavirus viral load may be employed. For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 P-1)·
Dosage amount and interval may be adjusted individually to provide the active ingredient at a sufficient amount to induce or suppress the biological effect (minimal effective concentration, MFC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drags or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
The present teachings further envisage treating with other anti-viral drags or anti- inflammatory drags or anti-coagulants as separate treatments or in a co-formulation.
Without being limited to COVID19 but for the sake of example, according to a specific embodiment, the antiviral drag is selected from the group consisting of remdesivir, an interferon, ribavirin, adefovir, tenofovir, acyclovir, brivudin, cidofovir, fomivirsen, foscamet, ganciclovir, penciclovir, amantadine, rimantadine and zanamivir.
Also contemplated are plasma treatments from infected persons who survived and/or anti- HIV drags such as lopinavir and ritonavir, as well as chloroquine.
Specific examples for drags that are routinely used for the treatment of COVID-19 include, but are not limited to, Lopinavir /Ritonavir, Nucleoside analogues, Neuraminidase inhibitors, Remdesivir, polypeptide (EK1), abidol, RNA synthesis inhibitors (such as TDF, 3TC), anti- inflammatory drags (such as hormones and other molecules), Chinese traditional medicine, such ShuFengJieDu Capsules and Lianhuaqingwen Capsule, could be the drag treatment options for COVID19.
According to another aspect of the invention, there is provided a method of diagnosing a Coronavirus infection in a subject in need thereof, the method comprising:
(a) contacting a biological sample which may comprise a SPIKE protein of a Coronavirus with antibodies as described herein under conditions which allow complex formation between the composition and the SPIKE;
(b) analyzing presence or level of said complex, wherein said presence and/or level is indicative of the Coronavirus infection.
As used herein the term "diagnosing" refers to classifying a disease, determining a severity of a disease (grade or stage), monitoring progression, forecasting an outcome of the disease and/or prospects of recovery.
The subject may be a healthy subject (e.g., human) undergoing a routine well-being check-up. Alternatively, the subject may be at risk of the disease or infection. Yet alternatively, the method may be used to monitor treatment efficacy.
As used herein “biological sample” refers to a sample of tissue or fluid isolated from a subject, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, sputum, and also samples of in vivo cell culture constituents. It should be noted that a “biological sample obtained from the subject” may also optionally comprise a sample that has not been physically removed from the subject (in vivo as opposed to in vitro).
Numerous well known tissue or fluid collection methods can be utilized to collect the biological sample from the subject in order to determine the level of Coronaviruses or infected cells in the sample. Regardless of the procedure employed, once a biopsy/sample is obtained the level of the variant can be determined and a diagnosis can thus be made.
As mentioned, the method of the present invention is effected under conditions sufficient to form protein-protein interactions i.e., complex (e.g. a complex between). Such conditions (e.g., appropriate concentrations, buffers, temperatures, reaction times) as well as methods to optimize such conditions are known to those skilled in the art, and examples are disclosed herein below.
The antibody-SPIKE complex may comprise e.g., be attached, to an identifiable moiety. Alternatively or additionally, the complex may be identified indirectly such as by using a secondary antibody.
According to one embodiment, diagnosis is corroborated using any diagnostic method known in the art, such as by measuring the viral load or titer, by antigen level measurement, antibody level measurement, virus isolation and/or genomic detection by reverse transcriptase- polymerase chain reaction (RT-PCR), etc. For example, a higher viral load or titre often correlates with the severity of an active viral infection. The quantity of virus per mL can be calculated for example by estimating the live amount of virus in an involved body fluid (e.g. serum sample or whole blood).
As used herein the term “about” refers to ± 10 %.
The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".
The term “consisting of’ means “including and limited to”.
The term "consisting essentially of' means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
EXAMPLES
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular C1oning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes Ι-ΙII Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular C1oning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes Ι-ΙΠ Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes Ι-ΙII Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); “Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. L, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular C1oning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1- 317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
MATERIALS AND METHODS
Human blood collection
Convalescent participants were recruited to this study after being confirmed as free of symptoms of COVID-19 and after being diagnosed as convalescent patients based on two sequential RT-PCR tests with a negative result. All participants provided informed consent before participating in this study, and the study was approved by the institutional review board of the Weizmann Institute of Science. Peripheral blood mononuclear cells (PBMCs) and plasma samples were obtained from 30ml blood extracted from recruited individuals by a certified physician and processed in an enhanced biosafety level 2 facility. Samples underwent a gradient separation protocol, in which 12ml of Ficoll-Paque (GE Healthcare) were added to the bottom of a 50-ml falcon tube containing 30ml of peripheral blood. Samples were centrifuged at 2000 RPM, 8°C, for 25 minutes. The plasma was aliquoted and preserved at -80°C. The buffy coat containing PBMCs was separated, washed twice, aliquoted at 5 million PBMCs per vial and frozen slowly to -80°C.
Mice
C57BL/6 mice were provided by Harlan. All experiments using mice were approved by the Weizmann Institute Animal Care and Use Committee.
Immunization and treatments
Mice were injected intraperitoneally with 200μ1 PBS containing either 10μg spike protein in alum (2:1), or immune complex containing 10μg spike protein with 60μg A7 or isotype control after 2h incubation at room temperature. Blood samples were collected prior to the immunization, followed by blood collection on day 3 and every 7 days up to 21 days after the injection. Blood samples were centrifuged at 3000 RPM, 4°C, for 10 minutes and plasma was aliquoted and frozen. Flow cytometry
Single-cell suspensions were obtained on days 7, 14 and 21 following the immunization, by forcing inguinal lymph nodes through a mesh into PBS containing 2% serum and ImM EDTA. Cell suspensions were washed and incubated with fluorescently labeled antibodies (Biolegend) for 30 min. Germinal center cells were gated as live/single, B220+CD38low FAS+. Analysis was performed using FlowJo vl0.7.
Phagocytosis assay
Antibody-dependent cellular phagocytosis was assessed by the measurement of the uptake of antibody-opsonized, antigen-coated fluorescent beads by a monocytic cell line. Briefly, 2μg of Biotinylated Spike protein was used to saturate the binding sites of 0.5mg 1μm fluorescent NeutrAvidin beads (Invitrogen). Excess antigen was removed by washing the beads, which were then blocked with 1% BSA. Next, the beads were washed and incubated with antibodies at a final concentration of 0.5μΜ or plasma diluted 1:100 for 2h at 37°C. Following opsonization, beads were washed, and unbound antibodies were removed. The beads were then either stained for IgG to determine the IgG coating or incubated with phagocytotic cells. For the phagocytosis assay ,THP-1 cells (ATCC) were added, and the cells were incubated for lh at 37°C to allow phagocytosis. The cells were then fixed, and the extent of phagocytosis was measured via flow cytometry (CytoFLEX). The data are reported as a phagocytic score, which takes into account the proportion of effector cells that phagocytosed and the degree of phagocytosis (integrated MFI: frequency x MFI) as previously described (Darrah, Patricia A., et al. "Multifunctional TH 1 cells define a correlate of vaccine-mediated protection against Leishmania major." Nature medicine 13.7 (2007): 843-850). For IgG staining purposes, the beads were incubated with anti-human IgG secondary antibody (Jackson Immuno Research) in blocking buffer at a 1:100 dilution for 30 min on ice. The beads were then washed, and the IgG was measured using the CytoFLEX flow cytometer.
Neutralization assay
Lentiviruses expressing S-Covidl9 spikes were produced by transfecting HEK293T cells with Luciferase-pLenti6, Δ19 S_covid-pCMV3 and ΔR89 Ψ vectors at 1:1:1 ratio, using Lipofectamine 2000 (Thermo Fisher). Media containing Lentiviruses was collected at 48h post- transfection, centrifuged at 600g for 5 min for clarifying from cells, and aliquots were frozen at - 80°C. For neutralization assays, HEK293T were transiently transfected with hACE2-pCDNA using Lipofectamine 2000. Following 18h post-transfection, cells were re-seeded on a poly-L- lysine pre-coated white, chimney 96-well plates (Greiner Bio-One). Cells were left to adhere for 8h, followed by the addition of S-covidl9 lentivirions, which were pie-incubated with 4-fold descending concentration series of monoclonal antibodies. Luminescence from the activity of luciferase was measured 48 h post-infection using a TECAN infinite M200 pro plate reader after applying Bright-Glo reagent (Promega) on cells.
Peptide array assay
CelluSpots HullB (Intavis) spike peptide arrays were used per the manufacturer’s instructions. Plasma samples were used at 1:10 dilution and monoclonal antibodies were used at lOnM final concentration.
ELISA
ELISA reactions to evaluate antibodies binding to SARS-CoV-2 RBD and trimeric spike proteins were carried out using flat-bottom MaxiSorp™ 96- well plates (Invitrogen). The plates were coated with 5 μg/ml protein solution in PBS at 100μΙ per well and left overnight at 4°C. The Plates were washed 5 times with washing buffer (lx PBS with 0.05% Tween-20 (Sigma- Aldrich)) and incubated with 100μΙ blocking buffer (lx PBS with 1% BSA) for lh at room temperature. The blocking solution was subsequently replaced by a serial dilutions of either monoclonal antibodies or plasma samples for 2.5h at RT. Plasma samples were assayed at a 1:10 starting dilution and 3 additional tenfold serial dilutions. Monoclonal antibodies were tested at 10μg/ml starting concentration and 8 additional fourfold serial dilutions. Plates were washed 6 times with washing buffer and then incubated with anti-human IgG or mouse IgG secondary antibody conjugated to horseradish peroxidase (HRP) (Jackson Immuno Research) in blocking buffer at a 1:5,000 dilution. Plates were developed by addition of the HRP substrate, TMB (Thermo Fisher) and absorbance was measured at 630nm with an ELISA microplate reader (Synergy HT, Biotek). To determine concentration of antibodies in plasma samples, a standard curve was used on each plate with a known amount of IgG antibodies. The standard curve was graphed and analyzed using four-parameter nonlinear regression (GraphPad Prism) and the total IgG was calculated using the O.D. values.
Single-cell immunoglobulin analysis
Spike reactive CD19+, CD27+, IgGl+, IgK* peripheral blood memory B cells were single cell sorted into 96 well plates. These in turn underwent nested PCR amplification and Sanger sequencing of their heavy and light chain transcripts, as previously described (PMID: 17996249). Upon collection of all immunoglobulin transcripts, data analysis was performed as detailed below. Determination of clonality and reconstruction of lineage trees
Ig Fasta sequences were aligned against the IMGT human heavy chain gene database (downloaded at Dec. 2019) and light chain gene database (downloaded at Feb. 2017) using NCBI IgBlast (version 1.14.0) (Ye et al., 2013). Post processing of IgBlast output, and clonal clustering were performed using Change-0 vO.4.6 (Gupta et al., 2015), Alakazam v0.3.0, SHazaM vO.2.3, and custom scripts within the R statistical computing environment, as follows. V(D)J sequences were assigned to clonal groups by partitioning sequences based on identity of IGHV gene annotations, IGHJ gene annotations, and junction region lengths. Within these groups, sequences differing from one another by a distance of more than 15 nucleotides between the V genes were defined as separate clones. The clonal distance threshold was determined by manual inspection using heatmaps of V genes hamming distance. Full-length germline sequences were reconstructed for each clonal cluster with D segment and N/P regions masked (replaced with Ns), with any ambiguous gene assignments within clonal groups resolved by the majority rule. Lineage trees were constructed for each clone having at least two unique sequences using PHYLIP (v3.697) (Felsenstein, 2005) and Alakazam. Selection quantification was calculated using BASELINe’s local test (Yaari et al., 2012).
Selection of antibody candidates, cloning and expression
Antibody transcripts were chosen for cloning and expression on the basis of several criteria. These included: relation of the candidate to an expanded B cell clone, a high degree of somatic hypermutation or homology to the CDR3 consensus sequence motif as generated by aggregating all sequences whose V gene appeared in more than 5% of the sample size. Candidate heavy and light chain transcripts including 5’ & 3’ vector homologous, 30 nucleotide long arms were ordered as gene blocks from IDT. These were cloned into IgGl/K expression vectors via the restriction free method using Phusion High-Fidelity DNA polymerase (NEB), according to the manufacturer’s protocol. Following PCR, the previous expression vector templates were degraded by incubating the products with the DPN1 restriction enzyme (NEB) for 16 hours at 37C. The products were then transformed into DH5a competent bacteria via the heat shock method (42°C, 90s). Selection of plasmid bearing bacteria was performed on the basis of Ampicillin resistance inherent to the vectors. 24 hours following transformation, single bacterial colonies were isolated. These underwent colony PCR and were re-plated on an index plate. The colony PCR products were sequenced using the Sanger method to confirm that their corresponding plasmids indeed contain the desired constructs in-frame and absent of any de- novo mutations. Colonies harboring successfully cloned plasmids were used to form glycerol stocks as well as a larger biomass of the desired vectors using a Maxiprep kit (Qiagen). The Maxiprep products were then sequenced again to confirm their accuracy. Antibody expression was performed in 293T HEK cells. Cells were grown to 90% confluence in 15cm plates, in complete growth medium (DMEM, 10% foetal bovine serum, IX MEM-Eagle non essential amino acids, 2mM glutamine, 1:100 Pen-Strep solution). Prior to transfection, the 293T HEK monolayers were carefully washed with PBS and complemented with serum free complete growth medium. The transfection mix was formed in 1ml of serum free medium per plate - and included 12.5μg of each vector (IgGl & IgK) and 50μg of linear, 25kDa linear polyethylenimine as a transfection reagent. Antibody purification was performed five days following introduction of the transfection mix to the plates. Supernatants were collected, filtered, and reacted overnight at 4°C with protein G sepharose beads (GE Healthcare). The beads were washed with PBS and eluted using IgG elution buffer (Thermo-scientific) into 1M Tris buffer after which they were dialyzed to PBS overnight. Measurement of the antibody concentration was performed using Nanodrop. SARS-CoV2 SPIKE Ectodomain Construct (SEQ ID Nos: 113-114)
EXAMPLE 1
Serum antibodies from convalescent patients support immune complex uptake through Antibody-Dependent Cellular Phagocytosis (ADCP)
ADCP is one of the major functions of antibodies that promote the internalization of antibody-coated pathogens or antigens by cells of the innate immune system. The present inventors examined the capacity of antibodies in serum derived from convalescent COVID-19 patients to mediate antigen capture. For this purpose, 10 recovered individuals that donated blood samples 6-10 weeks after recovery were recruited. Whereas SARS-CoV-2-neutralizing antibodies usually interact with the receptor-binding domain (RBD) of the spike, antibodies with ADCP potential are not necessarily restricted to this domain. Therefore, the ADCP potential of antibodies that bind spike triplex protein and not necessarily the RBD domain was examined. For this purpose, the SARS-CoV-2 spike protein complex was generated as well as the single- molecule RBD domain. To evaluate if anti-spike antibodies are present in the recovered patients, ELISA was performed using patient-derived sera. It was found that all of the recovered patients have anti-spike IgGl antibodies in their blood, however, the quantity of the antibodies varied significantly among the examined individuals. Thus, the study cohort of recovered patients has detectable IgGl antibodies that bind the spike protein in their sera. In order for an antibody to mediate ADCP, it must effectively bind its target in multiple sites to form a high-density surface coating. To test whether the amount of spike-specific antibodies in the recovered patient blood are sufficient to induce ADCP, serum IgG was first examined for the ability to form soluble immune complexes (ICs). For this purpose, spike complexes were biotinylated and conjugated to streptavidin-coated beads. The beads were incubated with recovered patient sera or with samples that were taken from healthy controls, for 2 hours followed by fluorescent anti-IgG staining. This assay revealed that IgG antibodies in the recovered patients can form ICs in various efficiencies whereas beads from healthy donors showed low background level of antibody coating (Figure 1A). Most of the recovered patients (7/9) formed IC uniformly whereas two subjects in the cohort showed very effective bead coating (Figure 1A). To examine if these complexes are able to support ADCP, immune complexes were generated with serum antibodies and incubated with THP-1 macrophage cell line. Cells that were incubated with beads that were incubated with serum derived from healthy controls showed IC uptake activity that was similar to macrophages that were incubated with beads without serum (FMO) (Figure IB). In contrast, Most of the ICs composed of patient- derived antibodies were internalized by THP-1 macrophages very effectively (Figure IB). Collectively, it can be concluded that serum antibodies from recovered patients 6-10 weeks after infection can mediate the internalization of ICs through ADCP. Thus, The left panel in Figure IB represents the mean fluorescent intensity observed in macrophages that phagocytosed SPIKE coated beads, whereas the right side represents the percent of macrophages that performed phagocytosis from the tested population. In other words, the left side is a quantitative measure of the amount of beads phagocytosed and the right side, a measure of how many macrophages participate in phagocytosis in general. Taken together, these results show that in the presence of serum from SARS CoV2 convalescent patients, more macrophages conduct phagocytosis and also that the process occurs more efficiently.
EXAMPLE 2 Cloning of spike-specific antibodies
The ADCP analysis of recovered patients was performed with polyclonal antibodies that most likely bind multiple sites on the spike trimeric complex. The aim is to expose and characterize monoclonal antibodies that effectively mediate ADCP in COVID-9 convalescent patients. For this purpose immunoglobulins of memory B cells from two patients were sequenced, one of which showed the highest ADCP activity in the IC-uptake assay. Memory B cells (MBC) were defined as class-switched (IgGl) CD 19+ CD27+ cells that bind fluorescently labeled spike trimeric complexes. For simplicity, the present inventors focused only on IgK expressing memory B cells. Spike-specific single MBCs were sorted and subjected to PCR amplification of their immunoglobulin genes followed by gene sequencing.
In order to test the functional activity of patient immunoglobulins, the recovered sequences were cloned into expressing vectors to produce monoclonal antibodies. Out of the 9 expressed antibodies, 4 bound the spike protein in ELISA (Figure 2A). Dashed lines show unspecific binding to BSA. To examine how effectively these monoclonal antibodies bind the spike protein, dilution ELISA was performed for all of the immunoglobulins that showed binding activity. Surprisingly, mAb WIS-A7 (or A7) which carried only 3 mutations (2 and 1 in the heavy and light chains respectively) compared to the germline version (also referred to herein as “gl”) in IMGT was the most effective in binding the spike protein, whereas the mutated antibodies interacted modestly with the spike (A9, C1 and C3) (Figure 2A). To examine if these 3 mutations are important for effective antigen binding, mAb A7 was reverted to its germline configuration and its binding capacity to the spike protein was tested. ELISA revealed that the germline version of WIS-A7 was 100-fold less effective in binding the spike (Figure 2B). Strikingly, a significant increase in antibody binding was due to a single mutation in either the heavy or the light chains (Figure 2C, SMI and SM3). Notably, the mutation on the light chain endowed the antibody with slightly more effective binding than the fully mutated version. Furthermore, no additive effect was observed as either mutation in the light or heavy chains increased antigen-binding nearly by 100-fold. Thus, a mutated spike-specific antibody with the potential to mediate effector functions such as ADCP was cloned. The sequence of the heavy chain and light chains of these antibodies are as set forth in SEQ ID NOs: are provided in Table 2 above. Essentially, all A7 family members are able to mediate ADCP efficiently with the mutated version being the most efficient. The germline version of A7 is the least efficient ADCP mediator in the family, but still - it is much more efficient compared to other antibodies which are not from the A7 clone, such as A9, C1 and C3.
EXAMPLE 3
Detection of the antibody binding sites
Antibodies can bind either a linear sequence or a 3D structure of a protein. In an attempt to find the antibody binding sites, an array of linear peptides that cover all of the spike sequences was used [CelluSpots HullB (Intavis)]. Whereas C1 showed a clear target binding of two overlapping peptides, A7 and C3 showed binding patterns that were similar to a control nonspecific antibody. A9 did not show any binding activity in this assay (not shown). Surprisingly, the germline version of A7 bound two specific overlapping peptides that were not detected in the mutated version of the antibody (Figure 3). This finding suggests that the mutations endowed the immunoglobulin with the effective binding of a 3D structure and caused the loss of binding of a linear sequence. Whereas the target of C1 was located outside the RBD domain, binding of the germline version of A7 was mapped to the edge of the RBD domain. These findings suggest that the A7 binding domain in the RBD might be available for interaction only when it is embedded in the full spike structure. Together, two short linear peptides (SEQ ID NOs: 1 and 2 of Figure 3 were found that serve as targets for spike-specific antibodies.
EXAMPLE 4
Patient-derived monoclonal antibodies support antibody-dependent cellular phagocytosis and virus neutralization
Example 1 above showed that patient-derived serum IgG can mediate ADCP. To examine if this function can be mediated by monoclonal antibodies, the present inventors first examined if the spike-specific immunoglobulins can form ICs. For this purpose, spike-coated beads were mixed with the same amount of different monoclonal antibodies. Flow cytometric analysis revealed that besides A9, all of the cloned antibodies, including the germline version of A7, bound all of the spike coated beads and formed IC (Figure 4A). Additional mean fluorescent intensity (MET) measurements revealed that A7 and its germline version were the most effective in coating the beads (Figue 4B). This assay indicates that many A7 antibodies bind one bead and possibly several antibodies bind one spike complex. Next, the present inventors examined if the ICs composed of these monoclonal antibodies can mediate ADCP in vitro. Whereas all of the monoclonal antibodies promoted phagocytic activity by THP-1 cells, A7 was the most effective immunoglobulin in mediating this process (Figure 4C). Notably, although the germline version of A7 was not very effective in binding the spike in ELISA, it mediated ADCP effectively (Figure 4C). Collectively, it can be concluded that monoclonal antibodies can induce ADCP, and their ability to form immune complexes determines the effectiveness of this process.
In addition to effector functions, the capacity of the monoclonal antibodies to neutralize pseudovirus invasion into epithelial cells that stably express ACE2 was determined. The A7 antibody inhibited the entry of viruses into cells (Figure 4D). Interestingly, although both C1 and C3 antibodies did not show RBD binding activity they were able to neutralize virus infection (Figure 4D). C1 effectively neutralized viral infection whereas C3 neutralized only 50% of the viral infections at maximal concentrations (Figure 4D). Thus, the cloned antibodies had dual functions, they were able to promote ADCP and neutralize virus invasion into cells.
EXAMPLE 5
Vaccination with immune complexes induce rapid antibody-mediated immune responses in mice
The A7 monoclonal antibody was very effective in the generation of ICs that promoted efficient antigen uptake by innate immune cells through ADCP. Immune complexes are very potent inducers of antibody generation and were previously used as vaccines. Whereas IgGl binds to activating FC receptors in humans, in mice this function is mediated by IgG2a/b. Thus, a mouse version of A7 was produced that has an IgG2a FC domain. To examine whether A7 can be used as IC for vaccination the present inventors generated immune complexes in vitro and used them for i.p. immunization of mice. As a negative control, the spike protein was mixed with unspecific IgG2a antibody and as a positive control for endogenous antibody generation, mice were immunized with traditional antigen in alum vaccine. Spike in alum immunization triggered robust IgGl antibody formation only at day 14 after administration of the vaccine whereas immunization with IC triggered endogenous antibody generation very rapidly that was already detected at day 7 of the response (Figures 5A-B). In some cases, IC immunization induced a stronger IgGl antibody response than the traditional vaccination at day 14 after administration (Figures 5A-B).
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
It is the intent of the applicants) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.

Claims

WHAT IS CLAIMED IS:
1. A composition comprising at least one antigenic determinant of Coronavirus and at least one antibody comprising an antigen binding domain which binds said at least one antigenic determinant of Coronavirus.
2. The composition of claim 1 for use in preventing or treating Coronavirus infection.
3. A method of preventing or treating Coronavirus infection in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of claim 1, thereby treating or preventing the Coronavirus infection in the subject.
4. A composition comprising a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 2 or analog or a fragment thereof, the peptide being shorter than 50 amino acids in length and capable of specifically binding an A7 antibody.
5. A composition comprising a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 1 or analog or a fragment thereof, the peptide being shorter than 50 amino acids in length and capable of specifically binding a C1 or C3 antibody.
6. The composition of any one of claims 4-5 for use in preventing or treating Coronavirus infection in a subject in need thereof.
7. A method of treating or preventing Coronavirus infection in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of any one of claims 4-5, thereby treating or preventing Coronavirus infection in the subject.
8. The composition or method of any one of claims 1-3, wherein said at least one antigenic determinant is a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 1 or analog or a fragment thereof, the peptide being shorter than 50 amino acids in length and capable of specifically binding a C1 or C3 antibody.
9. The composition or method of any one of claims 1-3, wherein said at least one antigenic determinant is a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 2 or analog or a fragment thereof, the peptide being shorter than 50 amino acids in length and capable of specifically binding an A7 antibody.
10. The composition or method of any one of claims 1-3, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9.
11. A human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus for use in preventing or treating Coronavirus infection in a subject in need thereof, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and
A9.
12. A method of preventing or treating Coronavirus infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9, thereby preventing or treating Coronavirus in the subject.
13. A method of producing an antibody capable of binding an antigenic determinant of Coronavirus, the method comprising:
(a) expressing in a host cell a heterologous polynucleotide encoding a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9; and optionally (b) recovering the antibody from the host cell.
14. A vaccine comprising an effective amount of a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus and an excipient, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and Ught chain of an antibody selected from the group consisting of A7, C1, C3 and A9.
15. A human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus attached to a heterologous effector moiety or carrier, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and Ught chain of an antibody selected from the group consisting of A7, C1, C3 and A9.
16. The antibody, method or vaccine of any one of claims 1-15, wherein said antibody is a recombinant antibody.
17. The antibody, method or vaccine of any one of claims 1-15, wherein said antibody is a monoclonal antibody.
18. The antibody, method or vaccine of any one of claims 1-15, wherein said antibody is capable of activating antibody-dependent ceUular phagocytosis.
19. The antibody, method or vaccine of any one of claims 1-15, wherein said antibody is of an IgGl serotype.
20. The composition, antibody, method or vaccine of any one of claims 1-16, wherein said antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of A7, C1 or C3.
21. The composition, antibody, method or vaccine of any one of claims 1-16, wherein said antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of A7.
22. The composition, antibody, method or vaccine of any one of claims 1-16, wherein said antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of C1 or C3.
23. The antibody, method or vaccine of any one of claims 1-22, wherein said human antibody comprising an antigen binding domain which binds said Coronavirus comprises a plurality of different human antibodies each comprising an antigen binding domain which binds a Coronavirus.
24. A method of detecting a Coronavirus infection, the method comprising contacting a biological sample suspected of being infected with Coronavirus with a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus under conditions which allow a specific immunocomplex formation between said antibody and said Spike, wherein a presence of said immunocomplex is indicative of Coronavirus infection, wherein an antigen binding domain of said antibody comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and A9.
25. The method of claim 24, wherein said antibody is labeled.
26. The method of any one of claims 24-25, wherein said contacting is effected in- vivo.
27. The method of any one of claims 24-25, wherein said contacting is effected ex- vivo.
28. A diagnostic kit for detecting a Coronavirus infection, the kit comprising a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus which allow a specific immunocomplex formation between said antibody and said Spike, wherein an antigen binding domain of said antibody comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of A7, C1, C3 and
A9.
29. The kit of claim 28, wherein said antibody is labeled.
30. The composition, antibody, method, vaccine or kit of any one of claims 1-29, wherein said Coronavirus is SAR-CoV-2, Middle East respiratory syndrome Coronavirus (MERS-CoV) or severe acute respiratory syndrome Coronavirus (SARS-CoV).
31. The composition, antibody, method, vaccine or kit of any one of claims 1-30, wherein said Coronavirus is SAR-CoV-2.
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