WO2023147251A1 - Anticorps spécifique de coronavirus et ses utilisations - Google Patents

Anticorps spécifique de coronavirus et ses utilisations Download PDF

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Publication number
WO2023147251A1
WO2023147251A1 PCT/US2023/060915 US2023060915W WO2023147251A1 WO 2023147251 A1 WO2023147251 A1 WO 2023147251A1 US 2023060915 W US2023060915 W US 2023060915W WO 2023147251 A1 WO2023147251 A1 WO 2023147251A1
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Prior art keywords
antibody
cov
amino acid
antigen
substantially similar
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PCT/US2023/060915
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English (en)
Inventor
Kuo-I Lin
Takashi ANGATA
Yi-Hsuan Chang
Wei-nan CHEN
Szu Teng MA
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Academia Sinica
Chou, Mei-Yin
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Publication of WO2023147251A1 publication Critical patent/WO2023147251A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present disclosure relates to an antibody or antigen-binding fragment thereof, which is specific to coronaviruses, and uses thereof.
  • the COVID-19 pandemic has drawn globally active efforts to develop effective strategies to control the spread of SARS-CoV-2 and ameliorate symptoms.
  • the infection causes symptoms of direct cytopathic effects and excessive inflammatory responses in the infected subject.
  • the lack of valid treatment causes high morbidity and mortality.
  • the emergence of these newly identified viruses highlights the need for the development of novel antiviral strategies.
  • the present disclosure provides novel therapeutic or detecting anti-Coronavirus (such as anti-SARS-CoV-2-Spike protein) antibodies and their use for treating or preventing or detecting viral infection.
  • anti-Coronavirus such as anti-SARS-CoV-2-Spike protein
  • the present disclosure provides an antibody or antigen-binding fragment thereof that is specific for an epitope in coronaviruses (CoVs), particularly, SARS-CoV-2.
  • CoVs coronaviruses
  • SARS-CoV-2 coronaviruses
  • the antibody according to the disclosure is thus useful for treating and/or preventing or detecting diseases and/or disorders caused by or related to CoVs, particularly SARS-CoV- 2.
  • the antibody of the disclosure is also useful for detecting CoVs (particularly, SARS- CoV-2).
  • the present disclosure provides an antibody or antigen-binding fragment thereof that is specific for an epitope in a CoV; wherein the antibody or antigen-binding fragment thereof comprises complementarity determining regions (CDRs) of a heavy chain variable region and CDRs of a light chain variable region, wherein the CDRs of the heavy chain variable region comprise: CDRH1 of an amino acid sequence of SEQ ID NO: 1, 6 or 11 or a substantially similar sequence thereof, CDRH2 of an amino acid sequence of SEQ ID NO: 2, 7 or 12 or a substantially similar sequence thereof, and CDRH3 of an amino acid sequence of SEQ ID NO: 3, 8 or 13 or a substantially similar sequence thereof; and wherein the CDRs of the light chain variable region comprise:
  • CDRL1 of an amino acid sequence of SEQ ID NO: 4, 9 or 14 or a substantially similar sequence thereof CDRL2 of an amino acid sequence of DVS, DAS or AAS or a substantially similar sequence thereof, and CDRL3 of an amino acid sequence of SEQ ID NO: 5, 10 or 15 or a substantially similar sequence thereof.
  • the disclosure provides an antibody (particularly 5SB12) or antigen-binding fragment thereof that is specific for an epitope in a CoV; wherein the antibody or antigen-binding fragment thereof comprises complementarity determining regions (CDRs) of a heavy chain variable region and CDRs of a light chain variable region, wherein the CDRs of the heavy chain variable region comprise:
  • the disclosure provides an antibody (particularly 1-2SA8) or antigen-binding fragment thereof that is specific for an epitope in a CoV; wherein the antibody or antigen-binding fragment thereof comprises CDRs of a heavy chain variable region and CDRs of a light chain variable region, wherein the CDRs of the heavy chain variable region comprise:
  • CDRH1 of an amino acid sequence of SEQ ID NO: 6 or a substantially similar sequence thereof CDRH2 of an amino acid sequence of SEQ ID NO: 7 or a substantially similar sequence thereof, and CDRH3 of an amino acid sequence of SEQ ID NO: 8 or a substantially similar sequence thereof; and wherein the CDRs of the light chain variable region comprise:
  • CDRL1 of an amino acid sequence of SEQ ID NO: 9 or a substantially similar sequence thereof CDRL2 of an amino acid sequence of DAS or a substantially similar sequence thereof, and CDRL3 of an amino acid sequence of SEQ ID NO: 10 or a substantially similar sequence thereof.
  • the disclosure provides an antibody (particularly 15SE6) or antigen-binding fragment thereof that is specific for an epitope in a CoV; wherein the antibody or antigen-binding fragment thereof comprises CDRs of a heavy chain variable region and CDRs of a light chain variable region, wherein the CDRs of the heavy chain variable region comprise:
  • CDRL1 of an amino acid sequence of SEQ ID NO: 14 or a substantially similar sequence thereof CDRL2 of an amino acid sequence of AAS or a substantially similar sequence thereof, and CDRL3 of an amino acid sequence of SEQ ID NO: 15 or a substantially similar sequence thereof.
  • the epitope is located in Spike protein. In some further embodiments of the disclosure, the epitope is located in a receptor-binding domain (RBD) of Spike protein.
  • the antibody is an Fab fragment, an F(ab')2 fragment, an ScFv fragment, a monoclonal antibody, a chimeric antibody, a nanobody, a humanized antibody or a human antibody.
  • the present disclosure provides a vector encoding the antibody or antigen-binding fragment thereof as disclosed herein.
  • the present disclosure provides a genetically engineered cell expressing the antibody or antigen-binding fragment thereof as disclosed herein or containing the vector as disclosed herein.
  • the present disclosure also provides a method for manufacturing the antibody or antigen-binding fragment thereof as disclosed herein, comprising: (a) introducing into a host cell one or more polynucleotides encoding said antibody or antigen-binding fragment; (b) culturing the host cell under conditions favorable to expression of the one or more polynucleotides; and (c) optionally, isolating the antibody or antigen-binding fragment from the host cell and/or a medium in which the host cell is grown.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as disclosed herein and a pharmaceutically acceptable carrier and, optionally, a further therapeutic agent.
  • the therapeutic agent examples include, but are not limited to, an antiviral agent.
  • the therapeutic agent is an anti-inflammatory agent or an antibody or antigen-binding fragment thereof that is specific for Spike protein of a CoV.
  • the subject is vaccinated.
  • the present disclosure provides a vessel or injection device comprising the antibody or antigen-binding fragment thereof as disclosed herein.
  • the present disclosure provides a method for treating or preventing infection with a coronavirus in a subject in need thereof, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof as disclosed herein.
  • the present disclosure provides a pharmaceutical composition for use in treating or preventing infection with a coronavirus in a subject in need thereof, comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof as disclosed herein and a pharmaceutically acceptable carrier and, optionally, a further therapeutic agent.
  • the CoV described herein includes, but is not limited to, SARS- CoV, MERS-CoV or SARS-CoV-2.
  • the CoV described herein is wild type (WT) CoV, D614G, alpha-CoV, beta-CoV, gamma-CoV, delta-CoV, or omicron-CoV.
  • the subject is administered one or more further therapeutic agents.
  • the present disclosure provides a method for neutralizing a coronavirus in a subject in need thereof, comprises administering to the subject the antibody or antigen-binding fragment thereof as disclosed herein.
  • the present disclosure provides a pharmaceutical composition for use in neutralizing a coronavirus in a subject in need thereof, comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof as disclosed herein and a pharmaceutically acceptable carrier and, optionally, a further therapeutic agent.
  • the present disclosure also provides a method for eliciting antibody-dependent cell- mediated cytotoxicity against a coronavirus in a subject in need thereof, comprising administering to the subject the antibody or antigen-binding fragment thereof as disclosed herein.
  • the present disclosure provides a pharmaceutical composition for use in eliciting antibody-dependent cell -mediated cytotoxicity against a coronavirus in a subject in need thereof, comprising a therapeutically effective amount of the antibody or antigenbinding fragment thereof as disclosed herein and a pharmaceutically acceptable carrier and, optionally, a further therapeutic agent.
  • the present disclosure provides a method for administering the antibody or antigenbinding fragment thereof as disclosed herein into the body of a subject, comprising injecting the antibody or antigen-binding fragment into the body of the subject.
  • the antibody or antigen-binding fragment is injected into the body of the subject subcutaneously, intravenously, or intramuscularly.
  • the present disclosure provides a method for detecting a coronavirus in a sample comprising contacting the sample with the antibody or antigen-binding fragment thereof as disclosed herein.
  • the present disclosure provides a kit for detecting a coronavirus in a sample, wherein the kit comprises the antibody or antigen-binding fragment thereof as disclosed herein. [0030]
  • the present disclosure is described in detail in the following sections. Other characteristics, purposes and advantages of the present disclosure can be found in the detailed description and claims.
  • FIGs. 1A to 1C show the binding of mAb with SARS-CoV-2 Spike protein.
  • FIG. 1A depicts cell-based assay showing the binding avidity of 5SB12, 1-2SA8 and 15SE6 to the S protein, as determined by FACS.
  • FIG. IB depicts ELISA showing the binding of 5SB12, 1-2SA8 and 15SE6 to the full-length Spike, RBD domain, SI and S2 domains of SARS- CoV-2 (WH01).
  • FIG. 1C depicts binding avidity of 5SB12, 1-2SA8 and 15SE6 to full- length S protein as measured by bio-layer interferometry.
  • FIGs 2A to 2C show that 1-2SA8, 15SE6 and 5SB12 bind to Spike protein of various variants of SARS-CoV-2.
  • Cell-based assay showing the binding and avidity of 1-2SA8 (FIG. 2A), 15SE6 (FIG. 2B) and 5SB12 (FIG. 2C) to HEK293T cells expressing WT (WH01) and various variants of concern (VOC) forms of Spike proteins of SARS-CoV-2, including D614G, B.l.1.7 [alpha], B.1.351 [beta], P.l [gamma] and B.1.617.2 [delta],
  • FIGs 3A to 3C show EC50 of 1-2SA8 (FIG. 3A), 15SE6 (FIG. 3B) and 5SB12 (FIG. 3C) to neutralize SARS-CoV-2 pseudoviruses of WT (WH01) or indicated variant of concern. EC50 of each antibody against various pseudoviruses was indicated.
  • WT (WH01), D614G, B. l.1.7 [alpha], B.1.351 [beta], P.l [gamma] and B.1.617.2 [delta] were tested.
  • FIGs. 3D and 3E show that mAb 1-2SA8 is able to recognize Omicron subvariants BA.4/5, but not BA.l.
  • FIGs. 3F and 3G show that mAb 1-2SA8 is able to neutralize Omicron subvariants BA.4/5 and BF.7.
  • IC50 was indicated.
  • FIG. 4 A shows antibody-dependent cell-mediated cytotoxicity (ADCC) ability of identified mAbs.
  • FIG. 4B shows that mAb 1-2SA8 has antibody-dependent cell-mediated cytotoxicity (ADCC) activity against Omicron subvariant Spike proteins expressing cells. ADCC ability of 1- 2SA8 against Omicron subvariants BA.l and BA.4/5 Spike protein expressing 293T cells.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FIG. 5 A and 5B show that 5 SB 12, but not 5SB12-LALA, protect hamsters from lethal dose challenge of SARSCoV-2 (WH01).
  • FIG. 5A shows flow chart of mAb treatment and viral challenge.
  • FIG. 5B shows viral titers in the lungs of mice with indicated treatment (3.5 mg/kg in PBS by ip) and infection. * p ⁇ 0.05, ** p ⁇ 0.01.
  • FIGs. 6A to 6D show that 1-2SA8 and 15SE6 protect SARS-CoV-2 infection in hamsters.
  • FIG. 6A shows experimental design. Hamsters were treated with mAb (3.5 mg/kg in PBS by ip at 24 hours before infection.
  • FIG. 6B shows weight changes after antibody administration and SARS-CoV-2 (WH01) challenge.
  • FIG. 6C shows the levels of SARS- CoV-2 viral genome as determined by RT-qPCR.
  • FIG. 6D depicts H&E stain showing the reduction of histopathological lung lesions after the treatment with 1-2SA8 and 15SE6. * p ⁇ 0.05, ** p ⁇ 0.01, *** ⁇ 0.001.
  • FIG. 7 shows that three mAbs prevent body weight loss in K18-hACE2 TG mice after challenge with lethal dose of alpha (UK) variant of SARS-CoV-2.
  • mAbs (15 mg/kg in PBS) were given by ip, followed by infection with alpha variant of SARS-CoV-2.
  • FIGs. 8A to 8C show different mode of actions of mAbs 5SB12, 1-2SA8 and 15SE6.
  • FIG. 8 A shows that 5 SB 12, but not 5SB12-LALA, pre-treatment (3.5 mg/kg in PBS by ip) is able to protect hamsters from SARS-CoV-2 infection-induced decrease of body weight, and reduce viral titers in the lungs.
  • FIG. 8B shows dose-dependent protection by 5SB12 pretreatment in SARS-CoV-2 infection in hamsters.
  • FIG. 8 A shows that 5 SB 12, but not 5SB12-LALA, pre-treatment (3.5 mg/kg in PBS by ip) is able to protect hamsters from SARS-CoV-2 infection-induced decrease of body weight, and reduce viral titers in the lungs.
  • FIG. 8B shows dose-dependent protection by 5SB12 pretreatment in SARS-CoV-2 infection in hamsters.
  • antibody refers to any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that is specific for or interacts with a particular antigen, such as SARS-CoV-2-Spike protein.
  • CDR complementarity determining region
  • the term “antibody” includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM).
  • Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, CHI, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region comprises one domain (CLI).
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the FRs of the anti-SARS-CoV-2-Spike protein antibody may be identical to the human germline sequences, or may be naturally or artificially modified.
  • An amino acid consensus sequence may be defined based on a side- by-side analysis of two or more CDRs.
  • antigen-binding portion of an antibody, "antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • epitope refers to the site on the antigen to which an antibody binds.
  • CDR complementarity determining region
  • the term “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • residue positions which do not differ by conservative amino acid substitutions Preferably, residue positions which do not differ by conservative amino acid substitutions.
  • a “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art.
  • groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysinearginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.
  • a conservative replacement is any change having a positive value in the PAM250 loglikelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445, herein incorporated by reference.
  • a "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
  • monoclonal antibody as used herein is not limited to antibodies produced through hybridoma technology.
  • a monoclonal antibody is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, or by any references to available or known in the art.
  • the term “nanobody” refers to an antibody comprising the small single variable domain (VHH of antibodies obtained from camelids and dromedaries.
  • VHH small single variable domain
  • Antibody proteins obtained from members of the camel and dromedary (Camelus baclrianus and Calelus dromaderius) family including new world members such as llama species (Lama paccos, Lama glama and Lama vicugna) have been characterized with respect to size, structural complexity and antigenicity for human subjects.
  • Certain IgG antibodies from this family of mammals as found in nature lack light chains, and are thus structurally distinct from the typical four chain quaternary structure having two heavy and two light chains, for antibodies from other animals.
  • Humanized forms of non-human antibodies are chimeric immunoglobulins that contain minimal sequences derived from non-human immunoglobulin.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the term "therapeutic agent” refers to any compound, substance, drug or active ingredient having a therapeutic or pharmacological effect that is suitable for administration to a mammal, for example a human.
  • immunoconjugate refers to an antigen-binding protein, e.g., an antibody or antigen-binding fragment, which is chemically or biologically linked to a radioactive agent, a cytokine, an interferon, a target or reporter moiety, an enzyme, a peptide or protein or a therapeutic agent.
  • the antigen-binding protein may be linked to the radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide or therapeutic agent at any location along the molecule so long as it is able to bind its target (CoV-S).
  • immunoconjugates include antibody-drug conjugates and antibody- toxin fusion proteins.
  • the agent may be a second, different antibody that binds specifically to CoV-S.
  • the type of therapeutic moiety that may be conjugated to the anti-CoV-S antigen-binding protein e.g., antibody or fragment
  • vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • genetically engineered or “genetic engineering” of cells refers to manipulating genes using genetic materials for the change of gene copies and/or gene expression level in the cell.
  • the genetic materials can be in the form of DNA or RNA.
  • the genetic materials can be transferred into cells by various means including viral transduction and non-viral transfection. After being genetically engineered, the expression level of certain genes in the cells can be altered permanently or temporarily.
  • coronavirus refers to any virus of the coronavirus family, including, but not limited to, SARS-CoV-2, MERS-CoV, and SARS-CoV.
  • SARS-CoV-2 refers to the newly-emerged coronavirus which is rapidly spreading to other areas of the globe. It binds via the viral spike protein to human host cell receptor angiotensin-converting enzyme 2 (ACE2). The spike protein also binds to and is cleaved by TMPRSS2, which activates the spike protein for membrane fusion of the virus.
  • ACE2 human host cell receptor angiotensin-converting enzyme 2
  • CoV-S also called “S” or “S protein” refers to the spike protein of a coronavirus, and can refer to specific S proteins such as SARS-CoV-2-S, MERS-CoV S, and SARS-CoV S.
  • coronavirus infection refers to infection with a coronavirus such as SARS-CoV-2, MERS-CoV, or SARS-CoV.
  • coronavirus respiratory tract infections often in the lower respiratory tract. Symptoms can include high fever, dry cough, shortness of breath, pneumonia, gastro-intestinal symptoms such as diarrhea, organ failure (kidney failure and renal dysfunction), septic shock, and death in severe cases.
  • the term "pharmaceutical composition” refers to a mixture containing a therapeutic agent administered to a mammal, for example a human, for preventing, treating, or eliminating a particular disease or pathological condition that the mammal suffers from.
  • a therapeutic agent administered to a mammal, for example a human
  • a therapeutic agent administered to a mammal, for example a human
  • a therapeutic agent administered to a mammal, for example a human
  • treatment cover any treatment of a disease in a mammal, particularly in a human, and include: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • preventing or “prevention” is recognized in the art, and when used in relation to a condition, it includes administering, prior to onset of the condition, an agent to reduce the frequency or severity of or to delay the onset of symptoms of a medical condition in a subject, relative to a subject which does not receive the agent.
  • the terms "individual,” “subject,” “host,” and “patient,” refer to a mammal, including, but not limited to, murines (rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc. Particularly, the subject is vaccinated.
  • the term "in need of treatment” refers to a judgment made by a caregiver (e.g., physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals) that a subject requires or will benefit from with regard to treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the subject is ill, or will be ill, as the result of a condition that is treatable by the compounds of the present disclosure.
  • a caregiver e.g., physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals
  • sample encompasses a variety of sample types obtained from an individual, subject or patient and can be used in a diagnostic or monitoring assay.
  • the definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof.
  • Neutralizing refers to a process wherein a molecule (e.g., antibody) inhibits an activity of a coronavirus to any detectable degree, e.g., inhibits the ability of coronavirus to bind to a receptor, to be cleaved by a protease, or to mediate viral entry into a host cell or viral reproduction in a host cell.
  • a molecule e.g., antibody
  • Coronaviruses infect humans and animals and cause a variety of diseases, including respiratory, enteric, renal, and neurological diseases.
  • CoV uses its spike glycoprotein (S), a main target for a neutralizing antibody, to bind its receptor, and mediate membrane fusion and virus entry.
  • S spike glycoprotein
  • the coronavirus spike protein is highly conserved among all human coronaviruses (CoVs) and is involved in receptor recognition, viral attachment, and entry into host cells.
  • SARS-CoV-2 S protein is also highly conserved with that of CoVs.
  • TM protease serine 2 (TMPRSS2), a type 2 TM serine protease located on the host cell membrane, promotes virus entry into the cell by activating the S protein.
  • TMPRSS2 TM protease serine 2
  • the viral RNA is released, polyproteins are translated from the RNA genome, and replication and transcription of the viral RNA genome occur via protein cleavage and assembly of the replicase-transcriptase complex.
  • Viral RNA is replicated, and structural proteins are synthesized, assembled, and packaged in the host cell, after which viral particles are released (Fehr AR, Perlman S. Coronaviruses: an overview of their replication and pathogenesis. Methods Mol Biol. 2015;1282:1-23).
  • the SARS-CoV-2-Spike protein is a 1273 amino acid type I membrane glycoprotein which assembles into trimers that constitute the spikes or peplomers on the surface of the enveloped coronavirus particle.
  • the protein has two essential functions, host receptor binding and membrane fusion, which are attributed to the N-terminal (SI) and C-terminal (S2) halves of the S protein.
  • CoV-S binds to its cognate receptor via a receptor binding domain (RBD) present in the SI subunit.
  • RBD receptor binding domain
  • the amino acid sequence of full-length SARS- CoV-2 spike protein is exemplified by the following amino acid sequence of SEQ ID NO: 19.
  • SI domain binds the receptor, it results in a conformational change of the S2 domain which facilitates the fusion between viral envelope and the plasma membrane of its target cell.
  • variants of SARS-CoV-2 Spike protein include, but are not limited to, D614G: D614G; B.1.1.7: 69-70 deletion, 144 deletion, N501Y, A570D, D614G, P681H, T716I, S982A and D1118H; B.1.351 : L18F, D80A, D215G, 242-244 deletion, R246I, K417N, E484K, N501Y, D614G and A701V.
  • CoV-S includes protein variants of CoV spike protein isolated from different CoV isolates as well as recombinant CoV spike protein or a fragment thereof.
  • the term also encompasses CoV spike protein or a fragment thereof coupled to, for example, a histidine tag, mouse or human Fc, or a signal sequence such as R0R1.
  • the present disclosure develops an antibody or antigen-binding fragment thereof that is specific for an epitope in a CoV, particularly, SARS-CoV-2-Spike protein.
  • monoclonal antibodies are isolated from patients recovered from SARS-CoV-2 infection by using single B cell screening platform.
  • FACS analysis shows that 5SB12, 1-2SA8 and 15SE6 mAbs all effectively recognize Spike protein of SARS-CoV-2 in a cell-based assay displaying full length SARS-CoV-2 Spike protein from Wuhan strain (WH01) at picomolar levels.
  • the present disclosure provides an antibody or antigen-binding fragment thereof that is specific for an epitope in a spike protein of a CoV; wherein the antibody or antigen-binding fragment thereof comprises CDRs of a heavy chain variable region and CDRs of a light chain variable region, wherein the CDRs of the heavy chain variable region comprise CDRH1, CDRH2, and CDRH3 regions, and the CDRs of the light chain variable region comprise CDRL1, CDRL2, and CDRL3 regions, and wherein: the CDRH1 region comprises the amino acid sequence of GYTFTGYQ (SEQ ID NO: 1) or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; the CDRH2 region comprises the amino acid sequence of INPNSGGT (SEQ ID NO: 2) or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; the CDRH3 region comprises the amino acid sequence of ARGPLRYGDYVLG
  • the antibody or antigen-binding fragment thereof comprising SEQ ID NOs: 1 to 5 and DVS is 5SB12.
  • Cell-based assay using cells expressing Spike protein from various variants of SARS-CoV-2 further revealed that 5SB12 appears to only recognize Spike proteins from wild type (WT) SARS-CoV-2 Wuhan strain (WH01), D614G, alpha (B. l.1.7), delta (B.1.617.2), and omicron (BA.l, 4/5 and BF.7) variants.
  • 5 SB 12 can only slightly neutralize WH01, D614G, alpha (Bl.1.7), delta (B.1.617.2), and omicron (BA. l, 4/5 and BF.7) variants.
  • the present disclosure provides an antibody or antigen-binding fragment thereof that is specific for an epitope in a spike protein of a CoV; wherein the antibody or antigen-binding fragment thereof comprises CDRs of a heavy chain variable region and CDRs of a light chain variable region, wherein the CDRs of the heavy chain variable region comprise CDRH1, CDRH2, and CDRH3 regions, and the CDRs of the light chain variable region comprise CDRL1, CDRL2, and CDRL3 regions, and wherein: the CDRH1 region comprises the amino acid sequence of GFFVSRNY (SEQ ID NO: 6) or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; the CDRH2 region comprises the amino acid sequence of IYSGGST (SEQ ID NO: 7) or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; the CDRH3 region comprises the amino acid sequence of ARDHVRSGM
  • the antibody or antigen-binding fragment thereof comprising SEQ ID NOs: 6 to 10 and DAS is 1-2SA8.
  • the isolated mAb, 1-2SA8 is demonstrated to recognize the receptor-binding domain (RBD) of Spike protein of SARS- CoV-2 in high affinity and shows protective efficacy against various SARS-CoV-2 variants.
  • Cell-based assay using cells expressing Spike protein from various variants of SARS-CoV- 2 further revealed that, 1-2SA8 recognizes a broad spectrum of Spike proteins, including wild type (WT) SARS-CoV-2 Wuhan strain (WH01), D614G, alpha (B.1.1.7), beta (B. 1.351), gamma (P.
  • 1- 2SA8 possesses the activity to neutralize WH01 as well as all the tested SARS-CoV-2 pseudovirus variants, including D614G, alpha (Bl.1.7), beta (B. 1.351), gamma (P.l), delta (B.1.617.2), and omicron (BA.l, 4/5 and BF.7) with IC50 values in the picomolar range.
  • the present disclosure provides an antibody or antigen-binding fragment thereof that is specific for an epitope in a spike protein of a CoV; wherein the antibody or antigen-binding fragment thereof comprises CDRs of a heavy chain variable region and CDRs of a light chain variable region, wherein the CDRs of the heavy chain variable region comprise CDRH1, CDRH2, and CDRH3 regions, and the CDRs of the light chain variable region comprise CDRL1, CDRL2, and CDRL3 regions, and wherein: the CDRH1 region comprises the amino acid sequence of GYTFTSYY (SEQ ID NO: 11) or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; the CDRH2 region comprises the amino acid sequence of INPSGGST (SEQ ID NO: 12) or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; the CDRH3 region comprises the amino acid sequence of ARWYDSLTY
  • the antibody or antigen-binding fragment thereof comprising SEQ ID NOs: 11 to 15 and AAS is 15SE6.
  • Cell-based assay using cells expressing Spike protein from various variants of SARS-CoV-2 further revealed that 15 SE6 also recognizes a broad spectrum of Spike proteins, including wild type (WT) SARS-CoV- 2 Wuhan strain (WH01), D614G, alpha (B.l.1.7), beta (B. 1.351), delta (B.1.617.2), and omicron (BA.l, 4/5 and BF.7) with EC50 values in the picomolar range.
  • 15SE6 is able to neutralize WH01, D614G, alpha (Bl.1.7), delta (B.1.617.2), and omicron (BA. l, 4/5 and BF.7) pseudoviruses in the picomolar range.
  • 5SB12 does not possess potent efficacy to neutralize SARS-CoV-2
  • 5SB 12 shows good antibody-dependent cell-mediated cytotoxicity (ADCC) activity as compared with 1-2SA8 and 15SE6, suggesting the potential utility of 5SB12 in vivo.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • 5SB12 when given to hamsters, 5SB12 prevents SARS-CoV-2 (WH01) infection, which is dependent on the engagement with effector cells through the Fc constant region of 5SB12.
  • 1-2SA8 and 15SE6 both are able to effectively protect hamsters from SARS-CoV-2 (WH01) infection, and the protective efficacy is independent of the effector function of mAbs.
  • the 5SB12, 1-2SA8 and 15SE6 monoclonal antibodies identified here are isolated from peripheral blood of convalescent COVID-19 patients by single B cell screening platform and recombinant antibody technology.
  • 1-2SA8 and 15SE6 possess neutralizing activity against various variants of SARS-CoV-2 pseudoviruses, and prophylactic efficacy against SARS-CoV-2 infection in hamsters.
  • 5SB12 does not possess strong neutralizing activity against SARS-CoV-2, but exhibits ADCC activity when engaging with NK cells, and shows prophylactic efficacy against SARS-CoV-2 infection in hamsters.
  • the antibody according to the disclosure can be full-length (for example, an IgGl or IgG4 antibody) or may comprise only an antigen-binding portion (for example, a Fab, F(ab')2 or scFv fragment), and may be modified to affect functionality as needed.
  • the antibody or antigen-binding fragment thereof is conjugated with an anti-CoV-S antigen-binding proteins, e.g., antibodies or antigen-binding fragments, conjugated to another moiety, e.g., a therapeutic moiety (an "immunoconjugate"), such as a toxoid or an antiviral agent to treat coronavirus infection.
  • an anti-CoV-S antibody or fragment is conjugated to any of the further therapeutic agents set forth herein.
  • Various techniques known to persons of ordinary skill in the art can be used to determine whether an antibody "specifically binds to one or more amino acids" within a polypeptide or protein.
  • Exemplary techniques include, e.g., a routine cross-blocking assay such as that described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., N.Y.), alanine scanning mutational analysis, peptide blots analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis.
  • methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer, 2000, Protein Science 9:487-496).
  • Another method that can be used to identify the amino acids within a polypeptide with which an antibody specifically binds is hydrogen/deuterium exchange detected by mass spectrometry.
  • the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium -lab eled protein. Next, the protein/antibody complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except for the residues protected by the antibody (which remain deuterium -lab eled).
  • the target protein After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium -lab eled residues which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.
  • test antibody If the test antibody is able to bind to SARS-CoV-2-Spike protein following saturation binding with the reference anti-SARS-CoV-2-Spike protein antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-SARS-CoV-2- Spike protein antibody. On the other hand, if the test antibody is unable to bind to the SARS- CoV-2-Spike protein molecule following saturation binding with the reference anti-SARS- CoV-2-Spike protein antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference anti-SARS-CoV-2-Spike protein antibody of the disclosure.
  • Additional routine experimentation e.g., peptide mutation and binding analyses
  • peptide mutation and binding analyses can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding.
  • steric blocking or another phenomenon
  • this sort can be performed using ELISA, RIA, Biacore, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art.
  • two antibodies bind to the same (or overlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay.
  • two antibodies are deemed to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
  • Two antibodies are deemed to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
  • the antibody also includes an antigen-binding fragment of a full antibody molecule.
  • An antigen-binding fragment of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains.
  • DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized.
  • the DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
  • Non-limiting examples of an antigen-binding fragment include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide.
  • CDR complementarity determining region
  • engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed by the expression "antigenbinding fragment," as used herein.
  • SMIPs small modular immunopharmaceuticals
  • An antigen-binding fragment of an antibody typically comprises at least one variable domain.
  • the variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences.
  • the VH and VL domains may be situated relative to one another in any suitable arrangement.
  • the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers.
  • the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
  • an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain.
  • variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure include: (i) VH-CHI; (ii) VH-CH2; (iii) VH- B; (iv) VH-CHI-CH2; (V) VH-CHI-CH2-CH3, (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CHI; (ix) VL-CH2; (X) VL- B; (xi) VL-CHI-CH2; (xii) VL-CHI-CH2-CH3; (xiii) VL- CH2-CH3; and (xiv) VL-CL.
  • variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region.
  • a hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule.
  • an antigen-binding fragment of an antibody of the present disclosure may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
  • the anti-SARS-CoV-2-Spike protein antibody disclosed herein may comprise one or more amino acid substitutions, insertions, and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases.
  • the present disclosure includes an antibody, and an antigen-binding fragment thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another mammalian germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations").
  • Germline mutations A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, could easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof.
  • all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived.
  • only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3.
  • one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived).
  • the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence.
  • antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired properties such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc.
  • Antibodies and antigen-binding fragments obtained in this general manner are encompassed by the present disclosure.
  • the present disclosure also includes an anti-SARS-CoV-2-Spike protein antibody comprising variants of any of the VH, VL, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions.
  • the present disclosure includes an anti-SARS-CoV-2-Spike protein antibody having VH, VL, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions relative to any of the VH, VL, and/or CDR amino acid sequences disclosed herein.
  • the antibody according to the disclosure is a humanized antibody.
  • some amino acid residues in the human framework region are replaced by the corresponding amino acid residues in the species of CDRs; e.g., a rodent.
  • the antibodies of the present disclosure may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide.
  • the anti-SARS-CoV-2-Spike protein antibodies of the present disclosure can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein.
  • another functional molecule e.g., another peptide or protein.
  • an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bispecific or a multispecific antibody with a second binding specificity.
  • the present disclosure includes bispecific antibodies wherein one arm of an immunoglobulin is specific for SARS-CoV-2-Spike protein or a fragment thereof, and the other arm of the immunoglobulin is specific for a second therapeutic target or is conjugated to a therapeutic moiety.
  • the present disclosure provides a genetically engineered cell expressing the antibody or antigen-binding fragment thereof or containing the vector.
  • the genetically engineered cell may be an immune cell.
  • the antibody or antigen-binding fragment thereof can be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems.
  • the expression system is a mammalian cell expression, such as a hybridoma, or a CHO cell expression system. Many such systems are widely available from commercial suppliers.
  • the VH and VL regions may be expressed using a single vector, e.g., in a di-cistronic expression unit, or under the control of different promoters.
  • the VH and VL region may be expressed using separate vectors.
  • a VH or VL region as described herein may optionally comprise a methionine at the N-terminus.
  • the genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody.
  • Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity (see, e.g., Kuby, Immunology (3. sup. rd ed. 1997)).
  • An example of a method for manufacturing the antibody or antigen-binding fragment comprises: (a) introducing into a host cell one or more polynucleotides encoding said antibody or antigen-binding fragment; (b) culturing the host cell under conditions favorable to expression of the one or more polynucleotides; and (c) optionally, isolating the antibody or antigen-binding fragment from the host cell and/or a medium in which the host cell is grown.
  • a vector can be used to introduce a polynucleotide encoding the antibody or antigenbinding fragment of the invention to a host cell.
  • one type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • compositions comprising the antibody or antigen-binding fragment thereof.
  • the pharmaceutical compositions of the disclosure are formulated with suitable diluents, carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like.
  • the compositions may be formulated for specific uses, such as for veterinary uses or pharmaceutical uses in humans.
  • the form of the composition and the excipients, diluents and/or carriers used will depend upon the intended uses of the antibody and, for therapeutic uses, the mode of administration. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.
  • formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN.TM., Life Technologies, Carlsbad, Calif.), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.
  • the pharmaceutical composition comprises a further therapeutic agent, such as an antiviral agent.
  • the antiviral agent may be an antibody to S protein of SARS-CoV-2; anti-inflammatory agent; an antibody to an NTD region of S protein of SARS-CoV-2; an antibody to a HR1 region of S protein of SARS-CoV-2; an antibody to an RBD region of S protein of SARS-CoV-2; a SARS-CoV monoclonal antibody; a MERS-CoV monoclonal antibody; a SARS-CoV-2 monoclonal antibody; a peptide; a protease inhibitor; a PIKfyve inhibitor; a TMPRSS2 inhibitor; and a cathepsin inhibitor; a furin inhibitor; an antiviral peptide; an antiviral protein; an antiviral chemical compound.
  • the antiviral agent may be at least one selected from the group consisting of: 1A9; 201; 311mab-31B5; 311mab- 32D4; 47D11; 4A8; 4C2; 80R; Apilimod; B38; camostat mesylate; Casirivimab; CR3014; CR3022; D12; E-64D; EK1; EK1C4; H4; HR2P; IBP02; Imdevimab; m336; MERS-27; MERS- 4; MI-701; n3088; n3130; P2B-2F6; P2C-1F11; PI8; S230; S309; SARS-CoV-2 S HR2P fragment (aal 168-1203); Tetrandrine; Viracept (nelfinavir mesylate); YM201636; a-l-PDX; favipiravir; IFN-a; IFN-al
  • the alpha-interferon species may be a mixture of at least seven species of alpha-interferon produced by human white blood cells, wherein the seven species are: interferon alpha 2; interferon alpha 4; interferon alpha 7; interferon alpha 8; interferon alpha 10; interferon alpha 16; and interferon alpha 17.
  • the dose of antibody administered to a patient may vary depending upon the age and size of the patient, target disease, conditions, route of administration, and the like.
  • the preferred dose is typically calculated according to body weight or body surface area.
  • an antibody of the present disclosure is used for treating a condition or disease associated with CoV in an adult patient, it may be advantageous to intravenously administer the antibody of the present disclosure.
  • the frequency and the duration of the treatment can be adjusted.
  • Effective dosages and schedules for administering the antibody may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly.
  • interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8: 1351).
  • Various delivery systems are known and can be used to administer the pharmaceutical composition of the disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432).
  • Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.
  • Administration can be systemic or local.
  • a pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a vessel or injection device such as standard needle and syringe.
  • a pen delivery device readily has applications in delivering a pharmaceutical composition of the present disclosure.
  • Such a pen delivery device can be reusable or disposable.
  • a reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once the entire pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused.
  • a disposable pen delivery device there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
  • the pharmaceutical composition can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).
  • polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla.
  • a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249: 1527-1533.
  • the injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described here in a sterile aqueous medium or an oily medium conventionally used for injections.
  • aqueous medium for injections there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc.
  • an alcohol e.g., ethanol
  • a polyalcohol e.g., propylene glycol, polyethylene glycol
  • a nonionic surfactant e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil
  • oily medium there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients.
  • dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.
  • the present disclosure provides a method for detecting a coronavirus in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof.
  • the present disclosure provides a method for neutralizing a coronavirus in a subject in need, comprising administering to the subject the antibody or antigen-binding fragment thereof.
  • the present disclosure also provides a method for eliciting antibody-dependent cell- mediated cytotoxicity against a coronavirus in a subject in need thereof, comprising administering to the subject the antibody or antigen-binding fragment thereof as disclosed herein.
  • the present disclosure provides a kit for detecting a coronavirus in a sample, wherein the kit comprises the antibody or antigen-binding fragment thereof.
  • the anti-SARS-CoV-2-Spike protein antibody of the present disclosure may also be used to detect and/or measure coronavirus, or SARS-CoV-2-Spike protein-expressing cells in a sample, e.g., for diagnostic purposes.
  • an anti-SARS-CoV-2-Spike protein antibody, or fragment thereof may be used to diagnose a condition or disease characterized by coronavirus infection.
  • Exemplary diagnostic assays for coronavirus may comprise, e.g., contacting a sample, obtained from a patient, with an anti-SARS-CoV-2-Spike protein antibody of the disclosure, wherein the anti-SARS-CoV-2-Spike protein antibody is labeled with a detectable label or reporter molecule.
  • an unlabeled anti-SARS-CoV- 2-Spike protein antibody can be used in diagnostic applications in combination with a secondary antibody which is itself detectably labeled.
  • the detectable label or reporter molecule can be a radioisotope, such as 3 H, 14 C, 32 P, 35 S, or 125 I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase.
  • Specific exemplary assays that can be used to detect or measure coronavirus in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).
  • Live single spike-specific memory B cells (CD 19 + CD27 + IgG + ) were sorted into 96-well PCR plates (Applied Biosystems) containing 10 pl/well catch buffer (10 mM Tris-HCl, pH 8, and 5 U/pl RNasin (Promega)) by BD FACSAria II.
  • Seminested second round PCR was performed using KAPA2G fast HS genotyping mix (KAPATM Biosystems) with 4 pl of unpurified first round PCR product at 94°C for 5 min followed by 49 cycles of 94°C for 30 s, 58°C (IgH/IgK) or 60°C (Ig ) for 30 s, 72°C for 45 s, and final incubation at 72°C for 5 min.
  • PCR products were then analyzed on 2% agarose gels and sequenced. The VH, V K and V ; . genes were identified by searching on the IMGT website (http://imgt.org/IMGT_vquest/input).
  • the genes were then amplified from second round PCR product with single gene-specific VH, V K and V ; . gene primers containing restriction sites for cloning into the vectors containing human IgH or IgL expression backbone.
  • the chimeric IgH and IgL expression constructions were co-transfected into Expi293 for antibody production.
  • Binding of antibody with S protein expressing 293T surface 293T cells were transfected with pcDNA6/Spike-P2A-eGFP. Transfected cells were selected under 10 pg/ml of blasticidin for 2-3 weeks. Selected cells were then sorted by FACSAria II to obtain eGFP + expressing cells. These cells were maintained in DMEM containing 10% FBS and 10 pg/ml of blasticidin. 2-3 xlO 5 cells were incubated with serially diluted antibodies in FACS buffer on ice for 1 h.
  • the S protein variants used here are: WH01 spike: original S protein; D614G: D614G; B.1.1.7: 69-70 deletion, 144 deletion, N501Y, A570D, D614G, P681H, T716I, S982A and D1118H; B.1.351 : L18F, D80A, D215G, 242-244 deletion, R246I, K417N, E484K, N501Y, D614G and A701V. [0115] Affinity and avidity determination using Octet (bio-layer interferometry).
  • Purified mAb IgG was loaded at 5 or 10 pg/ml in kinetics buffer (0.01% endotoxin-free BSA, 0.002% Tween-20, and 0.005% NaNs in PBS) onto Protein G biosensors (Molecular Devices, FORTEBIOTM). Association and dissociation of S protein (SARS-CoV-2 WH01) by IgG was performed in kinetics buffer at indicated concentrations for 5 min. KD values were calculated using a 1 : 1 global fit model (OCTETTM).
  • HEK293T cells stably expressing Spike-expression vectors from SARS- CoV-2 WH01 and human NK-92 cells were used as target cells and effector cells, respectively.
  • Spike expressing HEK293T cells or human NK-92 cells were cultured in complete DMEM or a-MEM medium and collected by centrifugation at 300g for 3 min.
  • Spike-expressing HEK293T cells were washed in PBS and labeled with 5(iM Calcein AM (INVITROGENTM) in PBS at 37° C with 5% CO2 for 20 min.
  • ELISA ELISA. Plates were first coated with 2 pg-50 ng/well (in 0.1 ml) of Spike protein or various subunits of Spike proteins purchased from Sino Biological Inc., and then blocked with blocking solution containing 2% BSA in PBS. Serially diluted mAbs with 2% BSA in PBS and HRP- conjugated secondary antibody (1 : 10,000, JACKSONTM ImmunoResearch Laboratories, Inc.) were sequentially added. After extensive washing with PBST (0.05% Tween-20), peroxidase substrate solution (TMB) and IM H2SO4 stop solution were used and absorbance (OD 450 nm) was read by a microplate reader. [0118] Pseudovirus neutralization assay.
  • the pseudotyped lentivirus carrying SARS-CoV-2 S protein was generated by transiently transfecting HEK293T cells with pCMV-AR&91, pLAS2w.Fluc. Ppuro and pcDNA3.1-nCoV-SA18 (or pcDNA3.1-nCoV-SA18 D614G).
  • HEK293T cells were seeded one day before transfection, and indicated plasmids were delivered into cells by using TransITR-LTl transfection reagent (MIRUSTM). The culture medium was refreshed at 16 h and harvested at 48 h and 72h post-transfection.
  • the transduction unit (TU) of SARS-CoV-2 peudotyped lentivirus was estimated by using cell viability assay in response to the limited dilution of lentivirus.
  • HEK293T cells stably expressing human ACE2 gene were plated on a 96-well plate one day before lentivirus transduction.
  • different amounts of lentivirus were added into the culture medium containing polybrene (final concentration 8 pg/ml). Spin infection was carried out at 1,100 xg in the 96-well plate for 30 min at 37°C.
  • the culture medium containing the virus and polybrene was removed and replaced with fresh complete DMEM containing 2.5 pg/ml puromycin. After treating puromycin for 48 h, the culture media was removed and the cell viability was detected by using 10% AlarmaBlue reagents according to the manufacturer's instruction. The survival rate of uninfected cells (without puromycin treatment) was set as 100%. The virus titer (transduction units) was determined by plotting the survival cells versus a diluted viral dose.
  • mAbs were serially diluted with desired dilution and incubated with 1,000 TU of SARS-CoV-2 pseudotyped lentivirus in DMEM (supplemented with 1% FBS and 100 U/ml Penicillin/Streptomycin) for 1 h at 37°C. The mixture was then inoculated with 10,000 HEK293T cells stably expressing human ACE2 gene in the 96-well plate. The culture medium was replaced with fresh complete DMEM (supplemented with 10% FBS and 100 U/ml Penicillin/ Streptomycin) at 16 h post-infection and cells were continuously cultured for another 48 h before performing luciferase assay.
  • DMEM supplied with 1% FBS and 100 U/ml Penicillin/Streptomycin
  • luciferase assay For luciferase assay, the expression level of luciferase gene was determined by using BRIGHT-GLOTM Luciferase Assay System (PROMEGATM). The relative light unit (RLU) was detected by Tecan i-control (INFINITETM 500). The percentage of inhibition was calculated as the ratio of RLU reduction in the presence of diluted serum to the RLU value of no serum control and the calculation formula is shown: (RLU 3ontro1 - RLU S5rum ) / RLU 3ontro1 .
  • the COVID-19 pandemic has drawn globally active efforts to develop effective strategies to control the spread of SARS-CoV-2 and ameliorate symptoms.
  • mAbs monoclonal antibodies
  • ELISA analysis showing that several clones are able to bind with the Spike (WH01)- expressing HEK293T cells. Particularly, some mAbs are able to recognize RBD domain of Spike (WH01).
  • FACS analysis shows that 5SB12, 1-2SA8 and 15SE6 mAbs all effectively recognize Spike protein of SARS-CoV-2 in a cell-based assay displaying full length SARS-CoV-2 Spike protein from Wuhan strain (WH01) at picomolar levels (FIG. 1A).
  • the avidity of 5SB12 is 3.41 x 10' 11 M; the avidity of 1-2SA8 is 2.52 x 10' 10 M; the avidity of 15SE6 is 6.42 x 10' 11 M, Particularly, 1-2SA8 and 15SE6 mAbs also bind with full length Spike protein and RBD domain of Spike protein at picomolar levels by ELISA (FIG. IB).
  • 1-2SA8 possesses the activity to neutralize WH01 as well as all the tested SARS-CoV-2 pseudovirus variants, including D614G, alpha (Bl.1.7), beta (B. 1.351), gamma (P.l) and delta (B.1.617.2) with IC50 values in the picomolar range (FIG. 3 A).
  • 15SE6 is able to neutralize several different types of variants in the picomolar range as well (FIG. 3B), while 5 SB 12 can only neutralize WH01 and the delta variant (FIG. 3C).
  • mAb 1-2SA8 is able to recognize Omicron subvariants BA. l and BA.4/5 (FIGs. 3D and 3E). Moreover, mAb 1-2SA8 is able to neutralize Omicron subvariants BA.4/5 and BF.7 (FIGs. 3F and 3G).
  • 5 SB 12 does not possess potent efficacy to neutralize SARS-CoV-2, 5 SB 12 possessed good antibody-dependent cell-mediated cytotoxicity (ADCC) activity as compared with 1-2SA8 and 15SE6, suggesting the potential utility of 5SB12 in vivo (FIG. 4A).
  • mAb 1-2SA8 has antibody-dependent cell-mediated cytotoxicity(ADCC) activity against Omicron subvariant Spike proteins expressing cells (FIG. 4B).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • 5SB12 prevents SARS-CoV-2 (WH01) infection, which is dependent on the engagement with effector cells through the Fc constant region of 5SB 12 (FIGs. 5A and 5B).
  • 1-2SA8 and 15SE6 both are able to effectively protect hamsters from SARS-CoV-2 (WH01) infection, and the protective efficacy is independent of the effector function of mAbs (FIGs. 6A to 6D).
  • KI 8-hACE2 TG mice we demonstrated that 1-2SA8 and 15SE6 are able to completely protect the lethal dose of alpha (Bl.1.7) strain challenge (FIGs. 7 and 8A to 8C8).
  • 1-2SA8 and 15SE6 showed potent neutralization activities across a broad spectrum of SARS-CoV-2 variants.
  • One mAb, 5 SB 12 despite not possessing superior neutralization activities, is able to protect SARS-CoV-2 infection in vivo through the effector functions.

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Abstract

La présente invention concerne un anticorps ou un fragment de Liaison à l'antigène de celui-ci qui est spécifique du SARS-CoV-2. La présente invention concerne également une composition pharmaceutique, une méthode de traitement et/ou de prévention de maladies et/ou de troubles provoqués par un coronavirus chez un sujet en ayant besoin, et une méthode de détection d'un coronavirus dans un échantillon.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10787501B1 (en) * 2020-04-02 2020-09-29 Regeneron Pharmaceuticals, Inc. Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments
US20210261650A1 (en) * 2020-02-26 2021-08-26 Vir Biotechnology, Inc. Antibodies against sars-cov-2 and methods of using the same
WO2021194188A1 (fr) * 2020-03-22 2021-09-30 (주)셀트리온 Molécule de liaison ayant une activité neutralisante contre le sras-coronavirus-2
WO2021226249A1 (fr) * 2020-05-06 2021-11-11 Sorrento Therapeutics, Inc. Anticorps neutralisants se liant à la protéine s de sars-cov-2

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210261650A1 (en) * 2020-02-26 2021-08-26 Vir Biotechnology, Inc. Antibodies against sars-cov-2 and methods of using the same
WO2021194188A1 (fr) * 2020-03-22 2021-09-30 (주)셀트리온 Molécule de liaison ayant une activité neutralisante contre le sras-coronavirus-2
US10787501B1 (en) * 2020-04-02 2020-09-29 Regeneron Pharmaceuticals, Inc. Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments
WO2021226249A1 (fr) * 2020-05-06 2021-11-11 Sorrento Therapeutics, Inc. Anticorps neutralisants se liant à la protéine s de sars-cov-2

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