WO2022187626A1 - Anticorps de glycoprotéine anti-sars-cov-2 à variante spike et fragments de liaison à l'antigène - Google Patents
Anticorps de glycoprotéine anti-sars-cov-2 à variante spike et fragments de liaison à l'antigène Download PDFInfo
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Classifications
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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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1002—Coronaviridae
- C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- sequence listing is submitted concurrently with the specification electronically via EFS-Web as an ASCII formatted sequence listing with a file name of “10915W001-Sequence.txt”, created on March 4, 2022, and having a size of 933,357 bytes.
- the sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
- the present invention relates to antibodies and antigen-binding fragments that bind specifically to coronavirus spike proteins and methods for treating or preventing coronavirus infections with said antibodies and fragments.
- SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2
- SARS-CoV-2 was first identified from an outbreak in Wuhan, China and as of March 20, 2020, the World Health Organization has reported 209,839 confirmed cases in 168 countries, areas, or territories, resulting in 8,778 deaths.
- Clinical features of COVID-19 include fever, dry cough, and fatigue, and the disease can cause respiratory failure resulting in death.
- SARS- CoV-2-S neutralizing therapeutic anti-SARS-CoV-2-Spike protein (SARS- CoV-2-S) antibodies and their use for treating or preventing viral infection.
- the present disclosure addresses this need, in part, by providing human anti-SARS-CoV-2-S antibodies, such as those of Table 1, and combinations thereof including, for example, combinations with other therapeutics (e.g., anti-inflammatory agents, antimalarial agents, antiviral agents, or other antibodies or antigen-binding fragments), and methods of use thereof for treating viral infections.
- therapeutics e.g., anti-inflammatory agents, antimalarial agents, antiviral agents, or other antibodies or antigen-binding fragments
- the present disclosure provides neutralizing human antigen-binding proteins that specifically bind to SARS-CoV-2-S, for example, antibodies or antigen-binding fragments thereof, and methods for modifying antibodies to enhance their binding and/or neutralizing properties, e.g., for variant spike proteins such as those containing an E484k mutation.
- the present disclosure provides a method for modifying an antibody or antigen-binding fragment thereof that binds to a wild-type SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 832, comprising: a) identifying a first amino acid in said antibody or antigen-binding fragment that is in proximity to amino acid E484 of the wild-type SARS-CoV-2 spike protein when the antibody or antigen-binding fragment is bound to the wild-type SARS-CoV-2 spike protein; and b) modifying said first amino acid, thereby generating a modified antibody or antigen-binding fragment thereof, wherein the binding of said modified antibody or antigen-binding fragment to a variant SARS- CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 851 is greater than the binding of said antibody or antigen-binding fragment to said variant SARS-CoV-2 spike protein prior to said modifying.
- the present disclosure provides a method for modifying an antibody or antigen-binding fragment thereof that binds to a variant SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 851, comprising: a) identifying a first amino acid in said antibody or antigen-binding fragment that is in proximity to amino acid K484 of the variant SARS-CoV-2 spike protein when the antibody or antigen-binding fragment is bound to the variant SARS-CoV-2 spike protein; and b) modifying said first amino acid, thereby generating a modified antibody or antigen-binding fragment thereof, wherein the binding of said modified antibody or antigen-binding fragment to the variant SARS-CoV-2 spike protein is greater than the binding of said antibody or antigen-binding fragment to said variant SARS-CoV- 2 spike protein prior to said modifying.
- a measure of said binding is binding affinity. In some embodiments, a measure of said binding is dissociative half- life.
- the present disclosure provides a method for modifying an antibody or antigen-binding fragment thereof that binds to a wild-type SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 832, comprising: a) identifying a first amino acid in said antibody or antigen-binding fragment that is in proximity to amino acid E484 of the wild-type SARS-CoV-2 spike protein when the antibody or antigen-binding fragment is bound to the wild-type SARS-CoV-2 spike protein; and b) modifying said first amino acid, thereby generating a modified antibody or antigen-binding fragment thereof, wherein said modified antibody or antigen-binding fragment has greater neutralization against a variant SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 851 than said antibody or antigen-binding fragment has neutralization against said variant SARS- CoV-2 spike protein prior to said modifying.
- the present disclosure provides a method for modifying an antibody or antigen-binding fragment thereof that binds to a variant SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 851, comprising: a) identifying a first amino acid in said antibody or antigen-binding fragment that is in proximity to amino acid K484 of the variant SARS-CoV-2 spike protein when the antibody or antigen-binding fragment is bound to the variant SARS-CoV-2 spike protein; and b) modifying said first amino acid, thereby generating a modified antibody or antigen-binding fragment thereof, wherein said modified antibody or antigen-binding fragment has greater neutralization against a variant SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 851 than said antibody or antigen-binding fragment has neutralization against said variant SARS-CoV-2 spike protein prior to said modifying.
- said neutralizing is determined by neutralization of a pseudotyped virus expressing SARS-CoV-2-S or neutralization of SARS-CoV-2 virus.
- said modifying comprises substituting a second amino acid for said first amino acid.
- said substituting comprises introducing a substitution mutation in a nucleic acid sequence encoding said amino acid.
- said first amino acid comprises a positively charged side chain at pH 7 0
- said first amino acid is selected from the group consisting of lysine, arginine, and histidine.
- said second amino acid comprises a negatively charged side chain at pH 70
- said second amino acid is selected from the group consisting of aspartate and glutamate.
- said modifying comprises chemically modifying said amino acid.
- said chemically modifying comprises introducing a negative charge.
- said proximity comprises 3.5 A to 4.5 A between said first amino acid and said amino acid at position 484 of said spike protein. In some embodiments, said proximity comprises about 4.0 A. In some cases, said fist amino acid forms a salt bridge with said amino acid at position 484 of said spike protein. [00016] In one aspect, the present disclosure provides a modified antibody or antigen-binding fragment prepared by any of the methods discussed above or herein.
- the modified antibody comprises an immunoglobulin constant region.
- the immunoglobulin constant region is an IgGl constant region.
- the modified antibody is a recombinant antibody. In some embodiments the modified antibody is multispecific.
- the present disclosure provides a polynucleotide encoding a heavy chain of the modified antibody or antigen-binding fragment discussed above or herein.
- the present disclosure provides a polynucleotide encoding a light chain of the modified antibody or antigen-binding fragment discussed above or herein. [00019] In one aspect, the present disclosure provides a vector comprising the polynucleotide discussed above.
- the present disclosure provides a host cell comprising the modified antibody or antigen-binding fragment thereof discussed above or herein, or the polynucleotide or vector discussed above.
- the present disclosure provides a pharmaceutical composition
- a pharmaceutical composition comprising the modified antibody or antigen-binding fragment thereof discussed above or herein, or the polynucleotide, the vector, or host cell discussed above.
- the present disclosure provides a complex comprising the modified antibody or antigen-binding fragment discussed above or herein bound to a SARS-CoV-2 spike protein.
- the present disclosure provides a method for making the modified antibody or antigen-binding fragment discussed above or 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 host cell is a Chinese hamster ovary cell.
- any of the features or components of embodiments discussed above or herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value discussed above or herein may be combined with another related value discussed above or herein to recite a range with the values representing the upper and lower ends of the range, and such ranges are encompassed within the scope of the present disclosure.
- 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 was identified as the cause of a serious outbreak starting in Wuhan, China, and which is rapidly spreading to other areas of the globe.
- SARS-CoV-2 has also been known as 2019-nCoV and Wuhan coronavirus. 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.
- 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 amino acid sequence provided in SEQ ID NO: 832.
- the term “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 ROR1.
- 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 present invention includes methods for treating or preventing a viral infection in a subject.
- virus includes any virus whose infection in the body of a subject is treatable or preventable by administration of an anti-CoV-S antibody or antigen-binding fragment thereof (e.g., wherein infectivity of the virus is at least partially dependent on CoV-S).
- a “virus” is any virus that expresses spike protein (e.g., CoV-S).
- virus also includes a CoV-S-dependent respiratory virus which is a virus that infects the respiratory tissue of a subject (e.g., upper and/or lower respiratory tract, trachea, bronchi, lungs) and is treatable or preventable by administration of an anti-CoV-S antibody or antigenbinding fragment thereof.
- virus includes coronavirus, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), SARS-CoV (severe acute respiratory syndrome coronavirus), and MERS-CoV (Middle East respiratory syndrome (MERS) coronavirus).
- Coronaviruses can include the genera of alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses.
- the antibodies or antigen-binding fragments provided herein can bind to and/or neutralize an alphacoronavirus, a betacoronavirus, a gammacoronavirus, and/or a deltacoronavirus. In certain embodiments, this binding and/or neutralization can be specific for a particular genus of coronavirus or for a particular subgroup of a genus.
- “Viral infection” refers to the invasion and multiplication of a virus in the body of a subject.
- Coronavirus virions are spherical with diameters of approximately 125 nm. The most prominent feature of coronaviruses is the club-shape spike projections emanating from the surface of the virion. These spikes are a defining feature of the virion and give them the appearance of a solar corona, prompting the name, coronaviruses. Within the envelope of the virion is the nucleocapsid. Coronaviruses have helically symmetrical nucleocapsids, which is uncommon among positive-sense RNA viruses, but far more common for negative-sense RNA viruses. SARS-CoV-2, MERS-CoV, and SARS-CoV belong to the coronavirus family.
- the initial attachment of the virion to the host cell is initiated by interactions between the S protein and its receptor.
- the sites of receptor binding domains (RBD) within the SI region of a coronavirus S protein vary depending on the virus, with some having the RBD at the C-terminus of SI.
- RBD receptor binding domains
- the S-protein/receptor interaction is the primary determinant for a coronavirus to infect a host species and also governs the tissue tropism of the virus.
- Many coronaviruses utilize peptidases as their cellular receptor. Following receptor binding, the virus must next gain access to the host cell cytosol.
- SEQ ID NO: 832 An exemplary spike protein sequence is given as SEQ ID NO: 832; a variant spike protein sequence containing an E484K mutation is given as SEQ ID NO: 851.
- the present invention provides antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, that specifically bind to CoV spike protein or an antigenic fragment thereof.
- antibody refers to immunoglobulin molecules comprising four polypeptide chains, two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds (i.e., "full antibody molecules"), as well as multimers thereof (e.g. IgM).
- exemplary antibodies include, for example, those listed in Table 1.
- Each heavy chain comprises a heavy chain variable region (“HCVR” or “VH”) and a heavy chain constant region (comprised of domains CH 1 , CH 2 and CH 3 ).
- Each light chain is comprised of a light chain variable region (“LCVR or “V L ”) and a light chain constant region (CL).
- VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
- CDR complementarity determining regions
- FR framework regions
- Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
- Heavy chain CDRs can also be referred to as HCDRs or CDR-Hs, and numbered as described above (e.g., HCDR1, HCDR2, and HCDR3 or CDR-H1, CDR-H2, and CDR-H3).
- light chain CDRs can be referred to as LCDRs or CDR-Ls, and numbered LCDR1, LCDR2, and LCDR3, or CDR-L1, CDR-L2, and CDR-L3.
- the FRs of the antibody are identical to the human germline sequences, or are naturally or artificially modified.
- Exemplary human germline sequences include, but are not limited to, VH3-66 and Vkl-33.
- anti-CoV-S antibodies or antigen- binding fragments thereof comprising HCDR and LCDR sequences of Table 1 within a VH3-66 or Vkl-33 variable heavy chain or light chain region.
- the present disclosure further provides anti-CoV-S antibodies or antigen-binding fragments thereof (e.g., anti-SARS-CoV-2-S antibodies or antigenbinding fragments thereof) comprising HCDR and LCDR sequences of Table 1 within a combination of a light chain selected from IgKV4-l, IgKV 1-5, IgKVl-9, IgKVl-12, IgKV3-15, IgKVl-16, IgKVl-17, IgKV3-20, IgLV3-21, IgKV2-24, IgKVl-33, IgKVl-39, IgLVl-40, IgLVl-44, IgLVl-51, IgLV3-l, IgKVl-6, IgLV2-8, IgKV3-ll, IgLV2-ll, IgLV2-14, IgLV2-23, or IgLV6-57, and a heavy chain selected from IgHVl-69, IgHV3-64, IgHV4-59,
- the present disclosure further provides anti-CoV-S antibodies or antigen-binding fragments thereof (e.g, anti-SARS-CoV-2-S antibodies or antigen-binding fragments thereof) comprising HCVR and LCVR sequences of Table 1 within a combination of a light chain selected from IgKV4-l, IgKV 1-5, IgKVl-9, IgKVl-12, IgKV3-15, IgKVl-16, IgKVl-17, IgKV3-20, IgLV3-21, IgKV2-24, IgKVl-33, IgKVl-39, IgLVl-40, IgLVl-44, IgLVl-51, IgLV3-l, IgKVl-6, IgLV2-8, IgKV3- 11, IgLV2-l 1, IgLV2-14, IgLV2-23, or IgLV6-57, and a heavy chain selected from IgHVl-69, IgHV3-64, IgHV4-59,
- variable domains of both the heavy and light immunoglobulin chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FR).
- CDRs complementarity determining regions
- FR framework regions
- both light and heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
- the assignment of amino acids to each domain is in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, etal.; National Institutes of Health, Bethesda, Md.; 5 th ed.; NIHPubl. No. 91-3242 (1991); Kabat (1978) Adv. Prot.
- the present invention includes monoclonal anti-CoV-S antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof, as well as monoclonal compositions comprising a plurality of isolated monoclonal antigen-binding proteins.
- monoclonal antibody refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts.
- a “plurality" of such monoclonal antibodies and fragments in a composition refers to a concentration of identical (i.e., as discussed above, in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts) antibodies and fragments which is above that which would normally occur in nature, e.g., in the blood of a host organism such as a mouse or a human.
- an anti-CoV-S antigen-binding protein e.g, antibody or antigen-binding fragment comprises a heavy chain constant domain, e.g., of the type IgA (e.g, IgAl or IgA2), IgD, IgE, IgG (e.g, IgGl, IgG2, IgG3 and IgG4) or IgM.
- an antigen-binding protein, e.g, antibody or antigen-binding fragment comprises a light chain constant domain, e.g., of the type kappa or lambda.
- human antigen-binding protein such as an antibody, as used herein, includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences whether in a human cell or grafted into a non-human cell, e.g., a mouse cell. See e.g., US8502018, US6596541 orUS5789215.
- the human mAh s of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and, in particular, CDR3.
- human antibody is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g, mouse) have been grafted onto human FR sequences.
- the term includes antibodies recombinantly produced in a non-human mammal or in cells of a non-human mammal.
- the term is not intended to include antibodies isolated from or generated in a human subject. See below.
- the present invention includes anti-CoV-S chimeric antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof, and methods of use thereof.
- a "chimeric antibody” is an antibody having the variable domain from a first antibody and the constant domain from a second antibody, where the first and second antibodies are from different species.
- the present invention includes anti-CoV-S hybrid antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof, and methods of use thereof.
- hybrid antibody is an antibody having the variable domain from a first antibody and the constant domain from a second antibody, wherein the first and second antibodies are from different animals, or wherein the variable domain, but not the constant region, is from a first animal.
- a variable domain can be taken from an antibody isolated from a human and expressed with a fixed constant region not isolated from that antibody.
- Exemplary hybrid antibodies are described in Example 1, which refers to antibody heavy chain variable region and light chain variable region derived PCR products that were cloned into expression vectors containing a heavy constant region and a light constant region, respectively.
- Hybrid antibodies are synthetic and non-natrually occurring because the variable and constant regions they contain are not isolated from a single natural source.
- antigen-binding proteins such as antibodies or antigen-binding fragments thereof, refers to such molecules created, expressed, isolated or obtained by technologies or methods known in the art as recombinant DNA technology which include, e.g, DNA splicing and transgenic expression.
- the term includes antibodies expressed in a nonhuman mammal (including transgenic non-human mammals, e.g, transgenic mice), or a cell (e.g, CHO cells) expression system, or a non-human cell expression system, or isolated from a recombinant combinatorial human antibody library.
- a recombinant antibody shares a sequence with an antibody isolated from an organism (e.g, a mouse or a human), but has been expressed via recombinant DNA technology.
- Such antibodies may have post-translational modifications (e.g, glycosylation) that differ from the antibody as isolated from the organism.
- Recombinant anti-CoV-S antigen-binding proteins e.g, antibodies and antigen-binding fragments, disclosed herein may also be produced in an E. colUTl expression system.
- nucleic acids encoding the anti-CoV-S antibody immunoglobulin molecules of the invention e.g ., as found in Table 1 may be inserted into a pET-based plasmid and expressed in the E. colUTl system.
- the present invention includes methods for expressing an antibody or antigen-binding fragment thereof or immunoglobulin chain thereof in a host cell (e.g., bacterial host cell such as E.
- a bacterial host cell such as an E. coli , includes a polynucleotide encoding the T7 RNA polymerase gene operably linked to a lac promoter and expression of the polymerase and the chain is induced by incubation of the host cell with IPTG (isopropyl-beta-D- thiogalactopyranoside).
- IPTG isopropyl-beta-D- thiogalactopyranoside
- Transformation can be by any known method for introducing polynucleotides (e.g., DNA or RNA, including mRNA) into a host cell.
- Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, lipid nanoparticle technology, biolistic injection and direct microinjection of the DNA into nuclei.
- nucleic acid molecules may be introduced into mammalian cells by viral vectors such as lentivirus or adeno-associated virus.
- an antibody or antigen-binding fragment thereof of the present disclosure can be introduced to a subject in nucleic acid form (e.g, DNA or RNA, including mRNA), such that the subject’s own cells produce the antibody.
- nucleic acid form e.g, DNA or RNA, including mRNA
- the present disclosure further provides modifications to nucleotide sequences encoding the anti-CoV-S antibodies described herein that result in increased antibody expression, increased antibody stability, increased nucleic acid (e.g., mRNA) stability, or improved affinity or specificity of the antibodies for the CoV spike protein.
- the present invention includes recombinant methods for making an anti-CoV-S antigen-binding protein, such as an antibody or antigen-binding fragment thereof of the present invention, or an immunoglobulin chain thereof, comprising (i) introducing one or more polynucleotides (e.g ., including the nucleotide sequence of any one or more of the sequences of Table 2) encoding light and/or heavy immunoglobulin chains, or CDRs, of the antigen-binding protein, e.g., of Table 1, for example, wherein the polynucleotide is in a vector; and/or integrated into a host cell chromosome and/or is operably linked to a promoter; (ii) culturing the host cell (e.g, CHO or Pichia or Pichia pastoris) under condition favorable to expression of the polynucleotide and, (iii) optionally, isolating the antigen-binding protein, (e.g,
- a polynucleotide can be integrated into a host cell chromosome through targeted insertion with a vector such as adeno-associated virus (AAV), e.g., after cleavage of the chromosome using a gene editing system (e.g., CRISPR (for example, CRISPR-Cas9), TALEN, megaTAL, zinc finger, or Argonaute).
- AAV adeno-associated virus
- CRISPR for example, CRISPR-Cas9
- TALEN for example, CRISPR-Cas9
- TALEN megaTAL
- zinc finger or Argonaute
- Targeted insertions can take place, for example, at host cell loci such as an albumin or immunoglopbulin genomic locus.
- insertion can be at a random locus, e.g., using a vector such as lentivirus.
- an antigen-binding protein e.g, antibody or antigen-binding fragment
- co-expression of the chains in a single host cell leads to association of the chains, e.g, in the cell or on the cell surface or outside the cell if such chains are secreted, so as to form the antigen-binding protein (e.g, antibody or antigen-binding fragment).
- the methods include those wherein only a heavy immunoglobulin chain or only a light immunoglobulin chain (e.g, any of those discussed herein including mature fragments and/or variable domains thereof) is expressed.
- the present invention also includes anti-CoV-S antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, comprising a heavy chain immunoglobulin (or variable domain thereof or comprising the CDRs thereof) encoded by a polynucleotide comprising a nucleotide sequence set forth in Table 2 and a light chain immunoglobulin (or variable domain thereof or comprising the CDRs thereof) encoded by a nucleotide sequence set forth in Table 2 which are the product of such production methods, and, optionally, the purification methods set forth herein.
- anti-CoV-S antigen-binding proteins such as antibodies and antigen-binding fragments thereof, comprising a heavy chain immunoglobulin (or variable domain thereof or comprising the CDRs thereof) encoded by a polynucleotide comprising a nucleotide sequence set forth in Table 2 and a light chain immunoglobulin (or variable domain thereof or comprising the CDRs thereof) encoded by a nucleot
- the product of the method is an anti-CoV-S antigen-binding protein which is an antibody or fragment comprising an HCVR comprising an amino acid sequence set forth in Table 1 and an LCVR comprising an amino acid sequence set forth in Table 1, wherein the HCVR and LCVR sequences are selected from a single antibody listed in Table 1.
- the product of the method is an anti-CoV-S antigen-binding protein which is an antibody or fragment comprising HCDR1, HCDR2, and HCDR3 comprising amino acid sequences set forth in Table 1 and LCDR1,
- the product of the method is an anti-CoV-S antigen-binding protein which is an antibody or fragment comprising a heavy chain comprising an HC amino acid sequence set forth in Table 1 and a light chain comprising an LC amino acid sequence set forth in Table 1.
- Eukaryotic and prokaryotic host cells may be used as hosts for expression of an anti-CoV-S antigen-binding protein.
- host cells are well known in the art and many are available from the American Type Culture Collection (ATCC). These host cells include, inter alia , Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g ., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines.
- Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells.
- Other cell lines that may be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni ), amphibian cells, bacterial cells, plant cells and fungal cells.
- Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris , Pichia finlandica, Pichia trehalophila , Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri ), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrys
- the present invention includes an isolated host cell (e.g, a CHO cell) comprising an antigen-binding protein, such as those of Table 1; or a polynucleotide encoding such a polypeptide thereof.
- the term “specifically binds” refers to those antigen-binding proteins (e.g ., mAbs) having a binding affinity to an antigen, such as a CoV-S protein (e.g., SARS-CoV-2-S), expressed as KD, of at least about 10 '8 M, as measured by real-time, label free bio-layer interferometry assay, for example, at 25°C or 37°C, e.g, an Octet® HTX biosensor, or by surface plasmon resonance, e.g, BIACORETM, or by solution-affinity ELISA.
- the present invention includes antigen-binding proteins that specifically bind to a CoV-S protein.
- antigen-binding portion or “antigen-binding fragment” of an antibody or antigen-binding protein, 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.
- Non-limiting examples of antigen-binding fragments 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, as defined in W008/020079 or WO09/138519) (e.g, monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.
- SMIPs small modular immunopharmaceuticals
- the antigen-binding fragment comprises three or more CDRs of an antibody of Table 1 (e.g, CDR-H1, CDR-H2 and CDR-H3; or CDR-L1, CDR-L2 and CDR-L3).
- An antigen-binding fragment of an antibody will, in an embodiment of the invention, comprise 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.
- variable region may be dimeric and contain VH - VH, VH - VL or VL - V L dimers.
- the antigen-binding fragment of an antibody may contain a monomeric VH or V L domain.
- an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain.
- Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigenbinding fragment of an antibody of the present invention include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (V) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (X) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-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 invention 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)).
- Antigen-binding proteins may be mono-specific or multi-specific (e.g, bi-specific). Multispecific antigen-binding proteins are discussed further herein.
- antibody or antibody fragments of the invention may be conjugated to a moiety such a ligand or a therapeutic moiety (“immunoconjugate”), such as an anti-viral drug, a second anti-influenza antibody, or any other therapeutic moiety useful for treating a viral infection, e.g, influenza viral infection. See below.
- the present invention also provides a complex comprising an anti-CoV-S antigenbinding protein, e.g, antibody or antigen-binding fragment, discussed herein complexed with CoV-S polypeptide or an antigenic fragment thereof and/or with a secondary antibody or antigen-binding fragment thereof (e.g., detectably labeled secondary antibody) that binds specifically to the anti-CoV-S antibody or fragment.
- an anti-CoV-S antigenbinding protein e.g, antibody or antigen-binding fragment
- the antibody or fragment is in vitro (e.g, is immobilized to a solid substrate) or is in the body of a subject.
- the CoV-S is in vitro (e.g, is immobilized to a solid substrate) or is on the surface of a virus or is in the body of a subject.
- Immobilized anti-CoV-S antibodies and antigen-binding fragments thereof which are covalently linked to an insoluble matrix material (e.g ., glass or polysaccharide such as agarose or sepharose, e.g, a bead or other particle thereof) are also part of the present invention; optionally, wherein the immobilized antibody is complexed with CoV-S or antigenic fragment thereof or a secondary antibody or fragment thereof.
- isolated antigen-binding proteins are at least partially free of other biological molecules from the cells or cell culture from which they are produced.
- biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other material such as cellular debris and growth medium.
- An isolated antibody or antigenbinding fragment may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof.
- isolated is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antibodies or fragments.
- epitope refers to an antigenic determinant (e.g, a CoV-S polypeptide) that interacts with a specific antigen-binding site of an antigen-binding protein, e.g, a variable region of an antibody molecule, known as a paratope.
- a specific antigen-binding site of an antigen-binding protein e.g, a variable region of an antibody molecule, known as a paratope.
- a single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects.
- epitope also refers to a site on an antigen to which B and/or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional.
- Epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction.
- Epitopes may be linear or conformational, that is, composed of non-linear amino acids.
- epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics.
- Methods for determining the epitope of an antigen-binding protein include alanine scanning mutational analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63), peptide cleavage analysis, crystallographic studies and NMR analysis.
- methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Prot. Sci. 9: 487-496).
- Another method that can be used to identify the amino acids within a polypeptide with which an antigen-binding protein e.g ., antibody or fragment or polypeptide
- an antigen-binding protein e.g ., antibody or fragment or polypeptide
- the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antigen-binding protein, e.g. , antibody or fragment or polypeptide, to the deuterium-labeled protein.
- the CoV-S protein/ antigen-binding protein complex is transferred to water and exchangeable protons within amino acids that are protected by the antibody complex undergo deuterium-to-hydrogen back-exchange at a slower rate than exchangeable protons within amino acids that are not part of the interface.
- amino acids that form part of the protein/ antigen-binding protein interface may retain deuterium and therefore exhibit relatively higher mass compared to amino acids not included in the interface.
- the target protein After dissociation of the antigen-binding protein (e.g, antibody or fragment or polypeptide), the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antigen-binding protein interacts. See, e.g, Ehring (1999) Analytical Biochemistry 267: 252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A.
- the antigen-binding protein e.g, antibody or fragment or polypeptide
- the term “competes” as used herein refers to an antigen-binding protein (e.g, antibody or antigen-binding fragment thereof) that binds to an antigen (e.g, CoV-S) and inhibits or blocks the binding of another antigen-binding protein (e.g, antibody or antigen-binding fragment thereof) to the antigen.
- the term also includes competition between two antigen-binding proteins e.g, antibodies, in both orientations, i.e., a first antibody that binds and blocks binding of second antibody and vice versa.
- the first antigen-binding protein (e.g, antibody) and second antigen-binding protein (e.g, antibody) may bind to the same epitope.
- the first and second antigen-binding proteins may bind to different, but, for example, overlapping epitopes, wherein binding of one inhibits or blocks the binding of the second antibody, e.g, via steric hindrance.
- Competition between antigen-binding proteins (e.g, antibodies) may be measured by methods known in the art, for example, by a realtime, label-free bio-layer interferometry assay.
- Epitope mapping (e.g., via alanine scanning or hydrogen-deuterium exchange (HDX)) can be used to determine whether two or more antibodies are non-competing (e.g., on a spike protein receptor binding domain (RBD) monomer), competing for the same epitope, or competing but with diverse micro-epitopes (e.g., identified through HDX).
- HDX hydrogen-deuterium exchange
- competition between a first and second anti- CoV-S antigen-binding protein is determined by measuring the ability of an immobilized first anti-CoV-S antigen-binding protein (e.g, antibody) (not initially complexed with CoV-S protein) to bind to soluble CoV-S protein complexed with a second anti-CoV-S antigen-binding protein (e.g, antibody).
- the degree of competition can be expressed as a percentage of the reduction in binding.
- Such competition can be measured using a real time, label-free biolayer interferometry assay, e.g, on an Octet RED384 biosensor (Pall ForteBio Corp.), ELISA (enzyme-linked immunosorbent assays) or SPR (surface plasmon resonance).
- Binding competition between anti-CoV-S antigen-binding proteins can be determined using a real time, label-free bio-layer interferometry assay on an Octet RED384 biosensor (Pall ForteBio Corp.).
- the anti-CoV-S mAb can be first captured onto anti-hFc antibody coated Octet biosensor tips (Pall ForteBio Corp., # 18-5060) by submerging the tips into a solution of anti-CoV-S mAb (subsequently referred to as “mAbl”).
- the antibody captured biosensor tips can then be saturated with a known blocking isotype control mAb (subsequently referred to as “blocking mAb”) by dipping into a solution of blocking mAb.
- blocking mAb a known blocking isotype control mAb
- the biosensor tips can then be subsequently dipped into a co-complexed solution of CoV-S polypeptide and a second anti-CoV-S mAb (subsequently referred to as “mAb2”), that had been pre-incubated for a period of time and binding of mAbl to the CoV-S polypeptide can be determined.
- the biosensor tips can be washed in buffer in between every step of the experiment. The real-time binding response can be monitored during the course of the experiment and the binding response at the end of every step can be recorded.
- the competition assay is conducted at 25 °C and pH about 7, e.g, 7.4, e.g, in the presence of buffer, salt, surfactant and a non-specific protein (e.g, bovine serum albumin).
- a non-specific protein e.g, bovine serum albumin.
- an antibody or antigen-binding fragment of the invention which is modified in some way retains the ability to specifically bind to CoV-S, e.g. , retains at least 10% of its CoV-S binding activity (when compared to the parental antibody) when that activity is expressed on a molar basis.
- an antibody or antigen-binding fragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the CoV-S binding affinity as the parental antibody. It is also intended that an antibody or antigen-binding fragment of the invention can include conservative or non-conservative amino acid substitutions (referred to as “conservative variants” or “function conserved variants” of the antibody) that do not substantially alter its biologic activity.
- a “variant” of a polypeptide, or a “modified” polypeptide, such as an immunoglobulin chain refers to a polypeptide comprising an amino acid sequence that is at least about 70-99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical or similar to a referenced amino acid sequence that is set forth herein; when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences (e.g, expect threshold: 10; word size: 3; max matches in a query range: 0; BLOSUM 62 matrix; gap costs: existence 11, extension 1; conditional compositional score matrix adjustment).
- a “variant” of a polynucleotide or a “modified” polynucleotide refers to a polynucleotide comprising a nucleotide sequence that is at least about 70-99.9% (e.g, at least about 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9%) identical to a referenced nucleotide sequence that is set forth herein (e.g, SEQ ID NO: 1, 9, 17, 19, 21, 29, 37, 39, 41, 49, 57, or 59); when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences (e.g, expect threshold: 10; word size: 28
- Anti-CoV-S antigen-binding proteins include a heavy chain immunoglobulin variable region having at least 70% (e.g, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) amino acid sequence identity to the HCVR amino acid sequences set forth in Table 1; and/or a light chain immunoglobulin variable region having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) amino acid sequence identity to the LCVR amino acid sequences set forth in Table 1.
- a heavy chain immunoglobulin variable region having at least 70% (e.g, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) amino acid sequence identity to the LCVR amino acid sequences set forth in Table 1.
- a modified anti-CoV-S antigen-binding protein may include a polypeptide comprising an amino acid sequence that is set forth herein except for one or more (e.g, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) mutations such as, for example, missense mutations (e.g, conservative substitutions), non-sense mutations, deletions, or insertions.
- the present invention includes antigen-binding proteins which include an immunoglobulin light chain variant comprising an LCVR amino acid sequence set forth in Table 1 but having one or more of such mutations and/or an immunoglobulin heavy chain variant comprising an HCVR amino acid sequence set forth in Table 1 but having one or more of such mutations.
- a variant anti-CoV-S antigen-binding protein includes an immunoglobulin light chain variant comprising CDR-L1, CDR-L2 and CDR-L3 wherein one or more (e.g., 1 or 2 or 3) of such CDRs has one or more of such mutations (e.g, conservative substitutions) and/or an immunoglobulin heavy chain variant comprising CDR-H1, CDR-H2 and CDR-H3 wherein one or more (e.g, 1 or 2 or 3) of such CDRs has one or more of such mutations (e.g., conservative substitutions).
- Substitutions can be in a CDR, framework, or constant region.
- Modified anti-SARS-CoV-2-S antibodies, or antigen-binding fragments thereof can be generated, for example, through structural analysis of the antibody in complex with the spike glycoprotein. In some embodiments, this structural analysis is through cryo-electron microscophy or protein crystallography. For example, amino acids of an antibody or antigenbinding fragment that interact with amino acid 484 of SEQ ID NO: 832 can be identified and mutated to generate a modified antibody or antigen-binding fragement with enhanced binding (e.g., binding affinity or dissociative half-life) or neutralization of an E484K variant. In some embodiments, this modification comprises a substitution mutation, e.g., in a polynucleic acid encoding the antibody or antigen-binding fragment.
- This mutation can, for example, comprise mutating a positively charged side chain at pH 7.0 such as lysine, arginine, or histidine to a negatively charged side chain at pH 7.0, such as aspartate or glutamate.
- a non-positively charged side chain can be mutated to a negatively-charged side chain (e.g., a nonpolar or polar side chain can be mutated to a negatively-charged side chain).
- this change could enhance binding by allowing a salt bridge to form between the negatively charged side chain and the lysine at position 484 of the variant spike protein, or by allowing one or more new hydrogen bonds to form.
- An antibody also can be modified, for example, by introducing a chemical modification to an amino acid.
- the invention further provides variant anti-CoV-S antigen-binding proteins, e.g ., antibodies or antigen-binding fragments thereof, comprising one or more variant CDRs (e.g, any one or more of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and/or CDR-H3) that are set forth herein with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity or similarity to, e.g, the heavy chain and light chain CDRs of Table 1.
- variant CDRs e.g., any one or more of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and/or CDR-H3
- Embodiments of the present invention also include variant antigen-binding proteins, e.g, anti-CoV-S antibodies and antigen-binding fragments thereof, that comprise immunoglobulin VHS and VLS; or HCs and LCs, which comprise an amino acid sequence having 70% or more (e.g, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) overall amino acid sequence identity or similarity to the amino acid sequences of the corresponding VHS, VLS, HCS or LCs specifically set forth herein, but wherein the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 of such immunoglobulins are not variants and comprise CDR amino acid sequence set forth in Table 1.
- the CDRs within variant antigen-binding proteins are not, themselves, variants.
- a “conservatively modified variant” or a “conservative substitution” refers to a variant wherein there is one or more substitutions of amino acids in a polypeptide with other amino acids having similar characteristics (e.g. charge, side- chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.). Such changes can frequently be made without significantly disrupting the biological activity of the antibody or fragment.
- Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g, Watson etal. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4 th Ed.)).
- substitutions of structurally or functionally similar amino acids are less likely to significantly disrupt biological activity.
- Examples of 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: cysteine and methionine.
- Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.
- a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet etal. (1992) Science 256: 1443 45.
- Function-conservative variants of the anti-CoV-S antibodies and antigen-binding fragments thereof are also part of the present invention. Any of the variants of the anti-CoV-S antibodies and antigen-binding fragments thereof (as discussed herein) may be “function- conservative variants”. Such function-conservative variants may, in some cases, also be characterized as conservatively modified variants. “Function-conservative variants,” as used herein, refers to variants of the anti-CoV-S antibodies or antigen-binding fragments thereof in which one or more amino acid residues have been changed without significantly altering one or more functional properties of the antibody or fragment. In an embodiment of the invention, a function-conservative variant anti-CoV-S antibody or antigen-binding fragment thereof of the present invention comprises a variant amino acid sequence and exhibits one or more of the following functional properties:
- coronavirus e.g., SARS-CoV-2, SARS-CoV, and/or MERS-CoV
- ACE2- and/or TMPRSS2-expressing cells e.g, Calu-3 cells
- mice Protects a mouse engineered to express the human TMPRSS2 and/or ACE2 protein from death caused by coronavirus infection (e.g, SARS-CoV-2, SARS-CoV, or MERS-CoV), for example, wherein the mice are infected with an otherwise lethal dose of the virus, optionally when combined with a second therapeutic agent.
- coronavirus infection e.g, SARS-CoV-2, SARS-CoV, or MERS-CoV
- mice Protects a mouse engineered to express the human TMPRSS2 and/or ACE2 protein from weight loss caused by coronavirus infection (e.g, SARS-CoV-2, SARS-CoV, or MERS-CoV), for example, wherein the mice are infected with a dose of the virus that would otherwise cause weighht loss, optionally when combined with a second therapeutic agent.
- coronavirus infection e.g, SARS-CoV-2, SARS-CoV, or MERS-CoV
- a “neutralizing” or “antagonist” anti-CoV-S antigen-binding protein refers to a molecule that inhibits an activity of CoV-S to any detectable degree, e.g. , inhibits the ability of CoV-S to bind to a receptor such as ACE2, to be cleaved by a protease such as TMPRSS2, or to mediate viral entry into a host cell or viral reproduction in a host cell.
- Table 1 refers to antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, that comprise the heavy chain or VH (or a variant thereof) and light chain or VL (or a variant thereof) as set forth below; or that comprise a VH that comprises the CDRs thereof (CDR-H1 (or a variant thereof), CDR-H2 (or a variant thereof) and CDR-H3 (or a variant thereof)) and a VL that comprises the CDRs thereof (CDR-L1 (or a variant thereof), CDR-L2 (or a variant thereof) and CDR-L3 (or a variant thereof)), e.g.
- the antibodies described herein also include embodiments wherein the VH is fused to a wild-type IgG4 (e.g., wherein residue 108 is S) or to IgG4 variants (e.g, wherein residue 108 is
- Antibodies and antigen-binding fragments of the present invention comprise immunoglobulin chains including the amino acid sequences set forth herein as well as cellular and in vitro post-translational modifications to the antibody.
- the present invention includes antibodies and antigen-binding fragments thereof that specifically bind to CoV-S comprising heavy and/or light chain amino acid sequences set forth herein (e.g, CDR-H1, CDR- H2, CDR-H3, CDR-L1, CDR-L2 and/or CDR-L3) as well as antibodies and fragments wherein one or more amino acid residues is glycosylated, one or more Asn residues is deamidated, one or more residues (e.g, Met, Trp and/or His) is oxidized, the N-terminal Gin is pyroglutamate (pyroE) and/or the C-terminal Lysine is missing.
- CoV-S comprising heavy and/or light chain amino acid sequences set forth herein (e.g, CDR-H1, CDR
- SARS-CoV-2-S anti-SARS-CoV-2-Spike protein
- the present invention provides methods for administering an anti-CoV-S antigenbinding protein of the present invention, e.g. , those of Table 1, comprising introducing the antigen-binding protein into the body of a subject (e.g., a human).
- the method comprises piercing the body of the subject with a needle of a syringe and injecting the antigenbinding protein into the body of the subject, e.g, into the vein, artery, tumor, muscular tissue or subcutis of the subject.
- the present invention provides a vessel (e.g, a plastic or glass vial, e.g, with a cap or a chromatography column, hollow bore needle or a syringe cylinder) comprising an anti-CoV-S antigen-binding protein of the present invention, e.g, those of Table 1.
- a vessel e.g, a plastic or glass vial, e.g, with a cap or a chromatography column, hollow bore needle or a syringe cylinder
- an anti-CoV-S antigen-binding protein of the present invention e.g, those of Table 1.
- the present invention also provides an injection device comprising one or more antigen-binding proteins (e.g, antibody or antigen-binding fragment) that bind specifically to CoV-S, e.g, those of Table 1, or a pharmaceutical composition thereof.
- the injection device may be packaged into a kit.
- An injection device is a device that introduces a substance into the body of a subject via a parenteral route, e.g, intramuscular, subcutaneous or intravenous.
- an injection device may be a syringe (e.g., pre-filled with the pharmaceutical composition, such as an auto-injector) which, for example, includes a cylinder or barrel for holding fluid to be injected (e.g, comprising the antibody or fragment or a pharmaceutical composition thereof), a needle for piecing skin and/or blood vessels for injection of the fluid; and a plunger for pushing the fluid out of the cylinder and through the needle bore.
- an injection device that comprises an antigen-binding protein, e.g, an antibody or antigen-binding fragment thereof, from a combination of the present invention, or a pharmaceutical composition thereof is an intravenous (IV) injection device.
- IV intravenous
- Such a device can include the antigen-binding protein or a pharmaceutical composition thereof in a cannula or trocar/needle which may be attached to a tube which may be attached to a bag or reservoir for holding fluid (e.g, saline) introduced into the body of the subject through the cannula or trocar/needle.
- the antibody or fragment or a pharmaceutical composition thereof may, in an embodiment of the invention, be introduced into the device once the trocar and cannula are inserted into the vein of a subject and the trocar is removed from the inserted cannula.
- the IV device may, for example, be inserted into a peripheral vein (e.g ., in the hand or arm); the superior vena cava or inferior vena cava , or within the right atrium of the heart (e.g., a central IV); or into a subclavian, internal jugular, or a femoral vein and, for example, advanced toward the heart until it reaches the superior vena cava or right atrium (e.g, a central venous line).
- an injection device is an autoinjector; a jet injector or an external infusion pump.
- a jet injector uses a high-pressure narrow jet of liquid which penetrate the epidermis to introduce the antibody or fragment or a pharmaceutical composition thereof to a subject’s body.
- External infusion pumps are medical devices that deliver the antibody or fragment or a pharmaceutical composition thereof into a subject’s body in controlled amounts. External infusion pumps may be powered electrically or mechanically.
- Different pumps operate in different ways, for example, a syringe pump holds fluid in the reservoir of a syringe, and a moveable piston controls fluid delivery, an elastomeric pump holds fluid in a stretchable balloon reservoir, and pressure from the elastic walls of the balloon drives fluid delivery.
- a set of rollers pinches down on a length of flexible tubing, pushing fluid forward.
- fluids can be delivered from multiple reservoirs at multiple rates.
- Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention to make human antibodies that specifically bind to CoV-S.
- An immunogen comprising any one of the following can be used to generate antibodies to CoV-S.
- the antibodies of the invention are obtained from mice immunized with a full length, native CoV-S, or with a live attenuated or inactivated virus, or with DNA encoding the protein or fragment thereof.
- the CoV-S protein or a fragment thereof may be produced using standard biochemical techniques and modified and used as immunogen.
- the immunogen is a recombinantly produced CoV-S protein or fragment thereof.
- the immunogen may be a CoV-S polypeptide vaccine. In certain embodiments, one or more booster injections may be administered. In certain embodiments, the immunogen may be a recombinant CoV-S polypeptide expressed in E. coli or in any other eukaryotic or mammalian cells such as Chinese hamster ovary (CHO) cells.
- VELOCIMMUNE® technology see, for example, US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®
- any other known method for generating monoclonal antibodies high affinity chimeric antibodies to CoV-S can be initially isolated having a human variable region and a mouse constant region.
- the VELOCIMMUNE® technology involves generation of a transgenic mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation.
- the DNA encoding the variable regions of the heavy and light chains of the antibody are isolated and operably linked to DNA encoding the human heavy and light chain constant regions.
- the DNA is then expressed in a cell capable of expressing the fully human antibody.
- lymphatic cells such as B-cells
- the lymphatic cells may be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest.
- DNA encoding the variable regions of the heavy chain and light chain may be isolated and linked to desirable isotypic constant regions of the heavy chain and light chain.
- Such an antibody protein may be produced in a cell, such as a CHO cell.
- DNA encoding the antigen-specific chimeric antibodies or the variable domains of the light and heavy chains may be isolated directly from antigen-specific lymphocytes.
- high affinity chimeric antibodies are isolated having a human variable region and a mouse constant region.
- the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc.
- the mouse constant regions are replaced with a desired human constant region to generate the fully human antibody of the invention, for example wild-type or modified IgGl or IgG4. While the constant region selected may vary according to specific use, high affinity antigenbinding and target specificity characteristics reside in the variable region.
- anti-CoV-S antigen-binding proteins e.g ., antibodies or antigen-binding fragments
- an Fc domain comprising one or more mutations, which, for example, enhance or diminish antibody binding to the FcRn receptor, e.g. , at acidic pH as compared to neutral pH.
- the present invention includes anti-CoV-S antibodies comprising a mutation in the CH2 or a CH3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g, in an endosome where pH ranges from about 5.5 to about 6.0).
- an acidic environment e.g, in an endosome where pH ranges from about 5.5 to about 6.0.
- Such mutations may result in an increase in serum half-life of the antibody when administered to an animal.
- Non-limiting examples of such Fc modifications include, e.g, a modification at position 250 (e.g, E or Q); 250 and 428 (e.g, L or F); 252 (e.g, L/Y/F/W or T), 254 (e.g, S or T), and 256 (e.g, S/R/Q/E/D or T); or a modification at position 428 and/or 433 (e.g, H/L/R/S/P/Q or K) and/or 434 (e.g, A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g, 308F, V308F), and 434.
- a modification at position 250 e.g, E or Q
- 250 and 428 e.g, L or F
- 252 e.g, L/
- the modification comprises a 428L (e.g, M428L) and 434S (e.g, N434S) modification; a 428L, 2591 (e.g, V259I), and 308F (e.g, V308F) modification; a 433K (e.g., H433K) and a 434 (e.g, 434Y) modification; a 252, 254, and 256 (e.g, 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g, T250Q and M428L); and a 307 and/or 308 modification (e.g, 308F or 308P).
- a 428L e.g, M428L
- 434S e.g, N434S
- 428L, 2591 e.g, V259I
- 308F e.g, V308F
- 433K e.g., H433K
- the modification comprises a 265 A (e.g, D265A) and/or a 297A (e.g, N297A) modification.
- the present invention includes anti-CoV-S antigen-binding proteins, e.g, antibodies or antigen-binding fragments, comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g, T250Q and M248L); 252Y, 254T and 256E (e.g, M252Y, S254T and T256E); 428L and 434S (e.g, M428L and N434S); 2571 and 31 II (e.g, P257I and Q31 II); 2571 and 434H (e.g, P257I and N434H); 376V and 434H (e.g, D376V and N434H); 307A, 380A and 434A (e.
- Anti-CoV-S antigen-binding proteins e.g., antibodies and antigen-binding fragments thereof, that comprise a VH and/or VL as set forth herein comprising any possible combinations of the foregoing Fc domain mutations, are contemplated within the scope of the present invention.
- the present invention also includes anti-CoV-S antigen-binding proteins, antibodies or antigen-binding fragments, comprising a VH set forth herein and a chimeric heavy chain constant (CH) region, wherein the chimeric CH region comprises segments derived from the CH regions of more than one immunoglobulin isotype.
- CH heavy chain constant
- the antibodies of the invention may comprise a chimeric CH region comprising part or all of a CH2 domain derived from a human IgGl, human IgG2 or human IgG4 molecule, combined with part or all of a CH3 domain derived from a human IgGl, human IgG2 or human IgG4 molecule.
- the antibodies of the invention comprise a chimeric CH region having a chimeric hinge region.
- a chimeric hinge may comprise an “upper hinge” amino acid sequence (amino acid residues from positions 216 to 227 according to EU numbering) derived from a human IgGl, a human IgG2 or a human IgG4 hinge region, combined with a “lower hinge” sequence (amino acid residues from positions 228 to 236 according to EU numbering) derived from a human IgGl, a human IgG2 or a human IgG4 hinge region.
- the chimeric hinge region comprises amino acid residues derived from a human IgGl or a human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge.
- An antibody comprising a chimeric CH region as described herein may, in certain embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, e.g., W02014/022540).
- the invention encompasses 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 anti-viral drug to treat influenza virus infection.
- an anti-CoV-S antibody or fragment is conjugated to any of the further therapeutic agents set forth herein.
- the term “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) will take into account the condition to be treated and the desired therapeutic effect to be achieved. See, e.g., Amon el al, “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, Monoclonal Antibodies And Cancer Therapy, Reisfeld etal. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al, “Antibodies For Drug Delivery”, Controlled Drug Delivery (2 nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc.
- the present invention includes anti-CoV-S antigen-binding proteins, e.g. , antibodies and antigen-binding fragments thereof, as well as methods of use thereof and methods of making such antigen-binding proteins.
- anti-CoV-S antigen-binding proteins, e.g., antibodies or antigen-binding fragments, includes multispecific (e.g, bispecific or biparatopic) molecules that include at least one first antigen-binding domain that specifically binds to CoV-S (e.g, an antigen-binding domain from an antibody of Table 1) and at least one second antigen-binding domain that binds to a different antigen or to an epitope in CoV-S which is different from that of the first antigen-binding domain.
- the first antigen-binding domain and the second antigen-binding domain are both selected from the antigen-binding domains of Table 1.
- the first and second epitopes overlap.
- the first and second epitopes do not overlap.
- a multispecific antibody is a bispecific IgG antibody (e.g, IgGl or IgG4) that includes a first antigen-binding domain that binds specifically to CoV-S including the heavy and light immunoglobulin chain of an antibody of Table 1, and a second antigen-binding domain that binds specifically to a different epitope of CoV-S.
- a bispecific IgG antibody (e.g, IgGl or IgG4) includes a first antigen-binding domain that binds specifically to CoV-S and a second binding domain that binds to a host cell protein, e.g., ACE2 or TMPRSS2.
- the antibodies of Table 1 include multispecific molecules, e.g. , antibodies or antigenbinding fragments, that include the CDR-Hs and CDR-Ls, VH and VL, or HC and LC of those antibodies, respectively (including variants thereof as set forth herein).
- an antigen-binding domain that binds specifically to CoV-S which may be included in a multispecific molecule, comprises:
- the multispecific antibody or fragment includes more than two different binding specificities (e.g, a trispecific molecule), for example, one or more additional antigen-binding domains which are the same or different from the first and/or second antigen-binding domain.
- more than two different binding specificities e.g, a trispecific molecule
- additional antigen-binding domains which are the same or different from the first and/or second antigen-binding domain.
- a bispecific antigen-binding fragment comprises a first scFv (e.g, comprising VH and VL sequences of Table 1) having binding specificity for a first epitope (e.g ., CoV-S) and a second scFv having binding specificity for a second, different epitope.
- the first and second scFv are tethered with a linker, e.g., a peptide linker (e.g., a GS linker such as (GGGGS)n (SEQ ID NO: 834) wherein n is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).
- a linker e.g., a peptide linker (e.g., a GS linker such as (GGGGS)n (SEQ ID NO: 834) wherein n is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).
- Other bispecific antigen-binding fragments include an F(a
- the present invention provides methods for treating or preventing viral infection (e.g., coronavirus infection) by administering a therapeutically effective amount of anti-CoV-S antigen-binding protein, e.g, antibody or antigen-binding fragment, (e.g, of Table 1) to a subject (e.g, a human) in need of such treatment or prevention.
- a therapeutically effective amount of anti-CoV-S antigen-binding protein e.g, antibody or antigen-binding fragment, (e.g, of Table 1)
- a subject e.g, a human
- Coronavirus infection may be treated or prevented, in a subject, by administering an anti-CoV-S antigen-binding protein of the present invention to a subject.
- An effective or therapeutically effective dose of anti-CoV-S antigen-binding protein, e.g, antibody or antigen-binding fragment (e.g, of Table 1), for treating or preventing a viral infection refers to the amount of the antibody or fragment sufficient to alleviate one or more signs and/or symptoms of the infection in the treated subject, whether by inducing the regression or elimination of such signs and/or symptoms or by inhibiting the progression of such signs and/or symptoms.
- the dose amount may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like.
- an effective or therapeutically effective dose of antibody or antigen-binding fragment thereof of the present invention, for treating or preventing viral infection, e.g, in an adult human subject is about 0.01 to about 200 mg/kg, e.g., up to about 150 mg/kg.
- the dosage is up to about 10.8 or 11 grams (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 grams).
- the frequency and the duration of the treatment can be adjusted.
- the antigen-binding protein of the present invention can be administered at an initial dose, followed by one or more secondary doses.
- the initial dose may be followed by administration of a second or a plurality of subsequent doses of antibody or antigen-binding fragment thereof in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.
- the term “subject” refers to a mammal (e.g ., rat, mouse, cat, dog, cow, pig, sheep, horse, goat, rabbit), preferably a human, for example, in need of prevention and/or treatment of a disease or disorder such as viral infection or cancer.
- the subject may have a viral infection, e.g., an influenza infection, or be predisposed to developing an infection.
- Subjects predisposed to developing an infection, or subjects who may be at elevated risk for contracting an infection include subjects with compromised immune systems because of autoimmune disease, subjects receiving immunosuppressive therapy (for example, following organ transplant), subjects afflicted with human immunodeficiency syndrome (HIV) or acquired immune deficiency syndrome (AIDS), subjects with forms of anemia that deplete or destroy white blood cells, subjects receiving radiation or chemotherapy, or subjects afflicted with an inflammatory disorder. Additionally, subjects of very young (e.g, 5 years of age or younger) or old age (e.g, 65 years of age or older) are at increased risk.
- immunosuppressive therapy for example, following organ transplant
- HIV human immunodeficiency syndrome
- AIDS acquired immune deficiency syndrome
- subjects with forms of anemia that deplete or destroy white blood cells subjects receiving radiation or chemotherapy, or subjects afflicted with an inflammatory disorder.
- subjects of very young e.g, 5 years of age or younger
- old age e.g, 65 years of age or older
- a subject may be at risk of contracting a viral infection due to proximity to an outbreak of the disease, e.g. subject resides in a densely-populated city or in close proximity to subjects having confirmed or suspected infections of a virus, or choice of employment, e.g. hospital worker, pharmaceutical researcher, traveler to infected area, or frequent flier.
- “Treat” or “treating” means to administer an anti-CoV-S antigen-binding protein, e.g, antibody or antigen-binding fragment of the present invention (e.g, of Table 1), to a subject having one or more signs or symptoms of a disease or infection, e.g, viral infection, for which the antigen-binding protein is effective when administered to the subject at an effective or therapeutically effective amount or dose (as discussed herein).
- an anti-CoV-S antigen-binding protein e.g, antibody or antigen-binding fragment of the present invention (e.g, of Table 1)
- a subject having one or more signs or symptoms of a disease or infection e.g, viral infection
- the present invention also encompasses prophylactically administering an anti-CoV-S antigen-binding protein, e.g, antibody or antigen-binding fragment thereof of the present invention (e.g, of Table 1), to a subject who is at risk of viral infection so as to prevent such infection.
- an anti-CoV-S antigen-binding protein e.g, antibody or antigen-binding fragment thereof of the present invention (e.g, of Table 1)
- Passive antibody-based immunoprophylaxis has proven an effective strategy for preventing subject from viral infection. See e.g, Berry el al, Passive broad-spectrum influenza immunoprophylaxis. Influenza Res Treat. 2014; 2014:267594. Epub 2014 Sep 22; and Jianqiang et at. , Passive immune neutralization strategies for prevention and control of influenza A infections, Immunotherapy. 2012 February; 4(2): 175-186; Prabhu et al, Antivir Ther.
- Prevent or “preventing” means to administer an anti-CoV-S antigen-binding protein, e.g ., antibody or antigen-binding fragment of the present invention (e.g, of Table 1), to a subject to inhibit the manifestation of a disease or infection (e.g., viral infection) in the body of a subject, for which the antigen-binding protein is effective when administered to the subject at an effective or therapeutically effective amount or dose (as discussed herein).
- an anti-CoV-S antigen-binding protein e.g ., antibody or antigen-binding fragment of the present invention (e.g, of Table 1)
- a disease or infection e.g., viral infection
- a sign or symptom of a viral infection in a subject is survival or proliferation of virus in the body of the subject, e.g, as determined by viral titer assay (e.g, coronavirus propagation in embryonated chicken eggs or coronavirus spike protein assay). Other signs and symptoms of viral infection are discussed herein.
- viral titer assay e.g, coronavirus propagation in embryonated chicken eggs or coronavirus spike protein assay.
- Other signs and symptoms of viral infection are discussed herein.
- the subject may be a non-human animal
- the antigen-binding proteins e.g, antibodies and antigen-binding fragments
- the non-human animals e.g, cats, dogs, pigs, cows, horses, goats, rabbits, sheep, and the like.
- the present invention provides a method for treating or preventing viral infection (e.g, coronavirus infection) or for inducing the regression or elimination or inhibiting the progression of at least one sign or symptom of viral infection such as:
- antigen-binding protein e.g ., antibodies and antigen-binding fragments thereof (e.g, of Table 1)
- antigen-binding protein is admixed with a pharmaceutically acceptable carrier or excipient.
- a pharmaceutically acceptable carrier or excipient See, e.g. , Remington’s Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984); Hardman, et al.
- the pharmaceutical composition is sterile. Such compositions are part of the present invention.
- compositions comprising an anti-CoV-S antigen-binding proteins, e.g. , antibody or antigenbinding fragment thereof (e.g, of Table 1), or a pharmaceutical composition thereof that includes a pharmaceutically acceptable carrier but substantially lacks water.
- an anti-CoV-S antigen-binding proteins e.g. , antibody or antigenbinding fragment thereof (e.g, of Table 1)
- a pharmaceutical composition thereof that includes a pharmaceutically acceptable carrier but substantially lacks water.
- a further therapeutic agent that is administered to a subject in association with an anti-CoV-S antigen-binding protein, e.g, antibody or antigen-binding fragment thereof (e.g, of Table 1), disclosed herein is administered to the subject in accordance with the Physicians’ Desk Reference 2003 (Thomson Healthcare; 57 th edition (Nov. 1, 2002)).
- an anti-CoV-S antigen-binding protein e.g, antibody or antigen-binding fragment thereof (e.g, of Table 1)
- the mode of administration can vary. Routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal or intra-arterial.
- the present invention provides methods for administering an anti-CoV-S antigenbinding protein, e.g ., antibody or antigen-binding fragment thereof (e.g, of Table 1), comprising introducing the protein into the body of a subject.
- the method comprises piercing the body of the subject with a needle of a syringe and injecting the antigen-binding protein into the body of the subject, e.g. , into the vein, artery, tumor, muscular tissue or subcutis of the subject.
- the present invention provides a vessel (e.g, a plastic or glass vial, e.g, with a cap or a chromatography column, hollow bore needle or a syringe cylinder) comprising any of the anti-CoV-S antigen-binding proteins, e.g, antibodies or antigen-binding fragments thereof (e.g, of Table 1), polypeptides (e.g, an HC, LC, VH or VL of Table 1) or polynucleotides (e.g, of Table 2) or vectors set forth herein or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier.
- a vessel e.g, a plastic or glass vial, e.g, with a cap or a chromatography column, hollow bore needle or a syringe cylinder
- any of the anti-CoV-S antigen-binding proteins e.g, antibodies or antigen-binding fragments thereof (e.g, of Table 1), polypeptides (e.g, an
- an anti-CoV-S antigen-binding protein e.g, antibody or antigen-binding fragment thereof of the present invention (e.g, of Table 1)
- a further therapeutic agent includes, but is not limited to: an anti-inflammatory agent, an antimalarial agent, a second antibody or antigen-binding fragment thereof that specifically binds TMPRSS2, and a second antibody or antigen-binding fragment thereof that specifically binds to CoV-S.
- an antimalarial agent is chloroquine or hydroxychloroquine.
- an anti-inflammatory agent is an antibody such as sarilumab, tocilizumab, or gimsilumab.
- the further therapeutic agent is a second antibody or antigen-binding fragment disclosed herein, e.g., of Table 1.
- one, two, three, four, or more antibodies, or antigen-binding fragments thereof, of Table 1 can be administered in combination (e.g,, concurrently or sequentially).
- a combination of antibodies can be selected from among those binding to different epitope clusters.
- certain antibodies described herein belong to epitope clusters as follows: Cluster 1, mAb 10987, mAb 10922, mAb 10936, and mAbl0934; Cluster 2, mAb 10989, mAh 10977, and mAb 10933; Cluster 3, mAb 10920; Cluster 4, mAb 10954, mAb 10986, and mAb 10964; and Cluster 5, mAblQ984.
- a combination of two antibodies can be selected from, for example, Cluster 1 and Cluster 2, Cluster 1 and Cluster 3, Cluster 1 and Cluster 4, Cluster 1 and Cluster 5, Cluster 2 and Cluster 3, Cluster 2 and Cluster 4, Cluster 2 and Cluster 5, Cluster 3 and Cluster 4, duster 3 and Cluster 5, and Cluster 4 and Cluster 5.
- an antibody that specifically binds TMPRSS2 is H1H7017N, as described in International Patent Pub. No. WO/2019/147831.
- anti-CoV-S antigen-binding proteins e.g ., anti-SARS-CoV- 2-S antibodies or antigen-binding fragments thereof
- the present invention includes a composition comprising two (or more) anti-SARS- CoV-2-S antibodies or antigen-binding fragments comprising variable domains from human subjects, wherein the two (or more) antibodies or antigen-binding fragments are derived from different subjects (e.g, two different human subjects).
- Antibody variable regions derived from human B cells are discussed, e.g.
- a composition may comprise a combination of an antibody or antigen-binding fragment thereof with variable domains derived from donor 1 and an antibody or antigen-binding fragment thereof with variable domains derived from donor 2.
- a composition may comprise a combination of an antibody or antigen-binding fragment thereof with variable domains derived from donor 1 and an antibody or antigen-binding fragment thereof with variable domains derived from donor 3.
- a composition may comprise a combination of an antibody or antigen-binding fragment thereof with variable domains derived from donor 2 and an antibody or antigen-binding fragment thereof with variable domains derived from donor 3.
- a composition may comprise a combination of mAb 10987 (e.g, an antibody comprising the CDRs, the variable regions, or the heavy and light chain sequences shown in Table 1) from Donor 1, and mAbl0989 (e.g, an antibody comprising the CDRs, the variable regions, or the heavy and light chain sequences shown in Table 1) from Donor 3.
- the further therapeutic agent is an anti-viral drug and/or a vaccine.
- anti-viral drug refers to any anti-infective drug or therapy used to treat, prevent, or ameliorate a viral infection in a subject.
- anti -viral drug includes, but is not limited to a cationic steroid antimicrobial, leupeptin, aprotinin, ribavirin, or interferon-alpha2b.
- Methods for treating or preventing virus (e.g., coronavirus) infection in a subject in need of said treatment or prevention by administering an antibody or antigen-binding fragment of Table 1 in association with a further therapeutic agent are part of the present invention.
- the further therapeutic agent is a vaccine, e.g. , a coronavirus vaccine.
- a vaccine is an inactivated/killed virus vaccine, a live attenuated virus vaccine or a virus subunit vaccine.
- the further therapeutic agent is:
- the anti -viral drug is an antibody or antigenbinding fragment that binds specifically to coronavirus, e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV.
- Exemplary anti-CoV-S antibodies include, but are not limited to: H4sH15188P; H1H15188P; H1H15211P; H1H15177P; H4sH15211P; H1H15260P2; H1H15259P2; H1H15203P; H4sH15260P2; H4sH15231P2; H1H15237P2; H1H15208P; H1H15228P2; H1H15233P2; H1H15264P2; H1H15231P2; H1H15253P2; H1H15215P; and H1H15249P2, as set forth in International patent application publication no.
- WO/2015/179535 or an antigenbinding fragment thereof, e.g., wherein the antibody or fragment comprises a light chain immunoglobulin that includes CDR-L1, CDR-L2 and CDR-L3 (e.g., the VL or light chain thereof); and a heavy chain that includes CDR-H1, CDR-H2 and CDR-H3 (e.g, the VH or heavy chain thereof) of any of the foregoing anti-CoV-S antibodies.
- a light chain immunoglobulin that includes CDR-L1, CDR-L2 and CDR-L3 (e.g., the VL or light chain thereof)
- a heavy chain that includes CDR-H1, CDR-H2 and CDR-H3 (e.g, the VH or heavy chain thereof) of any of the foregoing anti-CoV-S antibodies.
- the further therapeutic agent is not aprotinin, leupeptin, a cationic steroid antimicrobial, an influenza vaccine (e.g., killed, live, attenuated whole virus or subunit vaccine), or an antibody against influenza virus (e.g ., an antihemagglutinin antibody).
- influenza vaccine e.g., killed, live, attenuated whole virus or subunit vaccine
- antibody against influenza virus e.g ., an antihemagglutinin antibody
- an anti-CoV-S antigenbinding protein e.g., antibody or antigen-binding fragment thereof of the present invention
- another agent can be formulated into a single composition, e.g, for simultaneous delivery, or formulated separately into two or more compositions (e.g, a kit).
- Each component can be administered to a subject at a different time than when the other component is administered; for example, each administration may be given non-simultaneously (e.g, separately or sequentially) at intervals over a given period of time.
- the separate components may be administered to a subject by the same or by a different route (e.g, wherein an anti-CoV-S antibody or antigen-binding fragment thereof.
- kits comprising one or more components that include, but are not limited to, an anti-CoV-S antigen-binding protein, e.g, an antibody or antigen-binding fragment as discussed herein (e.g, of Table 1), in association with one or more additional components including, but not limited to, a further therapeutic agent, as discussed herein.
- an anti-CoV-S antigen-binding protein e.g, an antibody or antigen-binding fragment as discussed herein (e.g, of Table 1)
- additional components including, but not limited to, a further therapeutic agent, as discussed herein.
- the antigen-binding protein and/or the further therapeutic agent can be formulated as a single composition or separately in two or more compositions, e.g, with a pharmaceutically acceptable carrier, in a pharmaceutical composition.
- the kit includes an anti-CoV-S antigen-binding protein, e.g, an antibody or antigen-binding fragment thereof of the invention (e.g, of Table 1), or a pharmaceutical composition thereof in one container (e.g, in a sterile glass or plastic vial) and a further therapeutic agent in another container (e.g, in a sterile glass or plastic vial).
- an anti-CoV-S antigen-binding protein e.g, an antibody or antigen-binding fragment thereof of the invention (e.g, of Table 1)
- a pharmaceutical composition thereof in one container (e.g, in a sterile glass or plastic vial) and a further therapeutic agent in another container (e.g, in a sterile glass or plastic vial).
- the kit comprises a combination of the invention, including an anti-CoV-S antigen-binding protein, e.g, antibody or antigen-binding fragment thereof of the invention (e.g, of Table 1), or pharmaceutical composition thereof in combination with one or more further therapeutic agents formulated together, optionally, in a pharmaceutical composition, in a single, common container.
- an anti-CoV-S antigen-binding protein e.g, antibody or antigen-binding fragment thereof of the invention (e.g, of Table 1)
- pharmaceutical composition thereof in combination with one or more further therapeutic agents formulated together, optionally, in a pharmaceutical composition, in a single, common container.
- the kit can include a device (e.g, an injection device) for performing such administration.
- the kit can include one or more hypodermic needles or other injection devices as discussed above containing the anti-CoV-S antigen-binding protein, e.g ., antibody or antigen-binding fragment thereof of the present invention (e.g, of Table 1).
- the kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely.
- the following information regarding a combination of the invention may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer/distributor information and patent information.
- the anti-CoV-S antigen-binding proteins may be used to detect and/or measure CoV-S in a sample.
- Exemplary assays for CoV-S may include, e.g, contacting a sample with an anti-CoV-S antigen-binding protein of the invention, wherein the anti-CoV-S antigen-binding protein is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate CoV-S from samples.
- the presence of an anti-CoV-S antigen-binding protein complexed with CoV-S indicates the presence of CoV-S in the sample.
- an unlabeled anti-CoV-S antibody can be used 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, b- galactosidase, horseradish peroxidase, or luciferase.
- the present invention includes a method for detecting the presence of spike protein polypeptide in a sample comprising contacting the sample with an anti-CoV-S antigen-binding protein and detecting the presence of a CoV-S/anti-CoV-S antigen-binding protein wherein the presence of the complex indicates the presence of CoV-S.
- An anti-CoV-S antigen-binding protein of the invention (e.g ., of Table 1) may be used in a Western blot or immune-protein blot procedure for detecting the presence of CoV-S or a fragment thereof in a sample.
- Such a procedure forms part of the present invention and includes the steps of e.g. :
- a membrane or other solid substrate comprising a sample to be tested for the presence of CoV-S, e.g. , optionally including the step of transferring proteins from a sample to be tested for the presence of CoV-S (e.g, from a PAGE or SDS-PAGE electrophoretic separation of the proteins in the sample) onto a membrane or other solid substrate using a method known in the art (e.g, semi-dry blotting or tank blotting); and contacting the membrane or other solid substrate to be tested for the presence of CoV-S or a fragment thereof with an anti-CoV-S antigen-binding protein of the invention.
- a method known in the art e.g, semi-dry blotting or tank blotting
- Such a membrane may take the form, for example, of a nitrocellulose or vinyl-based (e.g, polyvinylidene fluoride (PVDF)) membrane to which the proteins to be tested for the presence of CoV-S in a non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or SDS- PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel have been transferred (e.g, following electrophoretic separation in the gel).
- PAGE polyacrylamide gel electrophoresis
- SDS- PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
- Detection of the bound antigen-binding protein indicates that the CoV-S protein is present on the membrane or substrate and in the sample. Detection of the bound antigen-binding protein may be by binding the antigen-binding protein with a secondary antibody (an antiimmunoglobulin antibody) which is detectably labeled and, then, detecting the presence of the secondary antibody label.
- a secondary antibody an antiimmunoglobulin antibody
- anti-CoV-S antigen-binding proteins e.g, antibodies and antigen-binding fragments (e.g, of Table 1)
- Such a method forms part of the present invention and comprises, e.g.,
- the antigen-binding protein itself is detectably labeled, it can be detected directly.
- the antigen-binding protein may be bound by a detectably labeled secondary antibody wherein the label is then detected.
- Example 1 Generation of human antibodies to SARS-CoV-2 spike protein (SARS-CoV-2-S) [000127] Human antibodies to SARS-CoV-2-Spike protein (SARS-CoV-2-S) were generated in a VELOCIMMUNE ® mouse comprising DNA encoding human immunoglobulin heavy and kappa light chain variable regions or human immunoglobulin heavy and lambda light chain variable regions.
- Each mouse was immunized with a vector expressing the SARS-CoV-2-S receptor binding domain (RBD) (amino acids 1-1273 ofNCBI accession number (MN908947.3), SEQ ID NO: 832), followed by a booster with a SARS-CoV-2-S vector or a SARS-CoV-2-S protein.
- the antibody immune response was monitored by a SARS-CoV-2-S-specific immunoassay.
- lymphocytes were harvested and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines.
- the hybridoma cell lines were screened and selected to identify cell lines that produce SARS-CoV-2- S-specific antibodies.
- Anti-SARS-CoV-2-S antibodies were also isolated directly from antigenpositive mouse B cells without fusion to myeloma cells, as described in U.S. Patent 7582298, herein specifically incorporated by reference in its entirety. Using this method, fully human anti- SARS-CoV-2-S antibodies (i.e., antibodies possessing human variable domains and human constant domains) were obtained.
- Antibody variable regions were also isolated from human blood samples. Whole blood was received from patients 3-4 weeks after a laboratory-confirmed PCR positive test for SARS-CoV- 2 and symptomatic COVID-19 disease. Red blood cells were lysed using an ammonium chloride based lysis buffer (Life Technologies) and B cells were enriched by negative selection. Single B cells that bound the SARS-CoV-2 spike protein were isolated by fluorescent-activated cell sorting (FACS). Isolated B cells were single-well plated and mixed with antibody light and heavy variable region-specific PCR primers. cDNAs for each single B cell were synthesized via a reverse transcriptase (RT) reaction.
- RT reverse transcriptase
- Each resulting RT product was then split and transferred into two corresponding wells for subsequent antibody heavy and light chain PCRs.
- One set of the resulting RT products was first amplified by PCR using a 5’ degenerate primer specific for antibody heavy variable region leader sequence or a 5’ degenerate primer specific for antibody light chain variable region leader sequence and a 3’ primer specific for antibody constant region, to form an amplicon.
- the amplicons were then amplified again by PCR using a 5’ degenerate primer specific for antibody heavy variable region framework 1 or a 5’ degenerate primer specific for antibody light chain variable region framework 1 and a 3’ primer specific for antibody constant region, to generate amplicons for cloning.
- the antibody heavy chain and light chain derived PCR products were cloned into expression vectors containing heavy constant region and light constant region, respectively, thereby producing expression vectors for hybrid antibodies.
- the expression vectors expressing full-length heavy and light chain pairs were transfected into CHO cells to produce antibody proteins for testing.
- Plasmids encoding modified anti-Sars-CoV-2-S antibodies are generated by cloning synthetically- synthesized double-stranded DNA fragments (gBlocks; Integrated DNA Technologies, Coralville, IA) representing the modified anti-Sars-Cov-2-S variable domains (either the heavy chain or the light chain variable domain) into human IgGl heavy chain or human kappa expression plasmids.
- Anti-Sars-Cov-2 antibodies are produced in CHO cells after transfection with two expression plasmids encoding a target antigen binding IgGl heavy chain and the target antigen binding light chain. Antibodies are purified by differential protein A affinity chromatography.
- Example 2 Heavy and light chain variable region amino acid and nucleotide sequences [000131] Table 1 sets forth the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs, as well as the heavy chain and light chain sequences, of exemplary anti-SARS-CoV-2-S antibodies. The corresponding nucleic acid sequence identifiers are set forth in Table 2.
- Antibodies disclosed herein have fully human variable regions but can have mouse constant regions (e.g., a mouse IgGl Fc or a mouse IgG2 Fc (a or b isotype)) or human constant regions (e.g., a human IgGl Fc or a human IgG4 Fc).
- mouse constant regions e.g., a mouse IgGl Fc or a mouse IgG2 Fc (a or b isotype)
- human constant regions e.g., a human IgGl Fc or a human IgG4 Fc.
- an antibody having a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., an antibody with a mouse IgGl Fc can be converted to an antibody with a human IgG4, etc.), but in any event, the variable domains (including the CDRs) - which are indicated by the numerical identifiers shown in Tables 1 and 2 will remain the same, and the binding properties to antigen are expected to be identical or substantially similar regardless of the nature of the constant domain.
- the antibodies were obtained from hybridomas generated from VELOCIMMUNE® mice, by direct isolation from antigen-positive VELOCIMMUNE® mouse B cells, or derived from variable regions cloned from antigen-positive human B cells. A summary of these sources is shown in Table 3.
- Example 3 Luminex binding of anti-SARS-CoV-2-S antibodies to wild-type and variant spike glycoproteins
- a Luminex binding assay was performed in order to determine the binding of 43 anti-SARS-COV-2-S antibodies to the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) with a C-terminal myc-myc-hexahistidine tag (SARS-CoV-2(RBD)(R319-F541).mmH) and SARS-CoV-2 spike glycoprotein RBD with an E484K substitution and a hexahistidine tag ((E484K)-His Recombinant Protein) (SinoBiologicals, Cat No. 40592-V08H84).
- proteins were amine-coupled to Luminex microspheres as follows: approximately 10 million MagPlex microspheres (MagPLex Microspheres, Luminex, Cat. No. MCI 0043 and MC10118), were resuspended by vortexing in 500 pL 0.1M NaPCri, pH 6.2 (activation buffer) and then magnetically separated to remove the supernatant. Microspheres were protected from light, as they are light sensitive. The microspheres were resuspended in 160 pL of activation buffer and the carboxylate groups (-COOH) were activated by addition of 20 pL of 50 mg/mL of N- hydroxysuccinimide (NHS, Thermo Scientific, Cat. No.
- the coupling reaction was quenched by the addition of 50 pL of 1M Tris-HCl, pH 8.0 and the microspheres were vortexed, magnetically separated, and washed three times with 800 pL of PBS 0.005% Tween200.05%), to remove uncoupled proteins and other reaction components. Microspheres were resuspended in 1 mL of PBS 2% BSA 0.05% Na Azide at 10 million microspheres/mL.
- Microspheres with amine-coupled proteins were mixed at 2700 beads/ml, and 75 pL of microspheres were plated per well on a 96 well filter plate (EMD Millipore, Cat. No: MSBVN1250) and mixed with 25 pL of individual anti-SARS-CoV-2 supernatant containing antibody. Samples and microspheres were incubated for two hours at 25oC and then washed twice with 200 pL of DPBS with 0.05% Tween 20.
- Table 4 shows that 21 anti-SARS-CoV-2 antibodies bound to SARS-CoV-2 RBD and RBD (E484K) proteins with similar binding signal intensities: mAbl0937, mAbl0935, mAbl0966, mAbll004, mAbl0956, mAbl0932, mAbllOlO, mAbl0957, mAbl0955, mAbl0954, mAbl0938, mAbl0984, mAbl0939, mAh 10971, mAh 10982, mAh 10967, mAh 10986, mAh 10969, mAh 10965, mAh 10985, mAbl0922.
- MFI median fluorescence intensity
- Table 5 shows that 17 anti-SARS-CoV-2 antibodies have enhanced binding for SARS-CoV2 RBD protein over the RBD (E484K) protein: mAh 10964, mAh 11008, mAh 11000, mAh 10998, mAbl0970, mAbl0915, mAbl0914, mAbl0941, mAbl0940, mAbl0930, mAh 10923, mAbl0921, mAbll006, mAbl0998, mAbl0930, mAbl0996 and mAbl0936.
- Table 6 shows that 5 anti-SARS-CoV-2 antibodies bound to SARS-CoV2 RBD but did not demonstrate binding to RBD (E484K) protein: mAbl 1002, mAb 10920, mAb 10977, mAb 10924 and mAbl0913.
- Example 4 Characterization of hybridoma supernatants by binding ELISA
- An ELISA binding assay is performed to identify antibody supernatants that bind to the SARS-CoV-2-Spike protein receptor binding domain (RBD), or to the E484K variant protein.
- a protein composed of the wild-type or E484K RBD of SARS-CoV-2 (amino acids 319- 541) expressed with a 6X histidine tag and two myc epitope tags at the C-terminus (SARS-CoV- 2-S-RBD-mmH; see also NCBI Accession Number MN908947.3) is coated at 1 pg/ml on a 96- well plate in PBS buffer overnight at 4°C.
- Nonspecific binding sites are subsequently blocked using a 0.5% (w/v) solution of BSA in PBS.
- Antibody supernatants or media alone are diluted 1 :40 or 1 :50 in the PSA+0.5% BSA blocking buffer and transferred to the washed microtiter plates. After one hour of incubation at room temperature, the wells are washed, and plate-bound supernatant is detected with either goat-anti-human IgG antibody conjugated with horseradish peroxidase (HRP) (Jackson Immunoresearch), or anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP) (Jackson Immunoresearch). The plates are then developed using TMB substrate solution (BD Biosciences) according to manufacturer’s recommendation and absorbance at 450nm was measured on a Victor X5 plate reader.
- HRP horseradish peroxidase
- HRP horseradish peroxidase
- Binding data are analyzed using a sigmoidal dose-response model within PrismTM software (GraphPad).
- the calculated IC50 value defined as the concentration of antibody required to block 50% of SARS-CoV-2 RBD-hFc or SARS-CoV-2 RBD(E484K)-hFc binding to plate-coated hACE2-His, is used as an indicator of blocking potency. Percent blocking is defined based on the background-corrected binding signal observed at the highest antibody concentration tested using this formula:
- Antibodies that block binding less than or equal to 50% at the highest concentration tested can be classified as non-blockers.
- Antibodies that have been modified to enhance binding to E484K variant spike protein show increased binding to the variant spike protein and/or reduced binding of the spike protein to hACE2 as compared to the corresponding antibodies prior to modification.
- Example 5 Antibody binding to SARS-CoV-2-S-expressing virus-like particle [000142] To investigate the ability of a panel of anti-SARS-CoV-2-S monoclonal antibodies to bind the SARS-CoV-2 spike glycoprotein, an in vitro binding assay utilizing SARS-CoV-2 spike protein-expressing viral-like particles (VLPs) in an electrochemiluminescence based detection platform (MSD) is used.
- VLPs SARS-CoV-2 spike protein-expressing viral-like particles
- MSD electrochemiluminescence based detection platform
- SARS-CoV-2 spike protein wild-type: NCBI Accession number MN908947.3, amino acids 16-1211; SEQ ID NO: 833; E484K variant: SEQ ID NO:
- VSV delta G Vesicular stomatitis virus lacking glycoprotein G
- VSV-SARS-CoV-2-S SARS-CoV-2 spike protein
- VSV-G VSV G protein
- the plates are then incubated for 1 hour at room temperature with shaking, after which the plates are washed with lx PBS to remove the unbound antibodies using an AquaMax2000 plate washer (MDS Analytical Technologies).
- the plate-bound antibodies are detected with a SULFO-TAGTM-conjugated anti-human IgG antibody (Jackson Immunoresearch) or a SULFO-TAGTM-conjugated anti-mouse IgG antibody (Jackson Immunoresearch) for 1 hour at room temperature.
- the plates are developed with the Read Buffer (MSD) according to manufacturer’s recommended procedure and the luminescent signals are recorded with a SECTOR Imager 600 (Meso Scale Development) instrument. Direct binding signals (in RLU) are captured, and a ratio of SARS- CoV-2-S-expressing VLPs to the irrelevant VLP was calculated.
- a signal observed from SARS-COV-2-S-expressing VLPs indicates binding, while comparison with negative VLPs provides a relative background.
- Media alone samples provide baseline signals of secondary antibody binding to samples with no supernatant.
- Antibodies modified to enhance binding to the E484K variant spike protein show increased binding as compared to the anitbodies from which they are modified.
- Example 6 Antibody neutralization of VSV-SARS-CoV-2-S pseudovirus infectivity
- VSV pseudotype viruses are generated by transiently transfecting 293T cells with a plasmid encoding for SARS-CoV-2 spike protein (wild-type or E484K).
- Cells are seeded in 15 cm plates at 1.2xl0 7 cells per plate in DMEM complete media one day prior to transfection with 15 pg/plate spike protein DNA using 125 pL Lipofectamine LTX, 30 pL PLUS reagent, and up to 3 mL Opti-Mem. 24 hours post transfection, the cells are washed with 10 mL PBS, then infected with an MOI of 0.1 VSV AG:mNcon virus in 10 mL Opti- Mem. Virus is incubated on cells for 1 hour, with gentle rocking every 10 minutes.
- VSV-SARS-CoV-2-S or VSV-SARS-CoV-2-S(E484K).
- Vero cells are seeded at 80% confluency in T225 flasks. To seed cells, media is removed from the cells, the cells are washed with 20mL PBS (Gibco: 20012-043), and 5mL TrypLE is added and incubated for ⁇ 5 minutes at 37 °C until the cells are dislodged. 5 mL of complete DMEM is added to inactivate the trypsin, and pipetted up and down to distribute the cells. To count the resuspended cells, 20,000 Vero cells are plated in 100 pL prewarmed Complete DMEM per well in a 96 Well Black Polystyrene Microplate (Coming: 3904).
- VSV-SARS-CoV-2-S and VSV-SARS-CoV-2-S(E484K) are thawed on ice and diluted 1 : 1 with infection media.
- a dilution of each supernatant is generated in 60ul infection media.
- 60 pi of diluted conditioned media is added to the wells.
- 60 pL of diluted VSV-SARS-CoV-2-S or VSV-SARS-CoV-2-S(E484K) is added to every well except the media control wells.
- 60 pL of infection media is added to those wells.
- Pseudoviruses are then incubated with supernatant dilutions for 30 minutes at room temperature.
- the ability of the anti-SARS-CoV-2-S antibodies to neutralize VSV-based SARS- CoV-2-S-expressing pseudotyped virus is assessed using a neutralization fluorescence focus assay.
- the neutralization potency of antibody at each dilution is represented as a percentage compared to mock supernatant control.
- Antibodies modified to enhance binding to the E484K variant spike protein showed increased binding as compared to the antibodies from which they are modified.
- VeroE6 media containing SARS-CoV-2 (WA-1) (1000 PFU/mL) is added to each serum dilution and to 250 pL media as an untreated control.
- the virus-antibody mixtures are incubated for 60 min at 37 °C.
- virus titers of the mixtures are determined by plaque assay.
- 50% plaque reduction neutralization titer (PRNT50) values (the serum dilutions at which plaque formation was reduced by 50% relative to that of the untreated control) are calculated using a 4- parameter logistic curve fit to the percent neutralization data (GraphPad Software, La Jolla, CA).
- Example 7 Biacore binding kinetics of anti-SARS-CoV-2-S monoclonal antibodies
- KD Equilibrium dissociation constants
- the Biacore CM5 sensor chip surface is first derivatized by amine coupling with either mouse anti-human Fc specific mAb or rabbit antimouse Fey monoclonal antibody (GE, Catalog # BR-1008-38) to capture anti-SARS-CoV-2 antibodies.
- Binding studies are performed on a human SARS-CoV-2 RBD extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (SARS-COV-2 RBD-MMH), SARS- CoV-2 RBD extracellular domain expressed with a C-terminal mouse IgG2a (SARS-COV-2 RBD-mFc), or SARS-CoV-2 RBD extracellular domain expressed with a C-terminal human IgGl (SARS-COV-2 RBD-hFc), each of which is generated with wild-type and E484K variant sequences.
- SARS-CoV-2 RBD-MMH C-terminal myc-myc-hexahistidine tag
- SARS-CoV-2 RBD-mFc SARS-CoV-2 RBD extracellular domain expressed with a C-terminal mouse IgG2a
- SARS-CoV-2 RBD-hFc SARS-CoV-2 RBD extracellular domain expressed with a C-terminal human
- the SARS-CoV-2 RBD antibody capture surface is regenerated using either a 10 sec injection of 20mM phosphoric acid for the mouse anti-human Fc specific monoclonal antibody surface or a 40 sec injection of lOmM Glycine, HC1, pH1.5 for the rabbit anti-mouse Fey specific polyclonal antibody.
- the association rate ( k a ) and dissociation rate (kd) are determined by fitting the real-time binding sensorgrams to a 1 : 1 binding model with mass transport limitation using BiaEvaluation software v3.1 or Biacore Insight Evaluation software v2.0. or curve-fitting software. Binding dissociation equilibrium constant (KD) and dissociative half-life (t1 ⁇ 2) were calculated from the kinetic rates as:
- Antibodies modified to enhance binding to the E484K variant spike protein show enhanced binding kinetics (e.g., a lower KD and/or a higher t 1 ⁇ 2) as compared to the antibodies from which they are modified.
- Example 8 Structure determination of antibody-bound spike protein [000158] To determine amino acids that are in proximity to amino acid 484 of the spike protein, structural analysis is performed via cryo-electron microscopy (cryoEM). Fab fragments are isolated using FabALACTICA kit (Genovis). 600 pg of the Fab is mixed with 300 pg of SARS-CoV-2-S RBD or SARS-CoV-2-S(E484K) RBD and incubated on ice for ⁇ 1 hour then injected into a Superdex 200 increase gel filtration column equilibrated to 50 mM Tris pH 7.5, 150 mM NaCl.
- cryoEM cryo-electron microscopy
- Peak fractions containing the mAbl0933 Fab - mAbl0987 Fab - RBD complex are collected and concentrated using a 10 kDa MWCO centrifugal filter.
- the protein sample is diluted to 1.5 mg/mL and 0.15% PMAL-C8 amphipol is added.
- 3.5 pL of protein is deposited onto a freshly plasma cleaned UltrHommeoil grid (1.2/1.3, 300 mesh). Excess solution is blotted away using filter paper and plunge-frozen into liquid ethane using a Vitrobot Mark IV.
- the cryoEM grid is transferred to a Titan Krios (Thermo Fisher) equipped with a K3 detector (Gatan).
- Movies are collected using EPU (Thermo Fisher) at 105,000x magnification, corresponding to a pixel size of 0.85 A. A dose rate of 15 electrons per pixel per second is used and each movie is 2 seconds, corresponding to a total dose of ⁇ 40 electrons per
- cryoEM data processing is carried out using cryoSPARE v2.14.2. Movies are aligned using patch motion correction and patch CTF estimation. Aligned micrographs are selected for further processing on the basis of estimated defocus values and CTF fit resolutions.
- An initial set of particles picked using blob picker are subjected to 2D classification to generate templates for template picking. Particles picked by template picking are subjected to multiple rounds of 2D classification to remove unbound fabs and particles containing an incomplete complex.
- Ab initio reconstruction with three classes generate a single class containing particles that correspond to the Fab-RBD complex. Heterogenous refinement of the particles in this class followed by non-uniform refinement results in a final resolution map containing particles that are used for model building. Into this map, models of the RBD (taken from PDB code 6M17) and the two Fabs are manually placed. These models are then manually rebuilt using Coot and real- space refined against the map using Phenix.
- CDR amino acids identified as being in proximity to amino acid 484 of the spike protein can then be modified as described previously herein, e.g., using gBlocks
- Example 9 Anti-SARS-CoV-2-S antibodies binding to spike protein-expressing cells [000161] To investigate the ability of a panel of anti-SAR.S-CoV-2-S monoclonal antibodies to bind to SAR.S-CoV-2-S expressing cells, an in vitro binding assay utilizing SAR.S-CoV-2-S or SARS-CoV-2-S(E484K) expressing cells in an electrochemiluminescence based detection platform (MSD) is used.
- MSD electrochemiluminescence based detection platform
- Jurkat/Tet3G/hCD20/Tet-3G inducible cells are engineered to transiently express the SARS-CoV-2 Spike Protein (Accession number MN908947.3, amino acids 16-1211, Jurkat/Tet3G/hCD20/Tet-On 3G Inducible COVID-19 Spike Protein High Sorted) (wild-type or E484K), and flow cytometry sorted for selection of high expression of the SARS-CoV-2 protein.
- Parental Jurkat/Tet3G/hCD20/Tet-3G are also included in the experiments as a negative binding control.
- anti-SARS-CoV-2 antibodies and a non-binding human IgGl control diluted in PBS + 0.5% BSA at a range of concentrations from 0.0008nM to 50nM, and buffer with no antibody are added in duplicate and the plates incubated for one hour at room temperature with shaking. The plates are then washed with IX PBS to remove the unbound antibodies using an AquaMax2000 plate washer (MDS Analytical Technologies). The plate-bound antibodies are detected with a SULFO-TAGTM-conjugated anti-human IgG antibody (Jackson Immunoresearch) for one hour at room temperature.
- the plates are developed with the Read Buffer (MSD) according to manufacturer’s recommended procedure and the luminescent signals were recorded with a SECTOR Imager 600 (Meso Scale Development) instrument.
- the direct binding signals (in RLU) are captured for SARS-CoV-2-S expressing cells and a negative control cell line.
- the ability of the anti-SARS-CoV-2 monoclonal antibodies to bind to SARS-CoV-2 spike protein-expressing cells and E484K variant SARS-CoV-2 spike protein-expressing cells compared with binding to parental cells is assessed using an immunobinding assay. Binding to the immobilized cells on 96-well high bind plates (MSD) is performed with a series of antibody dilutions and the bound antibodies were detected using SULFO-TAGTM-conjugated anti-human IgG. The binding signals from electrochemiluminescence are recorded on a Sector Imager 600 (MSD). All antibodies display a concentration-dependent binding and the ratio of the binding on spike expressing cells to the parental cells are analyzed at the concentration of 5.5nM and 0.20nM.
- Antibodies modified to enhance binding to the E484K variant spike protein show an increase in binding to E484K variant spike protein-expressing cells as compared to the antibodies from which they are modified.
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- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
La présente divulgation concerne des procédés de modification d'anticorps et de fragments de liaison à l'antigène correspondants pour améliorer la liaison à certains variants de la protéine spike de coronavirus de type sauvage (par exemple, un variant qui comprend une substitution E484K), ainsi que des procédés d'utilisation de tels anticorps et de tels fragments pour traiter ou prévenir des infections virales ( par exemple, des infections à coronavirus).
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