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WO2023288078A1 - Anticorps de coronavirus et leurs utilisations - Google Patents

Anticorps de coronavirus et leurs utilisations Download PDF

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Publication number
WO2023288078A1
WO2023288078A1 PCT/US2022/037330 US2022037330W WO2023288078A1 WO 2023288078 A1 WO2023288078 A1 WO 2023288078A1 US 2022037330 W US2022037330 W US 2022037330W WO 2023288078 A1 WO2023288078 A1 WO 2023288078A1
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amino acid
seq
acid sequence
antigen
antibody
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Inventor
Devin Sok
Joseph JARDINE
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International Aids Vaccine Initiative Inc
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International Aids Vaccine Initiative Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Coronavirus disease 19 (COVID-19) is an illness caused by the zoonotic SARS-CoV- 2 virus and its strains, which have caused a worldwide pandemic. The majority of infected individuals remain asymptomatic or experience mild symptoms, such as cough, fever, fatigue, or loss of smell.
  • the present application provides a neutralizing monoclonal antibody or an antigen- binding fragment thereof that binds a Spike protein of SARS-CoV-1 and/or SARS-CoV-2.
  • the neutralizing monoclonal antibody or antigen- binding fragment thereof binds a Spike protein of SARS-CoV-2 that comprises an amino acid sequence of SEQ ID No: 1 or 194.
  • the neutralizing monoclonal antibody or antigen-binding fragment thereof binds the S1 region of the Spike protein.
  • the S1 region targeted by the neutralizing monoclonal antibody or antigen-binding fragment thereof comprises an amino acid sequence within SEQ ID No: 1 or 194.
  • the S1 region of SARS-CoV-1 targeted by the neutralizing monoclonal antibody or antigen-binding fragment thereof comprises an amino acid sequence within SEQ ID No: 3.
  • the neutralizing monoclonal antibody or antigen-binding fragment thereof binds a receptor binding domain (RBD) of the Spike protein.
  • the RBD to which the neutralizing monoclonal antibody or antigen-binding fragment thereof binds, comprises an amino acid sequence of SEQ ID No: 2 or 195.
  • the neutralizing monoclonal antibody or antigen- binding fragment thereof inhibits binding of SARS-CoV-2 and/or SARS-CoV-1 to an ACE-2 receptor.
  • the neutralizing monoclonal antibody or antigen-binding fragment thereof has a binding affinity of 10 -6 to 10 -9 kD to the Spike protein of SARS-CoV- and/or SARS-CoV-2. In some embodiments, the antibody or antigen-binding fragment thereof has a binding affinity of 10 -9 to 10 -12 kD. In some embodiments, the neutralizing monoclonal antibody or antigen-binding fragment thereof does not cross-react with a human antigen.
  • the neutralizing monoclonal antibody or antigen-binding fragment thereof of the present application comprises: 1) a VH-CDR1 comprising an amino acid sequence of SEQ ID NO: 369, a VH-CDR2 comprising an amino acid sequence of SEQ ID NO: 370, and a VH- CDR3 comprising an amino acid sequence of SEQ ID NO: 371; 2) a VH-CDR1 comprising an amino acid sequence of SEQ ID NO: 374, a VH-CDR2 comprising an amino acid sequence of SEQ ID NO: 375, and a VH- CDR3 comprising an amino acid sequence of SEQ ID NO: 376; 3) a VH-CDR1 comprising an amino acid sequence of SEQ ID NO: 379, a VH-CDR2 comprising an amino acid sequence of SEQ ID NO: 380, and a VH- CDR3 comprising an amino acid sequence of SEQ ID NO: 381; 4) a VH-CDR1 comprising an amino acid sequence of S
  • the amino acid substitutions, deletions or insertions comprises a homologous substitution.
  • the VH chain comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of: SEQ ID NOs: 367, 372, 377, 382, and 387.
  • the VL chain comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of: SEQ ID NOs: 392, 397, 402, 407, 412, 417, 422, 427, 432, 437, 442, 447, 452, 457, 462, 467, 472, 477, 482, 487, 492, 497, 502, 507, and 512.
  • the neutralizing monoclonal antibody or antigen-binding fragment thereof of the present application is an antigen-binding fragment.
  • the antigen-binding fragment is a scFv. In some embodiments, the antigen- binding fragment is a Fab'. In some embodiments, the neutralizing monoclonal antibody or antigen-binding fragment of the present application is an antibody. In some embodiments, the antibody is an IgG antibody. In another aspect, this application provides a composition comprising a neutralizing monoclonal antibody or antigen-binding fragment thereof as described herein and a pharmaceutically acceptable carrier. In another aspect, this application provides a nucleic acid encoding a neutralizing monoclonal antibody or antigen-binding fragment thereof as described herein.
  • the present application provides a nucleic acid molecule encoding a VH chain comprising an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154, 164, 174, 184, 197, 207, 217, 227, 237, 247, 257, 267, 277, 287, 297, 307, 317, 327, 337, 347, 357, 367, 372, 377, 382, and 387.
  • the nucleic acid sequence is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185, 198, 208, 218, 228, 238, 248, 258, 268, 278, 288, 298, 308, 318, 328, 338, 348, 358, 368, 373, 378, 383, and 388.
  • the present application provides a nucleic acid molecule that encodes a VL chain comprising an amino acid sequence that is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, 119, 129, 139, 149, 159, 169, 179, 189, 202, 212, 222, 232, 242, 252, 262, 272, 282, 292, 302, 312, 322, 332, 342, 352, 362, 392, 397, 402, 407, 412, 417, 422, 427, 432, 437, 442, 447, 452, 457, 462, 467, 472, 477, 482, 487, 492, 497, 502, 507, and 512.
  • the nucleic acid sequence is at least 80%, 85%, 90%, 92%, 93%, 95%, 97%, 98%, 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 203, 213, 223, 233, 243, 253, 263, 273, 283, 293, 303, 313, 323, 333, 343, 353, 363, 393, 398, 403, 408, 413, 418, 423, 428, 433, 438, 443, 448, 453, 458, 463, 468, 473, 478, 483, 488, 493, 498, 503, 508, and 513.
  • this application provides a vector comprising a nucleic acid as described herein. In another aspect, this application provides a host cell comprising a vector as described herein. In some embodiments, the present application provides a lyophilized composition comprising a neutralizing monoclonal antibody or antigen-binding fragment thereof as described herein. In some embodiments, the present application provides a reconstituted lyophilized composition comprising a neutralizing monoclonal antibody or antigen-binding fragment thereof as described herein.
  • the composition of the present application is formulated for administration by lozenge, spray, oral administration, delayed release or sustained 25 release, transmucosal administration, syrup, mucoadhesive, buccal formulation, mucoadhesive tablet, topical administration, parenteral administration, injection, subdermal administration, oral solution, rectal administration, buccal administration or transdermal administration.
  • the present application provides a method of treating or preventing SARS-CoV-1 and SARS-CoV-2 infections comprising administering a therapeutically effective amount of a neutralizing monoclonal antibody or antigen-binding fragment thereof as described herein.
  • the appropriate dosage of the antibodies, or antibody fragments depend on various factors, such as the type of infection to be treated, the severity and course of the infection, the responsiveness of the infection, the generation of viral resistance to therapy, previous therapy, patient's clinical history, and so on.
  • the antibody can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the infection is achieved (e.g., reduction in viruria or viral damage to the kidney).
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient and will vary depending on the relative potency of an individual antibody or antibody fragment (e.g., antigen binding fragment).
  • dosage is from 0.01 mg to 10 mg (e.g., 0.01 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 7 mg, 8 mg, 9 mg, or 10 mg) per kg of body weight, and can be given once or more daily, weekly, monthly or yearly.
  • the antibody or antibody fragment (e.g., antigen binding fragment), of the present disclosure is given once every two weeks or once every three weeks.
  • the treating physician can estimate repetition rates for dosing based on measured half-life and concentrations of the antibody in bodily fluids or tissues.
  • the antibodies or antigen binding fragments thereof, disclosed herein have a half-life anywhere from 1 day to 5 weeks. In some embodiments, the antibodies or antigen binding fragments thereof have a half-life of 1 week to 3 weeks. In certain embodiments, the antibodies or antigen binding fragments thereof, disclosed herein have a half-life anywhere from 2 weeks to 3 weeks.
  • the present application provides a method of producing a neutralizing monoclonal antibody or antigen-binding fragment thereof as described herein, the method comprising the steps of: expressing the nucleic acid or set of nucleic acids encoding the antibody or antigen-binding fragment as described herein in a cultured cell, purifying the antibody or antigen-binding fragment.
  • Figure 1 depicts a graph illustrating the binding affinity of neutralizing monoclonal CR3022 variant antibodies against the Spike of SARS-CoV virus.
  • Figure 2 depicts a graph illustrating the binding affinity of neutralizing monoclonal CR3022 variant antibodies against the Spike of SARS-CoV-2 virus.
  • Figure 3A depicts a graph illustrating that neutralizing monoclonal CR3022 variant antibodies are not polyreactive against single stranded DNA (ssDNA).
  • Figure 3B depicts a graphs illustrating that neutralizing monoclonal CR3022 variant antibodies are not polyreactive against CHO SMP.
  • Figure 4A depicts a schemata illustrating the neutralization against SARS-CoV for monoclonal CR3022 variant antibodies.
  • Figure 4B depicts a schemata illustrating the neutralization against SARS-CoV-2 for monoclonal CR3022 variant antibodies.
  • Figure 5A depicts a schemata illustrating the neutralization against SARS-CoV-2 for monoclonal CR3022 variant antibodies and the RBD that are targeted are also indicated.
  • Figure 5B depicts a schemata illustrating the neutralization against SARS-CoV-1 for monoclonal CR3022 variant antibodies and the RBD that are targeted are also indicated.
  • Figure 6A and Figure 6B depict schematas illustrating that a wildtype CR3022 monoclonal antibody binds CoV1-RBD and CoV2-RBD, but does not neutralize SARS-CoV-2 using plasmon resonance to RBD target antigen.
  • Neutralization potencies are plotted compared to dissociation constants (KD, M) measured by surface plasmon resonance (SPR) to RBD target antigen.
  • Figure 7 depict schematas illustrating the SPR affinity of designed eCR3022 variant antibodies to SARS-1 RBD and SARS-2 RBD.
  • Figure 8A depicts a schemata illustrating the neutralization against SARS-CoV-1 pseudovirus for monoclonal CR3022 variant antibodies.
  • Figure 8B depicts a schemata illustrating the neutralization against SARS-CoV-2 pseudovirus for monoclonal CR3022 variant antibodies.
  • CoV coronavirus
  • CoVs tend to cause mild to moderate upper respiratory tract infections such as the common cold.
  • These CoV strains are extremely contagious, exhibit strong virulence and quickly transfer from human to human. Accordingly, it is an object of the present disclosure to provide methods for treating, preventing, or reducing the progression rate and/or severity of SARS-CoV- 1 and/or SARS- CoV-2 infections or COVID-19, particularly treating, preventing or reducing the progression rate and/or severity of one or more SARS-CoV- 1, SARS-CoV-2 or associated complications thereof.
  • this application discloses antibodies that are usful in treating, preventing, or reducing the progression rate and/or severity of SARS-CoV- 1 and/or SARS-CoV-2 infections.
  • treating, preventing or reducing the progression rate and/or severity of one or more associated complications include plural referents unless the context clearly dictates otherwise.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Bio-chemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • SARS-CoV-2 also called as “COVID-19” refers to the newly-emerged Severe Acute Respiratory Syndrome, which was first identified in Wuhan, China in 2019 (World Health Organization 2020). It belongs to the betacoronavirus lineage B and causes severe respiratory disease, similar to the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) that emerged in China in 2002.
  • SARS coronavirus 2 has been found to be closely related to coronaviruses found in bats (Perlman et al 2020, New England Journal of Medicine 382: 760-762) and pangolins (Zhang et al 2020, Current Biology. 30: 1346–1351).
  • SARS-CoV-2 binds via the viral spike protein to the human host cell.
  • the host cell receptor is the Angiotensin Converting Enzyme 2 (ACE-2) receptor.
  • ACE-2 Angiotensin Converting Enzyme 2
  • SARS-CoV-2 spike protein has been found to bind to ACE-2 receptor of other species, especially bats and pandolins (Hoffman et al 2020, Cell.181: 271-280).
  • ACE-2-2 Angiotensin Converting Enzyme 2
  • SARS-CoV-2 spike protein has been found to bind to ACE-2 receptor of other species, especially bats and pandolins (Hoffman et al 2020, Cell.181: 271-280).
  • SARS-CoV-2-S refers to the viral spike protein.
  • SARS-CoV-2-S includes protein variants of the SARS-CoV-2 spike protein isolated from different SARS-CoV-2 isolates (shown in, recombinant SARS-CoV-2 spike protein or fragments thereof. The term also encompasses SARS-CoV-2 spike protein or a fragment thereof coupled to various tags, such as for example, histidine tag, mouse or human Fc, or a signal sequence such as ROR1.
  • the SARS-CoV-2 spike protein is as set forth in SEQ ID No: 1.
  • the Spike protein is a type I membrane glycoprotein which assembles into trimers that constitute the spikes or peplomers on the surface of the enveloped MERS coronavirus particle.
  • the protein has two essential functions, host receptor binding and membrane fusion, which are attributed to the N-terminal (S1) and C-terminal (S2) halves of the S protein.
  • S1 and S2 halves of the S protein The term “Severe Acute Respiratory Syndrome-Coronavirus-2 Receptor Binding Domain”, “SARS-CoV-2-RBD,” as used herein, refers to a viral receptor binding domain of the Spike protein that is present in the S1 subunit of the Spike protein and comprises the sequence set forth in SEQ ID NO: 2 or 195, or biologically active fragments thereof.
  • the ACE-2 receptor refers to a type I transmembrane metallocarboxypeptidase with homology to ACE, an enzyme that plays a role in the Renin- Angiotensin system (RAS) and is generally considered to be a target for the treatment of hypertension.
  • RAS Renin- Angiotensin system
  • the ACE-2 receptor is mainly expressed in vascular endothelial cells, the renal tubular epithelium, and in Leydig cells in the testes.
  • ACE-2 is also expressed in the lung, kidney, and gastrointestinal tract, tissues shown to harbor SARS-CoV-2.
  • SARS-CoV-2 infection refers to the respiratory illness caused by the SARS-CoV-2 coronavirus.
  • SARS-CoV-1 Severe Acute Respiratory Syndrome, which was first identified in southern China in 2002 (World Health Organization 2020).
  • SARS coronavirus SARS-CoV
  • M membrane
  • N nucleocapsid
  • SARS-CoV-1-S severe Acute Respiratory Syndrome-Coronavirus-1 Spike
  • SARS-CoV-1-S refers to the viral spike protein.
  • SARS-CoV-1-S includes protein variants of the SARS-CoV-1 spike protein isolated from different SARS-CoV-1 isolates (shown in, recombinant SARS-CoV-1 spike protein or fragments thereof.
  • the term also encompasses SARS-CoV-1 spike protein or a fragment thereof coupled to various tags, such as for example, histidine tag, mouse or human Fc, or a signal sequence such as ROR1.
  • the Spike protein is a type I membrane glycoprotein which assembles into trimers that constitute the spikes or peplomers on the surface of the enveloped MERS coronavirus particle.
  • the protein has two essential functions, host receptor binding and membrane fusion, which are attributed to the N-terminal (S1) and C-terminal (S2) halves of the S protein.
  • S1 and S2 halves of the S protein The term "Severe Acute Respiratory Syndrome-Coronavirus-1 Receptor Binding Domain”, "SARS-CoV-1-RBD,” as used herein, refers to a viral receptor binding domain of the Spike protein that is present in the S1 subunit of the Spike protein and comprises the sequence set forth in SEQ ID NO: 3, or biologically active fragments thereof.
  • Antibodies and Antigen binding fragments of the disclosure refer to any one or more of the antibodies and antigen binding fragments provided herein.
  • Antibodies and antigen binding fragments of the disclosure comprise a heavy chain (VH) comprising a heavy chain variable domain and a light chain (VL) comprising a light chain variable domain.
  • VH domain comprises three CDRs, such as any of the CDRs provided herein and as defined or identified by the Chothia, Kabat or IMGT systems. These CDRs are typically interspersed with frame-work regions (FR), and together comprise the VH domain.
  • a VL comprises three CDRs, such as any of the CDRs provided herein and as defined by the Chothia, Kabat or IMGT systems. These CDRs are typically interspersed with framework regions (FR), and together comprise the VL domain.
  • the FR regions such as FRI, FR2, FR3, and/or FR4 can similarly be defined or identified by the Chothia, Kabat or IMGT systems.
  • CDRs are indicated as being, as identified or as defined by the Chothia, Kabat or IMGT systems, what is meant is that the CDRs are in accordance with that system (e.g., the Chothia CDRs, Kabat CDRs or the IMGT CDRs).
  • antibody also includes antigen-binding fragments of full antibody molecules.
  • antigen-binding portion of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suit-able standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains.
  • DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized.
  • the DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
  • the antibody name designations as used herein follow the formats: P0XA0Y or PXAY, P0XE0Y or PXEY, P0XD0Y or PXDY, P0XF0Y or PXFY, P0XH0Y or PXHY, P0XC0Y or PXCY, P0XG0Y or PXGY, P0XB0Y or PXBY, each denotes the same antibody.
  • the antibody name designation P04A05 is being used interchangeably with the designation P4A5, both designations denote the same antibody.
  • the disclosure provides for antibodies or antigen-binding fragments thereof that bind SARS-CoV-2.
  • the antibodies or antigen- binding fragments thereof bind the spike protein of SARS-CoV-2. In certain embodiments, the antibodies or antigen-binding fragments thereof bind the spike protein having an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1 or 194 or biologically active fragments thereof. In certain embodiments, the antibodies or antigen-binding fragments thereof bind the S1 region of the spike protein. In other embodiments, the antibodies or antigen binding fragments thereof bind the receptor-binding domain (RBD) of the spike protein.
  • RBD receptor-binding domain
  • the antibodies or antigen binding fragments thereof bind an RBD protein having an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2 or 195.
  • the antibodies and antigen binding fragments are neutralizing antibodies.
  • the antibodies and antigen binding fragments thereof do not cross react with human antigens.
  • the antibodies and antigen binding fragments thereof cross-react with the RBD of the SARS-CoV-1 RBD having an amino acid sequence as set forth in SEQ ID No: 3 or 196.
  • the antibodies or antigen binding fragments thereof bind the RBD of the spike protein, such as for example, RBD-A or RBD-B.
  • RBD-A examples include but are not limited to the following antibodies P09D05, P11A11, P11A06, P11G07, P09D07, or P04E05.
  • the neutralizing antibodies or antigen-binding fragments comprise a variable heavy chain (VH) and variable light chain (VL).
  • the VH chain is selected from the group comprising: 1) a VH-CDR1 comprising an amino acid sequence of SEQ ID NO: 369, a VH-CDR2 comprising an amino acid sequence of SEQ ID NO: 370, and a VH- CDR3 comprising an amino acid sequence of SEQ ID NO: 371; 2) a VH-CDR1 comprising an amino acid sequence of SEQ ID NO: 374, a VH-CDR2 comprising an amino acid sequence of SEQ ID NO: 375, and a VH- CDR3 comprising an amino acid sequence of SEQ ID NO: 376; 3) a VH-CDR1 comprising an amino acid sequence of SEQ ID NO: 379, a VH-CDR2 comprising an amino acid sequence of SEQ ID NO: 380, and a VH- CDR3 comprising an amino acid sequence of SEQ ID NO: 381; 4) a VH-CDR1 comprising an amino acid sequence of SEQ ID NO: 384
  • the VL chain is selected from the group comprising: (1) a VL-CDR1 comprising an amino acid sequence of SEQ ID NO: 394, a VL-CDR2 comprising an amino acid sequence of SEQ ID NO: 395, and a VL- CDR3 comprising an amino acid sequence of SEQ ID NO: 396; (2) a VL-CDR1 comprising an amino acid sequence of SEQ ID NO: 399, a VL-CDR2 comprising an amino acid sequence of SEQ ID NO: 400, and a VL- CDR3 comprising an amino acid sequence of SEQ ID NO: 401; (3) a VL-CDR1 comprising an amino acid sequence of SEQ ID NO: 404, a VL-CDR2 comprising an amino acid sequence of SEQ ID NO: 405, and a VL- CDR3 comprising an amino acid sequence of SEQ ID NO: 406; (4) a VL-CDR1 comprising an amino acid sequence of SEQ ID NO: 409, a
  • the disclosure provides for an antibody or antigen-binding fragment thereof comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises: i) a VH-CDRl having the amino acid sequence of SEQ ID NO: 26, but wherein 1, 2, 3, 4, or 5 amino acid substitutions, deletions or insertions are optionally present in the sequence of SEQ ID NO: 26; ii) a VH-CDR2 having the amino acid sequence of SEQ ID NO: 27, but wherein 1, 2, 3, 4, or 5 amino acid substitutions, deletions or insertions are optionally present in the sequence of SEQ ID NO: 27; and iii) a VH-CDR3 having the amino acid sequence of SEQ ID NO: 28, but wherein 1, 2, 3, 4, or 5 amino acid substitutions, deletions or insertions are optionally present in the sequence of SEQ ID NO: 28; and wherein the VL comprises: i) a VL-CDRl having the amino acid sequence of SEQ ID NO: 26, but
  • the present disclosure includes anti- SARS2-CoV-2 and/or SARS-CoV- 1 antibodies and antigen-binding fragments thereof that bind the SARS-CoV- 1-S spike protein and/or the SARS2-CoV-2-S or spike protein.
  • the antibody is a neutralizing and/or blocking anti- SARS2-CoV-2 antibody or antigen-binding fragment.
  • a “neutralizing” or “blocking” antibody or antigen-binding fragment is intended to refer to an antibody or antigen-binding fragment whose binding to the SARS-CoV- 1-S and SARS2- CoV-2-S or spike protein: (i) interferes with and/or blocks the interaction between the SARS- CoV- 1 and its receptor and SARS-CoV-2 and a ACE receptor, such as the human ACE-2 receptor and/or (ii) inhibits the rate of infection and or disease progression.
  • the neutralizing monoclonal antibody and antigen-binding fragments thereof bind SARS-CoV- 1 and/or SARS-CoV-2-S or Spike protein or fragments thereof.
  • an anti- SARS-CoV- 1/SARS-CoV-2 antibody or antigen binding fragment thereof binds to the S1 portion of SARS-CoV-2 Spike protein and the SARS-CoV-1 Spike protein. In some embodiments, an anti- SARS-CoV- 1/SARS-CoV-2 antibody or antigen binding fragment thereof binds RBD portion of the SARS-CoV- 1 and SARS-CoV-2 Spike proteins respectively.
  • the inhibition caused by an anti- SARS-CoV- 1/SARS-CoV-2 neutralizing or blocking antibody may or may not be complete so long as it is detectable using an appropriate assay.
  • any of the antibodies or antigen-binding fragments disclosed herein interferes with the interaction between SARS-CoV- 1 and SARS-CoV-2 and the RBD region of the SARS-CoV- 1-S and SARS-CoV-2-S proteins respectively.
  • the anti- SARS-CoV- 1/SARS-CoV2 antibodies or antigen-binding fragments block the interaction between SARS-CoV-2 and as ACE receptor, such as the human ACE-2 receptor, and SARS-CoV- 1 with its receptor with an IC50 value of less than about 15 nM, as measured by the assay such as that described in the Exemplification section.
  • the IC50 of the SARS-CoV- 1 and/or the anti-SARS-CoV-2 antibody or fragment thereof is measured in an epitope competition assay, such as the epitope competition assay described in the Exemplification section provided herein.
  • the antibodies or antigen-binding fragments of the present disclosure may possess one or more of the aforementioned biological characteristics, or any combinations thereof. Other biological characteristics of the antibodies of the present disclosure will be evident to a person of ordinary skill in the art from a review of the present disclosure including the Exemplification section provided herein.
  • the term "substantial similarity" or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity.
  • residue positions which are not identical differ by conservative amino acid substitutions.
  • any of the antibodies or antigen-binding fragments disclosed herein comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 conservative amino acid substitutions as compared to a reference sequence.
  • a "conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity).
  • R group side chain
  • a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art.
  • 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 are 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 et al. (1992) Science 256: 1443-1445.
  • a "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
  • antibodies immunoglobulins
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called e, h, i, k, and q, respectively.
  • the subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co., 2000).
  • An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.
  • antigen-binding fragments include: (i) Fab fragments; (ii) Fab' fragments; (iii) F(ab')2 fragments; (iv) Fd fragments; (v) Fv fragments; (vi) single-chain Fv (scFv) molecules; (vii) dAb fragments; and (viii) 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, cameliid antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), adnectins, small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment,” as used herein.
  • An antigen-binding fragment of an antibody will typically comprise at least one variable domain (e.g., at least one of a VH or VL).
  • variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences.
  • the VH and VL domains may be situated relative to one another in any suitable arrangement.
  • the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers.
  • the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
  • an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain.
  • Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure 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.
  • the hinge region comprises a glycine-serine linker.
  • an antigen-binding fragment of an antibody of the present disclosure may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
  • antigen-binding fragments may be monospecific or multispecific (e.g., bispecific).
  • a multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen.
  • the anti-SARS-CoV-2 antibodies of the disclosure are human antibodies.
  • the term "human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies of the disclosure 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 some embodiments, CDR3.
  • human antibody is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • the antibodies of the disclosure may, in some embodiments, be recombinant human antibodies.
  • recombinant human antibody is intended to include all human antibodies that are prepared, expressed, or created by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell or other methods that are well known in the art. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • Human antibodies can exist in two forms that are associated with hinge heterogeneity.
  • an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond.
  • the dimers are not linked via inter-chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody).
  • a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody).
  • These forms have been extremely difficult to separate, even after affinity purification.
  • the frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody.
  • a single amino acid substitution in the hinge region of the human lgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed using a human lgG1 hinge.
  • the current disclosure contemplates antibodies having one or more mutations in the hinge, CH2 or CH3 region which may be desirable, for example, in production, to improve the yield of the desired antibody form.
  • the antibodies of the disclosure may be isolated antibodies or isolated antigen- binding fragments.
  • An "isolated antibody” or “isolated antigen-binding fragment,” as used herein, means an antibody or antigen-binding fragment that has been identified and separated and/or recovered from at least one component of its natural environment.
  • an antibody or antigen-binding fragment that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced is an "isolated antibody” or an “isolated antigen-binding fragment” for purposes of the present disclosure.
  • An isolated antibody also includes an antibody in situ within a recombinant cell.
  • Isolated antibodies or antigen-binding fragments are antibodies or antigen-binding fragments that have been subjected to at least one purification or isolation step.
  • an isolated antibody or antigen-binding fragment may be substantially free of other cellular material and/or chemicals.
  • the anti- SARS-CoV- 1/SARS-CoV-2 antibodies or antigen-binding fragments disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived.
  • the present disclosure includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody or antigen- binding fragment was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations").
  • Germline mutations A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof.
  • all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived.
  • only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3.
  • one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived).
  • the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence.
  • antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc.
  • Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present disclosure.
  • the present disclosure also includes anti-SARS-CoV-2 antibodies comprising variants of any of the VH, VL, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions.
  • the present disclosure includes anti-SARS- CoV-2 antibodies having VH, VL, and/or CDR amino acid sequences with, e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conservative amino acid substitutions relative to any of the VH, VL, and/or CDR amino acid sequences disclosed herein.
  • epitope refers to an antigenic determinant that interacts with a specific antigen binding site in the 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. Epitopes may be either conformational or linear.
  • a conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain.
  • a linear epitope is one produced by adjacent amino acid residues in a polypeptide chain.
  • an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.
  • any portion of any of the antibodies or antigen-binding fragments of the disclosure may be similarly modified, such as with an epitope tag, a PEG moiety or moieties, and the like.
  • the antibodies or antigen-binding fragments may comprise more than one epitope tags, such as 2 epitope tags, or may include 0 epitope tags.
  • nucleic acid or fragment thereof indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below.
  • a nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.
  • Sequence similarity for polypeptides which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions.
  • GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof.
  • FASTA e.g., FASTA2 and FASTA3
  • Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters.
  • BLAST Altschul et al. (1990) J. Mol. Biol.215:403-410 and Altschul et al.
  • the sequences are compared using EMBOSS Needle pairwise sequence alignment.
  • Two antibodies or antigen-binding fragments are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single does or multiple dose.
  • antibodies or antigen-binding fragments will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.
  • two antibodies or antigen-binding fragments are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.
  • two antibodies or antigen-binding fragments are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.
  • two antibodies or antigen-binding fragments are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known. Bioequivalence may be demonstrated by in vivo and in vitro methods.
  • Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antibody.
  • Bioequivalent variants of anti-SARS-CoV-2 antibodies of the disclosure may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity.
  • cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation.
  • bioequivalent antibodies or antigen-binding fragments may include anti- SARS-CoV-2 antibody variants comprising amino acid changes which modify the glycosylation characteristics of the antibodies or antigen-binding fragments, e.g., mutations which eliminate or remove glycosylation.
  • the present disclosure provides anti-SARS- CoV1/SARS-CoV-2 antibodies or antigen-binding fragments that bind to SARS-CoV-2-S protein and the SARS-CoV-1-S protein.
  • the antibodies or antigen binding fragments thereof bind the RBD of SARS-CoV-2 and the RBD of SARS-CoV-1.
  • the present disclosure also includes anti- SARS-CoV1/SARS-CoV-2 antibodies that do not cross-react with human proteins.
  • the disclosure encompasses anti- SARS-CoV1/SARS-CoV-2 monoclonal antibodies conjugated to a therapeutic moiety ("immunoconjugate"), such as a cytotoxin or an antiviral agent.
  • a therapeutic moiety such as a cytotoxin or an antiviral agent.
  • the antibodies of the present disclosure may be used in combination therapy.
  • the antibodies of the present disclosure may be monospecific, bi-specific, or multispecific. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., 1991 , J. Immunol. 147:60-69; Kufer ei a/., 2004, Trends Biotechnol.22:238-244.
  • the anti-SARSCoV-2 antibodies or antigen-binding fragments of the present disclosure can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein.
  • an antibody or antigen-binding fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antigen-binding fragment to produce a bi-specific or a multispecific antibody with a second binding specificity.
  • the present disclosure includes bi-specific antibodies wherein one arm of an immunoglobulin is specific for SARS-CoV-2-S or a fragment thereof, such as the S1 region or the RBD region and the other arm of the immunoglobulin is specific for a second CoV target or is conjugated to a therapeutic moiety.
  • An exemplary bi-specific antibody or antigen-binding fragment format that can be used in the context of the present disclosure involves the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to its antigen as compared to a bi- specific antibody lacking the amino acid difference.
  • Ig immunoglobulin
  • second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to its antigen as compared to a bi- specific antibody lacking the amino acid difference.
  • bispecific formats that can be used in the context of the present disclosure include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG- scFv fusions, dual variable domain (DVD)-lg, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, lgG1/lgG2, dual acting Fab (DAF)-lgG, and Mab ⁇ 2>bispecific formats (see, e.g., Klein et al.
  • Bispecific antibodies or antigen-binding fragments can also be constructed using peptide/nucleic acid conjugation, e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates which then self-assemble into multimeric complexes with defined composition, valency and geometry. (See, e.g., Kazane et al., J. Am. C em. Soc. [Epub: Dec.4, 2012]).
  • nucleic Acids Encoding SARS-CoV1/SARS-CoV-2 Antibodies the disclosure provides for a nucleic acid capable of expressing any of the antibodies of antigen-binding fragments disclosed herein.
  • the nucleic acids may be single-stranded or double-stranded, DNA or RNA molecules.
  • the antibody or antigen-binding fragment nucleic acid sequences can be isolated, recombinant, and/or fused with a heterologous nucleotide sequence, or in a DNA library.
  • the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 198, 203, 208, 213, 218, 223, 228, 233, 238, 243, 248, 253, 258, 263, 268, 273, 278, 283, 288, 293, 298, 303, 308, 313, 318, 323, 328, 333, 338, 343, 348, 353, 358, 363, 393, 398, 403, 408, 413, 418, 423, 428, 433, 438,
  • nucleic acids encoding antibodies or antigen-binding fragments also include nucleotide sequences that hybridize under highly stringent conditions to a polynucleotide encoding any of the above-mentioned antibodies or antigen-binding fragments nucleotide sequence, or complement sequences thereof.
  • the nucleic acids hybridize under highly stringent conditions to a polynucleotide encoding an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 198, 203, 208, 213, 218, 223, 228, 233, 238, 243, 248, 253, 258, 263, 268, 273, 278, 283, 288, 293, 298, 303, 308, 313, 318, 323, 328, 333, 338, 343, 348, 353, 358, 363, 393, 398, 403, 408, 413
  • the nucleic acids hybridize under highly stringent conditions to a polynucleotide encoding an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 198, 203, 208, 213, 218, 223, 228, 233, 238, 243, 248, 253, 258, 263, 268, 273, 278, 283, 288, 293, 298, 303, 308, 313, 318, 323, 328, 333, 338, 343, 348, 353, 358, 363, 393, 398, 403, 408, 413
  • appropriate stringency conditions which promote DNA hybridization can be varied. For example, one could perform the hybridization at 6.0 x sodium chloride/sodium citrate (SSC) at about 45 ⁇ C, followed by a wash of 2.0 x SSC at 50 ⁇ C.
  • the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50 ⁇ C to a high stringency of about 0.2 x SSC at 50 ⁇ C.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22 ⁇ C, to high stringency conditions at about 65 ⁇ C. Both temperature and salt may be varied, or temperature or salt concentration may be held constant while the other variable is changed.
  • the disclosure provides nucleic acids which hybridize under low stringency conditions of 6 x SSC at room temperature followed by a wash at 2 x SSC at room temperature.
  • Isolated nucleic acids which differ from the nucleic acids encoding the antibody or antigen-binding fragment thereof due to degeneracy in the genetic code are also within the scope of the disclosure. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in “silent” mutations which do not affect the amino acid sequence of the protein.
  • the disclosure provides for a vector comprising any of the nucleic acids disclosed herein.
  • the disclosure provides for a host cell comprising any of the vectors disclosed herein.
  • an antibody of the disclosure is a full length antibody or an antigen binding fragment
  • antibodies and antigen binding fragments of the disclosure can be recombinantly expressed in cell lines.
  • sequences encoding particular antibodies or antigen binding fragments can be used for transformation of a suitable host cell, such as a mammalian host cell or yeast host cell.
  • transformation can be achieved using any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by transfection procedures known in the art.
  • the transformation procedure used may depend upon the host to be transformed.
  • Methods for introducing heterologous polynucleotides into mammalian cells include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
  • a nucleic acid molecule encoding the amino acid sequence of a heavy chain constant region (all or a portion), a heavy chain variable region of the disclosure, a light chain constant region, or a light chain variable region of the disclosure is inserted into an appropriate expression vector using standard ligation techniques.
  • the heavy or light chain constant region is appended to the C-terminus of the appropriate variable region and is ligated into an expression vector.
  • the vector is typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery such that amplification of the gene and/or expression of the gene can occur).
  • the heavy and light chain may be expressed from the same vector (e.g., from the same or different promoters present on the same vector) or the heavy and light chains may be expressed from different vectors.
  • the heavy and light chains are expressed from different vectors, which are transfected into the same host cell and co- expressed. Regardless of when the heavy and light chains are expressed in the same host cell from the same or a different vector, the chains can then associate to form an antibody (or antibody fragment, depending on the portions of the heavy and light chain being expressed).
  • expression vectors used in any of the host cells will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences.
  • flanking sequences in certain embodiments will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.
  • a promoter one or more enhancer sequences
  • an origin of replication a transcriptional termination sequence
  • a complete intron sequence containing a donor and acceptor splice site a sequence encoding a leader sequence for polypeptide secretion
  • ribosome binding site a sequence encoding a leader sequence for polypeptide secretion
  • polyadenylation sequence a polylinker region for inserting the nucleic acid encoding the poly
  • An origin of replication is typically a part of those prokaryotic expression vectors purchased commercially, and the origin aids in the amplification of the vector in a host cell. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence, and ligated into the vector.
  • the origin of replication from the plasmid pBR322 is suitable for most gram-negative bacteria and various viral origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV), or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells.
  • viral origins e.g., SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV), or papillomaviruses such as HPV or BPV
  • the origin of replication component is not needed for mammalian expression vectors (for example, the SV40 origin is often used only because it also contains the virus early promoter).
  • the expression and cloning vectors of the disclosure will typically contain a promoter that is recognized by the host organism and operably linked to the molecule encoding heavy and/or light chain. Promoters are untranscribed sequences located upstream (i.e., 5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription of the structural gene. Promoters are conventionally grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature.
  • Constitutive promoters initiate continual gene product production; that is, there is little or no control over gene expression.
  • a large number of promoters, recognized by a variety of potential host cells, are well known.
  • a suitable promoter is operably linked to the DNA encoding the heavy chain or light chain comprising an antibody or antigen binding fragment of the disclosure.
  • the same promoter is used for both the heavy and light chain.
  • different promoters are used for each.
  • Suitable promoters for use with yeast hosts are also well known in the art.
  • Yeast enhancers are advantageously used with yeast promoters.
  • Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B virus and most preferably Simian Virus 40 (SV40).
  • viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B virus and most preferably Simian Virus 40 (SV40).
  • adenovirus such as Adenovirus 2
  • bovine papilloma virus such as Adenovirus 2
  • avian sarcoma virus such as Adenovirus
  • Additional promoters which may be of interest include, but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-10); the CMV promoter; the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-97); the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.
  • elastase I gene control region that is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-46; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.
  • the vector may also include an enhancer sequence to increase transcription of DNA encoding light chain or heavy chain.
  • Expression vectors of the disclosure may be constructed from a starting vector such as a commercially available vector. Such vectors may or may not contain all of the desired flanking sequences. Where one or more of the flanking sequences described herein are not already present in the vector, they may be individually obtained and ligated into the vector. Methods used for obtaining each of the flanking sequences are well known to one skilled in the art.
  • the completed vector may be inserted into a suitable host cell for amplification and/or polypeptide expression.
  • the transformation of an expression vector into a selected host cell may be accomplished by well-known methods including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection, or other known techniques. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods are well known to the skilled worker.
  • the host cell when cultured under appropriate conditions, synthesizes the antibody or antigen binding fragment of the disclosure that can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted).
  • the selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule.
  • Mammalian cell lines available as host cells for expression are well known in the art and include, but are not limited to, many immortalized cell lines available from the American Type Culture Collection (A.T.C.C.), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines.
  • A.T.C.C. American Type Culture Collection
  • CHO Chinese hamster ovary
  • HeLa cells HeLa cells
  • BHK baby hamster kidney
  • COS monkey kidney cells
  • human hepatocellular carcinoma cells e.g., Hep G2
  • a heterologous antibody
  • a cell other than a mammalian cell is used, such as a yeast cell line (e.g., Pichia).
  • the cell line stably expresses an antibody or antigen binding fragment of the disclosure.
  • the cells transiently express an antibody or antigen binding fragment of the disclosure.
  • Pharmaceutical Compositions and Modes of Administration The antibodies or agents of the invention (also referred to herein as "active compounds"), and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody or agent and a pharmaceutically acceptable carrier.
  • the term "pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.4,522,811. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • compositions of the present application are useful for the treatment of a disease, disorder, or condition associated with COVID (e.g., SARS-CoV-1 and SARS-CoV-2 infections).
  • treatment is defined as the application or administration of a therapeutic agent to a patient, who has a disease or condition associated with COVID (e.g., SARS-CoV-1 and SARS-CoV-2 infections); or a symptom of, or a predisposition towards such disease or condition associated with COVID (e.g., SARS-CoV-1 and SARS-CoV-2 infections), with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, condition, symptoms thereof or the predisposition thereto.
  • a disease or condition associated with COVID e.g., SARS-CoV-1 and SARS-CoV-2 infections
  • a symptom of, or a predisposition towards such disease or condition associated with COVID e.g., SARS-CoV-1 and SARS-CoV-2 infections
  • the present application provides a method of treating one or more COVID-associated complications (e.g., SARS-CoV-1 and SARS-CoV-2 infections) by administrating an antibody or antigen-binding fragment thereof or a composition as described herein to a patient under conditions that generate a beneficial therapeutic response in the patient.
  • an antibody or antigen-binding fragment thereof as described herein may be administered at a therapeutically effective dose or amount to a patient with COVID infection (e.g., SARS-CoV-1 and SARS-CoV-2 infection).
  • the antibodies or antigen-binding fragments thereof, or compositions comprising any of the foregoing, as described herein are useful to treat subjects suffering from the severe and acute respiratory infection caused by COVID (e.g., SARS- CoV-1 and SARS-CoV-2).
  • COVID e.g., SARS- CoV-1 and SARS-CoV-2
  • the antibodies or antigen-binding fragments thereof, or compositions comprising any of the foregoing, as described herein are useful in decreasing viral titer or reducing viral load in the host.
  • the antibodies or antigen-binding fragments thereof, or compositions comprising any of the foregoing, as described herein are useful in preventing or reducing inflammation in the lung of a subject with COVID infection (e.g., SARS-CoV-1 and SARS-CoV-2 infection).
  • the antibodies or antigen-binding fragments thereof, or compositions comprising any of the foregoing, as described herein are useful in preventing or reducing interstitial, peribronchiolar or perivascular inflammation, alveolar damage and pleural changes in a subject with COVID infection (e.g., SARS-CoV-1 and SARS-CoV-2 infection).
  • the antibodies or antigen-binding fragments thereof, or compositions comprising any of the foregoing, as described herein may be used in or administerted to a subject in need thereof to relieve or prevent or ameliorate or decrease the severity of one or more of the symptoms or conditions of the disease or disorder.
  • the antibodies or antigen-bind fragements thereof, or compositions comprising any of the foregoing may be used to ameliorate or reduce the severity of at least one symptom of COVID infection (e.g., SARS-CoV-1 and SARS-CoV-2 infection), including, but not limited to fever, cough, shortness of breath, pneumonia, diarrhea, organ failure (e.g., kidney failure and renal dysfunction), septic shock, and death.
  • COVID infection e.g., SARS-CoV-1 and SARS-CoV-2 infection
  • the antibodies or antigen-binding fragments thereof, or compositions comprising any of the foregoing, as described in the present applicant may be used prophylactically in subjects at risk for developing COVID infection (e.g., SARS-CoV-1 and SARS-CoV-2 infection), such as immunocompromised individuals, elderly adults (more than 65 years of age), children younger than 2 years of age, travelers, healthcare workers, family members in close proximity to a COVID infection (e.g., SARS-CoV-1 and SARS- CoV-2 infection) patient, adults or children with contact with persons with confirmed or suspected COVID infection (e.g., SARS-CoV-1 and SARS-CoV-2 infection), and patients with a medical history (e.g., increased risk of pulmonary infection, heart disease or diabetes).
  • COVID infection e.g., SARS-CoV-1 and SARS-CoV-2 infection
  • a COVID infection e.g., SARS-CoV-1 and SARS-CoV-2 infection
  • the antibodies or antigen-binding fragments thereof, or compositions comprising any of the foregoing, as described in the present applicant may be used in the preparation of a medicament for treating patients suffering from COVID infection (e.g., SARS-CoV-1 and SARS-CoV-2 infection).
  • COVID infection e.g., SARS-CoV-1 and SARS-CoV-2 infection.
  • the antibodies or antigen-binding fragments thereof, or compositions comprising any of the foregoing, as described in the present application may be used as adjunct therapy with any other agent or any other therapy known to those skilled in the art useful for the treatment of COVID infection (e.g., SARS-CoV-1 and SARS-CoV-2 infection).
  • Target cell line HeLa-hACE2 and A549-hACE2 cells were generated through transduction of human ACE2 lentivirus.
  • pBOB-hACE2 construct was co-transfected into HEK293T cells along with lentiviral packaging plasmids pMDL, pREV, and pVSV-G (Addgene) by Lipofectamine 2000 (ThermoFischer Scientific, 11668019) according to manufacturer’s instructions.
  • Supernatants were collected 32 h after transfection, then were transducted to pre-seeded HeLa or A549 cells.12 h after transduction, stable cell lines were collected, and stored for neutralization assay.
  • the media in the flask was removed and 2 mL of SARS-CoV-2 strain USA-WA1/2020 (BEI Resources NR-52281) in complete DMEM was added to the flask at an MOI of 0.5 and was allowed to incubate for 30 TPU[ZLY HZ ,-e .” 7A2. After incubation, 30 mL of complete DMEM was added to the MSHYR' EOL MSHYR ]HY ZOLU WSHJLK PU H ,-e PUJ[IHZVX HZ .” 7A2 for 5 days. On day 5 post infection the supernatant was harvested and centrifuged at 1,000 ⁇ g for 5 minutes.
  • Neutralization ID50 or IC50 titers were calculated using “One-Site Fit LogIC50” regression in GraphPad Prism 8.0. The results from the neutralization assay are reproduced in table 2A and 2B below and the sequence information relating to each of the clones set forth below is incorporated by reference from U.S. Application Serial Nos: 63/021,086 ; 63/021,676; 63/024,512; 63/035,554; 63/036,405 and 63/038,093.
  • the plates are then washed three times with 100 ⁇ L of 1x PBS + 0.05% tween and subsequently 50 ul of a dilution series of monoclonal antibodies were added to the plate and incubated at RT for 1 hour.
  • the plates are washed again three times with 100 ⁇ L of 1x PBS + 0.05% tween before the addition of 50 ul of alkaline phosphatase conjugated goat anti-human Fc antibody (Jackson Immunoresearch 109- 055-098) diluted at 1:1000 and incubated at RT for 1 hour.
  • Example: 5 Whole Virus ELISA High binding plates may be coated with 12.5 ⁇ L of Galanthus Nivalis Lectin #;@>4 GLJZVX >HIVXHZVXPLY >&*+-)&.$ HZ *) aN(T> HUK PUJ[IHZLK V ⁇ LXUPNOZ HZ -b7' EOL ;@> was removed and 12.5 ⁇ L of SARS-CoV-2 was added to the plate at a concentration of 2x10 6 WM[(T> ZOLU PUJ[IHZLK MVX +- O HZ -b7' *+'.
  • the plate was then washed three times with 100 ⁇ L of 1x PBS supplemented with 0.05% tween.
  • 50 ⁇ L of 3% BSA were added to the plate and incubated at RT for 2 h.
  • the BSA was removed and 12.5 ⁇ L of plasma or mAb diluted in series was added to the plate then incubated at RT for 1.5 h.
  • the plate was then washed three times with 100 ⁇ L of 1x PBS supplemented with 0.05% tween.
  • Example: 6 Plasmid construction for full-length and recombinant soluble proteins To generate full-length SARS-CoV-1 (1255 amino acids; GenBank: AAP13567) and SARS- CoV-2 (1273 amino acids; GenBank: MN908947) spike genes were synthesized by GeneArt (Life Technologies) and cloned into the mammalian expression vector phCMV3 (Genlantis, USA) using PstI and BamH restriction sites. Expression plasmids for soluble S ectodomain protein SARS-CoV-1 (residue 1-1190) and SARS-CoV-2 (residue 1-1208) were constructed by PCR amplification and Gibson assembly cloning into vector phCMV3.
  • SARS-CoV-2 N-terminal domain-NTD receptor-binding domain-RBD (residue 332-527), RBD-SD1 (residue 320-591), and RBD-SD1-2 (residue 320- 681) subdomains
  • PCR-amplifications were carried out from the SARS-CoV-2 plasmid and gene fragments were cloned in frame with the original secretion signal or the Tissue Plasminogen Activator (TPA) leader sequence.
  • TPA Tissue Plasminogen Activator
  • Example: 7 Flow cytometry based cell surface SARS-CoV-1/CoV-2 spike binding assay Binding of mAbs/sera to the HEK293T cell-surface expressed SARS-CoV-1 and SARS- CoV-2 spikes was performed as described previously (18). Briefly, HEK293T cells were transfected with plasmids encoding full-length SARS-CoV-1 or SARS-CoV-2 spikes and incubated for 36-48 h at 37 o C.
  • Post incubation cells were trypsinized to prepare a single cell suspension and were distributed into 96-well plates.50 ⁇ l/well of 3-fold serial titrations of T5IY YZHXZPUN HZ *) dN(TS VX YLX[T YHTWSLY YZHXZPUN HZ *3,) KPS[ZPVU ]LXL HKKLK ZV transfected cells.
  • the Abs were incubated with cells for 1h on ice.
  • the plates were washed Z]PJL PU :57D I[MMLX #* ⁇ B6D% +" :6D% * T?
  • protein-encoding plasmids may be transfected into FreeStyle293F cells (Thermo Fisher) at a density of approximately 1 million cells/mL.
  • the premixed 40K PEI- transfectagroTM solution was gently poured into the filtered plasmid solution.
  • the solution was thoroughly mixed by inverting the tube several times.
  • the mixture rested at room temperature for 30 min and was poured into 1 L FreeStyle293F cell culture.
  • the cells were removed from the supernatant by centrifuging at 3500 rpm for 15 min.
  • the supernatant was filtered in a glass IVZZSL ]PZO H )'++ dT TLTIXHUL HUK RLWZ PU - o C storage before loading into the columns.
  • the His-tagged proteins were purified with the HisPur Ni-NTA Resin (Thermo Fisher).
  • each column was washed with at least 3 bed volumes of wash buffer (25 mM Imidazole, pH 7.4).
  • wash buffer 25 mM Imidazole, pH 7.4
  • To elute the purified proteins from the column we loaded 25 mL of the elution buffer (250 mM Imidazole, pH 7.4) at slow gravity speed ( ⁇ 4 sec/drop).
  • Amicon tubes we buffer exchanged the solution with PBS and concentrated the proteins.
  • the proteins were further purified by size-exclusion chromatography using Superdex 200 (GE Healthcare). The selected fractions were pooled and concentrated again for further use.
  • Detection was measured with alkaline phosphatase-conjugated goat anti-human IgG Fc ⁇ (Jackson ImmunoResearch) at 1:1000 dilution for lh. After the final wash, phosphatase substrate (Sigma- Aldrich) was added into wells. Absorption was measured at 405 nm. Non-linear regression curves were analyzed using Prism 8 software to calculate EC50 values.
  • 10 Antibody expression and purification Antibodies HC and LC constructs are transiently expressed with the Expi293 Expression System (Thermo fisher). After 4 days, 24-deep well culture supernatants were harvested to be directly tested for binding and neutralization.
  • Selected mAbs showing neutralizing activity in the HTP screening were re-expressed in small to medium scale cultures using individual colony plasmid DNA, and IgG purified on Protein A sepharose (GE Healthcare).
  • the antibodies targeting the RBD-A epitope compete best against the ACE2 receptor and the neutralization IC50 correlates well with the percent competition for ACE2 receptor binding for both S protein and for RBD.
  • the affinity of all RBD-specific antibodies to soluble RBD by surface plasmon resonance (SPR) was also assessed a poor correlation between affinity and neutralization potency was found (Table 3). However, the correlation is higher when limited to antibodies targeting the RBD-A epitope.
  • a library was constructed by sampling all possible single mutations across each of the CDR loops (highlighted in blue) excluding the addition of additional methionine and cysteine. The library size of each loop is ⁇ 150 individual variants.
  • Each of the individual CDR libraries was combined together resulting in a library with up to 3 mutations per chain (one per CDR loop) that contained ⁇ 3 million individual variants. Initially two separate libraries were created: the heavy chain library paired with the unmodified CR3022 light chain (HC library) and the light chain library paired with the unmodified CR3022 heavy chain (LC library).
  • Table: 4 Example: 14 Binding of mAbs to SARS1 and SARS2 RBD eCR3022 IgG was immobilized in a Biacore 8K and different concentrations of SARS1 or SARS2 RBD was flown as analyze to determine the affinities of the different antibody variants. Using similar methods described in the Examples herein, the binding of CR3022 variant mAbs to SARS1 and SARS2 RBD was determined as shown in Table 5A and Table 5B respectively. Table: 5A Table: 5B Example: 15 eCR3022 Pseudovirus Neutralization Assay Using similar methods described in Example 3, the eCR3022 mAbs were assayed for SARS-Cov-2 and SARS-Cov neutralization. Neutralization ID50 or IC50 titers were calculated using “One-Site Fit LogIC50” regression in GraphPad Prism 8.0. The results from the eCR3022 pseudovirus neutralization assay are reproduced in Table 6 below. Table: 6

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Abstract

Cette invention concerne des compositions et des procédés de traitement, de prévention ou de réduction du taux de progression et/ou de la gravité de la COVID-19, en particulier de traitement, de prévention ou de réduction de la vitesse de progression et/ou de la gravité d'une ou de plusieurs complications associées à la COVID-19.
PCT/US2022/037330 2021-07-16 2022-07-15 Anticorps de coronavirus et leurs utilisations Ceased WO2023288078A1 (fr)

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