WO2021203397A1 - Anti-sars-cov-2 antibodies and uses thereof - Google Patents

Anti-sars-cov-2 antibodies and uses thereof Download PDF

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Publication number
WO2021203397A1
WO2021203397A1 PCT/CN2020/084097 CN2020084097W WO2021203397A1 WO 2021203397 A1 WO2021203397 A1 WO 2021203397A1 CN 2020084097 W CN2020084097 W CN 2020084097W WO 2021203397 A1 WO2021203397 A1 WO 2021203397A1
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Prior art keywords
seq
antibody
sequence
cov
sars
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PCT/CN2020/084097
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French (fr)
Inventor
Zheng Zhang
Linqi Zhang
Lei Liu
Qi Zhang
Bin JU
Xuanling SHI
Qing Zhu
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Tsb Therapeutics (Beijing) Co. Ltd.
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Application filed by Tsb Therapeutics (Beijing) Co. Ltd. filed Critical Tsb Therapeutics (Beijing) Co. Ltd.
Priority to PCT/CN2020/084097 priority Critical patent/WO2021203397A1/en
Priority to US16/953,304 priority patent/US11365239B2/en
Priority to PCT/CN2021/081739 priority patent/WO2021185346A1/en
Priority to CN202180007473.2A priority patent/CN115315443A/en
Priority to TW110110113A priority patent/TW202200613A/en
Publication of WO2021203397A1 publication Critical patent/WO2021203397A1/en
Priority to US17/569,487 priority patent/US20220204591A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • 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
    • 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
    • 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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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

  • the present disclosure generally relates to novel anti-SARS-COV-2 antibodies.
  • SARS-CoV-2 is a positive-sense single-stranded RNA (+ssRNA) virus which belongs to the betacoronavirus family and shares substantial genetic and functional similarity with other pathogenic human betacoronaviruses, including Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV, also called SARS-CoV-1) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) .
  • SARS-CoV Severe Acute Respiratory Syndrome Coronavirus
  • SARS-CoV-1 Severe Acute Respiratory Syndrome Coronavirus
  • MERS-CoV Middle East Respiratory Syndrome Coronavirus
  • SARS-CoV-2 has four structural proteins, known as the S (spike) , E (envelope) , M (membrane) , and N (nucleocapsid) proteins; the S, E, and M proteins together create the viral envelope; inside the envelope is the N protein bounding to the RNA genome ( ⁇ 30 kb) in a continuous beads-on-a-string type conformation.
  • the spike protein is the protein responsible for allowing the SARS-CoV-2 virus to attach to the membrane of a host cell, the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 recognizes and attaches to the angiotensin-converting enzyme 2 (ACE2) receptor of host cells to use them as a mechanism of cell entry.
  • RBD receptor binding domain
  • ACE2 angiotensin-converting enzyme 2
  • the overall ACE2-binding mechanism is virtually the same between SARS-CoV-2 RBD and SARS-CoV RBD, indicating convergent ACE2-binding evolution between these two viruses. This suggests that disruption of the RBD and ACE2 interaction would block the entry of SARS-CoV-2 into the target cell. Indeed, a few such disruptive agents targeted to ACE2 have been shown to inhibit SARS-CoV infection.
  • Anti-RBD antibodies are therefore more favorable.
  • SARS-CoV-RBD or MERS-CoV RBD-based vaccine studies in experimental animals have also shown strong polyclonal antibody responses that inhibit viral entry. Such critical proof-of-concept findings indicate that anti-RBD antibodies might effectively block SARS-CoV-2 entry.
  • an antibody means one antibody or more than one antibody.
  • the present disclosure provides an isolated or recombinant antibody or an antigen-binding fragment thereof, which is capable of specifically binding to SARS-CoV-2, and exhibiting at least 50%less binding or non-detectable binding to SARS-CoV or MERS-CoV.
  • the present disclosure provides an isolated or recombinant antibody or an antigen-binding fragment thereof, having one or more features selected from the group consisting of: a) capable of specifically binding to spike protein of SARS-CoV-2 and exhibiting at least 50%less binding to spike protein of SARS-CoV or spike protein of MERS-CoV; b) capable of specifically binding to receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 comprising the amino acid sequence of SEQ ID NO: 128; c) exhibiting binding to RBD of spike protein of SARS-CoV comprising the amino acid sequence of SEQ ID NO: 124 at a level that is non-detectable or that is no more than 50%of the binding to the RBD of spike protein of SARS-CoV-2; d) exhibiting binding to RBD of spike protein of MERS-CoV comprising the amino acid sequence of SEQ ID NO: 126 at a level that is non-detectable or that is no more than 50%of the binding to RBD of the spike protein
  • the present disclosure provides an isolated or recombinant antibody or an antigen-binding fragment thereof capable of specifically binding to RBD of spike protein of SARS-CoV-2.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 98, SEQ ID NO: 99, and SEQ ID NO: 100.
  • the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110.
  • the antibody or antigen binding fragment of the present disclosure comprises a heavy chain CDR1 (HCDR1) comprising the sequence of SEQ ID NO: 1, a heavy chain CDR2 (HCDR2) comprising the sequence of SEQ ID NO: 2, a heavy chain CDR3 (HCDR3) comprising the sequence of SEQ ID NO: 3; a light chain CDR1 (LCDR1) comprising the sequence of SEQ ID NO: 4, a light chain CDR2 (LCDR2) comprising the sequence of SEQ ID NO: 5, and a light chain CDR3 (LCDR3) comprising the sequence of SEQ ID NO: 6.
  • HCDR1 comprising the sequence of SEQ ID NO: 1
  • HCDR2 comprising the sequence of SEQ ID NO: 2
  • HCDR3 HCDR3
  • LCDR1 light chain CDR1
  • LCDR2 light chain CDR2
  • LCDR3 light chain CDR3
  • the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 11, a HCDR2 comprising the sequence of SEQ ID NO: 12, a HCDR3 comprising the sequence of SEQ ID NO: 13, a LCDR1 comprising the sequence of SEQ ID NO: 14, a LCDR2 comprising the sequence of SEQ ID NO: 15, and a LCDR3 comprising the sequence of SEQ ID NO: 16.
  • the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 21, a HCDR2 comprising the sequence of SEQ ID NO: 22, a HCDR3 comprising the sequence of SEQ ID NO: 23, a LCDR1 comprising the sequence of SEQ ID NO: 24, a LCDR2 comprising the sequence of SEQ ID NO: 25, and a LCDR3 comprising the sequence of SEQ ID NO: 26.
  • the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 31, a HCDR2 comprising the sequence of SEQ ID NO: 32, a HCDR3 comprising the sequence of SEQ ID NO: 33, a LCDR1 comprising the sequence of SEQ ID NO: 34, a LCDR2 comprising the sequence of SEQ ID NO: 35, and a LCDR3 comprising the sequence of SEQ ID NO: 36.
  • the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 41, a HCDR2 comprising the sequence of SEQ ID NO: 42, a HCDR3 comprising the sequence of SEQ ID NO: 43, a LCDR1 comprising the sequence of SEQ ID NO: 44, a LCDR2 comprising the sequence of SEQ ID NO: 45, and a LCDR3 comprising the sequence of SEQ ID NO: 46.
  • the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 51, a HCDR2 comprising the sequence of SEQ ID NO: 52, a HCDR3 comprising the sequence of SEQ ID NO: 53, a LCDR1 comprising the sequence of SEQ ID NO: 54, a LCDR2 comprising the sequence of SEQ ID NO: 55, and a LCDR3 comprising the sequence of SEQ ID NO: 56.
  • the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 65, a HCDR2 comprising the sequence of SEQ ID NO: 66, a HCDR3 comprising the sequence of SEQ ID NO: 67, a LCDR1 comprising the sequence of SEQ ID NO: 68, a LCDR2 comprising the sequence of SEQ ID NO: 69, and a LCDR3 comprising the sequence of SEQ ID NO: 70.
  • the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 75, a HCDR2 comprising the sequence of SEQ ID NO: 76, a HCDR3 comprising the sequence of SEQ ID NO: 77, a LCDR1 comprising the sequence of SEQ ID NO: 78, a LCDR2 comprising the sequence of SEQ ID NO: 79, and a LCDR3 comprising the sequence of SEQ ID NO: 80.
  • the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 85, a HCDR2 comprising the sequence of SEQ ID NO: 86, a HCDR3 comprising the sequence of SEQ ID NO: 87, a LCDR1 comprising the sequence of SEQ ID NO: 88, a LCDR2 comprising the sequence of SEQ ID NO: 89, and a LCDR3 comprising the sequence of SEQ ID NO: 90.
  • the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 95, a HCDR2 comprising the sequence of SEQ ID NO: 96, a HCDR3 comprising the sequence of SEQ ID NO: 97, a LCDR1 comprising the sequence of SEQ ID NO: 98, a LCDR2 comprising the sequence of SEQ ID NO: 99, and a LCDR3 comprising the sequence of SEQ ID NO: 100.
  • the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 105, a HCDR2 comprising the sequence of SEQ ID NO: 106, a HCDR3 comprising the sequence of SEQ ID NO: 107, a LCDR1 comprising the sequence of SEQ ID NO: 108, a LCDR2 comprising the sequence of SEQ ID NO: 109, and a LCDR3 comprising the sequence of SEQ ID NO: 110.
  • the antibody or antigen binding fragment of the present disclosure comprises a heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 7, 17, 27, 37, 47, 57, 61, 71, 81, 91, 101, and 111, or a homologous sequence thereof having at least 80%sequence identity.
  • the antibody or antigen binding fragment of the present disclosure comprises a light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 8, 18, 28, 38, 48, 58, 62, 72, 82, 92, 102, and 112, or a homologous sequence thereof having at least 80%sequence identity.
  • the antibody or antigen binding fragment of the present disclosure comprises a pair of heavy chain variable region and light chain variable region sequences selected from the group consisting of: SEQ ID NOs: 7/8, 17/18, 27/28, 37/38, 47/48, 57/58, 61/62, 71/72, 81/82, 91/92, 101/102, and 111/112, or a pair of homologous sequences thereof having at least 80%sequence identity yet retaining specific binding affinity to RBD of spike protein of SARS-CoV-2.
  • the antibody or antigen binding fragment of the present disclosure further comprises an immunoglobulin constant region.
  • the immunoglobulin constant region is a constant region of human immunoglobulin.
  • the immunoglobulin constant region is a constant region of human IgG.
  • the antibody or antigen binding fragment of the present disclosure comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 or IgM.
  • the antibody or antigen binding fragment of the present disclosure comprises a heavy chain constant region of human IgG1.
  • the antibody or antigen binding fragment of the present disclosure comprises a constant region of human immunoglobulin kappa 1 light chain.
  • the antibody or antigen binding fragment of the present disclosure comprises a constant region of human immunoglobulin lambda light chain.
  • the antibody or antigen binding fragment of the present disclosure comprises one or more amino acid residue substitutions or modifications yet retains specific binding affinity to RBD of spike protein of SARS-CoV-2.
  • the antibody or antigen binding fragment is an affinity variant, a glycosylation variant, a cysteine-engineered variant, or an Fc variant.
  • the glycosylation variant comprises a mutation at N297 (e.g. N297A, N297Q, or N297G) , for example, to modify the glycosylation site.
  • N297 e.g. N297A, N297Q, or N297G
  • the Fc variant comprises one or more amino acid residue modifications or substitutions resulting in increased effector functions relative to a wildtype Fc.
  • the Fc variant comprises one or more amino acid substitution (s) at one or more of the positions selected from the group consisting of: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 246, 247, 248, 249, 252, 254, 255, 256, 258, 260, 262, 263, 264, 265, 267, 268, 269, 270, 272, 274, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 300, 301, 303, 304, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 345
  • the Fc variant comprises one or more amino acid substitution selected from the group consisting of 234Y, 235Q, 236A, 236W, 239D, 239E, 239M, 243L, 247I, 267E, 268D, 268E, 268F, 270E, 280H, 290S, 292P, 298A, 298D, 298V, 300L, 305I, 324T, 326A, 326D, 326W, 330L, 330M, 333S, 332D, 332E, 333A, 334A, 334E, 339D, 339Q, 345R, 396L, 430G, 440Y, and any combination thereof.
  • the Fc variant having increased effector function comprises a combination of mutations selected from the group consisting of: a) S239D, I332E, and A330L; b) F243L, R292P, Y300L, V305I and P396L; c) S239D and I332E; d) S239D, I332E and A330L; e) S298A, E333A and K334A; f) L234Y, L235Q, G236W, S239M, H268D, D270E and S298A (in one heavy chain) and D270E, K326D, A330M and K334E (in the opposing heavy chain) ; G236A, S239D and I332E; g) K326W and E333S; h) S267E, H268F and S324T; i) E345R, E430G and S440Y.
  • the Fc variant comprises one or more amino acid residue modifications or substitutions resulting in reduced effector functions relative to a wildtype Fc.
  • the Fc variant comprises one or more amino acid substitution (s) at a position selected from the group consisting of: 220, 226, 229, 233, 234, 235, 236, 237, 238, 267, 268, 269, 270, 297, 309, 318, 320, 322, 325, 328, 329, 330, and 331 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat.
  • the Fc variant comprises one or more amino acid substitution (s) selected from the group consisting of 220S, 226S, 228P, 229S, 233P, 234V, 234G, 234A, 234F, 234A, 235A, 235G, 235E, 236E, 236R, 237A, 237K, 238S, 267R, 268A, 268Q, 269R, 297A, 297Q, 297G, 309L, 318A, 322A, 325L, 328R, 330S, 331S and any combination thereof.
  • amino acid substitution selected from the group consisting of 220S, 226S, 228P, 229S, 233P, 234V, 234G, 234A, 234F, 234A, 235A, 235G, 235E, 236E, 236R, 237A, 237K, 238S, 267R, 268A, 268Q, 269R, 2
  • the Fc variant having reduced effector function comprises a combination of mutations selected from the group consisting of: a) K322A, L234A, and L235A; b) P331S, L234F, and L235E; c) L234A and L235A; c) N297A; d) N297Q; e) N297G; f) L235E; g) L234A and L235A (IgG1) ; h) F234A and L235A (IgG4) ; i) H268Q, V309L, A330S and P331S (IgG2) ; j) V234A, G237A, P238S, H268A, V309L, A330S and P331S (IgG2) .
  • the Fc variant comprises one or more amino acid residue modifications or substitutions resulting in improved binding affinity to neonatal Fc receptor (FcRn) at pH 6.0 while retaining minimal binding at pH 7.4, or increased serum half life of the antibody.
  • the Fc variant comprises one or more amino acid substitution (s) at a position selected from the group consisting of: 234 (e.g., with F) , 235 (e.g., with Q) , 238 (e.g., with D) , 250 (e.g., with E or Q) , 252 (e.g., with L/Y/F/W or T) , 254 (e.g., with S or T) , 256 (e.g., with S/R/Q/E/D or T) ; 259 (e.g., with I) ; 272 (e.g., with A) , 305 (e.g., with A) , 307 (e.g., with A or P
  • the Fc variant comprises one or more amino acid substitution (s) selected from the group consisting of 234F, 235Q, 238D, 250Q, 252T, 252Y, 254T, 256E, 259I, 272A, 305A, 307A, 308F, 311A, 322Q, 328E, 331S, 380A, 428L, 432C, 433K, 433S, 434S, 434Y, 434F, 434W, 434A, 435H, 436L, 437C and any combination thereof.
  • amino acid substitution selected from the group consisting of 234F, 235Q, 238D, 250Q, 252T, 252Y, 254T, 256E, 259I, 272A, 305A, 307A, 308F, 311A, 322Q, 328E, 331S, 380A, 428L, 432C, 433K, 433S, 434S, 434Y, 434F
  • the Fc variant having increased serum half life or improved pH-dependent binding to FcRn comprises a combination of mutations selected from the group consisting of: a) M428L and N434S; b) P238D and L328E; c) M252Y, S254T and T256E; d) L234F, L235Q, K322Q, M252T, S254T and T256E; e) M428L, V259I and V308F; f) H433K and N434Y; g) H433K and N434F; h) T250Q and M428L; i) T307A, E380A and N434A; and j) 432C, 433S, 434W, 435H, 436L, 437C.
  • At least one of the substitutions or modifications is in one or more of the CDR sequences. In some embodiments, at least one of the substitutions or modifications is in one or more of the non-CDR sequences of the heavy chain variable region or light chain variable region. In some embodiments, at least one of the substitutions is a conservative substitution.
  • the antibody or antigen binding fragment of the present disclosure is a monoclonal antibody, a bispecific antibody, a multi-specific antibody, a recombinant antibody, a labeled antibody, a bivalent antibody, an anti-idiotypic antibody, a fusion protein, or a dimerized or polymerized antibody, or a modified antibody (e.g. glycosylated antibody) .
  • the antibody or antigen binding fragment of the present disclosure is a diabody, a Fab, a Fab', a F (ab') 2 , a Fd, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2 , a bispecific dsFv (dsFv-dsFv') , a disulfide stabilized diabody (ds diabody) , a single-chain antibody molecule (scFv) , an scFv dimer (bivalent diabody) , a bispecific scFv dimer, a multispecific antibody, a heavy chain antibody, a camelized single domain antibody, a nanobody, a domain antibody, or a bivalent domain antibody.
  • the antibody or antigen binding fragment of the present disclosure is a full human antibody.
  • the antibody or antigen binding fragment of the present disclosure is linked to one or more conjugate moieties.
  • the conjugate moiety comprises a therapeutic agent, a radioactive isotope, a detectable label, a pharmacokinetic modifying moiety, or a purifying moiety.
  • the conjugate moiety is covalently attached either directly or via a linker.
  • the present disclosure provides an isolated or recombinant antibody or an antigen-binding fragment thereof, which competes for binding to RBD of spike protein of SARS-CoV-2 with the antibody or an antigen-binding fragment thereof described herein.
  • the present disclosure provides bispecific antibody molecules comprising an anti-SARS-CoV-2 antibody or antigen-binding fragment thereof as disclosed herein.
  • the bispecific or bivalent antibodies provided herein comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domains is derived from a monoclonal antibody selected from the group consisting of P2A-1A8, P2A-1A9, P2B-2G11, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, and P2C-1F11.
  • the second antigen-binding domain can be derived from any suitable antibody.
  • the bispecific antibodies provided herein comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first and the second antigen-binding domains are derived from any two monoclonal antibodies selected from the group consisting of P2A-1A8, P2A-1A9, P2B-2G11, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, and P2C-1F11.
  • the first and the second antigen-binding domains are derived from P2C-1F11 and P2B-2F6, respectively.
  • the bispecific antibody molecules have at least two distinct antigen-binding sites with different specificities.
  • the bispecific antibody molecules provided herein are capable of binding to different epitopes on the spike protein of SARS-CoV-2 virus. In some embodiments, the two or more antibodies bind to different epitopes in RBD of spike protein of SARS-CoV-2.
  • the bispecific antibody molecules provided herein has a first antigen-binding domains specificity directed to the RBD of the spike protein of SARS-CoV-2 virus and a second antigen-binding domains specificity directed to a second antigen.
  • the present disclosure provides an isolated polynucleotide encoding the antibody or antigen binding fragment thereof as described herein.
  • the isolated polynucleotide of the present disclosure comprises a nucleotide sequence selected from a group consisting of: SEQ ID NOs: 9-10, 19-20, 29-30, 39-40, 49-50, 59-60, 63-64, 73-74, 83-84, 93-94, 103-104, and 113-114, or a homologous sequence thereof having at least 80%sequence identity.
  • the homologue sequence encodes the same protein as encoded by any nucleotide sequence selected from the group consisting of SEQ ID NOs: 9-10, 19-20, 29-30, 39-40, 49-50, 59-60, 63-64, 73-74, 83-84, 93-94, 103-104, and 113-114.
  • the present disclosure provides a vector comprising the isolated polynucleotide of the present disclosure.
  • said vector is an expression vector.
  • the present disclosure provides a host cell comprising the vector of the present disclosure.
  • the present disclosure provides a method of producing the antibody or antigen binding fragment of the present disclosure.
  • the method comprises culturing the host cell of the present disclosure under the condition at which the expression vector of the present disclosure is expressed.
  • the method of the present disclosure further comprises purifying the antibody produced by the host cell.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen binding fragment of any of the preceding claims, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises a combination of two or more antibodies or antigen binding fragments of the present disclosure.
  • the pharmaceutical composition comprises a combination of two or more monoclonal antibodies, each of which comprises heavy chain CDR sequences and light chain CDR sequences derived from an antibody selected from the group consisting of P2A-1A8, P2A-1A9, P2B-2G11, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, and P2C-1F11.
  • the pharmaceutical composition comprises a first antibody comprising heavy chain CDR sequences and light chain CDR sequences derived from P2C-1F11, and a second antibody comprising heavy chain CDR sequences and light chain CDR sequences derived from antibody P2B-2F6.
  • the two or more antibodies or antigen binding fragments bind to different epitopes in RBD of spike protein of SARS-CoV-2.
  • the two or more antibodies comprise a first antibody which comprises P2C-1F11 or an antigen binding fragment thereof, and a second antibody which is selected from the group consisting of P2C-1A3, P2C-1C10, P2B-2F6, and P2A-1B3, or an antigen binding fragment thereof.
  • the two or more antibodies comprise a first antibody which comprises P2C-1A3 or an antigen binding fragment thereof, and a second antibody which is selected from the group consisting of P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof.
  • the two or more antibodies comprise a first antibody which comprises P2B-2F6 or an antigen binding fragment thereof, and a second antibody selected from the group consisting of P2C-1C10, P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof.
  • the two or more antibodies comprise a first antibody which comprises P2A-1B3 or an antigen binding fragment thereof, and a second antibody selected from the group consisting of P2C-1A3, P2C-1C10, P2C-1F11, P2B-2F6, and P2A-1A10, or an antigen binding fragment thereof.
  • the two or more antibodies comprise a first antibody which comprises P2C-1C10 or an antigen binding fragment thereof, and a second antibody selected from the group consisting of P2C-1A3, P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof.
  • the pharmaceutical compositions comprise the polynucleotides encoding the anti-SARS-CoV-2 antibodies or the antigen-binding fragments thereof, and one or more pharmaceutically acceptable carriers.
  • the present disclosure further provides pharmaceutical compositions comprising the polynucleotides encoding the combination of the two or more anti-SARS-CoV-2 antibodies or the antigen-binding fragments thereof, and one or more pharmaceutically acceptable carriers.
  • the polynucleotides comprise an expression vector.
  • the expression vector comprises a viral vector or a non-viral vector.
  • the expression vector is suitable for gene therapy in human.
  • the expression vector comprises a DNA vector or a RNA vector.
  • the pharmaceutical composition further comprises a second active agent, such as a second therapeutic agent or a second prophylactic agent.
  • the present disclosure provides a kit for detecting a SARS-CoV-2 antigen, comprising the antibody or antigen binding fragment of the present disclosure.
  • the kit of further comprises a control reagent comprising RBD of spike protein of the SARS-CoV-2.
  • the kit further comprises a set of reagents for detecting complex of the antibody or the antigen-binding fragment bound to the SARS-CoV-2 antigen.
  • the present disclosure provides a method of treating SARS-CoV-2 infection in a subject.
  • the present disclosure also provides methods of treating a disease, disorder or condition associated with SARs-CoV-2 infection in a subject.
  • the method comprises administering a therapeutically effective amount of one or more of the antibody, the antigen binding fragment, or one or more polynucleotides encoding one or more of the antibody or antigen-binding fragment thereof provided herein, or the pharmaceutical composition of the present disclosure to the subject.
  • the present disclosure provides a method of preventing SARS-CoV-2 infection in a subject.
  • the present disclosure also provides methods of preventing a disease, disorder or condition associated with SARs-CoV-2 infection in a subject.
  • the method comprises administering a prophylactically effective amount of one or more of the antibody or antigen binding fragment, or the pharmaceutical composition of the present disclosure to the subject.
  • the administration is via oral, nasal, intravenous, subcutaneous, or intramuscular administration.
  • the subject is human.
  • the polynucleotide provided herein can be administered to a subject by, for example, transfection techniques such as electroporation, or hydrodynamic injection.
  • the polynucleotides comprise viral vectors such as AAV, and can be administered via local injection (e.g. intramuscular, intranasal, intradermal, subcutaneous, etc. ) or systematic administration (e.g. intravenous administration) .
  • the method further comprises administering a therapeutically effective amount of a second active agent which can be a therapeutic agent or a prophylactic agent.
  • the second therapeutic agent is an anti-viral agent.
  • an anti-viral agent comprises an antiviral peptide, an anti-viral antibody, an anti-viral compound, an anti-viral cytokine, or an anti-viral oligonucleotide.
  • the second therapeutic agent is an RNA dependent RNA polymerase inhibitor, a non-nucleoside reverse transcriptase inhibitor (NNRTI) , nucleoside reverse transcriptase inhibitor (NRTI) , purine nucleoside, antiviral interferon, adamantine antiviral compound, or any other suitable antiviral agent.
  • NRTI non-nucleoside reverse transcriptase inhibitor
  • NRTI nucleoside reverse transcriptase inhibitor
  • purine nucleoside purine nucleoside
  • antiviral interferon adamantine antiviral compound
  • adamantine antiviral compound or any other suitable antiviral agent.
  • the second therapeutic agent is remdesivir, chloroquine, hydroxychloroquine, lopinavir, ritonavir, APN01, favilavir, mesalazine, toremifene, eplerenone, paroxetine, sirolimus, dactinomycin, irbesartan, emodin, mercaptopurine, melatonin, quinacrine, carvedilol, colchicine, camphor, equilin, oxymetholone, nafamosta, camostat, baricitinib, darunavir, ribavirin, galidesivir, BCX-4430, Arbidol, nitazoxanide, derivatives thereof, or any combination thereof.
  • the present disclosure provides a method of detecting presence or amount of SARS-CoV-2 virus antigen in a sample.
  • the method comprises contacting the sample with one or more of the antibody or antigen binding fragment of the present disclosure, and determining the presence or the amount of the SARS-CoV-2 virus antigen in the sample.
  • the present disclosure provides use of one or more of the antibody or antigen binding fragment of the present disclosure in the manufacture of a medicament for treating or preventing SARS-CoV-2 infection or a disease, disorder or condition associated with SARs-CoV-2 infection. In one aspect, the present disclosure provides use of one or more of the antibody or antigen binding fragment of the present disclosure in the manufacture of a medicament for preventing, managing, treating and/or ameliorating in a subject a disease or a disorder caused by or associated with coronavirus (e.g. SARs-COV-2) infection and/or a symptom or respiratory condition relating thereto.
  • coronavirus e.g. SARs-COV-2
  • the present disclosure provides use of one or more of the antibody or antigen binding fragment of the present disclosure in the manufacture of a diagnostic reagent for detecting SARS-CoV-2 infection.
  • the present disclosure provides a kit for detecting an antibody capable of specifically binding to receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, comprising a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 128.
  • the polypeptide is immobilized on a substrate.
  • the kit further comprises a set of reagents for detecting complex of the antibody bound to the polypeptide.
  • the present disclosure provides a method of detecting presence or amount of an antibody capable of specifically binding to RBD of the spike protein of SARS-CoV-2 in a sample, comprising contacting the sample with a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 128, and determining the presence or the level of the antibody in the sample.
  • the absence of the antibody in the sample or the level of the antibody in the sample being below a threshold indicates that the subject is more likely to suffer from disease progression.
  • the present disclosure provides a method of determining the likelihood of disease progression in a subject infected with SARS-CoV-2, the method comprising: contacting a sample obtained from the subject with a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 128, and detecting the presence or the level of an antibody in the sample wherein the antibody is capable of specifically binding to RBD of the spike protein of the SARS-CoV-2, wherein the subject is likely to experience disease progression when the antibody in the sample is absent or is below a threshold.
  • the present disclosure provides a method of monitoring treatment response in a subject infected with SARS-CoV-2 and received a treatment, the method comprising: (i) contacting a sample from the subject with a peptide comprising an amino acid sequence of SEQ ID NO: 128; (ii) detecting a first level of an antibody in the sample wherein the antibody is capable of specifically binding to RBD of the spike protein of the SARS-CoV-2; and (iii) comparing the first level of the antibody with a second level of the antibody detected in the subject prior to the treatment; wherein the first level being higher than the second level indicates that the subject is responsive to the treatment.
  • the present disclosure provides a method of neutralizing SARS-CoV-2 in a subject or in a sample in vitro, comprising administering a therapeutically effective amount of one or more of the antibody or antigen binding fragment thereof provided herein, or the pharmaceutical composition provided herein to the subject or to the sample.
  • FIG. 1 Analyses of plasma and B cell responses specific to SARS-CoV-2. Serial dilutions of plasma samples were analyzed for binding to the (A) RBDs or (B) trimeric Spikes of SARS-CoV-2, SARS-CoV and MERS-CoV by ELISA and (C) for neutralizing activity against pseudoviruses bearing envelope glycoprotein of SARS-CoV-2, SARS-CoV and MERS-CoV. Binding to SARS-CoV-2 NP protein was also evaluated (A) . All results were derived from at least two independent experiments. (D) Gating strategy for analysis and isolation of RBD-specific memory B cells and (E) their representation among the total and memory subpopulation of B cells in the eight study subjects. Samples were named as either A, B, or C depending on collection sequence. FSC-W, forward scatter width; FSC-A, forward scatter area; and SSC-A side scatter area.
  • FSC-W forward scatter width
  • FSC-A forward scatter area
  • FIG. 1 Heavy chain repertoires of SARS-CoV-2 RBD-specific antibodies analyzed (A) by individual subject or (B) across the eight subjects.
  • A Distribution and frequency of heavy chain variable (VH) genes usage in each subject shown along the horizontal bar. The same color scheme is used for each VH family across all study subjects. The VHs that dominate across isolated antibodies are indicated by actual frequencies in their respective color boxes. The number of RBD-binding antibodies versus total antibodies isolated are shown on the right.
  • B Clustering of VH genes and their association with ELISA binding activity across the eight subjects. Unrooted phylogenetic tree depicting the genetic relationships among all VH genes of the RBD-binding antibodies. Branch lengths are drawn to scale so that sequence relatedness can be readily assessed.
  • FIG. 3 Clonal expansion of specific heavy and light chain families in the P#2 antibody repertoire.
  • A Phylogenetic analysis of VH (left) and VL 20 (right) genes for all RBD-binding antibodies. Clonal expanded VH and VL clusters are paired and highlighted in three different colors. Branch lengths are drawn to scale so that sequence relatedness can be readily assessed.
  • B Clonal expansion in relation to members of other VH and VL families based on somatic hypermutations (SHM) and CDR3 loop lengths. For the pie charts of VH (left) and VL (right) genes, the radii represent the CDR3 loop length and the color scale indicates the degree of SHM. Heavy and light chain repertoires for each antibody are shown along the pie circles.
  • FIG. 4 Antibody neutralization analyzed by pseudovirus and live SARS-CoV-2.
  • A Quality control of antibody through ELISA analysis prior to neutralization assay. A serial dilution of each antibody was evaluated against SARS-CoV-2 RBD coated on the ELISA plate and their binding activity was recorded at an optical density (OD) of 450nm and 630nm.
  • B-C Antibody neutralization analyzed by pseudovirus (B) or live SARS-CoV-2 (C) . A serial dilution of each antibody was tested against pseudovirus while two dilutions against live SARS-CoV-2. Cytopathic effects (CPE) were observed daily and recorded on Day 2 post-exposure. Selected antibodies and their concentrations tested are indicated at the upper left corner.
  • CPE Cytopathic effects
  • FIG. 5 Analysis of plasma binding to cell surface expressed trimeric Spike protein.
  • HEK 293T cells transfected with expression plasmid encoding the full length spike of SARS-CoV-2, SARS-CoV or MERS-CoV were incubated with 1: 100 dilutions of convalescent plasma from the study subjects. The cells were then stained with PE labeled anti-human IgG Fc secondary antibody and analyzed by FACS. Positive control antibodies include S230 and m396 targeting the RBD of SARS-CoV Spike, and Mab-GD33 targeting the RBD of MERS-CoV Spike. VRC01 is negative control antibody targeting HIV-1 envelope glycoprotein.
  • RBD-specific memory B cells analyzed and isolated through FACS.
  • the recombinant RBD was labeled with either a Strep or His tag and used alone or in combination to identify and isolate RBD-specific single B cells through staining with the Streptavidin-APC and/or Streptavidin-PE, or anti-His-APC and anti-His-PE antibodies.
  • B cells to be isolated are highlighted in boxes or ovals. Samples were named as either A, B, or C depending on collection sequence.
  • FSC-W forward scatter width
  • FSC-A forward scatter area
  • SSC-A side scatter area SSC-A side scatter area.
  • FIG. 7 ELISA screening of SARS-CoV-2 RBD-specific antibodies in the supernatant of transfected cells. The study subjects and the date of sampling are indicated on the top. Samples were named as either A, B, or C depending on collection sequence. Antibodies tested for each sample are aligned in one vertical column whenever possible. For each evaluated antibody, at least two independent measurements were performed and are presented adjacently on the same row. Binding activities were assessed by OD 450 and indicated by the color scheme on the right. Negatives (no binding activity) are shown in gray for OD 450 values less than 0.1.
  • FIG. 8 Binding kinetics of isolated mAbs with SARS-CoV-2 RBD measured by SPR and ELISA respectively.
  • SPR the purified soluble SARS-CoV-2 RBD, SARS-CoV RBD and MERS-CoV2 RBD were covalently immobilized onto a CM5 sensor chip followed by injection of individual antibody at four or five different concentrations.
  • the black lines indicate the experimentally derived curves while the red lines represent fitted curves based on the SPR experimental data.
  • ELISA analysis recombinant SARS-CoV RBD and MERS-CoV2 RBD were coated on the ELISA plate, and a serial dilution of each antibody was evaluated against SARS-CoV RBD and MERS-CoV2 RBD coated plates respectively and their binding activity was recorded at an optical density (OD) of 450nm and 630nm.
  • OD optical density
  • S230 was used as a positive control antibody against SARS-COV
  • MAB-GD33 was used as a positive control antibody against MERS-COV
  • VRC01 was used as negative control antibody.
  • Figure 9 Antibody and ACE2 competition for binding to SARS-CoV-2 RBD measured by SPR.
  • the sensorgrams show distinct binding patterns of ACE2 to SARS-CoV-2 RBD with (red curve) or without (black curve) prior incubation with each testing antibody.
  • the competition capacity of each antibody is indicated by the level of reduction in response unit of ACE2 comparing with or without prior antibody incubation.
  • FIG. 10 Analysis of antibody binding to cell surface expressed trimeric Spike protein.
  • HEK 293T cells transfected with expression plasmid encoding the full length spike of SARS-CoV-2, SARS-CoV or MERS-CoV were incubated with 20ug/ml testing antibodies. The cells were then stained with PE labeled anti-human IgG Fc secondary antibody and analyzed by FACS. Positive control antibodies include S230 and m396 targeting the RBD of SARS-CoV Spike, and Mab-GD33 targeting the RBD of MERS-CoV Spike. VRC01 is the negative control antibody targeting HIV-1 envelope glycoprotein.
  • FIG. 11 Epitope mapping through competitive binding measured by SPR.
  • the sensorgrams show distinct binding patterns when pairs of testing antibodies were sequentially applied to the purified SARS-CoV-2 RBD covalently immobilized onto a CM5 sensor chip.
  • the level of reduction in response unit comparing with or without prior antibody incubation is the key criteria for determining the two mAbs recognize the separate or closely situated epitopes.
  • antibody as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, monovalent antibody, bivalent antibody, multivalent antibody, bispecific antibody, multi-specific antibody that binds to a specific antigen.
  • a native intact antibody comprises two heavy (H) chains and two light (L) chains.
  • Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each heavy chain consists of a variable region (VH) and a first, second, third, and optionally fourth constant region (CH1, CH2, CH3, CH4 respectively) ;
  • mammalian light chains are classified as ⁇ or ⁇ , while each light chain consists of a variable region (VL) and a constant region.
  • the antibody has a “Y” shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonding.
  • Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • the variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, HCDR3) .
  • CDRs complementarity determining regions
  • CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273 (4) , 927 (1997) ; Chothia, C. et al., J Mol Biol. Dec 5; 186 (3) : 651-63 (1985) ; Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901 (1987) ; Chothia, C. et al., Nature.
  • the three CDRs are interposed between flanking stretches known as framework regions (FRs) (light chain FRs including LFR1, LFR2, LFR3, and LFR4, heavy chain FRs including HFR1, HFR2, HFR3, and HFR4) , which are more highly conserved than the CDRs and form a scaffold to support the highly variable loops.
  • FRs framework regions
  • the constant regions of the heavy and light chains are not involved in antigen-binding, but exhibit various effector functions.
  • Antibodies are assigned to classes based on the amino acid sequences of the constant regions of their heavy chains.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively.
  • IgG1 gamma1 heavy chain
  • IgG2 gamma2 heavy chain
  • IgG3 gamma3 heavy chain
  • IgG4 gamma4 heavy chain
  • IgA1 (alpha1 heavy chain) or IgA2 (alpha2 heavy chain) .
  • antigen-binding fragment refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure.
  • antigen-binding fragment include, without limitation, a diabody, a Fab, a Fab', a F (ab') 2 , a Fd, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2 , a bispecific dsFv (dsFv-dsFv') , a disulfide stabilized diabody (ds diabody) , a single-chain antibody molecule (scFv) , an scFv dimer (bivalent diabody) , a bispecific scFv dimer, a single-chain Fv-Fc antibody (scFv-Fc) , a came
  • a “bispecific” antibody refers to an artificial antibody which has fragments derived from two different monoclonal antibodies.
  • a bispecific antibody may bind to overlapping epitopes or to two different epitopes. The two epitopes may present on the same antigen, or they may present on two different antigens.
  • the terms “multi-specific” antibody refers to an artificial antibody which has fragments derived from multiple different monoclonal antibodies, and may be capable of binding to more than one epitope.
  • chimeric means an antibody or antigen-binding fragment, having a portion of heavy and/or light chain derived from one species, and the rest of the heavy and/or light chain derived from a different species.
  • epitope refers to the specific group of atoms or amino acids on an antigen to which an antibody binds. Two antibodies may bind the same or a closely related epitope within an antigen if they exhibit competitive binding for the antigen.
  • An epitope can be linear or conformational (i.e. including amino acid residues spaced apart) .
  • an antibody or antigen-binding fragment blocks binding of a reference antibody to the antigen by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, or at least 95%, then the antibody or antigen-binding fragment may be considered to bind the same/closely related epitope as the reference antibody.
  • the capacity to block, or compete with, the binding of the antibody or the antigen-binding fragment of the present disclosure to SARS-CoV-2 typically indicates that an antibody or the antigen-binding fragment to be screened binds to an epitope or binding site on SARS-CoV-2 that structurally overlaps with the binding site on SARS-CoV-2 that is immunospecifically recognized by the antibody or the antigen-binding fragment of the present disclosure.
  • an antibody or an antigen-binding fragment of the present disclosure to be screened binds to an epitope or binding site that is sufficiently proximal to the binding site immunospecifically recognized by the antibody or the antigen-binding fragment of the present disclosure to sterically or otherwise inhibit binding of the antibodies or the antigen-binding fragment of the present disclosure to SARS-CoV-2.
  • Fab with regard to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond.
  • the heavy chain fragment of the Fab is known as “Fd” .
  • Fab' refers to a Fab fragment that includes a portion of the hinge region.
  • F (ab') 2 refers to a dimer of Fab’.
  • Fc with regard to an antibody (e.g. of IgG, IgA, or IgD isotype) refers to that portion of the antibody consisting of the second and third constant domains of a first heavy chain bound to the second and third constant domains of a second heavy chain via disulfide bonding.
  • Fc with regard to antibody of IgM and IgE isotype further comprises a fourth constant domain.
  • the Fc portion of the antibody is responsible for various effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) , Antibody-dependent cellular phagocytosis (ADCP) and complement dependent cytotoxicity (CDC) , but does not function in antigen binding.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP Antibody-dependent cellular phagocytosis
  • CDC complement dependent cytotoxicity
  • Fv with regard to an antibody refers to the smallest fragment of the antibody to bear the complete antigen binding site.
  • An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.
  • Single-chain Fv antibody or “scFv” refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one another directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85: 5879 (1988) ) .
  • ScFab refers to a fusion polypeptide with a Fd linked to a light chain via a polypeptide linker, resulting in the formation of a single chain Fab fragment (scFab) .
  • Single-chain Fv-Fc antibody or “scFv-Fc” refers to an engineered antibody consisting of a scFv connected to the Fc region of an antibody.
  • “Camelized single domain antibody, ” “heavy chain antibody, ” or “HCAb” refers to an antibody that contains two V H domains and no light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231 (1-2) : 25-38 (1999) ; Muyldermans S., J Biotechnol. Jun; 74 (4) : 277-302 (2001) ; WO94/04678; WO94/25591; U.S. Patent No. 6,005,079) .
  • Heavy chain antibodies were originally derived from Camelidae (camels, dromedaries, and llamas) .
  • variable domain of a heavy chain antibody represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. Nov; 21 (13) : 3490-8. Epub 2007 Jun 15 (2007) ) .
  • a “nanobody” refers to an antibody fragment that consists of a VHH domain from a heavy chain antibody and two constant domains, CH2 and CH3.
  • a “domain antibody” refers to an antibody fragment containing only the variable region of a heavy chain or the variable region of a light chain.
  • two or more V H domains are covalently joined with a peptide linker to create a bivalent or multivalent domain antibody.
  • the two V H domains of a bivalent domain antibody may target the same or different antigens.
  • valent refers to the presence of a specified number of antigen binding sites in a given molecule.
  • monovalent refers to an antibody or an antigen-binding fragment having only one single antigen-binding site; and the term “multivalent” refers to an antibody or an antigen-binding fragment having multiple antigen-binding sites.
  • bivalent denote the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antigen-binding molecule.
  • the antibody or antigen-binding fragment thereof is bivalent.
  • TriFabs refers to a trivalent, bispecific fusion protein composed of three units with Fab-functionalities. TriFabs harbor two regular Fabs fused to an asymmetric Fab-like moiety.
  • Fab-Fab refers to a fusion protein formed by fusing the Fd chain of a first Fab arm to the N-terminus of the Fd chain of a second Fab arm.
  • Fab-Fv refers to a fusion protein formed by fusing a heavy chain variable domain to the C-terminus of an Fd chain and a light chain variable domain to the C-terminus of a light chain.
  • a “Fab-dsFv” molecule can be formed by introducing an interdomain disulphide bond between the heavy chain variable domain and the heavy chain variable domain.
  • scFv dimer is a bivalent diabody or bispecific scFv (BsFv) comprising V H -V L (linked by a peptide linker) dimerized with another V H -V L moiety such that V H 's of one moiety coordinate with the V L 's of the other moiety and form two binding sites which can target the same antigens (or epitopes) or different antigens (or epitopes) .
  • a bispecific “scFv dimer” is a bispecific diabody comprising V H1 -V L2 (linked by a peptide linker) associated with V L1 -V H2 (also linked by a peptide linker) such that V H1 and V L1 coordinate and V H2 and V L2 coordinate and each coordinated pair has a different antigen specificity.
  • a “dsFv” refers to a disulfide-stabilized Fv fragment that the linkage between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.
  • a “ (dsFv) 2 ” or “ (dsFv-dsFv') ” comprises three peptide chains: two V H moieties linked by a peptide linker (e.g. a long flexible linker) and bound to two V L moieties, respectively, via disulfide bridges.
  • dsFv-dsFv' is bispecific in which each disulfide paired heavy and light chain has a different antigen specificity.
  • “Bibody” refers to a fusion protein formed by fusing a scFv to the C-terminus of either the light chain (Fab-L-scFv) or Fd (Fab-H-scFv) .
  • Tribody refers to a fusion protein formed by fusing a scFv to both light chain and heavy chain (Fab- (scFv) 2 ) .
  • MAb-Fv or “IgG-Fv” refers to a fusion protein formed by fusion of heavy chain variable domain (VH domain) to the C-terminus of one Fc chain and the VL domain either expressed separately or fused to the C-terminus of the other resulted in a bispecific, trivalent IgG-Fv (mAb-Fv) fusion protein, with the Fv stabilized by an interdomain disulphide bond.
  • VH domain heavy chain variable domain
  • mAb-Fv bispecific, trivalent IgG-Fv
  • ScFab-Fc-scFv 2 and “ScFab-Fc-scFv” refer to a fusion protein formed by fusion of a single-chain Fab with Fc and disulphide-stabilized Fv domains.
  • Appended IgG refers to a fusion protein with a Fab arm fused to an IgG to form the format of bispecific (Fab) 2 -Fc. It can form an “IgG-Fab” or a “Fab-IgG” , with a Fab fused to the C-terminus or N-terminus of an IgG molecule with or without a connector.
  • the appended IgG can be further modified to a format of IgG-Fab 4 (see, Brinkman et al., mAbs, 9 (2) , pp. 182–212 (2017) ) .
  • DVD-Ig refers to a dual-variable-domain antibody that is formed by fusion of an additional VH domain and VL domain of a second specificity to an IgG heavy chain and light chain.
  • CODV-Ig refers to a related format where the two VH domain and two VL domains are linked in a way that allows crossover pairing of the variable VH domain -VL domain, which are arranged either (from N-to C-terminus) in the order VH domain A-VH domain B and VL domain B-VL domain A, or in the order VH domain B-VH domain A and VL domain A-VL domain B.
  • a “CrossMab” refers to a technology of pairing of unmodified light chain with the corresponding unmodified heavy chain and pairing of the modified light chain with the corresponding modified heavy chain, thus resulting an antibody with reduced mispairing in the light chain.
  • a “WuxiBody” refers to is a bispecific antibody comprising a chimeric protein with variable domains of an antibody and the constant domains of TCR, wherein the subunits (such as alpha and beta domains) of TCR constant domains are associated by engineered disulfide bond (see, more details in WO2019057122A1) .
  • a “BiTE” is a bispecific T-cell engager molecule, comprising a first scFv with a first antigen specificity in the VL domain -VH domain orientation linked to a second scFv with a second specificity in the VH domain -VL domain orientation.
  • a “diabody” or “dAb” includes small antibody fragments with two antigen-binding sites, wherein the fragments comprise a V H domain connected to a V L domain in the same polypeptide chain (V H -V L or V L -V H ) (see, e.g. Holliger P. et al., Proc Natl Acad Sci USA. Jul 15; 90 (14) : 6444-8 (1993) ; EP404097; WO93/11161) .
  • the antigen-binding sites may target the same or different antigens (or epitopes) .
  • a “DART” is a diabody-like entity that has the VH of a first variable region linked to the VL of a second variable region, and the VH of the second variable region linked to the VL of the first variable region.
  • a “TandAb” is a bispecific fusion protein with four binding sites, two of which bind to a first antigen and the other two bind to a second antigen.
  • a “bispecific ds diabody” is a diabody target two different antigens (or epitopes) .
  • the term “fully human” when used with reference to an antibody refers to an antibody that are either directly derived from a human or based upon a human sequence. When an antibody is derived from or based on a human sequence and subsequently modified, it is still to be considered fully human as used throughout the specification. In other words, the term “fully human” when used with reference to an antibody, is intended to include binding molecules having variable and constant regions derived from human germline immunoglobulin sequences or based on variable or constant regions occurring in a human or human lymphocyte and modified in some form.
  • the fully human antibody may include amino acid residues not encoded by human germline immunoglobulin sequences, comprise substitutions and/or deletions (e.g., mutations introduced by, for instance, random or site-specific mutagenesis in vitro or by somatic mutation in vivo) .
  • substitutions and/or deletions e.g., mutations introduced by, for instance, random or site-specific mutagenesis in vitro or by somatic mutation in vivo.
  • “Based on” as used herein refers to the situation that a nucleic acid sequence may be exactly copied from a template, or with minor mutations, such as by error-prone PCR methods, or synthetically made matching the template exactly or with minor modifications.
  • Semi-synthetic molecules based on human sequences are also considered to be human as used herein.
  • affinity refers to the strength of non-covalent interaction between an immunoglobulin molecule (i.e. antibody) or fragment thereof and an antigen.
  • amino acid refers to an organic compound containing amine (-NH 2 ) and carboxyl (-COOH) functional groups, along with a side chain specific to each amino acid.
  • amine -NH 2
  • -COOH carboxyl
  • a “conservative substitution” with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties.
  • conservative substitutions can be made among amino acid residues with hydrophobic side chains (e.g. Met, Ala, Val, Leu, and Ile) , among residues with neutral hydrophilic side chains (e.g. Cys, Ser, Thr, Asn and Gln) , among residues with acidic side chains (e.g. Asp, Glu) , among amino acids with basic side chains (e.g. His, Lys, and Arg) , or among residues with aromatic side chains (e.g. Trp, Tyr, and Phe) .
  • conservative substitution usually does not cause significant change in the protein conformational structure, and therefore could retain the biological activity of a protein.
  • diagnosis refers to the identification of a pathological state, disease or condition, such as identification of SARS-CoV-2 infection, or refer to identification of a subject with SARS-CoV-2 infection who may benefit from a particular treatment regimen.
  • diagnosis contains the identification of presence or amount of SARS-CoV-2.
  • diagnosis refers to the identification of SARS-CoV-2 infection in a subject.
  • effector functions refer to biological activities attributable to the binding of Fc region of an antibody to its effectors such as C1 complex and Fc receptor.
  • exemplary effector functions include: complement dependent cytotoxicity (CDC) mediated by interaction of antibodies and C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by binding of Fc region of an antibody to Fc receptor on an effector cell; and phagocytosis. Effector functions can be evaluated using various assays such as Fc receptor binding assay, C1q binding assay, and cell lysis assay.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • nonspecific cytotoxic cells that express FcRs (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target ceil and subsequently cause lysis of the target cell.
  • FcRs e.g. Natural Killer (NK) cells, neutrophils, and macrophages
  • NK cells express Fc ⁇ RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991) .
  • binding or “specifically binds” in reference to the interaction of a binding molecule, e.g., an antibody, and its binding partner, e.g., an antigen, means that the interaction is dependent upon the presence of a particular structure, e.g., an antigenic determinant or epitope, on the binding partner.
  • the antibody preferentially binds or recognizes the binding partner even when the binding partner is present in a mixture of other molecules or organisms.
  • the binding may be mediated by covalent or non-covalent interactions or a combination of both.
  • Antibodies or fragments thereof that immunospecifically bind to an antigen may be cross-reactive with related antigens, carrying the same epitope.
  • K d value i.e., the dissociation constant between the antigen and antigen-binding molecule.
  • K d may be determined by using any conventional method known in the art, including but are not limited to radioimmunoassays (RIA) , enzyme-linked immunosorbent assays (ELISA) , surface plasmon resonance method, microscale thermophoresis method, HPLC-MS method and flow cytometry (such as FACS) method.
  • RIA radioimmunoassays
  • ELISA enzyme-linked immunosorbent assays
  • HPLC-MS microscale thermophoresis
  • flow cytometry such as FACS
  • ⁇ 5x10 -7 M, ⁇ 2x10 -7 M, ⁇ 10 -7 M, ⁇ 5x10 -8 M, ⁇ 2x10 -8 M, ⁇ 10 -8 M, ⁇ 5x10 -9 M, ⁇ 4x10 -9 M, ⁇ 3x10 -9 M, ⁇ 2x10 -9 M, or ⁇ 10 -9 M) can indicate specific binding between an antibody or antigen binding fragments thereof and SARS-CoV-2 (e.g. spike protein of SARS-CoV-2, or receptor binding domain of the spike protein of SARS-CoV-2) .
  • SARS-CoV-2 e.g. spike protein of SARS-CoV-2, or receptor binding domain of the spike protein of SARS-CoV-2
  • the ability to “compete for binding to RBD” as used herein refers to the ability of a SARS-CoV-2 antibody or antigen-binding fragment thereof to inhibit the binding interaction between RBD of spike protein of SARS-CoV-2 and its binding partner (e.g. a second SARS-CoV-2 antibody, or ACE2 receptor) to any detectable degree.
  • an antibody or antigen-binding fragment that compete for binding to SARS-CoV-2 inhibits the binding interaction between RBD of spike protein of SARS-CoV-2 and its binding partner by at least 85%, or at least 90%. In certain embodiments, this inhibition may be greater than 95%, or greater than 99%.
  • competitive inhibition is measured by means of an assay, wherein an antigen composition, i.e., a composition comprising SARS-CoV-2 or fragments thereof, is admixed with reference binding molecules, for example, the antibodies or antigen binding fragments of the present disclosure, or the ACE receptor (e.g. a recombinant binding moiety thereof) , and the antibodies or antigen binding fragments to be screened.
  • an antigen composition i.e., a composition comprising SARS-CoV-2 or fragments thereof
  • reference binding molecules for example, the antibodies or antigen binding fragments of the present disclosure, or the ACE receptor (e.g. a recombinant binding moiety thereof)
  • the antibodies or antigen binding fragments to be screened are present in excess. Protocols based upon ELISAs and Western blotting are suitable for use in such simple competition studies.
  • an antibody or antigen-binding fragment exhibits at least 30%competition at 1 ⁇ M, with 2 ⁇ M angiotensin converting enzyme 2 (ACE2) receptor for binding to the RBD of spike protein of SARS-CoV-2 immobilized at a resonance units (RU) of 250, as measured by SPR.
  • ACE2 angiotensin converting enzyme 2
  • homologous refers to nucleic acid sequences (or its complementary strand) or amino acid sequences that have sequence identity of at least 60%(e.g. at least 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to another sequences when optimally aligned.
  • host cell refers to a cell into which an exogenous polynucleotide and/or a vector can be or has been introduced.
  • isolated means one substance has been altered by the hand of man from the natural state. If an “isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living animal is not “isolated, ” but the same polynucleotide or polypeptide is “isolated” if it has been sufficiently separated from the coexisting materials of its natural state so as to exist in a substantially pure state.
  • An “isolated nucleic acid sequence” refers to the sequence of an isolated nucleic acid molecule.
  • an “isolated antibody or an antigen-binding fragment thereof” refers to the antibody or antigen-binding fragments thereof having a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%as determined by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis) , or chromatographic methods (such as ion exchange chromatography or reverse phase HPLC) .
  • an isolated antibody or antigen binding fragment is a recombinant protein or antigen binding fragment.
  • kit refers to a packaged combination of reagents in predetermined amounts with instructions for performing a therapeutics, or a diagnostic or detection assay.
  • neutralizing refers to antibody or the antigen binding fragment that inhibit SARS-CoV-2 virus from infecting a target cell for replication, regardless of the mechanism by which neutralization is achieved.
  • neutralization can, for example, be achieved by inhibiting the attachment or adhesion of SARS-CoV-2 virus or a pseudo SARS-CoV-2 virus bearing the spike protein to the cell surface, or by inhibition of the fusion of viral and cellular membranes following attachment of the virus to the target cell, and the like. Exemplary assays for determining neutralizing activity are described in the Examples provided herein.
  • the neutralizing activity of an antibody can be represented as half-maximal inhibitory concentrations (IC 50 ) of the antibody against the binding to ACE2.
  • nucleic acid or “polynucleotide” as used herein refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single-or double-stranded form. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) , alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19: 5081 (1991) ; Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985) ; and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994) ) .
  • Percent (%) sequence identity with respect to amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids) . Conservative substitution of the amino acid residues may or may not be considered as identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI) , see also, Altschul S.F.
  • polypeptide or “protein” means a string of at least two amino acids linked to one another by peptide bonds. Polypeptides and proteins may include moieties in addition to amino acids (e.g., may be glycosylated) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “polypeptide” or “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence) , or can be a functional portion thereof. Those of ordinary skill will further appreciate that a polypeptide or protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of a corresponding naturally-occurring amino acid and polymers.
  • pharmaceutically acceptable indicates that the designated carrier, vehicle, diluent, excipient (s) , and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.
  • recombinant when used with reference to a polypeptide (e.g., antibody, antigen) or a polynucleotide, refers to a polypeptide or polynucleotide that is produced by a recombinant method.
  • a “recombinant polypeptide” includes any polypeptide expressed from a recombinant polynucleotide.
  • a “recombinant polynucleotide” includes any polynucleotide which has been modified by the introduction of at least one exogenous (i.e., foreign, and typically heterologous) nucleotide or the alteration of at least one native nucleotide component of the polynucleotide, and need not include all of the coding sequence or the regulatory elements naturally associated with the coding sequence.
  • a “recombinant vector” refers to a non-naturally occurring vector, including, e.g., a vector comprising a recombinant polynucleotide sequence.
  • sample refers to a biological specimen that is obtained or derived from a subject of interest.
  • the sample contains a cellular and/or other molecular entity that is to be characterized and/or identified, for example based on physical, biochemical, chemical and/or physiological characteristics.
  • subject includes human and non-human animals.
  • Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians, and reptiles. Except when noted, the terms “patient” or “subject” are used herein interchangeably.
  • treating or “treatment” of a disease, disorder or condition as used herein includes alleviating a disease, disorder or condition, slowing the rate of development of a disease, disorder or condition, reducing or ending symptoms associated with a disease, disorder or condition, generating a complete or partial regression of a disease, disorder or condition, curing a disease, disorder or condition, or some combination thereof.
  • prevent or “preventing” of a disease, disorder or condition as used herein includes slowing the onset of a disease, disorder or condition, reducing the risk of developing a disease, disorder or condition, preventing or delaying the development of symptoms associated with a disease, disorder or condition, reducing the severity of a subsequent contraction or development of a disease, disorder or condition, ameliorating a related symptom, and inducing immunity to protect against a disease, disorder or condition.
  • SARS-CoV-2 virus antigen refers to a SARS-CoV-2 virus particle or an antigen found in a SARS-CoV-2 virus particle (e.g. a protein or protein fragments of envelop protein or spike protein (includes, extracellular domain of the spike protein, or RBD of the spike protein) and the like) .
  • Spike protein is composed of S1 protein (which contains RBD) and S2 protein, which are initially in one protein molecule until cleaved by protease into S1 and S2.
  • vector refers to a vehicle into which a genetic element may be operably inserted so as to bring about the expression of that genetic element, such as to produce the protein, RNA or DNA encoded by the genetic element, or to replicate the genetic element.
  • a vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell.
  • vectors examples include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses.
  • a vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes.
  • the vector may contain an origin of replication.
  • a vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating.
  • a vector can be an expression vector or a cloning vector.
  • the present disclosure provides vectors (e.g. expression vectors) containing the nucleic acid sequence provided herein encoding the antibody or an antigen-binding fragment thereof, at least one promoter (e.g. SV40, CMV, EF-1 ⁇ ) operably linked to the nucleic acid sequence, and at least one selection marker.
  • promoter e.g. SV40, CMV, EF-1 ⁇
  • the present disclosure in one aspect provides anti-SARS-CoV-2 antibodies and antigen-binding fragments thereof.
  • the anti-SARS-CoV-2 antibodies and antigen-binding fragments provided herein are capable of specifically binding to SARS-CoV-2. In certain embodiments, the antibodies and the antigen-binding fragments thereof provided herein specifically bind to SARS-CoV-2 at an Kd value of no more than 10 -7 M as measured by SPR.
  • the antibodies and the antigen-binding fragments thereof provided herein are capable of binding to the RBD of spike protein of SARS-CoV-2 at a Kd value of no more than 1x10 -7 M (e.g. no more than 5x10 -7 M, no more than 2x10 -7 M, no more than 10 -7 M, no more than 5x10 -8 M, no more than 2x10 -8 M, no more than 10 -8 M, no more than 5x10 -9 M, no more than 4x10 -9 M, no more than 3x10 -9 M, no more than 2x10 -9 M, or no more than 10 -9 M) as measured by SPR.
  • 1x10 -7 M e.g. no more than 5x10 -7 M, no more than 2x10 -7 M, no more than 10 -7 M, no more than 5x10 -8 M, no more than 2x10 -8 M, no more than 10 -8 M, no more than 5x10 -9 M, no more than 4x10 -9 M,
  • the antibodies and the antigen-binding fragments thereof provided herein bind to the RBD of spike protein of SARS-CoV or the RBD of spike protein of MERS-CoV at a significantly lower affinity or degree. In certain embodiments, the antibodies and the antigen-binding fragments thereof provided herein exhibit binding to the RBD of spike protein of SARS-CoV or the RBD of spike protein of MERS-CoV at a Kd value of at least 1x10 -6 M (e.g. at least 2x10 -6 M, at least 5x10 -6 M, at least 10 -5 M) as measured by SPR.
  • the antibodies and the antigen-binding fragments thereof provided herein do not detectably bind to SARS-CoV or MERS-CoV. In certain embodiments, the antibodies and the antigen-binding fragments thereof provided herein exhibits at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) less binding or non-detectable binding to SARS-CoV or MERS-CoV, than the binding to SARS-CoV-2 under equivalent assay conditions.
  • at least 50% e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%
  • the antibodies and the antigen-binding fragments thereof provided herein are capable of specifically binding to spike protein of SARS-CoV-2 and exhibiting at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) less binding to spike protein of SARS-CoV or spike protein of MERS-CoV, than the binding to spike protein of SARS-CoV-2 under equivalent assay conditions.
  • at least 50% e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%
  • the full length of spike protein of SARS-CoV-2 can comprise an amino acid sequence of SEQ ID NO: 134, optionally encoded by a polynucleotide sequence of SEQ ID NO: 135.
  • the antibodies and the antigen-binding fragments thereof provided herein are capable of specifically binding to receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 comprising the amino acid sequence of SEQ ID NO: 128.
  • the antibodies and the antigen-binding fragments thereof provided herein exhibit binding to RBD of spike protein of SARS-CoV comprising the amino acid sequence of SEQ ID NO: 124 at a level that is non-detectable or that is no more than 50% (e.g., no more than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, 1%) of the binding to the RBD of spike protein of SARS-CoV-2 under equivalent assay conditions.
  • the antibodies and the antigen-binding fragments thereof provided herein exhibit binding to RBD of spike protein of MERS-CoV comprising the amino acid sequence of SEQ ID NO: 126 at a level that is non-detectable or that is no more than 50% (e.g., no more than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, 1%) of the binding to RBD of the spike protein of SARS-CoV-2 under equivalent assay conditions..
  • the antibodies and the antigen-binding fragments thereof provided herein are capable of exhibiting at least 30%competition at 1 ⁇ M, with 2 ⁇ M ACE2 receptor for binding to the RBD of spike protein of SARS-CoV-2 immobilized at a (RU of 250, as measured by SPR.
  • SARS-CoV-2 RBD can be immobilized to a CM5 sensor chip via amine group for a final RU around 250.1 ⁇ M of the antibodies or the antigen-binding fragments thereof provided herein can be injected onto the chip until binding steady-state is reached.
  • 2 ⁇ M of human ACE2 or human ACE2 peptidase domain can be injected for 60 seconds. Blocking efficacy can be determined by comparison of response units with and without the antibody incubation.
  • Instruments and kits for SPR are commercially available as, for example, Biacore T200, GE Healthcare.
  • the antibodies and the antigen-binding fragments thereof provided herein are capable of binding to the RBD of spike protein of SARS-CoV-2 at an neutralizing activity at an IC 50 value of no more than 100 ⁇ g/ml (e.g., no more than 90 ⁇ g/ml, 80 ⁇ g/ml, 70 ⁇ g/ml, 60 ⁇ g/ml, 50 ⁇ g/ml, 40 ⁇ g/ml, 30 ⁇ g/ml, 20 ⁇ g/ml, 10 ⁇ g/ml, 5 ⁇ g/ml, 2 ⁇ g/ml, 1 ⁇ g/ml, 0.5 ⁇ g/ml, 0.2 ⁇ g/ml, 0.1 ⁇ g/ml, 0.05 ⁇ g/ml, 0.03 ⁇ g/ml) , as measured by pseudovirus neutralization assay.
  • IC 50 value of no more than 100 ⁇ g/ml (e.g., no more than 90 ⁇ g/ml, 80 ⁇ g/ml, 70 ⁇ g/ml, 60 ⁇ g/ml, 50 ⁇
  • Pseudovirus neutralization assay are known in the art, and in general involves generating a pseudovirus expressing a reporter gene and a viral protein of interest (such as the full length spike protein of SARS-CoV-2 of SEQ ID NO: 134) .
  • the antibodies and the antigen-binding fragments thereof provided herein can be incubated with the pseudovirus, and the titer of the pseudovirus can be determined via the report gene.
  • IC 50 is the concentration of the antibodies or the antigen-binding fragment thereof can inhibit 50%of the pseudovirus titer in the assay.
  • the present disclosure provides SARS-CoV-2 antibodies and antigen-binding fragments thereof comprising one or more (e.g. 1, 2, 3, 4, 5, or 6) CDRs comprising the sequences selected from the group consisting of SEQ ID NO: 1-6, 11-16, 21-26, 31-36, 41-46, 51-56, 65-70, 75-80, 85-90, 95-100, and 105-110.
  • SEQ ID NO: 1-6 11-16, 21-26, 31-36, 41-46, 51-56, 65-70, 75-80, 85-90, 95-100, and 105-110.
  • Antibody “P2A-1A8” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 7, and a light chain variable region having the sequence of SEQ ID NO: 8.
  • Antibody “P2A-1A9” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 17, and a light chain variable region having the sequence of SEQ ID NO: 18.
  • Antibody “P2A-1A10” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 27, and a light chain variable region having the sequence of SEQ ID NO: 28.
  • Antibody “P2A-1B3” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 37, and a light chain variable region having the sequence of SEQ ID NO: 38.
  • Antibody “P2B-2F6” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 47, and a light chain variable region having the sequence of SEQ ID NO: 48.
  • Antibody “P2B-2G4” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 57, and a light chain variable region having the sequence of SEQ ID NO: 58.
  • Antibody “P2B-2G11” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 61, and a light chain variable region having the sequence of SEQ ID NO: 62.
  • Antibody “P2C-1A3” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 71, and a light chain variable region having the sequence of SEQ ID NO: 72.
  • Antibody “P2C-1C8” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 81, and a light chain variable region having the sequence of SEQ ID NO: 82.
  • Antibody “P2C-1C10” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 91, and a light chain variable region having the sequence of SEQ ID NO: 92.
  • Antibody “P2C-1D5” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 101, and a light chain variable region having the sequence of SEQ ID NO: 102.
  • Antibody “P2C-1F11” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 111, and a light chain variable region having the sequence of SEQ ID NO: 112.
  • Table 1 below shows the CDR amino acid sequences of antibodies P2A-1A8, P2A-1A9, P2B-2G11, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, and P2C-1F11.
  • Table 2 shows the heavy chain and light chain variable region amino acid sequences of antibodies P2A-1A8, P2A-1A9, P2B-2G11, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, P2C-1F11, and the corresponding nucleic acid encoding sequence are shown in Table 3.
  • the antibodies or the antigen-binding fragments thereof provided herein further comprise an immunoglobulin (Ig) constant region, which optionally further comprises a heavy chain and/or a light chain constant region.
  • the heavy chain constant region comprises CH1, hinge, and/or CH2-CH3 regions (or optionally CH2-CH3-CH4 regions) .
  • the antibodies or the antigen-binding fragments thereof provided herein comprises heavy chain constant regions of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 or IgM.
  • the light chain constant region comprises C ⁇ or C ⁇ .
  • the constant region of the antibodies or the antigen-binding fragments thereof provided herein may be identical to the wild-type constant region sequence or be different in one or more mutations.
  • the heavy chain constant region comprises an Fc region.
  • Fc region is known to mediate effector functions such as antibody-dependent cellular cytotoxicity (ADCC) , Antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) of the antibody.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP Antibody-dependent cellular phagocytosis
  • CDC complement-dependent cytotoxicity
  • Fc regions of different Ig isotypes have different abilities to induce effector functions.
  • Fc regions of IgG1 and IgG3 have been recognized to induce both ADCC and CDC more effectively than those of IgG2 and IgG4.
  • the antibodies and antigen-binding fragments thereof provided herein comprises an Fc region of IgG1, or IgG3 isotype, which could induce ADCC or CDC.
  • the antibodies and antigen-binding fragments thereof provided herein comprise a constant region of IgG4 or IgG2 isotype, which has reduced or depleted effector function.
  • the anti-SARS-COV-2 antibodies or antigen-binding fragments thereof comprises a wild type human IgG1 Fc region comprising the sequence of SEQ ID NO: 115 or other wild type human IgG1 alleles.
  • Table 4 shows the amino acid sequences for the heavy chain and light chain constant regions of the monoclonal antibodies: P2A-1A8, P2A-1A9, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2B-2G11, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, and P2C-1F11, wherein the antibodies P2A-1A8, P2A-1A9, P2B-2F6, P2B-2G4, P2B-2G11, and P2C-1D5 have lambda light chains (with a lambda light chain constant region sequence of SEQ ID NO: 116) , the antibodies P2A-1A10, P2A-1B3, P2C-1A3, P2C-1C8, P2C-1C10, and P2C-1F11 have kappa light chains (with a kappa light chain constant region sequence of SEQ ID NO: 117) , and all 12 antibodies have the same heavy
  • signal peptide may be added when expressing the antibodies of the present disclosure, these signal peptides may be partially or full removed by host cells during the secretion of the antibody.
  • signal peptide SEQ ID NO: 130: MGWSCIILFLVATATGVHS
  • signal peptide SEQ ID NO: 131: MGWSCIILFLVATATGSWA
  • Table 10 which is appended at the end of the specification shows sequences and SEQ ID NOs mentioned or used in the present application.
  • the antibody or antigen binding fragments thereof provided herein comprise one or more modifications or substitutions in one or more of the CDR sequences provided in Table 1 above, one or more of the non-CDR sequences of the heavy chain variable region or light chain variable region provided in Table 2, and/or the constant region (e.g. Fc region) in Table 4, yet retaining specific binding affinity to RBD of spike protein of SARS-CoV-2.
  • at least one (or all) of the modifications (s) or substitution (s) comprises a conservative substitution.
  • the variants comprise 1, 2, or 3 CDR sequences having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) listed in Table 1 above, and in the meantime retain the binding affinity to SARS-COV-2 at a level similar to or even higher than its parent antibody.
  • the variants comprise one or more variable region sequences having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) listed in Table 2 above, and in the meantime retain the binding affinity to SARS-COV-2 at a level similar to or even higher than its parent antibody.
  • a total of 1 to 10 amino acids have been substituted, inserted, or deleted in a variable region sequence listed in Table 2 above.
  • the substitutions, insertions, or deletions occur in the non-CDR sequences (e.g. in the FRs) .
  • the variants of the antibodies or the antigen binding fragments thereof can retain their parent antibodies’ binding specificity to RBD of the spike protein of SARS-CoV-2, or may further have one or more desirable properties conferred by the modification (s) or substitution (s) .
  • the variants may have improved antigen-binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or depleted effector function (s) , improved FcRn receptor binding in a pH dependent manner, increased pharmacokinetic half-life, pH sensitivity, and/or compatibility to conjugation (e.g. one or more introduced cysteine residues) .
  • Such variants are also known as affinity variants, glycosylation variants, cysteine variants, Fc variants, and so on, which are described in more details as follows.
  • Affinity variant may contain modifications or substitutions in one or more CDR sequences as provided in Table 1 above, one or more framework (FR) sequences provided herein, or the heavy or light chain variable region sequences provided in Table 2 above.
  • FR sequences can be readily identified by a skilled person in the art based on the CDR sequences in Table 1 above and variable region sequences in Table 2 above, as it is well-known in the art that a CDR region is flanked by two FR regions in the variable region.
  • the affinity variants retain specific binding affinity to RBD of the spike protein of SARS-COV-2 of the parent antibody, or even have improved specific binding affinity to the RBD of the spike protein of SARS-CoV-2 over the parent antibody.
  • Various methods known in the art can be used to achieve this purpose.
  • a library of antibody variants (such as Fab or scFv variants) can be generated and expressed with phage display technology, and then screened for the binding affinity to the RBD of the spike protein of SARS-COV-2.
  • computer software can be used to virtually simulate the binding of the antibodies to the RBD of the spike protein of SARS-COV-2, and identify the amino acid residues on the antibodies which form the binding interface. Such residues may be either avoided in the substitution so as to prevent reduction in binding affinity, or targeted for substitution to provide for a stronger binding.
  • the affinity variant provided herein comprises one or more amino acid residue substitutions in one or more CDR sequences, and/or one or more FR sequences. In certain embodiments, an affinity variant comprises no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in the CDR sequences and/or FR sequences in total.
  • the anti-SARS-COV-2 antibodies and antigen-binding fragments provided herein also encompass a glycosylation variant, which can be obtained to either increase or decrease the extent of glycosylation of the antibody or antigen binding fragment thereof.
  • the antibody or antigen binding fragment thereof may comprise one or more modifications that introduces or removes a glycosylation site.
  • a glycosylation site is an amino acid residue with a side chain to which a carbohydrate moiety (e.g. an oligosaccharide structure) can be attached.
  • Glycosylation of antibodies is typically either N-linked or O-linked.
  • N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, for example, an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline.
  • O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly to serine or threonine. Removal of a native glycosylation site can be conveniently accomplished, for example, by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N-linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in the sequence in the is substituted. A new glycosylation site can be created in a similar way by introducing such a tripeptide sequence or serine or threonine residue.
  • the anti-SARS-COV-2 antibodies and antigen-binding fragments provided herein comprise a mutation at N297 (e.g. N297A, N297Q, or N297G) to remove the glycosylation site.
  • N297 e.g. N297A, N297Q, or N297G
  • the anti-SARS-COV-2 antibodies and antigen-binding fragments provided herein also encompass a cysteine-engineered variant, which comprises one or more introduced free cysteine amino acid residues.
  • a free cysteine residue is one which is not part of a disulfide bridge.
  • a cysteine-engineered variant is useful for conjugation with for example, a cytotoxic and/or imaging compound, a label, or a radioisoptype among others, at the site of the engineered cysteine, through for example a maleimide or haloacetyl.
  • Methods for engineering antibodies or antigen-binding fragments thereof to introduce free cysteine residues are known in the art, see, for example, WO2006/034488.
  • the anti-SARS-COV-2 antibodies and antigen-binding fragments provided herein also encompass an Fc variant, which comprises one or more amino acid residue modifications or substitutions at its Fc region and/or hinge region, for example, to provide for altered effector functions such as ADCC, ADCP and CDC.
  • Fc variant which comprises one or more amino acid residue modifications or substitutions at its Fc region and/or hinge region, for example, to provide for altered effector functions such as ADCC, ADCP and CDC.
  • Methods of altering ADCC activity by antibody engineering have been described in the art, see for example, Shields RL. et al., J Biol Chem. 2001.276 (9) : 6591-604; Idusogie EE. et al., J Immunol. 2000.164 (8) : 4178-84; Steurer W. et al., J Immunol. 1995, 155 (3) : 1165-74; Idusogie EE.
  • CDC activity of the antibodies provided herein can also be altered, for example, by improving or diminishing C1q binding and/or CDC (see, for example, WO99/51642; Duncan &Winter Nature 322: 738-40 (1988) ; U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821) ; and WO94/29351 concerning other examples of Fc region variants.
  • One or more amino acids selected from amino acid residues 329, 331 and 322 of the Fc region can be replaced with a different amino acid residue to alter Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC) (see, U.S. Pat. No. 6,194,551 by Idusogie et al) .
  • One or more amino acid substitution (s) can also be introduced to alter the ability of the antibody to fix complement (see PCT Publication WO 94/29351 by Bodmer et al.) .
  • ADCP antibody-dependent cellular phagocytosis
  • phagocytic immune cells e.g., macrophages, neutrophils and dendritic cells
  • Methods for altering the ADCP activity of antibodies by antibody engineering are known in the art, see for example, Kellner C et al., Transfus Med Hemother, (2017) 44: 327-336 and Chung AW et al., AIDS, (2014) 28: 2523-2530.
  • Fc variants are known in the art, see, for example, Wang et al., Protein Cell 2018, 9 (1) : 63-73 and Kang et al., Exp &Mol., Med. (2019) 51: 138, which are incorporated herein to their entirety.
  • the Fc variants provided herein has increased ADCC and/or increased affinity to an Fc ⁇ receptor (e.g. Fc ⁇ RI (CD64) , Fc ⁇ RII (CD32) and/or Fc ⁇ RIII (CD16) ) relative to a wildtype Fc (e.g. Fc of IgG1) .
  • Fc ⁇ receptor e.g. Fc ⁇ RI (CD64) , Fc ⁇ RII (CD32) and/or Fc ⁇ RIII (CD16)
  • an Fc variant comprises one or more amino acid substitution (s) at one or more of the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 246, 247, 248, 249, 252, 254, 255, 256, 258, 260, 262, 263, 264, 265, 267, 268, 269, 270, 272, 274, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 300, 301, 303, 304, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 345, 360, 373, 376, 378, 382, 388, 389, 396, 398, 414, 416, 419, 430, 433, 434
  • Fc variants with strongly enhanced binding to Fc ⁇ RIIIa include variant with S239D/I332E and S239D/I332E/A330L mutations, which showed the greatest increase in affinity for Fc ⁇ RIIIa, a decrease in Fc ⁇ RIIb binding, and strong cytotoxic activity, and variants with L235V, F243L, R292P, Y300L, V305I and P396L mutations, which exhibited enhancing Fc ⁇ RIIIa and concomitantly enhanced ADCC activity. (see Lazar et a . (2006) Proc. Nat'l Acad Sci. (USA) 103: 4005; Awan et al.
  • Modifications that increase binding to Clq can be introduced in order to enhance CDC activity.
  • exemplary modifications include, a K326 (e.g., K326W) and/or E333 modification in an IgG2, or a S267E/H268F/S324T modification, alone or in any combination, in an IgGl (see Idusogie et al. (2001) J. Immunol. 166: 2571, Moore et al. (2010) mAbs 2: 181) .
  • Other exemplary modifications include, K326W/E333S, S267E/H268F/S324T, and E345R/E430G/S440Y.
  • the Fc variants provided herein has reduced effector functions relative to a wildtype Fc (e.g. Fc of IgG1) , and comprise one or more amino acid substitution (s) at a position selected from the group consisting of: 220, 226, 229, 233, 234, 235, 236, 237, 238, 267, 268, 269, 270, 297, 309, 318, 320, 322, 325, 328, 329, 330, and 331 of the Fc region (see, WO2016/196228; Richards et al. (2008) Mol. Cancer Therap. 7: 2517; Moore et al.
  • a wildtype Fc e.g. Fc of IgG1
  • amino acid substitution amino acid substitution
  • substitutions for reduced effector functions include, without limitation, 220S, 226S, 228P, 229S, 233P, 234V, 234G, 234A, 234F, 234A, 235A, 235G, 235E, 236E, 236R, 237A, 237K, 238S, 267R, 268A, 268Q, 269R, 297A, 297Q, 297G, 309L, 318A, 322A, 325L, 328R, 330S, 331S, or any combination thereof (see, WO2016/196228; and Strohl (2009) Current Opinion in Biotechnology 20: 685-691) .
  • the Fc variant provided herein is of IgG1 isotype and comprises one or more amino acid substitution (s) selected from the group consisting of: L234A, L234F, L234V, F234A, V234A, L235A, L235E, G237A, P238S, H268Q, H268A, N297A, N297Q, N297G, V309L, A330S, and P331S, or any combination thereof (such as L234A/L235A) .
  • amino acid substitution selected from the group consisting of: L234A, L234F, L234V, F234A, V234A, L235A, L235E, G237A, P238S, H268Q, H268A, N297A, N297Q, N297G, V309L, A330S, and P331S, or any combination thereof (such as L234A/L235A) .
  • the Fc variant provided herein is of IgG2 isotype, and comprises one or more amino acid substitution (s) selected from the group consisting of: H268Q, V309L, A330S, P331S, V234A, G237A, P238S, H268A, and any combination thereof.
  • the Fc variant provided herein is of IgG4 isotype, and comprises one or more amino acid substitution (s) selected from the group consisting of: S228P, F234A, L235E, L235A, G237A, E318A, N297A, N297Q, N297G, and any combination thereof.
  • the anti-SARS-COV-2 antibodies and antigen-binding fragments provided herein is of IgG2/IgG4 cross isotype.
  • IgG2/IgG4 cross isotype is described in Rother RP et al, Nat Biotechnol 25: 1256–1264 (2007) .
  • the Fc variant comprises one or more amino acid substitution (s) that improves binding affinity to neonatal Fc receptor (FcRn) at pH 6.0 while retaining minimal binding at pH 7.4.
  • FcRn neonatal Fc receptor
  • Such a variant can have an extended pharmacokinetic half-life, as it binds to FcRn at acidic pH which allows it to escape from degradation in the lysosome and then be translocated and released out of the cell.
  • Methods of engineering an antibody and antigen-binding fragment thereof to improve binding affinity with FcRn are well-known in the art, see, for example, Vaughn, D. et al, Structure, 6 (1) : 63-73, 1998; Kontermann, R.
  • Non-limiting examples of Fc modifications that may result in an increase in serum half-life of the antibody when administered include, e.g., substitution (s) at one or more positions selected from: 234 (e.g., with F) , 235 (e.g., with Q) , 238 (e.g., with D) , 250 (e.g., with E or Q) , 252 (e.g., with L/Y/F/W or T) , 254 (e.g., with S or T) , 256 (e.g., with S/R/Q/E/D or T) ; 259 (e.g., with I) ; 272 (e.g., with A) , 305 (e.g., with A) , 307 (e.g., with A or P) , 308 (e.g., with F, C or P) , 311 (e.g., with A or R) , 312 (e.g., with A )
  • the Fc variant comprises one or more amino acid substitution (s) selected from the group consisting of 234F, 235Q, 238D, 250Q, 252T, 252Y, 254T, 256E, 259I, 272A, 305A, 307A, 308F, 311A, 322Q, 328E, 331S, 380A, 428L, 432C, 433K, 433S, 434S, 434Y, 434F, 434W, 434A, 435H, 436L, 437C and any combination thereof.
  • amino acid substitution selected from the group consisting of 234F, 235Q, 238D, 250Q, 252T, 252Y, 254T, 256E, 259I, 272A, 305A, 307A, 308F, 311A, 322Q, 328E, 331S, 380A, 428L, 432C, 433K, 433S, 434S, 434Y, 434F
  • the Fc modifications comprises one or pairs or groups of modifications selected from: a) a 428L (e.g., M428L) and 434S (e.g., N434S) substitution; a 428L, 259I (e.g., V259I) , and 308F (e.g., V308F) substitution; b) a 433K (e.g., H433K) and 434 (e.g., N434Y or N434F) substitution; c) a 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E) substitution; d) a 250Q and 428L substitution (e.g., T250Q and M428L) ; e) a 307A, 380A and 434A substitution (e.g., T307A, E380A and N434A) ; f) a P238D and L328
  • hybrid IgG isotypes may be used to increase FcRn binding and half-life of antibodies.
  • a hybrid Ig can be generated from two or more isotypes.
  • an IgGl/IgG3 hybrid variant may be constructed by substituting IgGl positions in the CH2 and/or CH3 region with the amino acids from IgG3 at positions where the two isotypes differ.
  • a hybrid Ig can comprises one or more modifications (e.g. substitutions) disclosed here.
  • the antibodies and antigen-binding fragments provided herein comprise all or a portion of the heavy chain variable domain and/or all or a portion of the light chain variable domain.
  • antigen-binding fragments are known in the art and can be developed based on the anti-SARS-CoV-2 antibodies provided herein, including for example, the exemplary antibodies whose CDR are shown in Tables 1 above, and variable sequences are shown in Tables 2 and 3, and their different variants (such as affinity variants, glycosylation variants, Fc variants, cysteine-engineered variants and so on) .
  • an anti-SARS-CoV-2 antigen-binding fragment is a diabody, a Fab, a Fab', a F (ab') 2 , a Fd, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2 , a bispecific dsFv (dsFv-dsFv') , a disulfide stabilized diabody (ds diabody) , a single-chain antibody molecule (scFv) , an scFv dimer (bivalent diabody) , a bispecific scFv dimer, a multispecific antibody, a heavy chain antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody.
  • Various techniques can be used for the production of such antigen-binding fragments.
  • Illustrative methods include, enzymatic digestion of intact antibodies (see, e.g. Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) ; and Brennan et al., Science, 229: 81 (1985) ) , recombinant expression by host cells such as E. Coli (e.g. for Fab, Fv and ScFv antibody fragments) , screening from a phage display library as discussed above (e.g.
  • the antigen-binding fragment is a scFv.
  • Generation of scFv is described in, for example, WO 93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458.
  • ScFv may be fused to an effector protein at either the amino or the carboxyl terminus to provide for a fusion protein (see, for example, Antibody Engineering, ed. Borrebaeck) .
  • the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof provided herein are bivalent, tetravalent, hexavalent, or multivalent. Any molecule being more than bivalent is considered multivalent, encompassing for example, trivalent, tetravalent, hexavalent, and so on.
  • a bivalent molecule can be monospecific if the two binding sites are both specific for binding to the same antigen or the same epitope. This, in certain embodiments, provides for stronger binding to the antigen or the epitope than a monovalent counterpart. Similar, a multivalent molecule may also be monospecific. In certain embodiments, in a bivalent or multivalent antigen-binding moiety, the first valent of binding site and the second valent of binding site are structurally identical (i.e. having the same sequences) , or structurally different (i.e. having different sequences albeit with the same specificity) .
  • a bivalent can also be bispecific, if the two binding sites are specific for different or overlapping antigens or epitopes. This also applies to a multivalent molecule.
  • a trivalent molecule can be bispecific when two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope) .
  • the present disclosure provides bispecific (or bivalent) antibody molecules comprising an anti-SARS-CoV-2 antibody or antigen-binding fragment thereof as disclosed herein.
  • the bispecific (or bivalent) antibodies provided herein comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domains is derived from a monoclonal antibody selected from the group consisting of P2A-1A8, P2A-1A9, P2B-2G11, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, and P2C-1F11.
  • the second antigen-binding domain can be derived from any suitable antibody.
  • the bispecific (or bivalent) antibodies provided herein comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first and the second antigen-binding domains are derived from any two monoclonal antibodies selected from the group consisting of P2A-1A8, P2A-1A9, P2B-2G11, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, and P2C-1F11.
  • the first and the second antigen-binding domains are derived from P2C-1F11 and P2B-2F6, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2A-1A8, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2A-1A9, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2B-2G11, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2A-1A10, respectively.
  • the first and the second antigen-binding domains are derived from P2C-1F11 and P2A-1B3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2B-2G4, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2C-1A3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2C-1C8, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2C-1C10, respectively.
  • the first and the second antigen-binding domains are derived from P2C-1F11 and P2C-1D5, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2C-1F11, respectively.
  • the first and the second antigen-binding domains are derived from P2A-1A8 and P2A-1A9, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A8 and P2B-2G11, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A8 and P2A-1A10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A8 and P2A-1B3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A8 and P2B-2F6, respectively.
  • the first and the second antigen-binding domains are derived from P2A-1A8 and P2B-2G4, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A8 and P2C-1A3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A8 and P2C-1C8, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A8 and P2C-1C10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A8 and P2C-1D5, respectively.
  • the first and the second antigen-binding domains are derived from P2A-1A9 and 2B-2G11, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A9 and P2A-1A10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A9 and P2A-1B3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A9 and P2B-2F6, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A9 and P2B-2G4, respectively.
  • the first and the second antigen-binding domains are derived from P2A-1A9 and P2C-1A3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A9 and P2C-1C8, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A9 and P2C-1C10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A9 and P2C-1D5, respectively.
  • the first and the second antigen-binding domains are derived from P2B-2G11 and P2A-1A10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G11 and P2A-1B3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G11 and P2B-2F6, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G11 and P2B-2G4, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G11 and P2C-1A3, respectively.
  • the first and the second antigen-binding domains are derived from P2B-2G11 and P2C-1C8, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G11 and P2C-1C10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G11 and P2C-1D5, respectively.
  • the first and the second antigen-binding domains are derived from P2A-1A10 and P2A-1B3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A10 and P2B-2F6, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A10 and P2B-2G4, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A10 and P2C-1A3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A10 and P2C-1C8, respectively.
  • the first and the second antigen-binding domains are derived from P2A-1A10 and P2C-1C10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A10 and P2C-1D5, respectively.
  • the first and the second antigen-binding domains are derived from P2A-1B3 and P2B-2F6, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1B3 and P2B-2G4, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1B3 and P2C-1A3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1B3 and P2C-1C8, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1B3 and P2C-1C10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1B3 and P2C-1D5, respectively.
  • the first and the second antigen-binding domains are derived from P2B-2F6 and P2B-2G4, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2F6 and P2C-1A3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2F6 and P2C-1C8, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2F6 and P2C-1C10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2F6 and P2C-1D5, respectively.
  • the first and the second antigen-binding domains are derived from P2B-2G4 and P2C-1A3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G4 and P2C-1C8, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G4 and P2C-1C10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G4 and P2C-1D5, respectively.
  • the first and the second antigen-binding domains are derived from P2C-1A3 and P2C-1C8, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1A3 and P2C-1C10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1A3 and P2C-1D5, respectively.
  • the first and the second antigen-binding domains are derived from P2C-1C8 and P2C-1C10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1C8 and P2C-1D5, respectively.
  • the first and the second antigen-binding domains are derived or from P2C-1C10 and P2C-1D5, respectively.
  • the bispecific antibody molecules can have at least two distinct antigen-binding sites with different specificities.
  • the bispecific antibody molecules provided herein are capable of binding to different epitopes on the spike protein of SARS-CoV-2 virus.
  • the bispecific antibody molecules provided herein comprises antigen-binding fragments derived from two or more antibodies provided herein.
  • the two or more antibodies bind to different epitopes in RBD of spike protein of SARS-CoV-2.
  • the two or more antibodies are no more than 70% (or no more than 60%, or no more than 50%) competitive against each other in binding to RBD of spike protein of SARS-CoV-2 virus.
  • the bispecific antibody comprises a first antigen-binding domain derived from P2C-1F11 and a second antigen-binding domain derived from an antibody selected from the group consisting of P2C-1A3, P2C-1C10, P2B-2F6, and P2A-1B3.
  • the bispecific antibody comprises a first antigen-binding domain derived from P2C-1A3 and a second antigen-binding domain derived from an antibody selected from the group consisting of P2C-1F11, and P2A-1B3.
  • the bispecific antibody comprises a first antigen-binding domain derived from P2B-2F6 and a second antigen-binding domain derived from an antibody selected from the group consisting of P2C-1C10, P2C-1F11, and P2A-1B3.
  • the bispecific antibody comprises a first antigen-binding domain derived from P2A-1B3 and a second antigen-binding domain derived from an antibody selected from the group consisting of P2C-1A3, P2C-1C10, P2C-1F11, P2B-2F6, and P2A-1A10.
  • the two or more antibodies comprise a first antibody which comprises P2C-1C10 or an antigen binding fragment thereof, and a second antibody selected from the group consisting of P2C-1A3, P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof.
  • antigen-binding domain comprise at least one heavy chain CDR sequence (e.g. comprising heavy chain CDR3, or three heavy chain CDRs) or at least one light chain CDR sequence (e.g. comprising light chain CDR3, or three heavy chain CDRs) of the specified monoclonal antibody.
  • the first and the second antigen-binding domains comprises the heavy chain CDR sequences of the specified monoclonal antibodies, and/or the light chain CDR sequences of the specified monoclonal antibodies.
  • the first and the second antigen-binding domains comprises the heavy chain variable region sequences of the specified monoclonal antibodies, and/or the light chain variable region sequences of the specified monoclonal antibodies. All the CDR sequences and variable region sequences of the specific monoclonal antibodies are provided in Tables 1 and 2 of the present disclosure.
  • the bispecific antibody molecules provided herein has a first antigen-binding domains specificity directed to the RBD of the spike protein of SARS-CoV-2 virus and a second antigen-binding domains specificity directed to a second antigen.
  • the second antigen can be for example, an epitope outside of RBD on the spike protein of SARS-CoV-2, S2 protein (i.e. which is cleaved from the spike protein) , nucleocapsid protein of SARS-CoV-2, or alternatively the second antigen can be an antigen on human immune cells such as T cell, macrophage cell, natural killer cells, or antigen-presenting cells.
  • the bispecific antibody molecules as provided herein are based on the format of a “whole” antibody, such as whole IgG or IgG-like molecules.
  • a bispecific IgG-like molecule can be an appended IgG, which is engineered by appending either the amino or carboxyl termini of either light or heavy chains of an IgG of a first specificity with additional antigen-binding units of a second specificity.
  • the appended antigen-binding units can be, for example, single domain antibodies (e.g. unpaired VL or VH, or VHH (i.e.
  • paired antibody variable domains e.g. Fv or scFv
  • engineered protein scaffolds e.g. Fv or scFv
  • appended IgG include, without limitation, Double-variable domain (DVD) -Ig, which has a second heavy chain variable domain (VH) fused to the VH of a first heavy chain and a second variable light chain domain (VL) fused to a first light chain of the IgG.
  • VH double-variable domain
  • VL variable light chain domain
  • a DVD-Ig can be bispecific when the first VH/VL and the second VH/VL are selected to bind to two different antigens.
  • a bispecific IgG or IgG-like molecules can be monovalent for each antigen and can be produced by co-expression of the two light and two heavy chains in a single host cell.
  • the bispecific antibody molecules as provided herein can be small recombinant bispecific formats based on variable domains, such as single domain antibody, Fv, and Fab, which may lack some or all of the antibody constant domains.
  • small recombinant bispecific formats include, without limitation, tandem single chain variable fragment molecules (taFvs) , diabodies (Dbs) , single chain diabodies (scDbs) and various other derivatives of these (see, bispecific antibody formats as described by Byrne H. et al. (2013) Trends Biotech, 31 (11) : 621-632, BiTE (bispecific T cell engager) , DARTs, and TandAb.
  • the two antigen-binding moieties can be linked by a peptide linker.
  • the bispecific antibody molecules as provided herein are in a bispecific format selected from bispecific IgG-like antibodies (BsIgG) comprising CrossMab; DAF (two-in-one) ; DAF (four-in-one) ; DutaMab; DT-IgG; Knobs-in-holes common LC; Knobs-in-holes assembly; Charge pair; Fab-arm exchange; SEEDbody; Triomab; LUZ-Y; Fcab; kappa-lamda-body; and Orthogonal Fab.
  • BsIgG bispecific IgG-like antibodies
  • the bispecific antibody molecules as provided herein are in a bispecific format selected from IgG-appended antibodies with an additional antigen-binding moiety consisting of DVD-IgG; IgG (H) -scFv; scFv- (H) IgG; IgG (L) -scFv; scFV- (L) IgG; IgG (L, H) -Fv; IgG (H) -V; V (H) -IgG; IgG (L) -V; V (L) -IgG; IgG-scFab; 2scFv-IgG; IgG-2scFv; scFv4-Ig; scFv4-Ig; and Zybody (see Id. ) .
  • the bispecific antibody molecules as provided herein are in a bispecific format selected from WuxiBody (WuXi Biologics, see, WO2019057122A1, incorporated herein to its entirety) ; Triomabs; hybrid hybridoma (quadroma) ; Multispecific anticalin platform (Pieris) ; Diabodies; Single chain diabodies; Tandem single chain Fv fragments; TandAbs, Trispecific Abs (Affimed) ; Darts (dual affinity retargeting; Macrogenics) ; Bispecific Xmabs (Xencor) ; Bispecific T cell engagers (Bites; Amgen; 55 kDa) ; Triplebodies; Tribody (Fab-scFv) ; Fusion Protein (CreativeBiolabs) ; multifunctional recombinant antibody derivates; Duobody platform (Genmab) ; Dock and lock platform; Knob into hole (KIH) platform; Humanized bispecific IgG antibody (RE
  • the bispecific antibody molecules as provided herein are in a format selected from bispecific antibody fragments comprising Nanobody; Nanobody-HAS; BiTE; Diabody; DART; TandAb; scDiabody; sc-Diabody-CH3; Diabody-CH3; Triple Body; Miniantibody; Minibody; TriBi minibody; scFv-CH3 KIH; Fab-scFv; scFv-CH-CL-scFv; F (ab') 2; F (ab') 2-scFv2; scFv-KIH; Fab-scFv-Fc; Tetravalent HCAb; scDiabody-Fc; Diabody-Fc; Tandem scFv-Fc; and Intrabody (see Id. ) .
  • the bispecific antibody molecules as provided herein are in a bispecific format such as Dock and Lock; ImmTAC; HSAbody; scDiabody-HAS; and Tandem scFv-Toxin (see Id. ) .
  • the bispecific antibody molecules as provided herein are based on a format selected from bispecific antibody conjugates comprising IgG-IgG; Cov-X-Body; and scFv1-PEG-scFv2 (see Id. ) .
  • bispecific antibody molecules provided herein can be made with any suitable methods known in the art.
  • two immunoglobulin heavy chain-light chain pairs having different antigen-binding specificities can be co-expressed in a host cell to produce bispecific antibodies in a recombinant way (see, for example, Milstein and Cuello, Nature, 305: 537 (1983) ) , followed by purification by affinity chromatography.
  • Recombinant approach may also be used, where sequences encoding the antibody heavy chain variable domains for the two specificities are respectively fused to immunoglobulin constant domain sequences, followed by insertion to an expression vector which is co-transfected with an expression vector for the light chain sequences to a suitable host cell for recombinant expression of the bispecific antibody (see, for example, WO 94/04690; Suresh et al., Methods in Enzymology, 121: 210 (1986) ) .
  • scFv dimers can also be recombinantly constructed and expressed from a host cell (see, e.g. Gruber et al., J. Immunol., 152: 5368 (1994) . )
  • Bispecific antibody molecule may be generated from a bispecific antibody, for example, by proteolytic cleavage, or by chemical linking.
  • an antigen-binding fragment e.g. Fab’
  • an antibody may be prepared and converted to Fab'-thiol derivative and then mixed and reacted with another converted Fab’ derivative having a different antigenic specificity to form a bispecific antibody molecule (see, for example, Brennan et al., Science, 229: 81 (1985) ) .
  • the bispecific antibody molecules may be engineered to promote heavy chain heterodimerization of the two different antigen-binding sites.
  • the Fc region is modified at the interface so that a knob-into-hole association can be formed to promote heterodimerization.
  • Knob-into-hole refers to an interaction between two polypeptides (such as CH3 domain) , where one polypeptide has a protuberance (i.e. “knob” ) due to presence of an amino acid residue having a bulky side chain (e.g. tyrosine or tryptophan) , and the other polypeptide has a cavity (i.e.
  • hole where a small side chain amino acid residue resides (e.g. alanine or threonine) , and the protuberance is positionable in the cavity so as to promote interaction of the two polypeptides to form a heterodimer or a complex.
  • a small side chain amino acid residue e.g. alanine or threonine
  • “charged pairs” can be introduced to the Fc polypeptides to electrostatically steer the formation towards heterodimerization.
  • Exemplary pairs include, D221E/P228E/L368E paired with D221R/P228R/K409R and C220E/P228E/368E paired with C220R/E224R/P228R/K409R (see Gunasekaran et al., 2010, J. Biol. Chem. 285 (25) : 19637. ) .
  • the binding interface of the two Fc polypeptide chains can be engineered such that in the heterodimer configuration, residues interact with residues of similar physical property (e.g., polar residues interacting with polar residues, or hydrophobic residues interact with hydrophobic residues) , while in the homodimer configuration residues interact with residues of different physical property.
  • residues e.g., polar residues interacting with polar residues, or hydrophobic residues interact with hydrophobic residues
  • Exemplary modifications include substitution at positions 364, 368, 399, 405, 409, 411, or any combination thereof (see, e.g, WO2014/145806, WO2014/110601, WO2016/086186, WO2016/086189, WO2016/086196, and WO2016/182751) .
  • the bispecific antibody molecules may be engineered to reduce random pairing of two different light chain variable regions with the two different heavy chain variable regions.
  • the bispecific antibody molecule comprise a common light chain capable of pairing with the two heavy chain variable regions.
  • CH1 domain of one heavy chain is exchanged with the constant region (CL) of the corresponding light chain (such as that applied in CrossMab technology) .
  • mutations are introduced into the CH1-CL interface and/or the VH-VL interface of the Fab fragments, so as to enforce correct pairing of the light chains with the corresponding heavy chains.
  • the CH1 domain and CL domain in one antigen-binding domain are replaced by TCR constant domains, so as to minimize mispairing between heavy chain of the first antigen-binding domain and light chain of the second antigen-binding domain (such as that applied in WuxiBody technology) .
  • the present disclosure provides an isolated or recombinant antibody or an antigen-binding fragment thereof, which competes for binding to RBD of spike protein of SARS-CoV-2 with the antibody or an antigen-binding fragment thereof described herein.
  • Antibodies or antigen binding fragments that competes with the antibody or antigen-binding fragment provided herein for binding to RBD of spike protein of SARS-CoV-2 include, but are not limited to, antibodies, antibody fragments and other binding agents that bind to an epitope or binding site bound by the antibody or antigen-binding fragment provided herein, or bind to a sufficiently proximal epitope or binding site.
  • competitive antibodies or antigen binding fragments of the disclosure will, when present in excess, inhibit specific binding of the antibody or antigen-binding fragment provided herein to RBD of the spike protein of SARS-CoV-2 by at least 10%, preferably by at least 25%, more preferably by at least 50%, and most preferably by at least 75%-90%or even greater.
  • the identification of one or more competitive antibodies or antigen binding fragments that bind to about, substantially, essentially or at the same epitope as the antibodies or antigen binding fragments of the present disclosure is a straightforward technical matter.
  • the identification of competitive binding molecules is determined in comparison to a reference binding molecule, for example, the antibodies or antigen binding fragments of the present disclosure, it will be understood that actually determining the epitope to which the reference binding molecule and the competitive binding molecule bind is not in any way required in order to identify a competitive binding molecule that binds to the same or substantially the same epitope as the reference binding molecule.
  • the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof further comprise one or more conjugate moieties.
  • a conjugate moiety is a moiety that can be attached to the antibodies or antigen-binding fragments thereof either directly or via a linker or through another conjugate moiety. It is contemplated that a variety of conjugate moieties may be linked to the antibodies or antigen-binding fragments thereof provided herein (see, for example, “Conjugate Vaccines” , Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr. (eds. ) , Carger Press, New York, (1989) ) . These conjugate moieties may be linked to the antibodies or antigen-binding fragments thereof by covalent binding, affinity binding, intercalation, coordinate binding, complexation, association, blending, or addition, among other methods.
  • the antibodies or antigen-binding fragments thereof provided herein may be engineered to contain specific sites outside the epitope binding portion that may be utilized for binding to one or more conjugate moieties.
  • a site may include one or more reactive amino acid residues, such as for example cysteine or histidine residues, to facilitate covalent linkage to a conjugate moiety.
  • conjugate moieties include but are not limited to, therapeutic agent, a radioactive isotope, a detectable label, a pharmacokinetic modifying moiety, or a purifying moiety.
  • the conjugate moiety comprises a clearance-modifying agent (e.g. a polymer such as PEG which extends half-life) , a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a detectable label (e.g. a luminescent label, a fluorescent label, an enzyme-substrate label) , a DNA-alkylator, a topoisomerase inhibitor, a tubulin-binder, a purification moiety or other anticancer drugs.
  • a clearance-modifying agent e.g. a polymer such as PEG which extends half-life
  • chemotherapeutic agent e.g. a toxin
  • a radioactive isotope e.g. a lanthan
  • detectable label may include a fluorescent labels (e.g. fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red) , enzyme-substrate labels (e.g. horseradish peroxidase, alkaline phosphatase, luceriferases, glucoamylase, lysozyme, saccharide oxidases or ⁇ -D-galactosidase) , radioisotopes (e.g.
  • the conjugate moiety can be a clearance-modifying agent which helps increase half-life of the antibody.
  • Illustrative example include water-soluble polymers, such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol/propylene glycol, and the like.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules.
  • the conjugate moiety can be a purification moiety such as a magnetic bead.
  • the antibodies or antigen-binding fragments thereof provided herein is used as a base for a conjugate.
  • the present disclosure provides isolated polynucleotides that encode the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof provided herein.
  • DNA encoding the monoclonal antibody is readily isolated, e.g., from B cells, and sequenced using conventional procedures (e.g. by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) .
  • the encoding DNA may also be obtained by synthetic methods.
  • the isolated polynucleotide that encodes the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof can be inserted into a vector for further cloning (amplification of the DNA) or for expression (i.e., expression vector) , using recombinant techniques known in the art.
  • Many vectors are available.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g. SV40, CMV, EF-1 ⁇ ) , and a transcription termination sequence.
  • the present disclosure provides vectors comprising the isolated polynucleotide provided herein.
  • the polynucleotide provided herein encodes the antibodies or antigen-binding fragments thereof, at least one promoter (e.g. SV40, CMV, EF-1 ⁇ ) operably linked to the nucleic acid sequence, and at least one selection marker.
  • promoter e.g. SV40, CMV, EF-1 ⁇
  • vectors include, but are not limited to, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g. herpes simplex virus) , poxvirus, baculovirus, papillomavirus, papovavirus (e.g.
  • SV40 lambda phage, and M13 phage, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.
  • RTM. pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos etc.
  • Vectors comprising the polynucleotide sequence encoding the antibody or antigen-binding fragment thereof can be introduced to a host cell for cloning or gene expression.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g. Salmonella typhimurium, Serratia, e.g. Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-SARS-CoV-2 antibody-encoding vectors.
  • Saccharomyces cerevisiae, or common baker’s yeast is the most commonly used among lower eukaryotic host microorganisms.
  • a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g. K. lactis, K. fragilis (ATCC 12, 424) , K. bulgaricus (ATCC 16,045) , K. wickeramii (ATCC 24, 178) , K.
  • waltii ATCC 56, 500
  • K. drosophilarum ATCC 36, 906
  • K. thermotolerans K. marxianus
  • yarrowia EP 402, 226)
  • Pichia pastoris EP 183, 070
  • Candida Trichoderma reesia
  • Neurospora crassa Neurospora crassa
  • Schwanniomyces such as Schwanniomyces occidentalis
  • filamentous fungi such as, e.g. Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
  • Suitable host cells for the expression of glycosylated antibodies or antigen-fragment thereof provided herein are derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar) , Aedes aegypti (mosquito) , Aedes albopictus (mosquito) , Drosophila melanogaster (fruiffly) , and Bombyx mori have been identified.
  • a variety of viral strains for transfection are publicly available, e.g.
  • the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
  • vertebrate cells have been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure.
  • useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651) ; human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36: 59 (1977) ) ; baby hamster kidney cells (BHK, ATCC CCL 10) ; Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.
  • mice sertoli cells TM4, Mather, Biol. Reprod. 23: 243-251 (1980) ) ; monkey kidney cells (CV1 ATCC CCL 70) ; African green monkey kidney cells (VERO-76, ATCC CRL-1587) ; human cervical carcinoma cells (HELA, ATCC CCL 2) ; canine kidney cells (MDCK, ATCC CCL 34) ; buffalo rat liver cells (BRL 3A, ATCC CRL 1442) ; human lung cells (W138, ATCC CCL 75) ; human liver cells (Hep G2, HB 8065) ; mouse mammary tumor (MMT 060562, ATCC CCL51) ; TRI cells (Mather et al., Annals N.
  • the host cell is a mammalian cultured cell line, such as CHO, BHK, NS0, 293 and their derivatives.
  • Host cells are transformed with the above-described expression or cloning vectors for anti-SARS-CoV-2 antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the antibody may be produced by homologous recombination known in the art.
  • the host cell is capable of producing the antibody or antigen-binding fragment thereof provided herein.
  • the present disclosure also provides a method of expressing the antibody or an antigen-binding fragment thereof provided herein, comprising culturing the host cell provided herein under the condition at which the vector of the present disclosure is expressed.
  • the host cells used to produce the antibodies or antigen-binding fragments thereof provided herein may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma) , Minimal Essential Medium (MEM) , (Sigma) , RPMI-1640 (Sigma) , and Dulbecco's Modified Eagle's Medium (DMEM) , Sigma) are suitable for culturing the host cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor) , salts (such as sodium chloride, calcium, magnesium, and phosphate) , buffers (such as HEPES) , nucleotides (such as adenosine and thymidine) , antibiotics (such as GENTAMYCIN TM drug) , trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) , and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to a person skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to a person skilled in the art.
  • the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5) , EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
  • sodium acetate pH 3.5
  • EDTA EDTA
  • PMSF phenylmethylsulfonylfluoride
  • Cell debris can be removed by centrifugation.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • Protein A immobilized on a solid phase is used for immunoaffinity purification of the antibody and antigen-binding fragment thereof.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
  • Protein A can be used to purify antibodies that are based on human gamma1, gamma2, or gamma4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983) ) .
  • Protein G is recommended for all mouse isotypes and for human gamma3 (Guss et al., EMBO J. 5: 1567 1575 (1986) ) .
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.
  • Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the antibody comprises a CH3 domain
  • the Bakerbond ABX TM resin J.T. Baker, Phillipsburg, N.J. ) is useful for purification.
  • the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g. from about 0-0.25M salt) .
  • the present disclosure further provides pharmaceutical compositions comprising the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof and one or more pharmaceutically acceptable carriers.
  • the pharmaceutical composition comprises a combination of two or more antibodies or the antigen binding fragments of the present disclosure.
  • the pharmaceutical composition comprises a combination of two or more monoclonal antibodies, each of which comprises heavy chain CDR sequences and light chain CDR sequences derived from an antibody selected from the group consisting of P2A-1A8, P2A-1A9, P2B-2G11, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, and P2C-1F11.
  • the pharmaceutical composition comprises a first antibody comprising heavy chain CDR sequences and light chain CDR sequences derived from P2C-1F11, and a second antibody comprising heavy chain CDR sequences and light chain CDR sequences derived from antibody P2B-2F6.
  • the two or more antibodies or the antigen binding fragments thereof bind to different epitopes in RBD of spike protein of SARS-CoV-2.
  • the pharmaceutical composition comprises a first antibody which comprises P2C-1F11 or an antigen binding fragment thereof, and a second antibody which is selected from the group consisting of P2C-1A3, P2C-1C10, P2B-2F6, and P2A-1B3, or an antigen binding fragment thereof.
  • the pharmaceutical composition comprises a first antibody which comprises P2C-1A3 or an antigen binding fragment thereof, and a second antibody which is selected from the group consisting of P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof.
  • the pharmaceutical composition comprises a first antibody which comprises P2B-2F6 or an antigen binding fragment thereof, and a second antibody selected from the group consisting of P2C-1C10, P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof.
  • the pharmaceutical composition comprises a first antibody which comprises P2A-1B3 or an antigen binding fragment thereof, and a second antibody selected from the group consisting of P2C-1A3, P2C-1C10, P2C-1F11, P2B-2F6, and P2A-1A10, or an antigen binding fragment thereof.
  • the pharmaceutical composition comprises a first antibody which comprises P2C-1C10 or an antigen binding fragment thereof, and a second antibody selected from the group consisting of P2C-1A3, P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof.
  • the present disclosure further provides pharmaceutical compositions comprising the polynucleotides encoding the anti-SARS-CoV-2 antibodies or the antigen-binding fragments thereof, and one or more pharmaceutically acceptable carriers.
  • the present disclosure further provides pharmaceutical compositions comprising the polynucleotides encoding the combination of the two or more anti-SARS-CoV-2 antibodies or the antigen-binding fragments thereof, and one or more pharmaceutically acceptable carriers.
  • compositions comprising an expression vector comprising the polynucleotides encoding the one or more of anti-SARS-CoV-2 antibodies or the antigen-binding fragments thereof, and one or more pharmaceutically acceptable carriers.
  • the expression vector comprises a viral vector or a non-viral vector.
  • viral vectors include, without limitation, adeno-associated virus (AAV) vector, lentivirus vector, retrovirus vector, and adenovirus vector.
  • non-viral vectors include, without limitation, naked DNA, plasmid, exosome, mRNA, and so on.
  • the expression vector is suitable for gene therapy in human. Suitable vectors for gene therapy include, for example, adeno-associated virus (AAV) , or adenovirus vector.
  • the expression vector comprises a DNA vector or a RNA vector.
  • the pharmaceutically acceptable carriers are polymeric excipients, such as without limitation, microspheres, microcapsules, polymeric micelles and dendrimers.
  • the polynucleotides, or polynucleotide vectors of the present disclosure may be encapsulated, adhered to, or coated on the polymer-based components by methods known in the art (see for example, W. Heiser, Nonviral gene transfer techniques, published by Humana Press, 2004; U.S. patent 6025337; Advanced Drug Delivery Reviews, 57 (15) : 2177-2202 (2005) ) .
  • the pharmaceutical composition further comprises a second active agent, such as a second therapeutic agent or a second prophylactic agent.
  • Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.
  • Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrins.
  • Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisol, butylated hydroxytoluene, and/or propyl gallate.
  • compositions comprising an antibody or antigen-binding fragment thereof and conjugates provided herein decreases oxidation of the antibody or antigen-binding fragment thereof. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving antibody stability and maximizing shelf-life. Therefore, in certain embodiments, pharmaceutical compositions are provided that comprise one or more antibodies or antigen-binding fragments thereof as disclosed herein and one or more antioxidants such as methionine.
  • pharmaceutical acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80) , sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (
  • Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride.
  • Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol.
  • Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.
  • compositions can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
  • compositions depends on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
  • the pharmaceutical compositions can be formulated for intravenous, oral, nasal, rectal, percutaneous, or intramuscular administration.
  • dosage forms for intravenous administration may be formulated as lyophilized powder or fluid formulation; dosage forms for nasal administration may conveniently be formulated as aerosols, solutions, drops, gels or dry powders.
  • the pharmaceutical compositions can be formulated in the form of tablets, capsule, pill, dragee, powder, granule, sachets, cachets, lozenges, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium) , spray, inhalant, or suppository.
  • the pharmaceutical compositions are formulated into an injectable composition.
  • the injectable pharmaceutical compositions may be prepared in any conventional form, such as for example liquid solution, suspension, emulsion, or solid forms suitable for generating liquid solution, suspension, or emulsion.
  • Preparations for injection may include sterile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions.
  • the solutions may be either aqueous or nonaqueous.
  • unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile and not pyretic, as is known and practiced in the art.
  • a sterile, lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment as disclosed herein in a suitable solvent.
  • the solvent may contain an excipient which improves the stability or other pharmacological components of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent.
  • the solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to a person skilled in the art at, in one embodiment, about neutral pH.
  • the resulting solution will be apportioned into vials for lyophilization.
  • Each vial can contain a single dosage or multiple dosages of the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof or composition thereof. Overfilling vials with a small amount above that needed for a dose or set of doses (e.g. about 10%) is acceptable so as to facilitate accurate sample withdrawal and accurate dosing.
  • the lyophilized powder can be stored under appropriate conditions, such as at about 4 °C to room temperature.
  • Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration.
  • the sterile and/or non-pyretic water or other liquid suitable carrier is added to lyophilized powder. The precise amount depends upon the selected therapy being given, and can be empirically determined.
  • the present disclosure also provides methods of treating SARs-CoV-2 infection or a disease, disorder or condition associated with SARs-CoV-2 infection in a subject, comprising administering to the subject a therapeutically effective amount of one or more of the antibody or antigen-binding fragment thereof provided herein, or one or more polynucleotides encoding one or more of the antibody or antigen-binding fragment thereof provided herein, or the pharmaceutical composition provided herein.
  • the therapeutically effective amount can be an amount effective to decrease SARs-COV-2 titers, or to alleviate one or more disease symptoms, viremia, or any other measurable manifestation of SARS-CoV-2 infection in the treated subject or population, whether by inducing the regression of or inhibiting the progression of symptom (s) associated with SARs-COV-2 infection by any clinically measurable degree.
  • Decrease in SARs-COV-2 titers can be measured in the lung, for example, by the concentration of SARs-COV-2 in sputum samples or a lavage from the lungs from a mammal.
  • Alleviation of a disease symptom can be assessed by any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of that symptom.
  • Exemplary symptoms associated with SARs-COV-2 infection include, without limitation, fever, dry cough, shortness in breath, pain or pressure in the chest, new confusion or inability to arouse, bluish lips or face, loss of sense of smell and/or loss of sense of taste.
  • a subject in need of treatment include, for example, those already infected with SARS-CoV-2 (symptomatic or asymptomatic) or inflicted with a condition resulting from infection of SARS-CoV-2.
  • Subjects partially or totally recovered from infection of SARS-CoV-2 might also be in need of treatment.
  • the subject is human.
  • the present disclosure also provides methods of preventing SARs-CoV-2 infection, or a disease, disorder or condition associated with SARs-COV-2 infection in a subject, comprising administering to the subject a prophylactically effective amount of one or more of the antibody or antigen-binding fragment thereof provided herein, or one or more polynucleotides encoding one or more of the antibody or antigen-binding fragment thereof provided herein, or the pharmaceutical composition provided herein.
  • Prevention encompasses inhibiting or reducing the spread of SARS-CoV-2 or inhibiting or reducing the onset, development or progression of one or more of the symptoms associated with infection with SARS-CoV-2.
  • the prophylactically effective amount can be an amount effective to neutralize SARs-COV-2 in the respiratory tract, lungs and/or other affected areas such as eyes, noses and mouth, in order block infection, or effective to ameliorate at least one symptom associated with SARs-COV-2 infection. Whether a symptom has been ameliorated can be assessed by any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of that symptom or in certain instances will ameliorate the need for hospitalization.
  • a subject in need of prevention include, for example, those in which infection with SARS-CoV-2 is to be prevented, or those who are at risk for SARS-CoV-2 infection.
  • the subject is human.
  • disease, disorder or condition associated with SARS-COV-2 infection include those that are caused by or related to SARs-COV-2 infection, such as, upper or lower respiratory tract infections, pharyngitis, pneumonia, tracheobronchitis, bronchiolitis, bronchitis, acute respiratory distress syndrome, diarrhea, and any related infections or inflammatory disorders.
  • the methods of treatment or prevention provided herein are also suitable for gene therapy by transfer of polynucleotide sequences encoding the antibody product or fragment thereof in a subject, such that the polynucleotide can be expressed in the subject to produce the antibody in vivo.
  • the polynucleotide provided herein can be administered to a subject by, for example, transfection techniques such as electroporation and hydrodynamic injection, which are suitable for administration of naked polynucleotides.
  • transfection techniques such as electroporation and hydrodynamic injection
  • polynucleotides in the form of viral vectors such as AAV it can be administered via local injection (e.g. intramuscular, intranasal, intradermal, subcutaneous, etc. ) or systematic administration (e.g. intravenous administration) .
  • the methods comprises administering to the subject a therapeutically effective amount or a prophylactically effective amount of a combination of two or more of the antibodies (or the antigen-binding fragment thereof) provided herein.
  • the two or more antibodies comprises a first antibody comprising heavy chain CDR sequences and light chain CDR sequences derived from P2C-1F11, and a second antibody comprising heavy chain CDR sequences and light chain CDR sequences derived from antibody P2B-2F6.
  • the two or more antibodies or the antigen binding fragments thereof bind to different epitopes in RBD of spike protein of SARS-CoV-2.
  • the two or more antibodies comprise a first antibody comprising P2C-1F11, and a second antibody which is selected from the group consisting of P2C-1A3, P2C-1C10, P2B-2F6, and P2A-1B3.
  • the two or more antibodies comprise a first antibody comprising P2C-1A3 and a second antibody which is selected from the group consisting of P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof.
  • the two or more antibodies comprise a first antibody comprising P2B-2F6 and a second antibody which is selected from the group consisting of P2C-1C10, P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof.
  • the two or more antibodies comprises a first antibody comprising P2A-1B3 and a second antibody which selected from the group consisting of P2C-1A3, P2C-1C10, P2C-1F11, P2B-2F6, and P2A-1A10, or an antigen binding fragment thereof.
  • the two or more antibodies comprise a first antibody which comprises P2C-1C10 or an antigen binding fragment thereof, and a second antibody selected from the group consisting of P2C-1A3, P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof.
  • the antibodies or antigen-binding fragments thereof provided herein may be administered by any route known in the art, such as for example parenteral (e.g. subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g. oral, intranasal, intraocular, sublingual, rectal, or topical) routes.
  • parenteral e.g. subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection
  • non-parenteral e.g. oral, intranasal, intraocular, sublingual, rectal, or topical routes.
  • the antibodies or antigen-binding fragments thereof provided herein may be administered alone or in combination a therapeutically effective amount of a second active agent.
  • the second active agent can be a therapeutic agent or a prophylactic agent.
  • the second therapeutic agent is an anti-viral agent.
  • the anti-viral agent comprises an antiviral peptide, an anti-viral antibody, an anti-viral compound, an anti-viral cytokine, or an anti-viral oligonucleotide.
  • the anti-viral agent is an RNA dependent RNA polymerase inhibitor, a non-nucleoside reverse transcriptase inhibitor (NNRTI) , nucleoside reverse transcriptase inhibitor (NRTI) , purine nucleoside, antiviral cytokines such as interferon, adamantine antiviral compound, anti-RBD antibody, anti-S1 antibody, anti-S2 antibody, siRNAs Targeting mRNA of coronavirus proteins M, N, or E (Chinese patent applications CN101173275 and CN1648249) , siRNAs targeting replicase and RNA polymerase region (US patent application US20050004063) , RNA Aptamers (Korean patent applications KR2009128837 and KR 2012139512) , ribozymes (Japanese patent application JP2007043942) , antisense oligonucleotides (PCT patent application WO2005023083) , or any other suitable anti
  • the anti-viral compound is selected from the group consisting of remdesivir, chloroquine, hydroxychloroquine, lopinavir, ritonavir, APN01, favilavir, mesalazine, toremifene, eplerenone, paroxetine, sirolimus, dactinomycin, irbesartan, emodin, mercaptopurine, melatonin, quinacrine, carvedilol, colchicine, camphor, equilin, oxymetholone, nafamosta, camostat, baricitinib, darunavir, ribavirin, galidesivir, BCX-4430, Arbidol, nitazoxanide, derivatives thereof, or any combination thereof. More examples of potentially useful anti-viral agents for SARS-CoV-2 reviewed by C. Liu et al, ACS Cent. Sci. 2020, 6, 3,
  • the second active agent e.g. prophylactic agent
  • the second active agent can be a SARS-CoV-2 vaccine (e.g. mRNA-1273 by Moderna, an AAV-based vaccine capable of expressing an SARS-CoV-2 immunogen) , an antibody (e.g. directed to SARS-CoV-2) , lymphokines, hematopoietic growth factors (such as IL-2, IL-3, IL-7, and IL-15) , which can for example serve to increase the number or activity of effector cells which interact with the antibodies.
  • SARS-CoV-2 vaccine e.g. mRNA-1273 by Moderna, an AAV-based vaccine capable of expressing an SARS-CoV-2 immunogen
  • an antibody e.g. directed to SARS-CoV-2
  • lymphokines e.g. directed to SARS-CoV-2
  • lymphokines e.g. directed to SARS-CoV-2
  • hematopoietic growth factors such as IL-2, IL
  • the second active agent can comprise hormonal therapy, immunotherapy, and anti-inflammatory agents.
  • an antibody or antigen-binding fragment thereof provided herein may be administered simultaneously with the one or more additional active agents, and in certain of these embodiments the antibody or antigen-binding fragment thereof and the additional therapeutic agent (s) may be administered as part of the same pharmaceutical composition.
  • an antibody or antigen-binding fragment thereof administered “in combination” with another active agent does not have to be administered simultaneously with or in the same composition as the agent.
  • An antibody or antigen-binding fragment thereof administered prior to or after another agent is considered to be administered “in combination” with that agent as the phrase is used herein, even if the antibody or antigen-binding fragment and the second agent are administered via different routes.
  • additional active agents administered in combination with the antibodies or antigen-binding fragments thereof disclosed herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to the Physicians'Desk Reference 2003 (Physicians'Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002) ) or protocols well known in the art.
  • the present disclosure provides a method of detecting presence or amount of SARS-CoV-2 virus antigen in a sample.
  • the SARS-CoV-2 virus antigen comprises spike protein, or comprises the SARS-CoV-2 virus particle.
  • the method comprises contacting the sample with the antibody or antigen binding fragment disclosed herein, and determining the presence or the amount of the SARS-CoV-2 virus antigen in the sample.
  • the anti-SARS-CoV-2 antibody disclosed herein is used in a method of diagnosing a subject suffering from a disorder (e.g., SARS-CoV-2 infection) , the method comprising: determining the presence or amount of SARS-CoV-2 virus antigen in a sample obtained from the subject by contacting the sample with an anti-SARS-CoV-2 antibody of the disclosure and detecting the presence of the bound antibody.
  • a disorder e.g., SARS-CoV-2 infection
  • a suitable sample can be obtained from respiratory tract of the subject, for example, an upper respiratory nasopharyngeal swab (NP) , oropharyngeal swabs (OP) , sputum, a lower respiratory tract aspirate, bronchoalveolar lavage sample, nasopharyngeal wash, nasopharyngeal aspirate, nasal aspirate, a nasal swap, a throat swap, a bronchoalveolar lavage fluid (BALF) , a cell or tissue sample from respiratory tract or from lung, and the like.
  • a suitable sample can be a body fluid sample such as a whole blood sample, a serum sample, or a plasma sample.
  • a suitable sample can be a urine sample or a stool sample.
  • the presence or level of SARS-CoV-2 virus antigen in a sample can be determined based on the detection of the presence or level of the complex of the virus antigen bound by the antibody or the antigen binding fragment thereof disclosed herein. Any suitable methods can be used for such detection, for example, by immunoassays such as immunohistochemistry (IHC) , immunofluorescence (IF) , immunoblotting (e.g., Western blotting) , flow cytometry (e.g., FACS TM ) , Enzyme-linked Immunosorbant Assay (ELISA) , enzyme immunoassay (EIA) , and radioimmunoassay (RIA) .
  • immunoassays such as immunohistochemistry (IHC) , immunofluorescence (IF) , immunoblotting (e.g., Western blotting) , flow cytometry (e.g., FACS TM ) , Enzyme-linked Immunosorbant Assay (ELISA) , enzyme immunoas
  • the antibodies or the antigen binding fragments thereof disclosed herein are detectably labeled, or are not labeled but can react with a second molecule which is detectably labeled (e.g. a detectably labeled secondary antibody) .
  • the antibodies or the antigen binding fragments thereof disclosed herein may be immobilized on a solid substrate.
  • the immobilization can be via covalent linking or non-covalent attachment (e.g. coating) .
  • solid substrate include porous and non-porous materials, latex particles, magnetic particles, microparticles, strips, beads, membranes, microtiter wells and plastic tubes. The choice of solid phase material and method of detectably labeling can be determined based upon desired assay format performance characteristics.
  • the level of the SARS-CoV-2 antigen can be determined, for example, by normalizing to a control value or to a standard curve.
  • the control value can be predetermined, or determined concurrently.
  • the assays and methods provided herein for the measurement of the level of the SARS-CoV-2 antigen can be adapted or optimized for use in automated and semi-automated systems, or point of care assay systems.
  • the present disclosure provides a method of detecting presence or amount of an antibody capable of specifically binding to RBD of the spike protein of SARS-CoV-2 in a sample, comprising contacting the sample with a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 128, and determining the presence or the level of the antibody in the sample.
  • the absence of the antibody in the sample or the level of the antibody in the sample being below a threshold indicates that the subject is more likely to suffer from disease progression.
  • the present disclosure provides a method of determining or predicting the likelihood of disease progression in a subject infected with SARS-CoV-2, the method comprising: contacting a sample obtained from the subject with a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 128, and detecting the presence or the level of an antibody in the sample wherein the antibody is capable of specifically binding to RBD of the spike protein of the SARS-CoV-2, wherein the subject is likely to experience disease progression when the antibody in the sample is absent or is below a threshold.
  • a subject infected with SARS-CoV-2 can produce antibodies against the SARS-CoV-2 antigens.
  • Such antibodies produced by human immune system are polyclonal, and can bind to different antigens or epitopes of SARS-CoV-2.
  • the presence or level of the antibodies specific to the RBD of the spike protein of the SARS-CoV-2 can be indicative of likelihood of disease progression in the subject.
  • Antibodies capable of specifically binding to the RBD of the spike protein of the SARS-CoV-2 are found by the inventors to be capable of effectively competing with ACE2 receptor for binding to the RBD, and also provide for SARS-CoV-2 virus neutralizing activity.
  • RBD-specific antibody can be associated with an effective immune response to the SARS-CoV-2, and the titer of such RBD-specific antibody in the body may correlate to the prognosis of the SARS-CoV-2 infection or a disease, disorder or condition associated with SARs-CoV-2 infection.
  • a threshold of the level of the RBD-specific antibodies can be predetermined.
  • the threshold refers to a level of the RBD-specific antibodies above which the sample is scored as being positive for RBD-specific antibodies.
  • the threshold can be a level above which the sample is scored as having sufficient neutralizing activity against the SARS-CoV-2. If the level of the RBD-specific antibodies is below the threshold, it could indicate insufficient protective immunity in the subject, and hence likelihood of disease progression. In contrast, if the level of the RBD-specific antibodies in the sample reaches or is above the threshold, it could indicate protective immunity in the subject, and hence less likely to suffer from disease progression.
  • a suitable sample can be obtained from blood, for example, a whole blood sample, a serum sample, or a plasma sample.
  • said sample is obtained from a subject suspected of having, inflicted with, or under treatment for SARS-CoV-2 infection, or a disease, disorder or condition associated with SARs-CoV-2 infection.
  • Polypeptides comprising the RBD of the spike protein of SARS-CoV-2 can be used in the methods provided to herein to detect presence or level of the RBD-specific antibodies in the subject.
  • the RBD of the spike protein of SARS-CoV-2 comprises an amino acid sequence comprising SEQ ID NO: 128.
  • the polypeptides can further comprise a tag. Exemplary tag include, without limitation, 6xHis tag or its fusion such SEQ ID NO: 132 or SEQ ID NO: 133.
  • the polypeptides comprising RBD may be produced by recombinant methods (e.g., by prokaryotic expression system or eukaryotic expression system) , or chemically synthesized (e.g.
  • polypeptides can be purified by methods known in the art. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymology, 182 (1990) ; Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982) .
  • the purification step (s) selected will depend, for example, on the nature of the production process used and the particular polypeptide of the present application produced.
  • the presence or level of RBD-specific antibodies in a sample can be determined based on the detection of the presence or level of the complex of the RBD bound by the RBD-specific antibodies. Any suitable methods can be used for such detection, for example, by immunoassays such as immunohistochemistry (IHC) , immunofluorescence (IF) , immunoblotting (e.g., Western blotting) , flow cytometry (e.g., FACS TM ) , Enzyme-linked Immunosorbant Assay (ELISA) , enzyme immunoassay (EIA) , and radioimmunoassay (RIA) , as described above.
  • immunoassays such as immunohistochemistry (IHC) , immunofluorescence (IF) , immunoblotting (e.g., Western blotting) , flow cytometry (e.g., FACS TM ) , Enzyme-linked Immunosorbant Assay (ELISA) , enzyme immunoassay (EIA)
  • the polypeptide comprising RBD of the spike protein of the SARS-CoV-2 may be immobilized on a solid substrate.
  • the immobilization can be via covalent linking or non-covalent attachment (e.g. coating) .
  • the sample suspected of containing the RBD-specific antibodies can be brought into contact with the bound polypeptide. After a suitable period of incubation, for a period of time sufficient to allow capture of the RBD-specific antibodies via formation of antibody-antigen complex. After washing away any unreacted materials, a detection antibody specific to the captured antibody can be added, which can produce a detectable signal to allow detection of the captured antibody.
  • the results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of the detectable signal.
  • the present disclosure provides a method of monitoring treatment response in a subject infected with SARS-CoV-2 and received a treatment, the method comprising: (i) contacting a sample from the subject with a peptide comprising an amino acid sequence comprising SEQ ID NO: 128; (ii) detecting a first level of an antibody in the sample wherein the antibody is capable of specifically binding to RBD of the spike protein of the SARS-CoV-2; and (iii) comparing the first level of the antibody with a second level of the antibody detected in the subject prior to the treatment; wherein the first level being higher than the second level indicates that the subject is responsive to the treatment.
  • a sample is obtained from a subject or patient prior to any treatment.
  • a test sample is obtained during or after treatment such as anti-viral treatment.
  • the present disclosure provides a kit for detecting an antibody capable of specifically binding to receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, comprising a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 128.
  • the polypeptide is immobilized on a substrate.
  • the kit further comprises a set of reagents for detecting complex of the antibody bound to the polypeptide.
  • the present disclosure provides a kit comprising one or more of the antibody or an antigen-binding fragment thereof provided herein.
  • the kit disclosed herein is a therapeutic kit.
  • the kit disclosed herein is a diagnostic kit.
  • kits can further include, if desired, one or more of various conventional kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers etc., as will be readily apparent to a person skilled in the art.
  • kit components such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers etc., as will be readily apparent to a person skilled in the art.
  • Instructions, either as inserts or a label, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.
  • the kit will include substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides the detectable chromophore or fluorophore) .
  • substrates and cofactors required by the enzyme e.g., a substrate precursor which provides the detectable chromophore or fluorophore
  • other additives may be included such as stabilizers, buffers (e.g., a block buffer or lysis buffer) and the like.
  • the relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay.
  • the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.
  • kits comprising one or more such reagents for use in a variety of detection assays, including for example, immunoassays such as ELISA (sandwich-type or competitive format) .
  • the kit's components may be pre-attached to a solid support, or may be applied to the surface of a solid support when the kit is used.
  • the signal generating means may come pre-associated with an antibody of the invention or may require combination with one or more components, e.g., buffers, antibody-enzyme conjugates, enzyme substrates, or the like, prior to use.
  • Kits may also include additional reagents, e.g., blocking reagents for reducing nonspecific binding to the solid phase surface, washing reagents, enzyme substrates, and the like.
  • the solid phase surface may be in the form of a tube, a bead, a microtiter plate, a microsphere, or other materials suitable for immobilizing proteins, peptides, or polypeptides.
  • an enzyme that catalyzes the formation of a chemiluminescent or chromogenic product or the reduction of a chemiluminescent or chromogenic substrate is a component of the signal generating means. Such enzymes are well known in the art.
  • Kits may comprise any of the capture agents and detection reagents described herein.
  • the kit may also comprise instructions for carrying out the methods of the invention.
  • the detection kits disclosed herein may also be prepared that comprise at least one of the antibodies or antigen-binding fragments disclosed herein and instructions for using the composition as a detection reagent.
  • Containers for use in such kits may typically comprise at least one vial, test tube, flask, bottle, syringe or other suitable container, into which one or more of the detection composition (s) may be placed, and preferably suitably aliquoted.
  • the kits disclosed herein will also typically include a means for containing the vial (s) in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vial (s) are retained.
  • the labeling agent may be provided either in the same container as the detection composition itself, or may alternatively be placed in a second distinct container means into which this second composition may be placed and suitably aliquoted.
  • the detection reagent may be prepared in a single container means, and in most cases, the kit will also typically include a means for containing the vial (s) in close confinement for commercial sale and/or convenient packaging and delivery.
  • a device or apparatus for carrying out the detection or monitoring methods described herein may include a chamber or tube into which sample can be input, a fluid handling system optionally including valves or pumps to direct flow of the sample through the device, optionally filters to separate plasma or serum from blood, mixing chambers for the addition of capture agents or detection reagents, and optionally a detection device for detecting the amount of detectable label bound to the capture agent immunocomplex.
  • a fluid handling system optionally including valves or pumps to direct flow of the sample through the device, optionally filters to separate plasma or serum from blood, mixing chambers for the addition of capture agents or detection reagents, and optionally a detection device for detecting the amount of detectable label bound to the capture agent immunocomplex.
  • the flow of sample may be passive (e.g., by capillary, hydrostatic, or other forces that do not require further manipulation of the device once sample is applied) or active (e.g., by application of force generated via mechanical pumps, electroosmotic pumps, centrifugal force, or increased air pressure) , or by a combination of active and passive forces.
  • PBMCs peripheral blood mononuclear cells
  • Recombinant RBDs and trimeric Spike from SARS-CoV-2, SARS-CoV, and MERS-CoV and receptor ACE2 Recombinant RBDs and trimeric Spike for MERS-CoV, SARS-CoV, and SARS-CoV-2 and the N-terminal peptidase domain of human ACE2 (residues Ser19-Asp615) were expressed using the Bac-to-Bac baculovirus system (Invitrogen) as previously described (Gui, M. et al. Cell Res 27, 119-129 (2017) ; Song, W. et al. PLoS Pathog 14, e1007236-e1007236 (2016) ; Wang, N. et al.
  • Amino acid sequence for RBD of spike protein for MERS-CoV is shown in SEQ ID NO: 126, and the polynucleotide sequence is shown in SEQ ID NO: 127.
  • Amino acid sequence for extracellular domain of the spike protein for MERS-CoV is shown in SEQ ID NO: 123.
  • Amino acid sequence for RBD of spike protein for SARS-CoV is shown in SEQ ID NO: 124, and the polynucleotide sequence is shown in SEQ ID NO: 125.
  • Amino acid sequence for extracellular domain of the spike protein for SARS-CoV is shown in SEQ ID NO: 122.
  • Amino acid sequence for RBD of spike protein for SARS-CoV-2 is shown in SEQ ID NO: 128, and the polynucleotide sequence is shown in SEQ ID NO: 129.
  • Amino acid sequence for extracellular domain of the spike protein for SARS-CoV-2 is shown in SEQ ID NO: 121. Extracellular domains of the spike protein were fused to an artificial sequence to enable formation of a trimeric Spike structure in vitro.
  • SARS-CoV-2 RBD (residues Arg319-Phe541) containing the gp67 secretion signal peptide (SEQ ID NO: 130) and a C-terminal 6 ⁇ His tag (SEQ ID NO: 132) was inserted into pFastBac-Dual vectors (Invitrogen) and transformed into DH10Bac component cells.
  • the bacmid was extracted and further transfected into Sf9 cells using Cellfectin II Reagents (Invitrogen) .
  • the recombinant viruses were harvested from the transfected supernatant and amplified to generate high-titer virus stock. Viruses were then used to infect Hi5 cells for RBD and trimeric Spike expression.
  • RBD recombinant RBDs and trimeric Spike derived from SARS-CoV-2, SARS-CoV and MERS-CoV and the SARS-CoV-2 NP protein (Sino Biological, Beijing) were diluted to final concentrations of 0.5 ⁇ g/ml or 2 ⁇ g/ml, then coated onto 96-well plates and incubated at 4°C overnight. Samples were washed with PBS-T (PBS containing 0.05%Tween 20) and blocked with blocking buffer (PBS containing 5%skim milk and 2%BSA) at RT for 1h.
  • PBS-T PBS containing 0.05%Tween 20
  • blocking buffer PBS containing 5%skim milk and 2%BSA
  • RBD-specific single B cells were sorted as previously described (Kong, L. et al. Immunity 44, 939-950 (2016) ; Wu, X. et al. Science 329, 856-861 (2010) ) .
  • PBMCs from infected and convalescent individuals were collected and incubated with an antibody and RBD cocktail for identification of RBD-specific B cells.
  • the cocktail consisted of CD19-PE-Cy7, CD3-Pacific Blue, CD8-Pacific Blue, CD14- Pacific Blue, CD27-APC-H7, IgG-FITC (BD Biosciences) and the recombinant RBD-Strep or RBD-His described above.
  • PBS phosphate-buffered saline
  • the stained cells were washed and resuspended in PBS before being strained through a 70 ⁇ m cell mesh (BD Biosciences) .
  • RBD-specific single B cells were gated as CD19+CD3-CD8-CD14-IgG+RBD+ and sorted into 96-well PCR plates containing 20 ⁇ l of lysis buffer (5 ⁇ l of 5 x first strand buffer, 0.5 ⁇ l of RNase out, 1.25 ⁇ l of 0.1 M DTT (Invitrogen) per well and 0.0625 ⁇ l of Igepal (Sigma) . Plates were then snap-frozen on dry ice and stored at -80 °C until RT reaction.
  • the IgG heavy and light chain variable genes were amplified by nested PCR and cloned into linear expression cassettes or expression vectors to produce full IgG1 antibodies as previously described (Liao, H. -X. et al. J Virol Methods, 2009; Tiller, T. et al. J. Immunol Methods, 2008) .
  • all second round PCR primers containing tag sequences were used to produce the linear Ig expression cassettes by overlapping PCR.
  • Overlapping PCR products of paired heavy and light chain expression cassettes were co-transfected into 293T cells (ATCC) grown in 24-well plates.
  • Antigen-specific ELISA was used to detect the binding capacity of transfected culture supernatants to SARS-CoV-2 RBD.
  • Monoclonal antibodies were produced by transient transfection of 293F cells (Life Technologies) with equal amounts of paired heavy and light chain plasmids.
  • Table 4 shows the encoding DNA sequences and the amino acid sequences for the heavy chain and light chain variable regions of the monoclonal antibodies including P2A-1A8, P2A-1A9, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2B-2G11, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, and P2C-1F11.
  • Antibodies P2A-1A8, P2A-1A9, P2B-2F6, P2B-2G4, P2B-2G11, and P2C-1D5 have lambda light chains, and the encoding DNA sequences and the amino acid sequences for the lambda constant region is shown in SEQ ID NO: 116 and SEQ ID NO: 119, respectively.
  • Antibodies P2A-1A10, P2A-1B3, P2C-1A3, P2C-1C8, P2C-1C10, and P2C-1F11 have kappa light chains, and the encoding DNA sequences and the amino acid sequences for the kappa constant region is shown in SEQ ID NO: 117 and SEQ ID NO: 120, respectively.
  • Antibodies in the culture supernatant was purified by affinity chromatography using Protein A beads columns (National Engineering Research Center for Biotechnology, Beijing) according to the manufacturer’s protocol. Concentrations were determined by BCA Protein Assay Kits (Thermo Scientific) . SARS-CoV, MERS-CoV, and HIV-1 mAbs were also included as controls. SARS-CoV antibodies (S230 and m396) previously isolated by others (Zhu, Z. et al. Proc Natl Acad Sci USA 104, 12123-12128 (2007) ) were synthesized and sequences verified before expression in 293T cells and purification by protein A chromatography. MERS-CoV antibodies (Mab-GD33) were derived from previously reported (Niu, P. et al. J Infect Dis 218, 1249-1260 (2016) ) . HIV-1 antibody VRC01 was a broadly neutralizing antibody directly isolated from a patient targeting the CD4 binding site of envelope glycoprotein 40.
  • Antibody binding kinetics, epitope mapping, and competition with receptor ACE2 measured by SPR were analyzed by SPR (Biacore T200, GE Healthcare) . Specifically, purified RBDs were covalently immobilized to a CM5 sensor chip via amine groups in 10mM sodium acetate buffer (pH 5.0) for a final RU around 250. SPR assays were run at a flow rate of 30ml/min in HEPE buffer. The sensograms were fit in a 1: 1 binding model with BIA Evaluation software (GE Healthcare) .
  • HEK 293T cells were transfected with expression plasmid encoding the full length spike of SARS-CoV-2, SARS-CoV or MERS-CoV and incubated at 37 °C for 36 h. The cells were digested with trypsin and distributed into 96 well plates for the individual staining. Cells were washed twice with 200 ⁇ l staining buffer (PBS with 2%heated-inactivated FBS) between each following steps. The cells were stained at room temperature for 30 minutes in 100 ⁇ l staining buffer with 1: 100 dilutions of plasma or 20 ⁇ g/ml monoclonal antibodies.
  • the cells were then stained with PE labeled anti-human IgG Fc secondary antibody (Biolegend) at a 1: 20 dilution in 50 ⁇ l staining buffer at room temperature for 30 minutes. Finally, the cells were re-suspended and analyzed with FACS Calibur instrument (BD Biosciences, USA) and FlowJo 10 software (FlowJo, USA) . HEK 293T cells without transfection were also stained as background control. S230 and m396 targeting the RBD of SARS-CoV spike (Zhu, Z. et al. Proc Natl Acad Sci USA 104, 12123-12128 (2007) ) and Mab-GD33 targeting the RBD of MERS-CoV spike (Niu, P.
  • SARS-CoV-2, SARS-CoV and MERS-CoV pseudovirus were generated by co-transfection of human immunodeficiency virus backbones expressing firefly luciferase (pNL43R-E-luciferase) and pcDNA3.1 (Invitrogen) expression vectors encoding the respective full length S proteins into 293T cells (ATCC) (Wang, N. et al. Cell Res 23, 986-993 (2013) ; Jiang, L. et al. Sci Transl Med 6, 234ra259-234ra259 (2014) ; Jia, W. et al.
  • Viral supernatants were collected 48 h later. Viral titers were measured as luciferase activity in relative light units (Bright-Glo Luciferase Assay Vector System, Promega Biosciences) . Control envelope glycoproteins derived from human immunodeficiency virus (HIV) -1 and their corresponding pseudoviruses were produced in the same manner. Control mAbs included VRC01 against HIV-1 40; S230 and m396 against SARS-CoV (Zhu, Z. et al.
  • Antibody production The production of antibodies was conducted as previously described (Jiang, L. et al. Sci Transl Med 6, 234ra259-234ra259 (2014) ; Zhang, Q. et al. Sci Rep 6, 25856-25856 (2016) ) .
  • the genes encoding the heavy and light chains of isolated antibodies were separately cloned into expression vectors containing IgG1 constant regions and the vectors were transiently transfected into HEK293T or 293F cells using polyethylenimine (PEI) (Sigma) .
  • PEI polyethylenimine
  • the antibodies secreted into the supernatant were collected and captured by protein A Sepharose (GE Healthcare) .
  • the bound antibodies were eluted and further purified by gel-filtration chromatography using a Superdex 200 High Performance column (GE Healthcare) .
  • the purified antibodies were either used in binding and neutralizing assays.
  • This example illustrates the identification of human plasma and B cell that responses specific to SARS-CoV-2 RBD.
  • serial plasma dilutions were applied to enzyme-linked immunosorbent assay (ELISA) plates coated with either recombinant RBD or trimeric Spike derived from SARS-CoV-2, SARS-CoV, and MERS-CoV or recombinant NP from SARS-CoV-2.
  • ELISA enzyme-linked immunosorbent assay
  • Binding activity was visualized using anti-human IgG secondary antibodies at an optical density (OD) of 450nm. Varying degrees of binding were found across individuals and among samples from the same individual. Samples from P#1, P#2, P#5, and P#16 demonstrated higher binding to both SARS-CoV-2 RBD and NP than the rest ( Figure 1 (A) ) .
  • sample P#1A demonstrated the lowest RBD-specific B cell response despite high-level plasma binding.
  • P#1 was the only patient succumb to disease, it is possible that this dichotomy of high plasma binding activity and low levels of RBD-specific B cells is a surrogate marker of rapid disease progression.
  • This example illustrates the cloning and analysis of single B cell antibody against SARS-CoV-2 RBD.
  • the RBD-binding B cells identified in EXAMPLE 2 were isolated into single cell suspension for cloning and evaluation of the mAb response ( Figure 1 (D) and Figure 6) .
  • Immunoglobulin heavy and light chains were amplified by RT-PCR using nested primers.
  • the amplified products were cloned into linear expression cassettes to produce full IgG1 antibodies as previously described (Kong, L. et al. Immunity 44, 939-950 (2016) ; Liao, H. -X. et al. J Virol Methods 158, 171-179) .
  • the number of B cell clones varied from 10 to 10 6 among the subjects and each clone has been differentially represented (Figure 7) .
  • the corresponding light-chain kappa (Igk) belongs to 2-40*01/2D-40*01, 3-20*01, and light-chain lambda (Igl) to 2-14*02 with the respective joining segment kappa 4 (Jk4) , Jk5 and joining segment lambda 1 (Jl1) (Table 9) . More importantly, these clonally expanded antibodies were identified in all three samples indicating that they are strongly selected for during infection. When comparing their representation within each cluster, VH1-2*06 and VH3-9*01 appeared to increase from approximately 33 to 45%, whereas VH3-48*02 decreased from 33 to 9%over the three time points, although the number of clones was too small for statistical significance.
  • This example illustrates the binding properties of the antibodies against SARS-CoV-2 RBD.
  • SHM did not appear to correlate with Kd; some germline clones with 0%divergence in both VH and VL genes (P2A-1A10, P2B-2G4, P2C-1A3, and P2C-1E1) had Kd values comparable to clones with higher levels of SHM.
  • each antibody for competition with ACE2 for binding to the SARS-CoV-2 RBD were measured.
  • the RBD was covalently immobilized on a CM5 sensor chip and first saturated by antibody and then flowed through with soluble ACE2. Competing capacity of each antibody was measured as percent reduction in ACE2 binding with the RBD (Table 7 and Figure 9) . As shown in Table 7, the evaluated antibodies demonstrated various competing capacity with ACE2. The most powerful were P2C-1F11 and P2B-2F6, which reduced ACE2 binding about 99.2%and 98.5%, respectively. Two of the three representative antibodies from the clonal expanded clusters (P2A-1A10 and P2A-1B3) had slightly over 80%and 90%reduction, respectively. The third representative (P2A-1A8) only showed 57%reduction.
  • This example illustrates the neutralizing properties of the antibodies against pseudoviruses bearing the Spike protein of SARS-CoV-2.
  • P2C-1F11, P2B-2F6, and P2C-1A3 for neutralizing activity analyses against live SARS-CoV-2. Consistent with their respective pseudovirus assay findings, P2C-1F11 and P2B-2F6 demonstrated potent neutralization activity while that of P2C-1A3 was somewhat lower, although it needs to be noted that CPE assay is not particularly quantitative ( Figure 4 (C) ) .
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • Antibodies having an IC50 ⁇ 50 ⁇ g/ml are defined as specific neutralizing antibody.
  • the program IMGT/V-QUEST was applied to analyze gene germline, complementarity determining region (CDR) 3 length, and somatic hypermutation (SHM) .
  • CDR3 length was calculated from amino acids sequences.
  • SHM frequency was calculated from the mutated nucleotides.
  • Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450-454, doi: 10.1038/nature02145 (2003) .

Abstract

Provided herein are novel anti-SARS-COV-2 antibodies or antigen binding fragments thereof, pharmaceutical composition and kits comprising the same, and the uses thereof.

Description

ANTI-SARS-COV-2 ANTIBODIES AND USES THEREOF FIELD OF THE INVENTION
The present disclosure generally relates to novel anti-SARS-COV-2 antibodies.
BACKGROUND
The recent outbreak of the new coronavirus, SARS-CoV-2 poses a serious global health emergency. SARS-CoV-2 is a positive-sense single-stranded RNA (+ssRNA) virus which belongs to the betacoronavirus family and shares substantial genetic and functional similarity with other pathogenic human betacoronaviruses, including Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV, also called SARS-CoV-1) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) . Like other coronaviruses, SARS-CoV-2 has four structural proteins, known as the S (spike) , E (envelope) , M (membrane) , and N (nucleocapsid) proteins; the S, E, and M proteins together create the viral envelope; inside the envelope is the N protein bounding to the RNA genome (～30 kb) in a continuous beads-on-a-string type conformation.
The spike protein is the protein responsible for allowing the SARS-CoV-2 virus to attach to the membrane of a host cell, the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 recognizes and attaches to the angiotensin-converting enzyme 2 (ACE2) receptor of host cells to use them as a mechanism of cell entry. The overall ACE2-binding mechanism is virtually the same between SARS-CoV-2 RBD and SARS-CoV RBD, indicating convergent ACE2-binding evolution between these two viruses. This suggests that disruption of the RBD and ACE2 interaction would block the entry of SARS-CoV-2 into the target cell. Indeed, a few such disruptive agents targeted to ACE2 have been shown to inhibit SARS-CoV infection. However, given the important physiological roles of ACE2 in vivo, these agents may have undesired side effects. Anti-RBD antibodies, on the other hand, are therefore more favorable. Furthermore, SARS-CoV-RBD or MERS-CoV RBD-based vaccine studies in experimental animals have also shown strong polyclonal antibody responses that inhibit viral entry. Such critical proof-of-concept findings indicate that anti-RBD antibodies might effectively block SARS-CoV-2 entry.
No SARS-CoV-2-specific treatments or vaccine are currently available, and the currently existing detective measures for SARS-CoV-2 infection are time-consuming and insensitive. Hence, there is an urgent need for novel anti-SARS-CoV-2 antibodies.
BRIEF SUMMARY OF THE INVENTION
Throughout the present disclosure, the articles “a, ” “an, ” and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an antibody” means one antibody or more than one antibody.
In one aspect, the present disclosure provides an isolated or recombinant antibody or an antigen-binding fragment thereof, which is capable of specifically binding to SARS-CoV-2, and exhibiting at least 50%less binding or non-detectable binding to SARS-CoV or MERS-CoV.
In another aspect, the present disclosure provides an isolated or recombinant antibody or an antigen-binding fragment thereof, having one or more features selected from the group consisting of: a) capable of specifically binding to spike protein of SARS-CoV-2 and exhibiting at least 50%less binding to spike protein of SARS-CoV or spike protein of MERS-CoV; b) capable of specifically binding to receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 comprising the amino acid sequence of SEQ ID NO: 128; c) exhibiting binding to RBD of spike protein of SARS-CoV comprising the amino acid sequence of SEQ ID NO: 124 at a level that is non-detectable or that is no more than 50%of the binding to the RBD of spike protein of SARS-CoV-2; d) exhibiting binding to RBD of spike protein of MERS-CoV comprising the amino acid sequence of SEQ ID NO: 126 at a level that is non-detectable or that is no more than 50%of the binding to RBD of the spike protein of SARS-CoV-2; e) capable of binding to the RBD of spike protein of SARS-CoV-2 at a K d value of no more than 1x10 -7M as measured by Surface Plasmon resonance (SPR) ; f) exhibiting binding to the RBD of spike protein of SARS-CoV or the RBD of spike protein of MERS-CoV at a K d value of at least 1x10 -6 M as measured by SPR; g) capable of exhibiting at least 30%competition at 1μM, with 2 μM angiotensin converting enzyme 2 (ACE2) receptor, for binding to the RBD of spike protein of SARS-CoV-2 immobilized at a resonance units (RU) of 250, as measured by SPR; and h) capable of binding to the RBD of spike protein of SARS-CoV-2 at an neutralizing activity at an IC 50 value of no more than 100μg/ml (for example, no more than 50μg/ml, no more than 40μg/ml, no more than 30μg/ml,  no more than 25μg/ml, no more than 20μg/ml, no more than 15μg/ml, no more than 10μg/ml, no more than 8μg/ml, no more than 6μg/ml, no more than 4μg/ml, no more than 2μg/ml, or no more than 1μg/ml) , as measured by pseudovirus neutralization assay.
In yet another aspect, the present disclosure provides an isolated or recombinant antibody or an antigen-binding fragment thereof capable of specifically binding to RBD of spike protein of SARS-CoV-2.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 98, SEQ ID NO: 99, and SEQ ID NO: 100.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises a heavy chain CDR1 (HCDR1) comprising the sequence of SEQ ID NO: 1, a heavy chain CDR2 (HCDR2) comprising the sequence of SEQ ID NO: 2, a heavy chain CDR3 (HCDR3) comprising the sequence of SEQ ID NO: 3; a light chain CDR1 (LCDR1) comprising the sequence of SEQ ID NO: 4, a light chain CDR2 (LCDR2) comprising the sequence of SEQ ID NO: 5, and a light chain CDR3 (LCDR3) comprising the sequence of SEQ ID NO: 6.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 11, a HCDR2 comprising the sequence of SEQ ID NO: 12, a HCDR3 comprising the sequence of SEQ ID NO: 13, a LCDR1 comprising the sequence of SEQ ID NO: 14, a LCDR2 comprising the sequence of SEQ ID NO: 15, and a LCDR3 comprising the sequence of SEQ ID NO: 16.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 21, a HCDR2 comprising the sequence of SEQ ID NO: 22, a HCDR3 comprising the sequence of SEQ ID NO: 23, a LCDR1 comprising the sequence of SEQ ID NO: 24, a LCDR2 comprising the sequence of SEQ ID NO: 25, and a LCDR3 comprising the sequence of SEQ ID NO: 26.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 31, a HCDR2  comprising the sequence of SEQ ID NO: 32, a HCDR3 comprising the sequence of SEQ ID NO: 33, a LCDR1 comprising the sequence of SEQ ID NO: 34, a LCDR2 comprising the sequence of SEQ ID NO: 35, and a LCDR3 comprising the sequence of SEQ ID NO: 36.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 41, a HCDR2 comprising the sequence of SEQ ID NO: 42, a HCDR3 comprising the sequence of SEQ ID NO: 43, a LCDR1 comprising the sequence of SEQ ID NO: 44, a LCDR2 comprising the sequence of SEQ ID NO: 45, and a LCDR3 comprising the sequence of SEQ ID NO: 46.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 51, a HCDR2 comprising the sequence of SEQ ID NO: 52, a HCDR3 comprising the sequence of SEQ ID NO: 53, a LCDR1 comprising the sequence of SEQ ID NO: 54, a LCDR2 comprising the sequence of SEQ ID NO: 55, and a LCDR3 comprising the sequence of SEQ ID NO: 56.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 65, a HCDR2 comprising the sequence of SEQ ID NO: 66, a HCDR3 comprising the sequence of SEQ ID NO: 67, a LCDR1 comprising the sequence of SEQ ID NO: 68, a LCDR2 comprising the sequence of SEQ ID NO: 69, and a LCDR3 comprising the sequence of SEQ ID NO: 70.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 75, a HCDR2 comprising the sequence of SEQ ID NO: 76, a HCDR3 comprising the sequence of SEQ ID NO: 77, a LCDR1 comprising the sequence of SEQ ID NO: 78, a LCDR2 comprising the sequence of SEQ ID NO: 79, and a LCDR3 comprising the sequence of SEQ ID NO: 80.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 85, a HCDR2 comprising the sequence of SEQ ID NO: 86, a HCDR3 comprising the sequence of SEQ ID NO: 87, a LCDR1 comprising the sequence of SEQ ID NO: 88, a LCDR2 comprising the sequence of SEQ ID NO: 89, and a LCDR3 comprising the sequence of SEQ ID NO: 90.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 95, a HCDR2 comprising the sequence of SEQ ID NO: 96, a HCDR3 comprising the sequence of SEQ ID  NO: 97, a LCDR1 comprising the sequence of SEQ ID NO: 98, a LCDR2 comprising the sequence of SEQ ID NO: 99, and a LCDR3 comprising the sequence of SEQ ID NO: 100.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises a HCDR1 comprising the sequence of SEQ ID NO: 105, a HCDR2 comprising the sequence of SEQ ID NO: 106, a HCDR3 comprising the sequence of SEQ ID NO: 107, a LCDR1 comprising the sequence of SEQ ID NO: 108, a LCDR2 comprising the sequence of SEQ ID NO: 109, and a LCDR3 comprising the sequence of SEQ ID NO: 110.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises a heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 7, 17, 27, 37, 47, 57, 61, 71, 81, 91, 101, and 111, or a homologous sequence thereof having at least 80%sequence identity.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises a light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 8, 18, 28, 38, 48, 58, 62, 72, 82, 92, 102, and 112, or a homologous sequence thereof having at least 80%sequence identity.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises a pair of heavy chain variable region and light chain variable region sequences selected from the group consisting of: SEQ ID NOs: 7/8, 17/18, 27/28, 37/38, 47/48, 57/58, 61/62, 71/72, 81/82, 91/92, 101/102, and 111/112, or a pair of homologous sequences thereof having at least 80%sequence identity yet retaining specific binding affinity to RBD of spike protein of SARS-CoV-2.
In some embodiments, the antibody or antigen binding fragment of the present disclosure further comprises an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is a constant region of human immunoglobulin. In some embodiments, the immunoglobulin constant region is a constant region of human IgG. In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 or IgM. In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises a heavy chain constant region of human IgG1. In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises a constant region of human immunoglobulin kappa 1 light chain. In some embodiments, the antibody or  antigen binding fragment of the present disclosure comprises a constant region of human immunoglobulin lambda light chain.
In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises one or more amino acid residue substitutions or modifications yet retains specific binding affinity to RBD of spike protein of SARS-CoV-2.
In some embodiments, the antibody or antigen binding fragment is an affinity variant, a glycosylation variant, a cysteine-engineered variant, or an Fc variant.
In some embodiments, the glycosylation variant comprises a mutation at N297 (e.g. N297A, N297Q, or N297G) , for example, to modify the glycosylation site.
In some embodiments, the Fc variant comprises one or more amino acid residue modifications or substitutions resulting in increased effector functions relative to a wildtype Fc. In some embodiments, the Fc variant comprises one or more amino acid substitution (s) at one or more of the positions selected from the group consisting of: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 246, 247, 248, 249, 252, 254, 255, 256, 258, 260, 262, 263, 264, 265, 267, 268, 269, 270, 272, 274, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 300, 301, 303, 304, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 345, 360, 373, 376, 378, 382, 388, 389, 396, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438, 439 and 440 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat. In some embodiments, the Fc variant comprises one or more amino acid substitution selected from the group consisting of 234Y, 235Q, 236A, 236W, 239D, 239E, 239M, 243L, 247I, 267E, 268D, 268E, 268F, 270E, 280H, 290S, 292P, 298A, 298D, 298V, 300L, 305I, 324T, 326A, 326D, 326W, 330L, 330M, 333S, 332D, 332E, 333A, 334A, 334E, 339D, 339Q, 345R, 396L, 430G, 440Y, and any combination thereof. In some embodiments, the Fc variant having increased effector function comprises a combination of mutations selected from the group consisting of: a) S239D, I332E, and A330L; b) F243L, R292P, Y300L, V305I and P396L; c) S239D and I332E; d) S239D, I332E and A330L; e) S298A, E333A and K334A; f) L234Y, L235Q, G236W, S239M, H268D, D270E and S298A (in one heavy chain) and D270E, K326D, A330M and K334E (in the opposing heavy chain) ; G236A, S239D and I332E; g) K326W and E333S; h) S267E, H268F and S324T; i) E345R, E430G and S440Y.
In some embodiments, the Fc variant comprises one or more amino acid residue modifications or substitutions resulting in reduced effector functions relative to a  wildtype Fc. In some embodiments, the Fc variant comprises one or more amino acid substitution (s) at a position selected from the group consisting of: 220, 226, 229, 233, 234, 235, 236, 237, 238, 267, 268, 269, 270, 297, 309, 318, 320, 322, 325, 328, 329, 330, and 331 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat. In some embodiments, the Fc variant comprises one or more amino acid substitution (s) selected from the group consisting of 220S, 226S, 228P, 229S, 233P, 234V, 234G, 234A, 234F, 234A, 235A, 235G, 235E, 236E, 236R, 237A, 237K, 238S, 267R, 268A, 268Q, 269R, 297A, 297Q, 297G, 309L, 318A, 322A, 325L, 328R, 330S, 331S and any combination thereof. In some embodiments, the Fc variant having reduced effector function comprises a combination of mutations selected from the group consisting of: a) K322A, L234A, and L235A; b) P331S, L234F, and L235E; c) L234A and L235A; c) N297A; d) N297Q; e) N297G; f) L235E; g) L234A and L235A (IgG1) ; h) F234A and L235A (IgG4) ; i) H268Q, V309L, A330S and P331S (IgG2) ; j) V234A, G237A, P238S, H268A, V309L, A330S and P331S (IgG2) .
In some embodiments, the Fc variant comprises one or more amino acid residue modifications or substitutions resulting in improved binding affinity to neonatal Fc receptor (FcRn) at pH 6.0 while retaining minimal binding at pH 7.4, or increased serum half life of the antibody. In some embodiments, the Fc variant comprises one or more amino acid substitution (s) at a position selected from the group consisting of: 234 (e.g., with F) , 235 (e.g., with Q) , 238 (e.g., with D) , 250 (e.g., with E or Q) , 252 (e.g., with L/Y/F/W or T) , 254 (e.g., with S or T) , 256 (e.g., with S/R/Q/E/D or T) ; 259 (e.g., with I) ; 272 (e.g., with A) , 305 (e.g., with A) , 307 (e.g., with A or P) , 308 (e.g., with F, C or P) , 311 (e.g., with A or R) , 312 (e.g., with A ) , 322 (e.g., Q) , 328 (e.g. E) , 331 (e.g., with A) , 378 (e.g., with A) , 380 (e.g., with A) , 382 (e.g., with A) , 428 (e.g., with L or F) , 432 (e.g., with C) , 433 (e.g., with H/L/R/S/P/Q or K) , 434 (e.g., with H/F or Y or S or A or W) , 435 (e.g. with H) , 436 (e.g., with L) and 437 (e.g., with C) ) (all positions by EU numbering) . In some embodiments, the Fc variant comprises one or more amino acid substitution (s) selected from the group consisting of 234F, 235Q, 238D, 250Q, 252T, 252Y, 254T, 256E, 259I, 272A, 305A, 307A, 308F, 311A, 322Q, 328E, 331S, 380A, 428L, 432C, 433K, 433S, 434S, 434Y, 434F, 434W, 434A, 435H, 436L, 437C and any combination thereof. In some embodiments, the Fc variant having increased serum half life or improved pH-dependent binding to FcRn comprises a combination of mutations selected from the group consisting of: a) M428L and N434S; b) P238D and L328E; c) M252Y, S254T and T256E; d) L234F, L235Q, K322Q, M252T, S254T and T256E; e)  M428L, V259I and V308F; f) H433K and N434Y; g) H433K and N434F; h) T250Q and M428L; i) T307A, E380A and N434A; and j) 432C, 433S, 434W, 435H, 436L, 437C.
In some embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences. In some embodiments, at least one of the substitutions or modifications is in one or more of the non-CDR sequences of the heavy chain variable region or light chain variable region. In some embodiments, at least one of the substitutions is a conservative substitution.
In some embodiments, the antibody or antigen binding fragment of the present disclosure is a monoclonal antibody, a bispecific antibody, a multi-specific antibody, a recombinant antibody, a labeled antibody, a bivalent antibody, an anti-idiotypic antibody, a fusion protein, or a dimerized or polymerized antibody, or a modified antibody (e.g. glycosylated antibody) . In some embodiments, the antibody or antigen binding fragment of the present disclosure is a diabody, a Fab, a Fab', a F (ab')  2, a Fd, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv)  2, a bispecific dsFv (dsFv-dsFv') , a disulfide stabilized diabody (ds diabody) , a single-chain antibody molecule (scFv) , an scFv dimer (bivalent diabody) , a bispecific scFv dimer, a multispecific antibody, a heavy chain antibody, a camelized single domain antibody, a nanobody, a domain antibody, or a bivalent domain antibody. In some embodiments, the antibody or antigen binding fragment of the present disclosure is a full human antibody.
In some embodiments, the antibody or antigen binding fragment of the present disclosure is linked to one or more conjugate moieties. In some embodiments, the conjugate moiety comprises a therapeutic agent, a radioactive isotope, a detectable label, a pharmacokinetic modifying moiety, or a purifying moiety. In some embodiments, the conjugate moiety is covalently attached either directly or via a linker.
In one aspect, the present disclosure provides an isolated or recombinant antibody or an antigen-binding fragment thereof, which competes for binding to RBD of spike protein of SARS-CoV-2 with the antibody or an antigen-binding fragment thereof described herein.
In another aspect, the present disclosure provides bispecific antibody molecules comprising an anti-SARS-CoV-2 antibody or antigen-binding fragment thereof as disclosed herein.
In certain embodiments, the bispecific or bivalent antibodies provided herein comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domains is derived from a monoclonal antibody selected from the group consisting of P2A-1A8, P2A-1A9, P2B-2G11, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, and P2C-1F11. The second antigen-binding domain can be derived from any suitable antibody.
In certain embodiments, the bispecific antibodies provided herein comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first and the second antigen-binding domains are derived from any two monoclonal antibodies selected from the group consisting of P2A-1A8, P2A-1A9, P2B-2G11, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, and P2C-1F11. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2B-2F6, respectively.
In certain embodiments, the bispecific antibody molecules have at least two distinct antigen-binding sites with different specificities.
In certain embodiments, the bispecific antibody molecules provided herein are capable of binding to different epitopes on the spike protein of SARS-CoV-2 virus. In some embodiments, the two or more antibodies bind to different epitopes in RBD of spike protein of SARS-CoV-2.
In certain embodiments, the bispecific antibody molecules provided herein has a first antigen-binding domains specificity directed to the RBD of the spike protein of SARS-CoV-2 virus and a second antigen-binding domains specificity directed to a second antigen.
In another aspect, the present disclosure provides an isolated polynucleotide encoding the antibody or antigen binding fragment thereof as described herein.
In some embodiments, the isolated polynucleotide of the present disclosure comprises a nucleotide sequence selected from a group consisting of: SEQ ID NOs: 9-10, 19-20, 29-30, 39-40, 49-50, 59-60, 63-64, 73-74, 83-84, 93-94, 103-104, and 113-114, or a homologous sequence thereof having at least 80%sequence identity.
In some embodiments, the homologue sequence encodes the same protein as encoded by any nucleotide sequence selected from the group consisting of SEQ ID NOs: 9-10, 19-20, 29-30, 39-40, 49-50, 59-60, 63-64, 73-74, 83-84, 93-94, 103-104, and 113-114.
In one aspect, the present disclosure provides a vector comprising the isolated polynucleotide of the present disclosure. In some embodiments, said vector is an expression vector.
In one aspect, the present disclosure provides a host cell comprising the vector of the present disclosure.
In one aspect, the present disclosure provides a method of producing the antibody or antigen binding fragment of the present disclosure. In some embodiments, the method comprises culturing the host cell of the present disclosure under the condition at which the expression vector of the present disclosure is expressed. In some embodiments, the method of the present disclosure further comprises purifying the antibody produced by the host cell.
In another aspect, the present disclosure provides a pharmaceutical composition comprising the antibody or antigen binding fragment of any of the preceding claims, and a pharmaceutically acceptable carrier.
In some embodiments, the pharmaceutical composition comprises a combination of two or more antibodies or antigen binding fragments of the present disclosure. In some embodiments, the pharmaceutical composition comprises a combination of two or more monoclonal antibodies, each of which comprises heavy chain CDR sequences and light chain CDR sequences derived from an antibody selected from the group consisting of P2A-1A8, P2A-1A9, P2B-2G11, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, and P2C-1F11. In certain embodiments, the pharmaceutical composition comprises a first antibody comprising heavy chain CDR sequences and light chain CDR sequences derived from P2C-1F11, and a second antibody comprising heavy chain CDR sequences and light chain CDR sequences derived from antibody P2B-2F6.
In some embodiments, the two or more antibodies or antigen binding fragments bind to different epitopes in RBD of spike protein of SARS-CoV-2. In some embodiments, the two or more antibodies comprise a first antibody which comprises P2C-1F11 or an antigen binding fragment thereof, and a second antibody which is selected  from the group consisting of P2C-1A3, P2C-1C10, P2B-2F6, and P2A-1B3, or an antigen binding fragment thereof. In some embodiments, the two or more antibodies comprise a first antibody which comprises P2C-1A3 or an antigen binding fragment thereof, and a second antibody which is selected from the group consisting of P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof. In some embodiments, the two or more antibodies comprise a first antibody which comprises P2B-2F6 or an antigen binding fragment thereof, and a second antibody selected from the group consisting of P2C-1C10, P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof. In some embodiments, the two or more antibodies comprise a first antibody which comprises P2A-1B3 or an antigen binding fragment thereof, and a second antibody selected from the group consisting of P2C-1A3, P2C-1C10, P2C-1F11, P2B-2F6, and P2A-1A10, or an antigen binding fragment thereof. In some embodiments, the two or more antibodies comprise a first antibody which comprises P2C-1C10 or an antigen binding fragment thereof, and a second antibody selected from the group consisting of P2C-1A3, P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof.
In some embodiments, the pharmaceutical compositions comprise the polynucleotides encoding the anti-SARS-CoV-2 antibodies or the antigen-binding fragments thereof, and one or more pharmaceutically acceptable carriers. The present disclosure further provides pharmaceutical compositions comprising the polynucleotides encoding the combination of the two or more anti-SARS-CoV-2 antibodies or the antigen-binding fragments thereof, and one or more pharmaceutically acceptable carriers. In certain embodiments, the polynucleotides comprise an expression vector. In certain embodiments, the expression vector comprises a viral vector or a non-viral vector. In certain embodiments, the expression vector is suitable for gene therapy in human. In certain embodiments, the expression vector comprises a DNA vector or a RNA vector.
In some embodiments, the pharmaceutical composition further comprises a second active agent, such as a second therapeutic agent or a second prophylactic agent.
In one aspect, the present disclosure provides a kit for detecting a SARS-CoV-2 antigen, comprising the antibody or antigen binding fragment of the present disclosure. In some embodiments, the kit of further comprises a control reagent comprising RBD of spike protein of the SARS-CoV-2. In some embodiments, the kit further comprises a set of reagents for detecting complex of the antibody or the antigen-binding fragment bound to the SARS-CoV-2 antigen.
In one aspect, the present disclosure provides a method of treating SARS-CoV-2 infection in a subject. The present disclosure also provides methods of treating a disease, disorder or condition associated with SARs-CoV-2 infection in a subject. In some embodiments, the method comprises administering a therapeutically effective amount of one or more of the antibody, the antigen binding fragment, or one or more polynucleotides encoding one or more of the antibody or antigen-binding fragment thereof provided herein, or the pharmaceutical composition of the present disclosure to the subject.
In one aspect, the present disclosure provides a method of preventing SARS-CoV-2 infection in a subject. The present disclosure also provides methods of preventing a disease, disorder or condition associated with SARs-CoV-2 infection in a subject. In some embodiments, the method comprises administering a prophylactically effective amount of one or more of the antibody or antigen binding fragment, or the pharmaceutical composition of the present disclosure to the subject.
In some embodiments, the administration is via oral, nasal, intravenous, subcutaneous, or intramuscular administration. In some embodiments, the subject is human. In some embodiments, the polynucleotide provided herein can be administered to a subject by, for example, transfection techniques such as electroporation, or hydrodynamic injection. In some embodiments, the polynucleotides comprise viral vectors such as AAV, and can be administered via local injection (e.g. intramuscular, intranasal, intradermal, subcutaneous, etc. ) or systematic administration (e.g. intravenous administration) .
In some embodiments, the method further comprises administering a therapeutically effective amount of a second active agent which can be a therapeutic agent or a prophylactic agent. In some embodiments, the second therapeutic agent is an anti-viral agent. In some embodiments, an anti-viral agent comprises an antiviral peptide, an anti-viral antibody, an anti-viral compound, an anti-viral cytokine, or an anti-viral oligonucleotide. In some embodiments, the second therapeutic agent is an RNA dependent RNA polymerase inhibitor, a non-nucleoside reverse transcriptase inhibitor (NNRTI) , nucleoside reverse transcriptase inhibitor (NRTI) , purine nucleoside, antiviral interferon, adamantine antiviral compound, or any other suitable antiviral agent. In some embodiments, the second therapeutic agent is remdesivir, chloroquine, hydroxychloroquine, lopinavir, ritonavir, APN01, favilavir, mesalazine, toremifene, eplerenone, paroxetine, sirolimus, dactinomycin, irbesartan, emodin, mercaptopurine, melatonin, quinacrine, carvedilol, colchicine, camphor, equilin, oxymetholone, nafamosta, camostat, baricitinib, darunavir,  ribavirin, galidesivir, BCX-4430, Arbidol, nitazoxanide, derivatives thereof, or any combination thereof.
In one aspect, the present disclosure provides a method of detecting presence or amount of SARS-CoV-2 virus antigen in a sample. In some embodiments, the method comprises contacting the sample with one or more of the antibody or antigen binding fragment of the present disclosure, and determining the presence or the amount of the SARS-CoV-2 virus antigen in the sample.
In one aspect, the present disclosure provides use of one or more of the antibody or antigen binding fragment of the present disclosure in the manufacture of a medicament for treating or preventing SARS-CoV-2 infection or a disease, disorder or condition associated with SARs-CoV-2 infection. In one aspect, the present disclosure provides use of one or more of the antibody or antigen binding fragment of the present disclosure in the manufacture of a medicament for preventing, managing, treating and/or ameliorating in a subject a disease or a disorder caused by or associated with coronavirus (e.g. SARs-COV-2) infection and/or a symptom or respiratory condition relating thereto.
In one aspect, the present disclosure provides use of one or more of the antibody or antigen binding fragment of the present disclosure in the manufacture of a diagnostic reagent for detecting SARS-CoV-2 infection.
In one aspect, the present disclosure provides a kit for detecting an antibody capable of specifically binding to receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, comprising a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 128. In some embodiments, the polypeptide is immobilized on a substrate. In some embodiments, the kit further comprises a set of reagents for detecting complex of the antibody bound to the polypeptide.
In one aspect, the present disclosure provides a method of detecting presence or amount of an antibody capable of specifically binding to RBD of the spike protein of SARS-CoV-2 in a sample, comprising contacting the sample with a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 128, and determining the presence or the level of the antibody in the sample. In some embodiments, the absence of the antibody in the sample or the level of the antibody in the sample being below a threshold indicates that the subject is more likely to suffer from disease progression.
In another aspect, the present disclosure provides a method of determining the likelihood of disease progression in a subject infected with SARS-CoV-2, the method comprising: contacting a sample obtained from the subject with a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 128, and detecting the presence or the level of an antibody in the sample wherein the antibody is capable of specifically binding to RBD of the spike protein of the SARS-CoV-2, wherein the subject is likely to experience disease progression when the antibody in the sample is absent or is below a threshold.
In yet another aspect, the present disclosure provides a method of monitoring treatment response in a subject infected with SARS-CoV-2 and received a treatment, the method comprising: (i) contacting a sample from the subject with a peptide comprising an amino acid sequence of SEQ ID NO: 128; (ii) detecting a first level of an antibody in the sample wherein the antibody is capable of specifically binding to RBD of the spike protein of the SARS-CoV-2; and (iii) comparing the first level of the antibody with a second level of the antibody detected in the subject prior to the treatment; wherein the first level being higher than the second level indicates that the subject is responsive to the treatment.
In yet another aspect, the present disclosure provides a method of neutralizing SARS-CoV-2 in a subject or in a sample in vitro, comprising administering a therapeutically effective amount of one or more of the antibody or antigen binding fragment thereof provided herein, or the pharmaceutical composition provided herein to the subject or to the sample.
BRIEF DESCRIPTION OF FIGURES
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Figure 1. Analyses of plasma and B cell responses specific to SARS-CoV-2. Serial dilutions of plasma samples were analyzed for binding to the (A) RBDs or (B) trimeric Spikes of SARS-CoV-2, SARS-CoV and MERS-CoV by ELISA and (C) for neutralizing activity against pseudoviruses bearing envelope glycoprotein of SARS-CoV-2, SARS-CoV and MERS-CoV. Binding to SARS-CoV-2 NP protein was also evaluated (A) . All results were derived from at least two independent experiments. (D) Gating strategy for analysis and isolation of RBD-specific memory B cells and (E) their representation among the total and memory subpopulation of B cells in the eight study subjects. Samples were named as either A,  B, or C depending on collection sequence. FSC-W, forward scatter width; FSC-A, forward scatter area; and SSC-A side scatter area.
Figure 2. Heavy chain repertoires of SARS-CoV-2 RBD-specific antibodies analyzed (A) by individual subject or (B) across the eight subjects. (A) Distribution and frequency of heavy chain variable (VH) genes usage in each subject shown along the horizontal bar. The same color scheme is used for each VH family across all study subjects. The VHs that dominate across isolated antibodies are indicated by actual frequencies in their respective color boxes. The number of RBD-binding antibodies versus total antibodies isolated are shown on the right. (B) Clustering of VH genes and their association with ELISA binding activity across the eight subjects. Unrooted phylogenetic tree depicting the genetic relationships among all VH genes of the RBD-binding antibodies. Branch lengths are drawn to scale so that sequence relatedness can be readily assessed. Sequences from the same study subject are shown in the same color at the branch tips. Colored circles represent the proportion (light orange, > 80%; light yellow, 60%-80%; light green < 60%) of VH clusters that bind to SARS-CoV-2 RBD with OD 450 values larger than 3. The VH gene families for the highest binding clusters are shown.
Figure 3. Clonal expansion of specific heavy and light chain families in the P#2 antibody repertoire. (A) Phylogenetic analysis of VH (left) and VL 20 (right) genes for all RBD-binding antibodies. Clonal expanded VH and VL clusters are paired and highlighted in three different colors. Branch lengths are drawn to scale so that sequence relatedness can be readily assessed. (B) Clonal expansion in relation to members of other VH and VL families based on somatic hypermutations (SHM) and CDR3 loop lengths. For the pie charts of VH (left) and VL (right) genes, the radii represent the CDR3 loop length and the color scale indicates the degree of SHM. Heavy and light chain repertoires for each antibody are shown along the pie circles.
Figure 4. Antibody neutralization analyzed by pseudovirus and live SARS-CoV-2. (A) Quality control of antibody through ELISA analysis prior to neutralization assay. A serial dilution of each antibody was evaluated against SARS-CoV-2 RBD coated on the ELISA plate and their binding activity was recorded at an optical density (OD) of 450nm and 630nm. (B-C) Antibody neutralization analyzed by pseudovirus (B) or live SARS-CoV-2 (C) . A serial dilution of each antibody was tested against pseudovirus while two dilutions against live SARS-CoV-2. Cytopathic effects (CPE) were observed daily and recorded on  Day 2 post-exposure. Selected antibodies and their concentrations tested are indicated at the upper left corner.
Figure 5. Analysis of plasma binding to cell surface expressed trimeric Spike protein. HEK 293T cells transfected with expression plasmid encoding the full length spike of SARS-CoV-2, SARS-CoV or MERS-CoV were incubated with 1: 100 dilutions of convalescent plasma from the study subjects. The cells were then stained with PE labeled anti-human IgG Fc secondary antibody and analyzed by FACS. Positive control antibodies include S230 and m396 targeting the RBD of SARS-CoV Spike, and Mab-GD33 targeting the RBD of MERS-CoV Spike. VRC01 is negative control antibody targeting HIV-1 envelope glycoprotein.
Figure 6. RBD-specific memory B cells analyzed and isolated through FACS. The recombinant RBD was labeled with either a Strep or His tag and used alone or in combination to identify and isolate RBD-specific single B cells through staining with the Streptavidin-APC and/or Streptavidin-PE, or anti-His-APC and anti-His-PE antibodies. B cells to be isolated are highlighted in boxes or ovals. Samples were named as either A, B, or C depending on collection sequence. FSC-W, forward scatter width; FSC-A, forward scatter area; and SSC-A side scatter area.
Figure 7. ELISA screening of SARS-CoV-2 RBD-specific antibodies in the supernatant of transfected cells. The study subjects and the date of sampling are indicated on the top. Samples were named as either A, B, or C depending on collection sequence. Antibodies tested for each sample are aligned in one vertical column whenever possible. For each evaluated antibody, at least two independent measurements were performed and are presented adjacently on the same row. Binding activities were assessed by OD 450 and indicated by the color scheme on the right. Negatives (no binding activity) are shown in gray for OD 450 values less than 0.1.
Figure 8. Binding kinetics of isolated mAbs with SARS-CoV-2 RBD measured by SPR and ELISA respectively. For SPR, the purified soluble SARS-CoV-2 RBD, SARS-CoV RBD and MERS-CoV2 RBD were covalently immobilized onto a CM5 sensor chip followed by injection of individual antibody at four or five different concentrations. The black lines indicate the experimentally derived curves while the red lines represent fitted curves based on the SPR experimental data. For ELISA analysis, recombinant SARS-CoV RBD and MERS-CoV2 RBD were coated on the ELISA plate, and a serial dilution of each  antibody was evaluated against SARS-CoV RBD and MERS-CoV2 RBD coated plates respectively and their binding activity was recorded at an optical density (OD) of 450nm and 630nm. For ELISA analysis, S230 was used as a positive control antibody against SARS-COV, MAB-GD33 was used as a positive control antibody against MERS-COV, and VRC01 was used as negative control antibody.
Figure 9. Antibody and ACE2 competition for binding to SARS-CoV-2 RBD measured by SPR. The sensorgrams show distinct binding patterns of ACE2 to SARS-CoV-2 RBD with (red curve) or without (black curve) prior incubation with each testing antibody. The competition capacity of each antibody is indicated by the level of reduction in response unit of ACE2 comparing with or without prior antibody incubation.
Figure 10. Analysis of antibody binding to cell surface expressed trimeric Spike protein. HEK 293T cells transfected with expression plasmid encoding the full length spike of SARS-CoV-2, SARS-CoV or MERS-CoV were incubated with 20ug/ml testing antibodies. The cells were then stained with PE labeled anti-human IgG Fc secondary antibody and analyzed by FACS. Positive control antibodies include S230 and m396 targeting the RBD of SARS-CoV Spike, and Mab-GD33 targeting the RBD of MERS-CoV Spike. VRC01 is the negative control antibody targeting HIV-1 envelope glycoprotein.
Figure 11. Epitope mapping through competitive binding measured by SPR. The sensorgrams show distinct binding patterns when pairs of testing antibodies were sequentially applied to the purified SARS-CoV-2 RBD covalently immobilized onto a CM5 sensor chip. The level of reduction in response unit comparing with or without prior antibody incubation is the key criteria for determining the two mAbs recognize the separate or closely situated epitopes.
DETAILED DESCRIPTION OF THE INVENTION
The following description of the disclosure is merely intended to illustrate various embodiments of the disclosure. As such, the specific modifications discussed are not to be construed as limitations on the scope of the disclosure. It will be apparent to a person skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the disclosure, and it is understood that such equivalent embodiments are to be included herein. All references cited herein, including publications, patents and patent applications are incorporated herein by reference in their entirety.
Definitions
Antibody related Terms
The term “antibody” as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, monovalent antibody, bivalent antibody, multivalent antibody, bispecific antibody, multi-specific antibody that binds to a specific antigen. A native intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each heavy chain consists of a variable region (VH) and a first, second, third, and optionally fourth constant region (CH1, CH2, CH3, CH4 respectively) ; mammalian light chains are classified as λ or κ, while each light chain consists of a variable region (VL) and a constant region. The antibody has a “Y” shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonding. Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, HCDR3) . CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273 (4) , 927 (1997) ; Chothia, C. et al., J Mol Biol. Dec 5; 186 (3) : 651-63 (1985) ; Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901 (1987) ; Chothia, C. et al., Nature. Dec 21-28; 342 (6252) : 877-83 (1989) ; Kabat E.A. et al., Sequences of Proteins of immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) ; Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27: 55-77 (2003) ; Marie-Paule Lefranc et al., Immunome Research, 1 (3) , (2005) ; Marie-Paule Lefranc, Molecular Biology of B cells (second edition) , chapter 26, 481-514, (2015) ) . The three CDRs are interposed between flanking stretches known as framework regions (FRs) (light chain FRs including LFR1, LFR2, LFR3, and LFR4, heavy chain FRs including HFR1, HFR2, HFR3, and HFR4) , which are more highly conserved than the CDRs and form a scaffold to support the highly variable loops. The constant regions of the heavy and light chains are not involved in antigen-binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequences of the constant regions of their heavy chains. The  five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgG1 (gamma1 heavy chain) , IgG2 (gamma2 heavy chain) , IgG3 (gamma3 heavy chain) , IgG4 (gamma4 heavy chain) , IgA1 (alpha1 heavy chain) , or IgA2 (alpha2 heavy chain) .
The term “antigen-binding fragment” as used herein refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure. Examples of antigen-binding fragment include, without limitation, a diabody, a Fab, a Fab', a F (ab')  2, a Fd, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv)  2, a bispecific dsFv (dsFv-dsFv') , a disulfide stabilized diabody (ds diabody) , a single-chain antibody molecule (scFv) , an scFv dimer (bivalent diabody) , a bispecific scFv dimer, a single-chain Fv-Fc antibody (scFv-Fc) , a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody binds.
As used herein, a “bispecific” antibody refers to an artificial antibody which has fragments derived from two different monoclonal antibodies. A bispecific antibody may bind to overlapping epitopes or to two different epitopes. The two epitopes may present on the same antigen, or they may present on two different antigens. As such, the terms “multi-specific” antibody refers to an artificial antibody which has fragments derived from multiple different monoclonal antibodies, and may be capable of binding to more than one epitope.
The term “chimeric” as used herein, means an antibody or antigen-binding fragment, having a portion of heavy and/or light chain derived from one species, and the rest of the heavy and/or light chain derived from a different species.
The term “epitope” as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody binds. Two antibodies may bind the same or a closely related epitope within an antigen if they exhibit competitive binding for the antigen. An epitope can be linear or conformational (i.e. including amino acid residues spaced apart) . For example, if an antibody or antigen-binding fragment blocks binding of a reference antibody to the antigen by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, or at least 95%, then the antibody or  antigen-binding fragment may be considered to bind the same/closely related epitope as the reference antibody. The capacity to block, or compete with, the binding of the antibody or the antigen-binding fragment of the present disclosure to SARS-CoV-2 typically indicates that an antibody or the antigen-binding fragment to be screened binds to an epitope or binding site on SARS-CoV-2 that structurally overlaps with the binding site on SARS-CoV-2 that is immunospecifically recognized by the antibody or the antigen-binding fragment of the present disclosure. Alternatively, this can indicate that an antibody or an antigen-binding fragment of the present disclosure to be screened binds to an epitope or binding site that is sufficiently proximal to the binding site immunospecifically recognized by the antibody or the antigen-binding fragment of the present disclosure to sterically or otherwise inhibit binding of the antibodies or the antigen-binding fragment of the present disclosure to SARS-CoV-2.
“Fab” with regard to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond. The heavy chain fragment of the Fab is known as “Fd” .
“Fab'” refers to a Fab fragment that includes a portion of the hinge region.
“F (ab')  2” refers to a dimer of Fab’.
“Fc” with regard to an antibody (e.g. of IgG, IgA, or IgD isotype) refers to that portion of the antibody consisting of the second and third constant domains of a first heavy chain bound to the second and third constant domains of a second heavy chain via disulfide bonding. Fc with regard to antibody of IgM and IgE isotype further comprises a fourth constant domain. The Fc portion of the antibody is responsible for various effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) , Antibody-dependent cellular phagocytosis (ADCP) and complement dependent cytotoxicity (CDC) , but does not function in antigen binding.
“Fv” with regard to an antibody refers to the smallest fragment of the antibody to bear the complete antigen binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.
“Single-chain Fv antibody” or “scFv” refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one  another directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85: 5879 (1988) ) .
“ScFab” refers to a fusion polypeptide with a Fd linked to a light chain via a polypeptide linker, resulting in the formation of a single chain Fab fragment (scFab) .
“Single-chain Fv-Fc antibody” or “scFv-Fc” refers to an engineered antibody consisting of a scFv connected to the Fc region of an antibody.
“Camelized single domain antibody, ” “heavy chain antibody, ” or “HCAb” refers to an antibody that contains two V H domains and no light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231 (1-2) : 25-38 (1999) ; Muyldermans S., J Biotechnol. Jun; 74 (4) : 277-302 (2001) ; WO94/04678; WO94/25591; U.S. Patent No. 6,005,079) . Heavy chain antibodies were originally derived from Camelidae (camels, dromedaries, and llamas) . Although devoid of light chains, camelized antibodies have an authentic antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3; 363 (6428) : 446-8 (1993) ; Nguyen VK. et al. Immunogenetics. Apr; 54 (1) : 39-47 (2002) ; Nguyen VK. et al. Immunology. May; 109 (1) : 93-101 (2003) ) . The variable domain of a heavy chain antibody (VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. Nov; 21 (13) : 3490-8. Epub 2007 Jun 15 (2007) ) .
A “nanobody” refers to an antibody fragment that consists of a VHH domain from a heavy chain antibody and two constant domains, CH2 and CH3.
A “domain antibody” refers to an antibody fragment containing only the variable region of a heavy chain or the variable region of a light chain. In certain instances, two or more V H domains are covalently joined with a peptide linker to create a bivalent or multivalent domain antibody. The two V H domains of a bivalent domain antibody may target the same or different antigens.
The term “valent” as used herein refers to the presence of a specified number of antigen binding sites in a given molecule. The term “monovalent” refers to an antibody or an antigen-binding fragment having only one single antigen-binding site; and the term “multivalent” refers to an antibody or an antigen-binding fragment having multiple antigen-binding sites. As such, the terms “bivalent” , “tetravalent” , and “hexavalent” denote the presence of two binding sites, four binding sites, and six binding sites, respectively, in an  antigen-binding molecule. In some embodiments, the antibody or antigen-binding fragment thereof is bivalent.
“TriFabs” refers to a trivalent, bispecific fusion protein composed of three units with Fab-functionalities. TriFabs harbor two regular Fabs fused to an asymmetric Fab-like moiety.
“Fab-Fab” refers to a fusion protein formed by fusing the Fd chain of a first Fab arm to the N-terminus of the Fd chain of a second Fab arm.
“Fab-Fv” refers to a fusion protein formed by fusing a heavy chain variable domain to the C-terminus of an Fd chain and a light chain variable domain to the C-terminus of a light chain. A “Fab-dsFv” molecule can be formed by introducing an interdomain disulphide bond between the heavy chain variable domain and the heavy chain variable domain.
An “scFv dimer” is a bivalent diabody or bispecific scFv (BsFv) comprising V H-V L (linked by a peptide linker) dimerized with another V H-V L moiety such that V H's of one moiety coordinate with the V L's of the other moiety and form two binding sites which can target the same antigens (or epitopes) or different antigens (or epitopes) .
A bispecific “scFv dimer” is a bispecific diabody comprising V H1-V L2 (linked by a peptide linker) associated with V L1-V H2 (also linked by a peptide linker) such that V H1 and V L1 coordinate and V H2 and V L2 coordinate and each coordinated pair has a different antigen specificity.
A “dsFv” refers to a disulfide-stabilized Fv fragment that the linkage between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some embodiments, a “ (dsFv)  2” or “ (dsFv-dsFv') ” comprises three peptide chains: two V H moieties linked by a peptide linker (e.g. a long flexible linker) and bound to two V L moieties, respectively, via disulfide bridges. In some embodiments, dsFv-dsFv'is bispecific in which each disulfide paired heavy and light chain has a different antigen specificity.
“Bibody” refers to a fusion protein formed by fusing a scFv to the C-terminus of either the light chain (Fab-L-scFv) or Fd (Fab-H-scFv) .
“Tribody” refers to a fusion protein formed by fusing a scFv to both light chain and heavy chain (Fab- (scFv)  2) .
“MAb-Fv” or “IgG-Fv” refers to a fusion protein formed by fusion of heavy chain variable domain (VH domain) to the C-terminus of one Fc chain and the VL domain either expressed separately or fused to the C-terminus of the other resulted in a bispecific, trivalent IgG-Fv (mAb-Fv) fusion protein, with the Fv stabilized by an interdomain disulphide bond.
“ScFab-Fc-scFv 2” and “ScFab-Fc-scFv” refer to a fusion protein formed by fusion of a single-chain Fab with Fc and disulphide-stabilized Fv domains.
“Appended IgG” refers to a fusion protein with a Fab arm fused to an IgG to form the format of bispecific (Fab)  2-Fc. It can form an “IgG-Fab” or a “Fab-IgG” , with a Fab fused to the C-terminus or N-terminus of an IgG molecule with or without a connector. In certain embodiments, the appended IgG can be further modified to a format of IgG-Fab 4 (see, Brinkman et al., mAbs, 9 (2) , pp. 182–212 (2017) ) .
“DVD-Ig” refers to a dual-variable-domain antibody that is formed by fusion of an additional VH domain and VL domain of a second specificity to an IgG heavy chain and light chain. “CODV-Ig” refers to a related format where the two VH domain and two VL domains are linked in a way that allows crossover pairing of the variable VH domain -VL domain, which are arranged either (from N-to C-terminus) in the order VH domain A-VH domain B and VL domain B-VL domain A, or in the order VH domain B-VH domain A and VL domain A-VL domain B.
A “CrossMab” refers to a technology of pairing of unmodified light chain with the corresponding unmodified heavy chain and pairing of the modified light chain with the corresponding modified heavy chain, thus resulting an antibody with reduced mispairing in the light chain.
A “WuxiBody” refers to is a bispecific antibody comprising a chimeric protein with variable domains of an antibody and the constant domains of TCR, wherein the subunits (such as alpha and beta domains) of TCR constant domains are associated by engineered disulfide bond (see, more details in WO2019057122A1) .
A “BiTE” is a bispecific T-cell engager molecule, comprising a first scFv with a first antigen specificity in the VL domain -VH domain orientation linked to a second scFv with a second specificity in the VH domain -VL domain orientation.
A “diabody” or “dAb” includes small antibody fragments with two antigen-binding sites, wherein the fragments comprise a V H domain connected to a V L domain in the same polypeptide chain (V H-V L or V L-V H) (see, e.g. Holliger P. et al., Proc Natl Acad Sci USA. Jul 15; 90 (14) : 6444-8 (1993) ; EP404097; WO93/11161) . By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain, thereby creating two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or epitopes) .
A “DART” is a diabody-like entity that has the VH of a first variable region linked to the VL of a second variable region, and the VH of the second variable region linked to the VL of the first variable region.
A “TandAb” is a bispecific fusion protein with four binding sites, two of which bind to a first antigen and the other two bind to a second antigen.
A “bispecific ds diabody” is a diabody target two different antigens (or epitopes) .
The term “fully human” when used with reference to an antibody, refers to an antibody that are either directly derived from a human or based upon a human sequence. When an antibody is derived from or based on a human sequence and subsequently modified, it is still to be considered fully human as used throughout the specification. In other words, the term “fully human” when used with reference to an antibody, is intended to include binding molecules having variable and constant regions derived from human germline immunoglobulin sequences or based on variable or constant regions occurring in a human or human lymphocyte and modified in some form. Thus, the fully human antibody may include amino acid residues not encoded by human germline immunoglobulin sequences, comprise substitutions and/or deletions (e.g., mutations introduced by, for instance, random or site-specific mutagenesis in vitro or by somatic mutation in vivo) . “Based on” as used herein refers to the situation that a nucleic acid sequence may be exactly copied from a template, or with minor mutations, such as by error-prone PCR methods, or synthetically made matching the template exactly or with minor modifications. Semi-synthetic molecules based on human sequences are also considered to be human as used herein.
Other Terms
The term “affinity” as used herein refers to the strength of non-covalent interaction between an immunoglobulin molecule (i.e. antibody) or fragment thereof and an antigen.
The term “amino acid” as used herein refers to an organic compound containing amine (-NH 2) and carboxyl (-COOH) functional groups, along with a side chain specific to each amino acid. The names of amino acids are also represented as standard single letter or three-letter codes in the present disclosure, which are summarized as follows.
A “conservative substitution” with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative substitutions can be made among amino acid residues with hydrophobic side chains (e.g. Met, Ala, Val, Leu, and Ile) , among residues with neutral hydrophilic side chains (e.g. Cys, Ser, Thr, Asn and Gln) , among residues with acidic side chains (e.g. Asp, Glu) , among amino acids with basic side chains (e.g. His, Lys, and Arg) , or among residues with aromatic side chains (e.g. Trp, Tyr, and Phe) . As known in the art, conservative substitution usually does not cause significant change in the protein conformational structure, and therefore could retain the biological activity of a protein.
The term “diagnosis” , “diagnose” or “diagnosing” refers to the identification of a pathological state, disease or condition, such as identification of SARS-CoV-2 infection, or refer to identification of a subject with SARS-CoV-2 infection who may benefit from a particular treatment regimen. In some embodiments, diagnosis contains the identification of presence or amount of SARS-CoV-2. In some embodiments, diagnosis refers to the identification of SARS-CoV-2 infection in a subject.
“Effector functions” as used herein refer to biological activities attributable to the binding of Fc region of an antibody to its effectors such as C1 complex and Fc receptor. Exemplary effector functions include: complement dependent cytotoxicity (CDC) mediated by interaction of antibodies and C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by binding of Fc region of an antibody to Fc receptor on an effector cell; and phagocytosis. Effector functions can be evaluated using various assays such as Fc receptor binding assay, C1q binding assay, and cell lysis assay.
The term “Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express FcRs (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target ceil and subsequently cause lysis of the target cell. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991) .
The term “specific binding” or “specifically binds” in reference to the interaction of a binding molecule, e.g., an antibody, and its binding partner, e.g., an antigen, means that the interaction is dependent upon the presence of a particular structure, e.g., an antigenic determinant or epitope, on the binding partner. In other words, the antibody preferentially binds or recognizes the binding partner even when the binding partner is present in a mixture of other molecules or organisms. The binding may be mediated by covalent or non-covalent interactions or a combination of both. Antibodies or fragments thereof that immunospecifically bind to an antigen may be cross-reactive with related antigens, carrying the same epitope. Specific binding can be characterized in binding affinity, for example, represented by K d value, i.e., the dissociation constant between the antigen and antigen-binding molecule. K d may be determined by using any conventional method known in the art, including but are not limited to radioimmunoassays (RIA) , enzyme-linked immunosorbent assays (ELISA) , surface plasmon resonance method, microscale thermophoresis method, HPLC-MS method and flow cytometry (such as FACS) method. A K d value of ≤10 -6 M (e.g. ≤5x10 -7 M, ≤2x10 -7 M, ≤10 -7 M, ≤5x10 -8 M, ≤2x10 -8 M, ≤10 -8 M, ≤5x10 -9 M, ≤4x10 -9M, ≤3x10 -9M, ≤2x10 -9 M, or ≤10 -9 M) can indicate specific binding between an antibody or antigen binding fragments thereof and SARS-CoV-2 (e.g. spike protein of SARS-CoV-2, or receptor binding domain of the spike protein of SARS-CoV-2) .
The ability to “compete for binding to RBD” as used herein refers to the ability of a SARS-CoV-2 antibody or antigen-binding fragment thereof to inhibit the binding interaction between RBD of spike protein of SARS-CoV-2 and its binding partner (e.g. a second SARS-CoV-2 antibody, or ACE2 receptor) to any detectable degree. In certain embodiments, an antibody or antigen-binding fragment that compete for binding to SARS-CoV-2 inhibits the binding interaction between RBD of spike protein of SARS-CoV-2  and its binding partner by at least 85%, or at least 90%. In certain embodiments, this inhibition may be greater than 95%, or greater than 99%. In general, competitive inhibition is measured by means of an assay, wherein an antigen composition, i.e., a composition comprising SARS-CoV-2 or fragments thereof, is admixed with reference binding molecules, for example, the antibodies or antigen binding fragments of the present disclosure, or the ACE receptor (e.g. a recombinant binding moiety thereof) , and the antibodies or antigen binding fragments to be screened. Usually, the antibodies or antigen binding fragments to be screened are present in excess. Protocols based upon ELISAs and Western blotting are suitable for use in such simple competition studies.
In certain embodiments, an antibody or antigen-binding fragment exhibits at least 30%competition at 1μM, with 2 μM angiotensin converting enzyme 2 (ACE2) receptor for binding to the RBD of spike protein of SARS-CoV-2 immobilized at a resonance units (RU) of 250, as measured by SPR.
The term “homologous” as used herein refers to nucleic acid sequences (or its complementary strand) or amino acid sequences that have sequence identity of at least 60%(e.g. at least 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to another sequences when optimally aligned.
The phrase “host cell” as used herein refers to a cell into which an exogenous polynucleotide and/or a vector can be or has been introduced.
The term “isolated” means one substance has been altered by the hand of man from the natural state. If an “isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated, ” but the same polynucleotide or polypeptide is “isolated” if it has been sufficiently separated from the coexisting materials of its natural state so as to exist in a substantially pure state. An “isolated nucleic acid sequence” refers to the sequence of an isolated nucleic acid molecule. In certain embodiments, an “isolated antibody or an antigen-binding fragment thereof” refers to the antibody or antigen-binding fragments thereof having a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%as determined by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis) , or chromatographic methods (such as ion  exchange chromatography or reverse phase HPLC) . In some embodiment, an isolated antibody or antigen binding fragment is a recombinant protein or antigen binding fragment.
The term “kit” as used herein refers to a packaged combination of reagents in predetermined amounts with instructions for performing a therapeutics, or a diagnostic or detection assay.
The term “neutralizing” as used herein in relation to the antibody or the antigen binding fragment of the present disclosure refers to antibody or the antigen binding fragment that inhibit SARS-CoV-2 virus from infecting a target cell for replication, regardless of the mechanism by which neutralization is achieved. Thus, neutralization can, for example, be achieved by inhibiting the attachment or adhesion of SARS-CoV-2 virus or a pseudo SARS-CoV-2 virus bearing the spike protein to the cell surface, or by inhibition of the fusion of viral and cellular membranes following attachment of the virus to the target cell, and the like. Exemplary assays for determining neutralizing activity are described in the Examples provided herein.
In some embodiments, the neutralizing activity of an antibody can be represented as half-maximal inhibitory concentrations (IC 50) of the antibody against the binding to ACE2.
The term “nucleic acid” or “polynucleotide” as used herein refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single-or double-stranded form. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) , alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19: 5081 (1991) ; Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985) ; and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994) ) .
“Percent (%) sequence identity” with respect to amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids) . Conservative  substitution of the amino acid residues may or may not be considered as identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI) , see also, Altschul S.F. et al., J. Mol. Biol., 215: 403–410 (1990) ; Stephen F. et al., Nucleic Acids Res., 25: 3389–3402 (1997) ) , ClustalW2 (available on the website of European Bioinformatics Institute, see also, Higgins D.G. et al., Methods in Enzymology, 266: 383-402 (1996) ; Larkin M.A. et al., Bioinformatics (Oxford, England) , 23 (21) : 2947-8 (2007) ) , and ALIGN or Megalign (DNASTAR) software. A person skilled in the art may use the default parameters provided by the tool, or may customize the parameters as appropriate for the alignment, such as for example, by selecting a suitable algorithm.
The term “polypeptide” or “protein” means a string of at least two amino acids linked to one another by peptide bonds. Polypeptides and proteins may include moieties in addition to amino acids (e.g., may be glycosylated) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “polypeptide” or “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence) , or can be a functional portion thereof. Those of ordinary skill will further appreciate that a polypeptide or protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of a corresponding naturally-occurring amino acid and polymers.
The term “pharmaceutically acceptable” indicates that the designated carrier, vehicle, diluent, excipient (s) , and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.
The term “recombinant” when used with reference to a polypeptide (e.g., antibody, antigen) or a polynucleotide, refers to a polypeptide or polynucleotide that is produced by a recombinant method. A “recombinant polypeptide” includes any polypeptide expressed from a recombinant polynucleotide. A “recombinant polynucleotide” includes any polynucleotide which has been modified by the introduction of at least one exogenous (i.e., foreign, and typically heterologous) nucleotide or the alteration of at least one native nucleotide component of the polynucleotide, and need not include all of the coding sequence  or the regulatory elements naturally associated with the coding sequence. A “recombinant vector” refers to a non-naturally occurring vector, including, e.g., a vector comprising a recombinant polynucleotide sequence.
As used herein, the term “sample” used herein refers to a biological specimen that is obtained or derived from a subject of interest. The sample contains a cellular and/or other molecular entity that is to be characterized and/or identified, for example based on physical, biochemical, chemical and/or physiological characteristics.
The term “subject” includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians, and reptiles. Except when noted, the terms “patient” or “subject” are used herein interchangeably.
The term “treating” or “treatment” of a disease, disorder or condition as used herein includes alleviating a disease, disorder or condition, slowing the rate of development of a disease, disorder or condition, reducing or ending symptoms associated with a disease, disorder or condition, generating a complete or partial regression of a disease, disorder or condition, curing a disease, disorder or condition, or some combination thereof.
The term “prevent” or “preventing” of a disease, disorder or condition as used herein includes slowing the onset of a disease, disorder or condition, reducing the risk of developing a disease, disorder or condition, preventing or delaying the development of symptoms associated with a disease, disorder or condition, reducing the severity of a subsequent contraction or development of a disease, disorder or condition, ameliorating a related symptom, and inducing immunity to protect against a disease, disorder or condition.
The term “SARS-CoV-2 virus antigen” as used herein refers to a SARS-CoV-2 virus particle or an antigen found in a SARS-CoV-2 virus particle (e.g. a protein or protein fragments of envelop protein or spike protein (includes, extracellular domain of the spike protein, or RBD of the spike protein) and the like) . Spike protein is composed of S1 protein (which contains RBD) and S2 protein, which are initially in one protein molecule until cleaved by protease into S1 and S2.
The term “vector” as used herein refers to a vehicle into which a genetic element may be operably inserted so as to bring about the expression of that genetic element, such as to produce the protein, RNA or DNA encoded by the genetic element, or to replicate the genetic element. A vector may be used to transform, transduce, or transfect a host cell so  as to bring about expression of the genetic element it carries within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. A vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating. A vector can be an expression vector or a cloning vector. The present disclosure provides vectors (e.g. expression vectors) containing the nucleic acid sequence provided herein encoding the antibody or an antigen-binding fragment thereof, at least one promoter (e.g. SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.
Anti-SARS-CoV-2 Antibodies
The present disclosure in one aspect provides anti-SARS-CoV-2 antibodies and antigen-binding fragments thereof.
In some embodiments, the anti-SARS-CoV-2 antibodies and antigen-binding fragments provided herein are capable of specifically binding to SARS-CoV-2. In certain embodiments, the antibodies and the antigen-binding fragments thereof provided herein specifically bind to SARS-CoV-2 at an Kd value of no more than 10 -7 M as measured by SPR.
In certain embodiments, the antibodies and the antigen-binding fragments thereof provided herein are capable of binding to the RBD of spike protein of SARS-CoV-2 at a Kd value of no more than 1x10 -7M (e.g. no more than 5x10 -7 M, no more than 2x10 -7 M, no more than 10 -7 M, no more than 5x10 -8 M, no more than 2x10 -8 M, no more than 10 -8 M, no more than 5x10 -9 M, no more than 4x10 -9M, no more than 3x10 -9M, no more than 2x10 -9 M, or no more than 10 -9 M) as measured by SPR.
In certain embodiments, the antibodies and the antigen-binding fragments thereof provided herein bind to the RBD of spike protein of SARS-CoV or the RBD of spike protein of MERS-CoV at a significantly lower affinity or degree. In certain embodiments, the antibodies and the antigen-binding fragments thereof provided herein exhibit binding to the RBD of spike protein of SARS-CoV or the RBD of spike protein of MERS-CoV at a Kd  value of at least 1x10 -6M (e.g. at least 2x10 -6 M, at least 5x10 -6 M, at least 10 -5 M) as measured by SPR.
In certain embodiments, the antibodies and the antigen-binding fragments thereof provided herein do not detectably bind to SARS-CoV or MERS-CoV. In certain embodiments, the antibodies and the antigen-binding fragments thereof provided herein exhibits at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) less binding or non-detectable binding to SARS-CoV or MERS-CoV, than the binding to SARS-CoV-2 under equivalent assay conditions. In certain embodiments, the antibodies and the antigen-binding fragments thereof provided herein are capable of specifically binding to spike protein of SARS-CoV-2 and exhibiting at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) less binding to spike protein of SARS-CoV or spike protein of MERS-CoV, than the binding to spike protein of SARS-CoV-2 under equivalent assay conditions. In certain embodiments, the full length of spike protein of SARS-CoV-2 can comprise an amino acid sequence of SEQ ID NO: 134, optionally encoded by a polynucleotide sequence of SEQ ID NO: 135. In certain embodiments, the antibodies and the antigen-binding fragments thereof provided herein are capable of specifically binding to receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 comprising the amino acid sequence of SEQ ID NO: 128. In certain embodiments, the antibodies and the antigen-binding fragments thereof provided herein exhibit binding to RBD of spike protein of SARS-CoV comprising the amino acid sequence of SEQ ID NO: 124 at a level that is non-detectable or that is no more than 50% (e.g., no more than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, 1%) of the binding to the RBD of spike protein of SARS-CoV-2 under equivalent assay conditions. In certain embodiments, the antibodies and the antigen-binding fragments thereof provided herein exhibit binding to RBD of spike protein of MERS-CoV comprising the amino acid sequence of SEQ ID NO: 126 at a level that is non-detectable or that is no more than 50% (e.g., no more than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, 1%) of the binding to RBD of the spike protein of SARS-CoV-2 under equivalent assay conditions..
In certain embodiments, the antibodies and the antigen-binding fragments thereof provided herein are capable of exhibiting at least 30%competition at 1μM, with 2 μM ACE2 receptor for binding to the RBD of spike protein of SARS-CoV-2 immobilized at  a (RU of 250, as measured by SPR. For example, SARS-CoV-2 RBD can be immobilized to a CM5 sensor chip via amine group for a final RU around 250.1 μM of the antibodies or the antigen-binding fragments thereof provided herein can be injected onto the chip until binding steady-state is reached. 2 μM of human ACE2 or human ACE2 peptidase domain can be injected for 60 seconds. Blocking efficacy can be determined by comparison of response units with and without the antibody incubation. Instruments and kits for SPR are commercially available as, for example, Biacore T200, GE Healthcare.
In certain embodiments, the antibodies and the antigen-binding fragments thereof provided herein are capable of binding to the RBD of spike protein of SARS-CoV-2 at an neutralizing activity at an IC 50 value of no more than 100μg/ml (e.g., no more than 90μg/ml, 80μg/ml, 70μg/ml, 60μg/ml, 50μg/ml, 40μg/ml, 30μg/ml, 20μg/ml, 10μg/ml, 5μg/ml, 2μg/ml, 1μg/ml, 0.5μg/ml, 0.2μg/ml, 0.1μg/ml, 0.05μg/ml, 0.03μg/ml) , as measured by pseudovirus neutralization assay. Pseudovirus neutralization assay are known in the art, and in general involves generating a pseudovirus expressing a reporter gene and a viral protein of interest (such as the full length spike protein of SARS-CoV-2 of SEQ ID NO: 134) . The antibodies and the antigen-binding fragments thereof provided herein can be incubated with the pseudovirus, and the titer of the pseudovirus can be determined via the report gene. IC 50 is the concentration of the antibodies or the antigen-binding fragment thereof can inhibit 50%of the pseudovirus titer in the assay.
Illustrative Anti-SARS-CoV-2 Antibodies
In certain embodiments, the present disclosure provides SARS-CoV-2 antibodies and antigen-binding fragments thereof comprising one or more (e.g. 1, 2, 3, 4, 5, or 6) CDRs comprising the sequences selected from the group consisting of SEQ ID NO: 1-6, 11-16, 21-26, 31-36, 41-46, 51-56, 65-70, 75-80, 85-90, 95-100, and 105-110.
Antibody “P2A-1A8” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 7, and a light chain variable region having the sequence of SEQ ID NO: 8.
Antibody “P2A-1A9” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 17, and a light chain variable region having the sequence of SEQ ID NO: 18.
Antibody “P2A-1A10” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 27, and a light chain variable region having the sequence of SEQ ID NO: 28.
Antibody “P2A-1B3” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 37, and a light chain variable region having the sequence of SEQ ID NO: 38.
Antibody “P2B-2F6” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 47, and a light chain variable region having the sequence of SEQ ID NO: 48.
Antibody “P2B-2G4” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 57, and a light chain variable region having the sequence of SEQ ID NO: 58.
Antibody “P2B-2G11” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 61, and a light chain variable region having the sequence of SEQ ID NO: 62.
Antibody “P2C-1A3” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 71, and a light chain variable region having the sequence of SEQ ID NO: 72.
Antibody “P2C-1C8” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 81, and a light chain variable region having the sequence of SEQ ID NO: 82.
Antibody “P2C-1C10” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 91, and a light chain variable region having the sequence of SEQ ID NO: 92.
Antibody “P2C-1D5” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 101, and a light chain variable region having the sequence of SEQ ID NO: 102.
Antibody “P2C-1F11” as used herein refers to a monoclonal fully human antibody having a heavy chain variable region having the sequence of SEQ ID NO: 111, and a light chain variable region having the sequence of SEQ ID NO: 112.
Table 1 below shows the CDR amino acid sequences of antibodies P2A-1A8, P2A-1A9, P2B-2G11, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, and P2C-1F11.
Table 1. CDR amino acid sequences of 12 antibodies
Figure PCTCN2020084097-appb-000001
Figure PCTCN2020084097-appb-000002
Table 2 below shows the heavy chain and light chain variable region amino acid sequences of antibodies P2A-1A8, P2A-1A9, P2B-2G11, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, P2C-1F11, and the corresponding nucleic acid encoding sequence are shown in Table 3.
Table 2. Variable region amino acid sequences of 12 antibodies
Figure PCTCN2020084097-appb-000003
Figure PCTCN2020084097-appb-000004
Figure PCTCN2020084097-appb-000005
Table 3. Variable region nucleotide sequences of 12 antibodies
Figure PCTCN2020084097-appb-000006
Figure PCTCN2020084097-appb-000007
Figure PCTCN2020084097-appb-000008
Figure PCTCN2020084097-appb-000009
Figure PCTCN2020084097-appb-000010
Figure PCTCN2020084097-appb-000011
In certain embodiments, the antibodies or the antigen-binding fragments thereof provided herein further comprise an immunoglobulin (Ig) constant region, which optionally further comprises a heavy chain and/or a light chain constant region. In certain embodiments, the heavy chain constant region comprises CH1, hinge, and/or CH2-CH3 regions (or optionally CH2-CH3-CH4 regions) . In certain embodiments, the antibodies or the antigen-binding fragments thereof provided herein comprises heavy chain constant regions of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 or IgM. In certain embodiments, the light chain constant region comprises Cκ or Cλ. The constant region of the antibodies or the antigen-binding fragments thereof provided herein may be identical to the wild-type constant region sequence or be different in one or more mutations.
In certain embodiments, the heavy chain constant region comprises an Fc region. Fc region is known to mediate effector functions such as antibody-dependent  cellular cytotoxicity (ADCC) , Antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) of the antibody. Fc regions of different Ig isotypes have different abilities to induce effector functions. For example, Fc regions of IgG1 and IgG3 have been recognized to induce both ADCC and CDC more effectively than those of IgG2 and IgG4. In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprises an Fc region of IgG1, or IgG3 isotype, which could induce ADCC or CDC. Alternatively, the antibodies and antigen-binding fragments thereof provided herein comprise a constant region of IgG4 or IgG2 isotype, which has reduced or depleted effector function. In certain embodiments, the anti-SARS-COV-2 antibodies or antigen-binding fragments thereof comprises a wild type human IgG1 Fc region comprising the sequence of SEQ ID NO: 115 or other wild type human IgG1 alleles.
Table 4 shows the amino acid sequences for the heavy chain and light chain constant regions of the monoclonal antibodies: P2A-1A8, P2A-1A9, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2B-2G11, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, and P2C-1F11, wherein the antibodies P2A-1A8, P2A-1A9, P2B-2F6, P2B-2G4, P2B-2G11, and P2C-1D5 have lambda light chains (with a lambda light chain constant region sequence of SEQ ID NO: 116) , the antibodies P2A-1A10, P2A-1B3, P2C-1A3, P2C-1C8, P2C-1C10, and P2C-1F11 have kappa light chains (with a kappa light chain constant region sequence of SEQ ID NO: 117) , and all 12 antibodies have the same heavy chain constant region (SEQ ID NO: 115) .
Table 4. Amino acid and nucleic acid sequences of constant regions
Figure PCTCN2020084097-appb-000012
Figure PCTCN2020084097-appb-000013
In some embodiments, signal peptide may be added when expressing the antibodies of the present disclosure, these signal peptides may be partially or full removed by host cells during the secretion of the antibody. In certain embodiments, for expressing the12 exemplary antibodies of the present disclosure, signal peptide (SEQ ID NO: 130:  MGWSCIILFLVATATGVHS) is included when expressing the heavy chain, signal peptide (SEQ ID NO: 131: MGWSCIILFLVATATGSWA) is included when expressing the light chain.
Table 10 which is appended at the end of the specification shows sequences and SEQ ID NOs mentioned or used in the present application.
Antibody Variants
In certain embodiments, the antibody or antigen binding fragments thereof provided herein comprise one or more modifications or substitutions in one or more of the CDR sequences provided in Table 1 above, one or more of the non-CDR sequences of the heavy chain variable region or light chain variable region provided in Table 2, and/or the constant region (e.g. Fc region) in Table 4, yet retaining specific binding affinity to RBD of spike protein of SARS-CoV-2. These are also referred to as variants of antibodies P2A-1A8, P2A-1A9, P2B-2G11, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, P2C-1F11, or the antigen binding fragments thereof. In certain embodiments, at least one (or all) of the modifications (s) or substitution (s) comprises a conservative substitution.
In certain embodiments, the variants comprise 1, 2, or 3 CDR sequences having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) listed in Table 1 above, and in the meantime retain the binding affinity to SARS-COV-2 at a level similar to or even higher than its parent antibody.
In certain embodiments, the variants comprise one or more variable region sequences having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) listed in Table 2 above, and in the meantime retain the binding affinity to SARS-COV-2 at a level similar to or even higher than its parent antibody. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, or deleted in a variable region sequence listed in Table 2 above. In some embodiments, the substitutions, insertions, or deletions occur in the non-CDR sequences (e.g. in the FRs) .
The variants of the antibodies or the antigen binding fragments thereof can retain their parent antibodies’ binding specificity to RBD of the spike protein of  SARS-CoV-2, or may further have one or more desirable properties conferred by the modification (s) or substitution (s) . For example, the variants may have improved antigen-binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or depleted effector function (s) , improved FcRn receptor binding in a pH dependent manner, increased pharmacokinetic half-life, pH sensitivity, and/or compatibility to conjugation (e.g. one or more introduced cysteine residues) . Such variants are also known as affinity variants, glycosylation variants, cysteine variants, Fc variants, and so on, which are described in more details as follows.
a) Affinity variant
Affinity variant may contain modifications or substitutions in one or more CDR sequences as provided in Table 1 above, one or more framework (FR) sequences provided herein, or the heavy or light chain variable region sequences provided in Table 2 above. FR sequences can be readily identified by a skilled person in the art based on the CDR sequences in Table 1 above and variable region sequences in Table 2 above, as it is well-known in the art that a CDR region is flanked by two FR regions in the variable region.
The affinity variants retain specific binding affinity to RBD of the spike protein of SARS-COV-2 of the parent antibody, or even have improved specific binding affinity to the RBD of the spike protein of SARS-CoV-2 over the parent antibody. Various methods known in the art can be used to achieve this purpose. For example, a library of antibody variants (such as Fab or scFv variants) can be generated and expressed with phage display technology, and then screened for the binding affinity to the RBD of the spike protein of SARS-COV-2. For another example, computer software can be used to virtually simulate the binding of the antibodies to the RBD of the spike protein of SARS-COV-2, and identify the amino acid residues on the antibodies which form the binding interface. Such residues may be either avoided in the substitution so as to prevent reduction in binding affinity, or targeted for substitution to provide for a stronger binding.
In certain embodiments, the affinity variant provided herein comprises one or more amino acid residue substitutions in one or more CDR sequences, and/or one or more FR sequences. In certain embodiments, an affinity variant comprises no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in the CDR sequences and/or FR sequences in total.
b) Glycosylation variant
The anti-SARS-COV-2 antibodies and antigen-binding fragments provided herein also encompass a glycosylation variant, which can be obtained to either increase or decrease the extent of glycosylation of the antibody or antigen binding fragment thereof.
The antibody or antigen binding fragment thereof may comprise one or more modifications that introduces or removes a glycosylation site. A glycosylation site is an amino acid residue with a side chain to which a carbohydrate moiety (e.g. an oligosaccharide structure) can be attached. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, for example, an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly to serine or threonine. Removal of a native glycosylation site can be conveniently accomplished, for example, by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N-linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in the sequence in the is substituted. A new glycosylation site can be created in a similar way by introducing such a tripeptide sequence or serine or threonine residue.
In certain embodiments, the anti-SARS-COV-2 antibodies and antigen-binding fragments provided herein comprise a mutation at N297 (e.g. N297A, N297Q, or N297G) to remove the glycosylation site.
c) Cysteine-engineered variant
The anti-SARS-COV-2 antibodies and antigen-binding fragments provided herein also encompass a cysteine-engineered variant, which comprises one or more introduced free cysteine amino acid residues.
A free cysteine residue is one which is not part of a disulfide bridge. A cysteine-engineered variant is useful for conjugation with for example, a cytotoxic and/or imaging compound, a label, or a radioisoptype among others, at the site of the engineered cysteine, through for example a maleimide or haloacetyl. Methods for engineering antibodies or antigen-binding fragments thereof to introduce free cysteine residues are known in the art, see, for example, WO2006/034488.
d) Fc Variant
The anti-SARS-COV-2 antibodies and antigen-binding fragments provided herein also encompass an Fc variant, which comprises one or more amino acid residue modifications or substitutions at its Fc region and/or hinge region, for example, to provide for altered effector functions such as ADCC, ADCP and CDC. Methods of altering ADCC activity by antibody engineering have been described in the art, see for example, Shields RL. et al., J Biol Chem. 2001.276 (9) : 6591-604; Idusogie EE. et al., J Immunol. 2000.164 (8) : 4178-84; Steurer W. et al., J Immunol. 1995, 155 (3) : 1165-74; Idusogie EE. et al., J Immunol. 2001, 166 (4) : 2571-5; Lazar GA. et al., PNAS, 2006, 103 (11) : 4005-4010; Ryan MC. et al., Mol. Cancer Ther., 2007, 6: 3009-3018; Richards JO, . et al., Mol Cancer Ther. 2008, 7 (8) : 2517-27; Shields R. L. et al, J. Biol. Chem, 2002, 277: 26733-26740; Shinkawa T. et al, J. Biol. Chem, 2003, 278: 3466-3473.
CDC activity of the antibodies provided herein can also be altered, for example, by improving or diminishing C1q binding and/or CDC (see, for example, WO99/51642; Duncan &Winter Nature 322: 738-40 (1988) ; U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821) ; and WO94/29351 concerning other examples of Fc region variants. One or more amino acids selected from amino acid residues 329, 331 and 322 of the Fc region can be replaced with a different amino acid residue to alter Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC) (see, U.S. Pat. No. 6,194,551 by Idusogie et al) . One or more amino acid substitution (s) can also be introduced to alter the ability of the antibody to fix complement (see PCT Publication WO 94/29351 by Bodmer et al.) .
The term “Antibody-dependent cellular phagocytosis” and “ADCP” refer to a process by which antibody-coated cells or particles are internalized, either in whole or in part, by phagocytic immune cells (e.g., macrophages, neutrophils and dendritic cells) that bind to an immunoglobulin Fc region. Methods for altering the ADCP activity of antibodies by antibody engineering are known in the art, see for example, Kellner C et al., Transfus Med Hemother, (2017) 44: 327-336 and Chung AW et al., AIDS, (2014) 28: 2523-2530.
Examples of Fc variants are known in the art, see, for example, Wang et al., Protein Cell 2018, 9 (1) : 63-73 and Kang et al., Exp &Mol., Med. (2019) 51: 138, which are incorporated herein to their entirety.
i) Fc variant with enhanced effector functions
In certain embodiments, the Fc variants provided herein has increased ADCC and/or increased affinity to an Fcγ receptor (e.g. FcγRI (CD64) , FcγRII (CD32) and/or FcγRIII (CD16) ) relative to a wildtype Fc (e.g. Fc of IgG1) . In certain embodiments, an Fc variant comprises one or more amino acid substitution (s) at one or more of the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 246, 247, 248, 249, 252, 254, 255, 256, 258, 260, 262, 263, 264, 265, 267, 268, 269, 270, 272, 274, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 300, 301, 303, 304, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 345, 360, 373, 376, 378, 382, 388, 389, 396, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438, 439 and 440 of the Fc region (see WO 00/42072 by Presta, WO2006/019447 by Lazar, and WO2016/196228, incorporated herein to its entirety) , wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat (see, Kabat E. A. et al., Sequences of Proteins of immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) ) . Exemplary substitutions for increased effector functions include, without limitation, 234Y, 235Q, 236A, 236W, 239D, 239E, 239M, 243L, 247I, 268D, 267E, 268D, 268E, 268F, 270E, 280H, 290S, 292P, 298A, 298D, 298V, 300L, 305I, 324T, 326A, 326D, 326W, 330L, 330M, 333S, 332D, 332E, 298A, 333A, 334A, 334E, 326A, 247I, 339D, 339Q, 345R, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 305I, 396L, 430G, 440Y, or any combination thereof (such as 239D/332E, 239D/332E/330L, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F, 267E/324T, and 267E/268F/324T) (see, WO2016/196228; Richards et al. (2008) Mol. Cancer Therap. 7: 2517; Moore et al. (2010) mAbs 2: 181; and Strohl (2009) Current Opinion in Biotechnology 20: 685-691) .
Specific mutations at positions 256, 290, 298, 333, 334 and 339 were shown to improve binding to FcγRIII. Additionally, the following combination mutants were shown to improve FcγRIII binding: T256A/S298A, S298A/E333A, S298A/K224A, F243L/R292P/Y300L/V305I/P396L, S298A/E333A/K334A and L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in one heavy chain and D270E/K326D/A330M/K334E in the opposing heavy chain (having enhanced FcγRIII binding and ADCC activity) . Other Fc variants with strongly enhanced binding to FcγRIIIa include variant with S239D/I332E and S239D/I332E/A330L mutations, which showed the greatest increase in affinity for FcγRIIIa, a decrease in FcγRIIb binding, and strong cytotoxic activity, and variants with L235V, F243L, R292P, Y300L, V305I and P396L mutations, which exhibited enhancing FcγRIIIa and concomitantly enhanced ADCC activity. (see Lazar  et a . (2006) Proc. Nat'l Acad Sci. (USA) 103: 4005; Awan et al. (2010) Blood 115: 1204; Desjarlais &Lazar (2011) Exp. Cell Res, Stavenhagen et al. (2007) Cancer Res 67: 8882) . Modifications that increase binding to Clq can be introduced in order to enhance CDC activity. Exemplary modifications include, a K326 (e.g., K326W) and/or E333 modification in an IgG2, or a S267E/H268F/S324T modification, alone or in any combination, in an IgGl (see Idusogie et al. (2001) J. Immunol. 166: 2571, Moore et al. (2010) mAbs 2: 181) . Other exemplary modifications include, K326W/E333S, S267E/H268F/S324T, and E345R/E430G/S440Y.
ii) Fc with reduced effector functions
In certain embodiments, the Fc variants provided herein has reduced effector functions relative to a wildtype Fc (e.g. Fc of IgG1) , and comprise one or more amino acid substitution (s) at a position selected from the group consisting of: 220, 226, 229, 233, 234, 235, 236, 237, 238, 267, 268, 269, 270, 297, 309, 318, 320, 322, 325, 328, 329, 330, and 331 of the Fc region (see, WO2016/196228; Richards et al. (2008) Mol. Cancer Therap. 7: 2517; Moore et al. (2010) mAbs 2: 181; and Strohl (2009) Current Opinion in Biotechnology 20: 685-691) , wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat. Exemplary substitutions for reduced effector functions include, without limitation, 220S, 226S, 228P, 229S, 233P, 234V, 234G, 234A, 234F, 234A, 235A, 235G, 235E, 236E, 236R, 237A, 237K, 238S, 267R, 268A, 268Q, 269R, 297A, 297Q, 297G, 309L, 318A, 322A, 325L, 328R, 330S, 331S, or any combination thereof (see, WO2016/196228; and Strohl (2009) Current Opinion in Biotechnology 20: 685-691) .
In certain embodiments, the Fc variant provided herein is of IgG1 isotype and comprises one or more amino acid substitution (s) selected from the group consisting of: L234A, L234F, L234V, F234A, V234A, L235A, L235E, G237A, P238S, H268Q, H268A, N297A, N297Q, N297G, V309L, A330S, and P331S, or any combination thereof (such as L234A/L235A) . In certain embodiments, the Fc variant provided herein is of IgG2 isotype, and comprises one or more amino acid substitution (s) selected from the group consisting of: H268Q, V309L, A330S, P331S, V234A, G237A, P238S, H268A, and any combination thereof. In certain embodiments, the Fc variant provided herein is of IgG4 isotype, and comprises one or more amino acid substitution (s) selected from the group consisting of: S228P, F234A, L235E, L235A, G237A, E318A, N297A, N297Q, N297G, and any combination thereof. In certain embodiments, the anti-SARS-COV-2 antibodies and antigen-binding fragments provided herein is of IgG2/IgG4 cross isotype. Examples of  IgG2/IgG4 cross isotype is described in Rother RP et al, Nat Biotechnol 25: 1256–1264 (2007) .
iii) Fc with altered binding to FcRn
In certain embodiments, the Fc variant comprises one or more amino acid substitution (s) that improves binding affinity to neonatal Fc receptor (FcRn) at pH 6.0 while retaining minimal binding at pH 7.4. Such a variant can have an extended pharmacokinetic half-life, as it binds to FcRn at acidic pH which allows it to escape from degradation in the lysosome and then be translocated and released out of the cell. Methods of engineering an antibody and antigen-binding fragment thereof to improve binding affinity with FcRn are well-known in the art, see, for example, Vaughn, D. et al, Structure, 6 (1) : 63-73, 1998; Kontermann, R. et al, Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al, Cancer Research, 70: 3269-3277 (2010) ; Hinton, P. et al, J. Immunology, 176: 346-356 (2006) ; Petkova et al. (2006) Int. Immunol. 18: 1759, Ball Acqua et al. Journal of Immunology 2002, 169: 5171-5180, Dall’A cqua WF. et al., J Biol Chem. 281: 23514–23524 (2006) ; Zalevsky J, et al, Nat Biotechnol.; 28: 157–159 (2010) ; WO 2009/086320; US 6,277,375; US 6,821,505; WO 97/34631; and WO 2002/060919.
Non-limiting examples of Fc modifications that may result in an increase in serum half-life of the antibody when administered include, e.g., substitution (s) at one or more positions selected from: 234 (e.g., with F) , 235 (e.g., with Q) , 238 (e.g., with D) , 250 (e.g., with E or Q) , 252 (e.g., with L/Y/F/W or T) , 254 (e.g., with S or T) , 256 (e.g., with S/R/Q/E/D or T) ; 259 (e.g., with I) ; 272 (e.g., with A) , 305 (e.g., with A) , 307 (e.g., with A or P) , 308 (e.g., with F, C or P) , 311 (e.g., with A or R) , 312 (e.g., with A ) , 322 (e.g., Q) , 328 (e.g. E) , 331 (e.g., with A) , 378 (e.g., with A) , 380 (e.g., with A) , 382 (e.g., with A) , 428 (e.g., with L or F) , 432 (e.g., with C) , 433 (e.g., with H/L/R/S/P/Q or K) , 434 (e.g., with H/F or Y or S or A or W) , 435 (e.g. with H) , 436 (e.g., with L) and 437 (e.g., with C) (all positions by EU numbering) (see, WO2016049000A2; WO2020052692; WO2016196228) . In some embodiments, the Fc variant comprises one or more amino acid substitution (s) selected from the group consisting of 234F, 235Q, 238D, 250Q, 252T, 252Y, 254T, 256E, 259I, 272A, 305A, 307A, 308F, 311A, 322Q, 328E, 331S, 380A, 428L, 432C, 433K, 433S, 434S, 434Y, 434F, 434W, 434A, 435H, 436L, 437C and any combination thereof. In some embodiments, the Fc modifications comprises one or pairs or groups of modifications selected from: a) a 428L (e.g., M428L) and 434S (e.g., N434S) substitution; a 428L, 259I (e.g., V259I) , and  308F (e.g., V308F) substitution; b) a 433K (e.g., H433K) and 434 (e.g., N434Y or N434F) substitution; c) a 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E) substitution; d) a 250Q and 428L substitution (e.g., T250Q and M428L) ; e) a 307A, 380A and 434A substitution (e.g., T307A, E380A and N434A) ; f) a P238D and L328E substitution; g) a L234F, L235Q, K322Q, M252T, S254T and T256E substitution; and h) and a L432C, H433S, N434W, Y436L and T437C substitution.
In some embodiments, hybrid IgG isotypes may be used to increase FcRn binding and half-life of antibodies. A hybrid Ig can be generated from two or more isotypes. For example, an IgGl/IgG3 hybrid variant may be constructed by substituting IgGl positions in the CH2 and/or CH3 region with the amino acids from IgG3 at positions where the two isotypes differ. In some embodiments, a hybrid Ig can comprises one or more modifications (e.g. substitutions) disclosed here.
Antigen-binding Fragments
Provided herein are also anti-SARS-CoV-2 antigen-binding fragments. In some embodiments, the antibodies and antigen-binding fragments provided herein comprise all or a portion of the heavy chain variable domain and/or all or a portion of the light chain variable domain.
Various types of antigen-binding fragments are known in the art and can be developed based on the anti-SARS-CoV-2 antibodies provided herein, including for example, the exemplary antibodies whose CDR are shown in Tables 1 above, and variable sequences are shown in Tables 2 and 3, and their different variants (such as affinity variants, glycosylation variants, Fc variants, cysteine-engineered variants and so on) .
In certain embodiments, an anti-SARS-CoV-2 antigen-binding fragment provided herein is a diabody, a Fab, a Fab', a F (ab')  2, a Fd, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv)  2, a bispecific dsFv (dsFv-dsFv') , a disulfide stabilized diabody (ds diabody) , a single-chain antibody molecule (scFv) , an scFv dimer (bivalent diabody) , a bispecific scFv dimer, a multispecific antibody, a heavy chain antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody.
Various techniques can be used for the production of such antigen-binding fragments. Illustrative methods include, enzymatic digestion of intact antibodies (see, e.g.  Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) ; and Brennan et al., Science, 229: 81 (1985) ) , recombinant expression by host cells such as E. Coli (e.g. for Fab, Fv and ScFv antibody fragments) , screening from a phage display library as discussed above (e.g. for ScFv) , and chemical coupling of two Fab'-SH fragments to form F (ab')  2 fragments (Carter et al., Bio/Technology 10: 163-167 (1992) ) . Other techniques for the production of antibody fragments will be apparent to a person skilled in the art.
In certain embodiments, the antigen-binding fragment is a scFv. Generation of scFv is described in, for example, WO 93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. ScFv may be fused to an effector protein at either the amino or the carboxyl terminus to provide for a fusion protein (see, for example, Antibody Engineering, ed. Borrebaeck) .
In certain embodiments, the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof provided herein are bivalent, tetravalent, hexavalent, or multivalent. Any molecule being more than bivalent is considered multivalent, encompassing for example, trivalent, tetravalent, hexavalent, and so on.
A bivalent molecule can be monospecific if the two binding sites are both specific for binding to the same antigen or the same epitope. This, in certain embodiments, provides for stronger binding to the antigen or the epitope than a monovalent counterpart. Similar, a multivalent molecule may also be monospecific. In certain embodiments, in a bivalent or multivalent antigen-binding moiety, the first valent of binding site and the second valent of binding site are structurally identical (i.e. having the same sequences) , or structurally different (i.e. having different sequences albeit with the same specificity) .
A bivalent can also be bispecific, if the two binding sites are specific for different or overlapping antigens or epitopes. This also applies to a multivalent molecule. For example, a trivalent molecule can be bispecific when two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope) .
Bispecific (or Bivalent) Antibody or Antigen-binding Fragments
In another aspect, the present disclosure provides bispecific (or bivalent) antibody molecules comprising an anti-SARS-CoV-2 antibody or antigen-binding fragment thereof as disclosed herein. In certain embodiments, the bispecific (or bivalent) antibodies provided herein comprises a first antigen-binding domain and a second antigen-binding  domain, wherein the first antigen-binding domains is derived from a monoclonal antibody selected from the group consisting of P2A-1A8, P2A-1A9, P2B-2G11, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, and P2C-1F11. The second antigen-binding domain can be derived from any suitable antibody.
In certain embodiments, the bispecific (or bivalent) antibodies provided herein comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first and the second antigen-binding domains are derived from any two monoclonal antibodies selected from the group consisting of P2A-1A8, P2A-1A9, P2B-2G11, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, and P2C-1F11.
In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2B-2F6, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2A-1A8, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2A-1A9, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2B-2G11, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2A-1A10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2A-1B3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2B-2G4, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2C-1A3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2C-1C8, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2C-1C10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2C-1D5, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1F11 and P2C-1F11, respectively.
In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A8 and P2A-1A9, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A8 and P2B-2G11, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A8 and P2A-1A10, respectively. In certain embodiments, the first and the second  antigen-binding domains are derived from P2A-1A8 and P2A-1B3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A8 and P2B-2F6, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A8 and P2B-2G4, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A8 and P2C-1A3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A8 and P2C-1C8, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A8 and P2C-1C10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A8 and P2C-1D5, respectively.
In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A9 and 2B-2G11, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A9 and P2A-1A10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A9 and P2A-1B3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A9 and P2B-2F6, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A9 and P2B-2G4, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A9 and P2C-1A3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A9 and P2C-1C8, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A9 and P2C-1C10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A9 and P2C-1D5, respectively.
In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G11 and P2A-1A10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G11 and P2A-1B3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G11 and P2B-2F6, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G11 and P2B-2G4, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G11 and P2C-1A3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G11 and P2C-1C8, respectively. In certain embodiments, the  first and the second antigen-binding domains are derived from P2B-2G11 and P2C-1C10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G11 and P2C-1D5, respectively.
In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A10 and P2A-1B3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A10 and P2B-2F6, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A10 and P2B-2G4, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A10 and P2C-1A3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A10 and P2C-1C8, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A10 and P2C-1C10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1A10 and P2C-1D5, respectively.
In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1B3 and P2B-2F6, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1B3 and P2B-2G4, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1B3 and P2C-1A3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1B3 and P2C-1C8, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1B3 and P2C-1C10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2A-1B3 and P2C-1D5, respectively.
In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2F6 and P2B-2G4, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2F6 and P2C-1A3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2F6 and P2C-1C8, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2F6 and P2C-1C10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2F6 and P2C-1D5, respectively.
In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G4 and P2C-1A3, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G4 and P2C-1C8, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G4 and P2C-1C10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2B-2G4 and P2C-1D5, respectively.
In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1A3 and P2C-1C8, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1A3 and P2C-1C10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1A3 and P2C-1D5, respectively.
In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1C8 and P2C-1C10, respectively. In certain embodiments, the first and the second antigen-binding domains are derived from P2C-1C8 and P2C-1D5, respectively.
In certain embodiments, the first and the second antigen-binding domains are derived or from P2C-1C10 and P2C-1D5, respectively.
In certain embodiments, the bispecific antibody molecules can have at least two distinct antigen-binding sites with different specificities. In certain embodiments, the bispecific antibody molecules provided herein are capable of binding to different epitopes on the spike protein of SARS-CoV-2 virus. In some embodiments, the bispecific antibody molecules provided herein comprises antigen-binding fragments derived from two or more antibodies provided herein. In some embodiments, the two or more antibodies bind to different epitopes in RBD of spike protein of SARS-CoV-2. In some embodiments, the two or more antibodies are no more than 70% (or no more than 60%, or no more than 50%) competitive against each other in binding to RBD of spike protein of SARS-CoV-2 virus. In certain embodiments, the bispecific antibody comprises a first antigen-binding domain derived from P2C-1F11 and a second antigen-binding domain derived from an antibody selected from the group consisting of P2C-1A3, P2C-1C10, P2B-2F6, and P2A-1B3. In certain embodiments, the bispecific antibody comprises a first antigen-binding domain derived from P2C-1A3 and a second antigen-binding domain derived from an antibody selected from the group consisting of P2C-1F11, and P2A-1B3. In certain embodiments,  the bispecific antibody comprises a first antigen-binding domain derived from P2B-2F6 and a second antigen-binding domain derived from an antibody selected from the group consisting of P2C-1C10, P2C-1F11, and P2A-1B3. In certain embodiments, the bispecific antibody comprises a first antigen-binding domain derived from P2A-1B3 and a second antigen-binding domain derived from an antibody selected from the group consisting of P2C-1A3, P2C-1C10, P2C-1F11, P2B-2F6, and P2A-1A10. In some embodiments, the two or more antibodies comprise a first antibody which comprises P2C-1C10 or an antigen binding fragment thereof, and a second antibody selected from the group consisting of P2C-1A3, P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof.
The term “derived from” as used herein with respect to antigen-binding domain, means that the antigen-binding domain comprise at least one heavy chain CDR sequence (e.g. comprising heavy chain CDR3, or three heavy chain CDRs) or at least one light chain CDR sequence (e.g. comprising light chain CDR3, or three heavy chain CDRs) of the specified monoclonal antibody. In certain embodiments, the first and the second antigen-binding domains comprises the heavy chain CDR sequences of the specified monoclonal antibodies, and/or the light chain CDR sequences of the specified monoclonal antibodies. In certain embodiments, the first and the second antigen-binding domains comprises the heavy chain variable region sequences of the specified monoclonal antibodies, and/or the light chain variable region sequences of the specified monoclonal antibodies. All the CDR sequences and variable region sequences of the specific monoclonal antibodies are provided in Tables 1 and 2 of the present disclosure.
In certain embodiments, the bispecific antibody molecules provided herein has a first antigen-binding domains specificity directed to the RBD of the spike protein of SARS-CoV-2 virus and a second antigen-binding domains specificity directed to a second antigen. In certain embodiments, the second antigen can be for example, an epitope outside of RBD on the spike protein of SARS-CoV-2, S2 protein (i.e. which is cleaved from the spike protein) , nucleocapsid protein of SARS-CoV-2, or alternatively the second antigen can be an antigen on human immune cells such as T cell, macrophage cell, natural killer cells, or antigen-presenting cells.
In certain embodiments, the bispecific antibody molecules as provided herein are based on the format of a “whole” antibody, such as whole IgG or IgG-like molecules. Examples of such bispecific antibody include but are not limited to, those produced by a  quadroma cell line. In another embodiment, a bispecific IgG-like molecule can be an appended IgG, which is engineered by appending either the amino or carboxyl termini of either light or heavy chains of an IgG of a first specificity with additional antigen-binding units of a second specificity. The appended antigen-binding units can be, for example, single domain antibodies (e.g. unpaired VL or VH, or VHH (i.e. heavy chain variable domain of a heavy chain antibody) ) , paired antibody variable domains (e.g. Fv or scFv) or engineered protein scaffolds. Examples of appended IgG include, without limitation, Double-variable domain (DVD) -Ig, which has a second heavy chain variable domain (VH) fused to the VH of a first heavy chain and a second variable light chain domain (VL) fused to a first light chain of the IgG. A DVD-Ig can be bispecific when the first VH/VL and the second VH/VL are selected to bind to two different antigens. In certain embodiments, a bispecific IgG or IgG-like molecules can be monovalent for each antigen and can be produced by co-expression of the two light and two heavy chains in a single host cell.
In certain embodiments, the bispecific antibody molecules as provided herein can be small recombinant bispecific formats based on variable domains, such as single domain antibody, Fv, and Fab, which may lack some or all of the antibody constant domains. Examples of small recombinant bispecific formats include, without limitation, tandem single chain variable fragment molecules (taFvs) , diabodies (Dbs) , single chain diabodies (scDbs) and various other derivatives of these (see, bispecific antibody formats as described by Byrne H. et al. (2013) Trends Biotech, 31 (11) : 621-632, BiTE (bispecific T cell engager) , DARTs, and TandAb. In certain embodiments, the two antigen-binding moieties can be linked by a peptide linker.
In certain embodiments, the bispecific antibody molecules as provided herein are in a bispecific format selected from bispecific IgG-like antibodies (BsIgG) comprising CrossMab; DAF (two-in-one) ; DAF (four-in-one) ; DutaMab; DT-IgG; Knobs-in-holes common LC; Knobs-in-holes assembly; Charge pair; Fab-arm exchange; SEEDbody; Triomab; LUZ-Y; Fcab; kappa-lamda-body; and Orthogonal Fab. For detailed description of the bispecific antibody formats please see Spiess C., Zhai Q. and Carter P. J. (2015) Molecular Immunology 67: 95-106, which is incorporated herein by reference to its entirety.
In certain embodiments, the bispecific antibody molecules as provided herein are in a bispecific format selected from IgG-appended antibodies with an additional antigen-binding moiety consisting of DVD-IgG; IgG (H) -scFv; scFv- (H) IgG; IgG (L) -scFv;  scFV- (L) IgG; IgG (L, H) -Fv; IgG (H) -V; V (H) -IgG; IgG (L) -V; V (L) -IgG; IgG-scFab; 2scFv-IgG; IgG-2scFv; scFv4-Ig; scFv4-Ig; and Zybody (see Id. ) .
In certain embodiments, the bispecific antibody molecules as provided herein are in a bispecific format selected from WuxiBody (WuXi Biologics, see, WO2019057122A1, incorporated herein to its entirety) ; Triomabs; hybrid hybridoma (quadroma) ; Multispecific anticalin platform (Pieris) ; Diabodies; Single chain diabodies; Tandem single chain Fv fragments; TandAbs, Trispecific Abs (Affimed) ; Darts (dual affinity retargeting; Macrogenics) ; Bispecific Xmabs (Xencor) ; Bispecific T cell engagers (Bites; Amgen; 55 kDa) ; Triplebodies; Tribody (Fab-scFv) ; Fusion Protein (CreativeBiolabs) ; multifunctional recombinant antibody derivates; Duobody platform (Genmab) ; Dock and lock platform; Knob into hole (KIH) platform; Humanized bispecific IgG antibody (REGN1979) (Regeneron) ; Mab 2 bispecific antibodies (F-Star) ; DVD-Ig (dual variable domain immunoglobulin) (Abbvie) ; kappa-lambda bodies; TBTI (tetravalent bispecific tandem Ig) ; and CrossMab.
In certain embodiments, the bispecific antibody molecules as provided herein are in a format selected from bispecific antibody fragments comprising Nanobody; Nanobody-HAS; BiTE; Diabody; DART; TandAb; scDiabody; sc-Diabody-CH3; Diabody-CH3; Triple Body; Miniantibody; Minibody; TriBi minibody; scFv-CH3 KIH; Fab-scFv; scFv-CH-CL-scFv; F (ab') 2; F (ab') 2-scFv2; scFv-KIH; Fab-scFv-Fc; Tetravalent HCAb; scDiabody-Fc; Diabody-Fc; Tandem scFv-Fc; and Intrabody (see Id. ) .
In certain embodiments, the bispecific antibody molecules as provided herein are in a bispecific format such as Dock and Lock; ImmTAC; HSAbody; scDiabody-HAS; and Tandem scFv-Toxin (see Id. ) .
In certain embodiments, the bispecific antibody molecules as provided herein are based on a format selected from bispecific antibody conjugates comprising IgG-IgG; Cov-X-Body; and scFv1-PEG-scFv2 (see Id. ) .
The bispecific antibody molecules provided herein can be made with any suitable methods known in the art. In a conventional approach, two immunoglobulin heavy chain-light chain pairs having different antigen-binding specificities can be co-expressed in a host cell to produce bispecific antibodies in a recombinant way (see, for example, Milstein and Cuello, Nature, 305: 537 (1983) ) , followed by purification by affinity chromatography.
Recombinant approach may also be used, where sequences encoding the antibody heavy chain variable domains for the two specificities are respectively fused to immunoglobulin constant domain sequences, followed by insertion to an expression vector which is co-transfected with an expression vector for the light chain sequences to a suitable host cell for recombinant expression of the bispecific antibody (see, for example, WO 94/04690; Suresh et al., Methods in Enzymology, 121: 210 (1986) ) . Similarly, scFv dimers can also be recombinantly constructed and expressed from a host cell (see, e.g. Gruber et al., J. Immunol., 152: 5368 (1994) . )
Bispecific antibody molecule may be generated from a bispecific antibody, for example, by proteolytic cleavage, or by chemical linking. For example, an antigen-binding fragment (e.g. Fab’) of an antibody may be prepared and converted to Fab'-thiol derivative and then mixed and reacted with another converted Fab’ derivative having a different antigenic specificity to form a bispecific antibody molecule (see, for example, Brennan et al., Science, 229: 81 (1985) ) .
In certain embodiments, the bispecific antibody molecules may be engineered to promote heavy chain heterodimerization of the two different antigen-binding sites. In certain embodiments, the Fc region is modified at the interface so that a knob-into-hole association can be formed to promote heterodimerization. “Knob-into-hole” as used herein, refers to an interaction between two polypeptides (such as CH3 domain) , where one polypeptide has a protuberance (i.e. “knob” ) due to presence of an amino acid residue having a bulky side chain (e.g. tyrosine or tryptophan) , and the other polypeptide has a cavity (i.e. “hole” ) where a small side chain amino acid residue resides (e.g. alanine or threonine) , and the protuberance is positionable in the cavity so as to promote interaction of the two polypeptides to form a heterodimer or a complex. Methods of generating polypeptides with knobs-into-holes are known in the art, e.g., as described in U.S. Pat. No. 5,731,168.
In some embodiments, “charged pairs” can be introduced to the Fc polypeptides to electrostatically steer the formation towards heterodimerization. Exemplary pairs include, D221E/P228E/L368E paired with D221R/P228R/K409R and C220E/P228E/368E paired with C220R/E224R/P228R/K409R (see Gunasekaran et al., 2010, J. Biol. Chem. 285 (25) : 19637. ) .
In some embodiments, the binding interface of the two Fc polypeptide chains can be engineered such that in the heterodimer configuration, residues interact with residues  of similar physical property (e.g., polar residues interacting with polar residues, or hydrophobic residues interact with hydrophobic residues) , while in the homodimer configuration residues interact with residues of different physical property. Exemplary modifications include substitution at positions 364, 368, 399, 405, 409, 411, or any combination thereof (see, e.g, WO2014/145806, WO2014/110601, WO2016/086186, WO2016/086189, WO2016/086196, and WO2016/182751) .
In some embodiments, the bispecific antibody molecules may be engineered to reduce random pairing of two different light chain variable regions with the two different heavy chain variable regions. In some embodiments, the bispecific antibody molecule comprise a common light chain capable of pairing with the two heavy chain variable regions. In some other embodiments, CH1 domain of one heavy chain is exchanged with the constant region (CL) of the corresponding light chain (such as that applied in CrossMab technology) . In some other embodiments, mutations are introduced into the CH1-CL interface and/or the VH-VL interface of the Fab fragments, so as to enforce correct pairing of the light chains with the corresponding heavy chains. In some other embodiments, the CH1 domain and CL domain in one antigen-binding domain are replaced by TCR constant domains, so as to minimize mispairing between heavy chain of the first antigen-binding domain and light chain of the second antigen-binding domain (such as that applied in WuxiBody technology) .
Competitive Binding
In one aspect, the present disclosure provides an isolated or recombinant antibody or an antigen-binding fragment thereof, which competes for binding to RBD of spike protein of SARS-CoV-2 with the antibody or an antigen-binding fragment thereof described herein.
Antibodies or antigen binding fragments that competes with the antibody or antigen-binding fragment provided herein for binding to RBD of spike protein of SARS-CoV-2 include, but are not limited to, antibodies, antibody fragments and other binding agents that bind to an epitope or binding site bound by the antibody or antigen-binding fragment provided herein, or bind to a sufficiently proximal epitope or binding site. Preferably, competitive antibodies or antigen binding fragments of the disclosure will, when present in excess, inhibit specific binding of the antibody or antigen-binding fragment provided herein to RBD of the spike protein of SARS-CoV-2 by at least 10%, preferably by at least 25%, more preferably by at least 50%, and most preferably  by at least 75%-90%or even greater. The identification of one or more competitive antibodies or antigen binding fragments that bind to about, substantially, essentially or at the same epitope as the antibodies or antigen binding fragments of the present disclosure is a straightforward technical matter. As the identification of competitive binding molecules is determined in comparison to a reference binding molecule, for example, the antibodies or antigen binding fragments of the present disclosure, it will be understood that actually determining the epitope to which the reference binding molecule and the competitive binding molecule bind is not in any way required in order to identify a competitive binding molecule that binds to the same or substantially the same epitope as the reference binding molecule.
Conjugates
In some embodiments, the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof further comprise one or more conjugate moieties. A conjugate moiety is a moiety that can be attached to the antibodies or antigen-binding fragments thereof either directly or via a linker or through another conjugate moiety. It is contemplated that a variety of conjugate moieties may be linked to the antibodies or antigen-binding fragments thereof provided herein (see, for example, “Conjugate Vaccines” , Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr. (eds. ) , Carger Press, New York, (1989) ) . These conjugate moieties may be linked to the antibodies or antigen-binding fragments thereof by covalent binding, affinity binding, intercalation, coordinate binding, complexation, association, blending, or addition, among other methods.
In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein may be engineered to contain specific sites outside the epitope binding portion that may be utilized for binding to one or more conjugate moieties. For example, such a site may include one or more reactive amino acid residues, such as for example cysteine or histidine residues, to facilitate covalent linkage to a conjugate moiety.
Examples of such conjugate moieties include but are not limited to, therapeutic agent, a radioactive isotope, a detectable label, a pharmacokinetic modifying moiety, or a purifying moiety. In some embodiments, the conjugate moiety comprises a clearance-modifying agent (e.g. a polymer such as PEG which extends half-life) , a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a detectable label (e.g. a luminescent label, a fluorescent label, an enzyme-substrate label) , a DNA-alkylator, a topoisomerase inhibitor, a tubulin-binder, a purification moiety or other anticancer drugs.
Examples of detectable label may include a fluorescent labels (e.g. fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red) , enzyme-substrate labels (e.g. horseradish peroxidase, alkaline phosphatase, luceriferases, glucoamylase, lysozyme, saccharide oxidases or β-D-galactosidase) , radioisotopes (e.g.  123I,  124I,  125I,  131I,  35S,  3H,  111In,  112In,  14C,  64Cu,  67Cu,  86Y,  88Y,  90Y,  177Lu,  211At,  186Re,  188Re,  153Sm,  212Bi, and  32P, other lanthanides) , luminescent labels, chromophoric moieties, digoxigenin, biotin/avidin, DNA molecules or gold for detection.
In certain embodiments, the conjugate moiety can be a clearance-modifying agent which helps increase half-life of the antibody. Illustrative example include water-soluble polymers, such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol/propylene glycol, and the like. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In certain embodiments, the conjugate moiety can be a purification moiety such as a magnetic bead. In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein is used as a base for a conjugate.
Polynucleotides and Recombinant Methods
The present disclosure provides isolated polynucleotides that encode the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof provided herein. DNA encoding the monoclonal antibody is readily isolated, e.g., from B cells, and sequenced using conventional procedures (e.g. by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) . The encoding DNA may also be obtained by synthetic methods.
The isolated polynucleotide that encodes the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof can be inserted into a vector for further cloning (amplification of the DNA) or for expression (i.e., expression vector) , using recombinant techniques known in the art. Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g. SV40, CMV, EF-1α) , and a transcription termination sequence.
The present disclosure provides vectors comprising the isolated polynucleotide provided herein. In certain embodiments, the polynucleotide provided herein encodes the  antibodies or antigen-binding fragments thereof, at least one promoter (e.g. SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g. herpes simplex virus) , poxvirus, baculovirus, papillomavirus, papovavirus (e.g. SV40) , lambda phage, and M13 phage, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT. RTM., pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos etc.
Vectors comprising the polynucleotide sequence encoding the antibody or antigen-binding fragment thereof can be introduced to a host cell for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g. Salmonella typhimurium, Serratia, e.g. Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-SARS-CoV-2 antibody-encoding vectors. Saccharomyces cerevisiae, or common baker’s yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g. K. lactis, K. fragilis (ATCC 12, 424) , K. bulgaricus (ATCC 16,045) , K. wickeramii (ATCC 24, 178) , K. waltii (ATCC 56, 500) , K. drosophilarum (ATCC 36, 906) , K. thermotolerans, and K. marxianus; yarrowia (EP 402, 226) ; Pichia pastoris (EP 183, 070) ; Candida; Trichoderma reesia (EP 244, 234) ; Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g. Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
Suitable host cells for the expression of glycosylated antibodies or antigen-fragment thereof provided herein are derived from multicellular organisms.  Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar) , Aedes aegypti (mosquito) , Aedes albopictus (mosquito) , Drosophila melanogaster (fruiffly) , and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g. the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651) ; human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36: 59 (1977) ) ; baby hamster kidney cells (BHK, ATCC CCL 10) ; Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980) ) ; mouse sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980) ) ; monkey kidney cells (CV1 ATCC CCL 70) ; African green monkey kidney cells (VERO-76, ATCC CRL-1587) ; human cervical carcinoma cells (HELA, ATCC CCL 2) ; canine kidney cells (MDCK, ATCC CCL 34) ; buffalo rat liver cells (BRL 3A, ATCC CRL 1442) ; human lung cells (W138, ATCC CCL 75) ; human liver cells (Hep G2, HB 8065) ; mouse mammary tumor (MMT 060562, ATCC CCL51) ; TRI cells (Mather et al., Annals N. Y. Acad. Sci. 383: 44-68 (1982) ) ; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2) . In some embodiments, the host cell is a mammalian cultured cell line, such as CHO, BHK, NS0, 293 and their derivatives.
Host cells are transformed with the above-described expression or cloning vectors for anti-SARS-CoV-2 antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. In another embodiment, the antibody may be produced by homologous recombination known in the art. In certain embodiments, the host cell is capable of producing the antibody or antigen-binding fragment thereof provided herein.
The present disclosure also provides a method of expressing the antibody or an antigen-binding fragment thereof provided herein, comprising culturing the host cell  provided herein under the condition at which the vector of the present disclosure is expressed. The host cells used to produce the antibodies or antigen-binding fragments thereof provided herein may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma) , Minimal Essential Medium (MEM) , (Sigma) , RPMI-1640 (Sigma) , and Dulbecco's Modified Eagle's Medium (DMEM) , Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58: 44 (1979) , Barnes et al., Anal. Biochem. 102: 255 (1980) , U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30, 985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor) , salts (such as sodium chloride, calcium, magnesium, and phosphate) , buffers (such as HEPES) , nucleotides (such as adenosine and thymidine) , antibiotics (such as GENTAMYCIN TM drug) , trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) , and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to a person skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to a person skilled in the art.
When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5) , EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
The anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof prepared from the cells can be purified using, for example, hydroxylapatite chromatography,  gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.
In certain embodiments, Protein A immobilized on a solid phase is used for immunoaffinity purification of the antibody and antigen-binding fragment thereof. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human gamma1, gamma2, or gamma4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983) ) . Protein G is recommended for all mouse isotypes and for human gamma3 (Guss et al., EMBO J. 5: 1567 1575 (1986) ) . The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX TM resin (J.T. Baker, Phillipsburg, N.J. ) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE TM chromatography on an anion or cation exchange resin (such as a polyaspartic acid column) , chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
Following any preliminary purification step (s) , the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g. from about 0-0.25M salt) .
Pharmaceutical Composition
The present disclosure further provides pharmaceutical compositions comprising the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof and one or more pharmaceutically acceptable carriers.
In some embodiments, the pharmaceutical composition comprises a combination of two or more antibodies or the antigen binding fragments of the present disclosure. In some embodiments, the pharmaceutical composition comprises a combination of two or more monoclonal antibodies, each of which comprises heavy chain  CDR sequences and light chain CDR sequences derived from an antibody selected from the group consisting of P2A-1A8, P2A-1A9, P2B-2G11, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, and P2C-1F11. In some embodiments, the pharmaceutical composition comprises a first antibody comprising heavy chain CDR sequences and light chain CDR sequences derived from P2C-1F11, and a second antibody comprising heavy chain CDR sequences and light chain CDR sequences derived from antibody P2B-2F6.
In some embodiments, the two or more antibodies or the antigen binding fragments thereof bind to different epitopes in RBD of spike protein of SARS-CoV-2. In certain embodiments, the pharmaceutical composition comprises a first antibody which comprises P2C-1F11 or an antigen binding fragment thereof, and a second antibody which is selected from the group consisting of P2C-1A3, P2C-1C10, P2B-2F6, and P2A-1B3, or an antigen binding fragment thereof. In certain embodiments, the pharmaceutical composition comprises a first antibody which comprises P2C-1A3 or an antigen binding fragment thereof, and a second antibody which is selected from the group consisting of P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof. In certain embodiments, the pharmaceutical composition comprises a first antibody which comprises P2B-2F6 or an antigen binding fragment thereof, and a second antibody selected from the group consisting of P2C-1C10, P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof. In certain embodiments, the pharmaceutical composition comprises a first antibody which comprises P2A-1B3 or an antigen binding fragment thereof, and a second antibody selected from the group consisting of P2C-1A3, P2C-1C10, P2C-1F11, P2B-2F6, and P2A-1A10, or an antigen binding fragment thereof. In some embodiments, the pharmaceutical composition comprises a first antibody which comprises P2C-1C10 or an antigen binding fragment thereof, and a second antibody selected from the group consisting of P2C-1A3, P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof.
The present disclosure further provides pharmaceutical compositions comprising the polynucleotides encoding the anti-SARS-CoV-2 antibodies or the antigen-binding fragments thereof, and one or more pharmaceutically acceptable carriers. The present disclosure further provides pharmaceutical compositions comprising the polynucleotides encoding the combination of the two or more anti-SARS-CoV-2 antibodies or the antigen-binding fragments thereof, and one or more pharmaceutically acceptable carriers.
The present disclosure further provides pharmaceutical compositions comprising an expression vector comprising the polynucleotides encoding the one or more of anti-SARS-CoV-2 antibodies or the antigen-binding fragments thereof, and one or more pharmaceutically acceptable carriers.
In certain embodiments, the expression vector comprises a viral vector or a non-viral vector. Examples of viral vectors include, without limitation, adeno-associated virus (AAV) vector, lentivirus vector, retrovirus vector, and adenovirus vector. Examples of non-viral vectors include, without limitation, naked DNA, plasmid, exosome, mRNA, and so on. In certain embodiments, the expression vector is suitable for gene therapy in human. Suitable vectors for gene therapy include, for example, adeno-associated virus (AAV) , or adenovirus vector. In certain embodiments, the expression vector comprises a DNA vector or a RNA vector. In certain embodiments, the pharmaceutically acceptable carriers are polymeric excipients, such as without limitation, microspheres, microcapsules, polymeric micelles and dendrimers. The polynucleotides, or polynucleotide vectors of the present disclosure may be encapsulated, adhered to, or coated on the polymer-based components by methods known in the art (see for example, W. Heiser, Nonviral gene transfer techniques, published by Humana Press, 2004; U.S. patent 6025337; Advanced Drug Delivery Reviews, 57 (15) : 2177-2202 (2005) ) .
In some embodiments, the pharmaceutical composition further comprises a second active agent, such as a second therapeutic agent or a second prophylactic agent.
Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.
Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisol, butylated hydroxytoluene, and/or propyl gallate. As disclosed herein, inclusion of one or more  antioxidants such as methionine in a composition comprising an antibody or antigen-binding fragment thereof and conjugates provided herein decreases oxidation of the antibody or antigen-binding fragment thereof. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving antibody stability and maximizing shelf-life. Therefore, in certain embodiments, pharmaceutical compositions are provided that comprise one or more antibodies or antigen-binding fragments thereof as disclosed herein and one or more antioxidants such as methionine. Further provided are methods for preventing oxidation of, extending the shelf-life of, and/or improving the efficacy of an antibody or antigen-binding fragment provided herein by mixing the antibody or antigen-binding fragment with one or more antioxidants such as methionine.
To further illustrate, pharmaceutical acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80) , sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid) , ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.
The pharmaceutical compositions can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder. Oral formulations can include  standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
The form of pharmaceutical compositions depends on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered. The pharmaceutical compositions can be formulated for intravenous, oral, nasal, rectal, percutaneous, or intramuscular administration. For example, dosage forms for intravenous administration, may be formulated as lyophilized powder or fluid formulation; dosage forms for nasal administration may conveniently be formulated as aerosols, solutions, drops, gels or dry powders. In accordance to the desired route of administration, the pharmaceutical compositions can be formulated in the form of tablets, capsule, pill, dragee, powder, granule, sachets, cachets, lozenges, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium) , spray, inhalant, or suppository.
In certain embodiments, the pharmaceutical compositions are formulated into an injectable composition. The injectable pharmaceutical compositions may be prepared in any conventional form, such as for example liquid solution, suspension, emulsion, or solid forms suitable for generating liquid solution, suspension, or emulsion. Preparations for injection may include sterile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions. The solutions may be either aqueous or nonaqueous.
In certain embodiments, unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile and not pyretic, as is known and practiced in the art.
In certain embodiments, a sterile, lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment as disclosed herein in a suitable solvent. The solvent may contain an excipient which improves the stability or other pharmacological components of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to a person skilled in the art at, in one embodiment, about neutral pH. Subsequent sterile filtration of the solution  followed by lyophilization under standard conditions known to a person skilled in the art provides a desirable formulation. In one embodiment, the resulting solution will be apportioned into vials for lyophilization. Each vial can contain a single dosage or multiple dosages of the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof or composition thereof. Overfilling vials with a small amount above that needed for a dose or set of doses (e.g. about 10%) is acceptable so as to facilitate accurate sample withdrawal and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4 ℃ to room temperature.
Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, for reconstitution the sterile and/or non-pyretic water or other liquid suitable carrier is added to lyophilized powder. The precise amount depends upon the selected therapy being given, and can be empirically determined.
Methods of Treatment or Prevention
The present disclosure also provides methods of treating SARs-CoV-2 infection or a disease, disorder or condition associated with SARs-CoV-2 infection in a subject, comprising administering to the subject a therapeutically effective amount of one or more of the antibody or antigen-binding fragment thereof provided herein, or one or more polynucleotides encoding one or more of the antibody or antigen-binding fragment thereof provided herein, or the pharmaceutical composition provided herein.
In certain embodiments, the therapeutically effective amount can be an amount effective to decrease SARs-COV-2 titers, or to alleviate one or more disease symptoms, viremia, or any other measurable manifestation of SARS-CoV-2 infection in the treated subject or population, whether by inducing the regression of or inhibiting the progression of symptom (s) associated with SARs-COV-2 infection by any clinically measurable degree. Decrease in SARs-COV-2 titers can be measured in the lung, for example, by the concentration of SARs-COV-2 in sputum samples or a lavage from the lungs from a mammal. Alleviation of a disease symptom can be assessed by any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of that symptom. Exemplary symptoms associated with SARs-COV-2 infection include, without limitation, fever, dry cough, shortness in breath, pain or pressure in the chest, new  confusion or inability to arouse, bluish lips or face, loss of sense of smell and/or loss of sense of taste.
A subject in need of treatment include, for example, those already infected with SARS-CoV-2 (symptomatic or asymptomatic) or inflicted with a condition resulting from infection of SARS-CoV-2. Subjects partially or totally recovered from infection of SARS-CoV-2 might also be in need of treatment. In certain embodiments, the subject is human.
The present disclosure also provides methods of preventing SARs-CoV-2 infection, or a disease, disorder or condition associated with SARs-COV-2 infection in a subject, comprising administering to the subject a prophylactically effective amount of one or more of the antibody or antigen-binding fragment thereof provided herein, or one or more polynucleotides encoding one or more of the antibody or antigen-binding fragment thereof provided herein, or the pharmaceutical composition provided herein. Prevention encompasses inhibiting or reducing the spread of SARS-CoV-2 or inhibiting or reducing the onset, development or progression of one or more of the symptoms associated with infection with SARS-CoV-2.
In certain embodiments, the prophylactically effective amount can be an amount effective to neutralize SARs-COV-2 in the respiratory tract, lungs and/or other affected areas such as eyes, noses and mouth, in order block infection, or effective to ameliorate at least one symptom associated with SARs-COV-2 infection. Whether a symptom has been ameliorated can be assessed by any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of that symptom or in certain instances will ameliorate the need for hospitalization.
A subject in need of prevention include, for example, those in which infection with SARS-CoV-2 is to be prevented, or those who are at risk for SARS-CoV-2 infection. In certain embodiments, the subject is human.
The term “disease, disorder or condition associated with SARS-COV-2 infection” as used herein include those that are caused by or related to SARs-COV-2 infection, such as, upper or lower respiratory tract infections, pharyngitis, pneumonia, tracheobronchitis, bronchiolitis, bronchitis, acute respiratory distress syndrome, diarrhea, and any related infections or inflammatory disorders.
The methods of treatment or prevention provided herein are also suitable for gene therapy by transfer of polynucleotide sequences encoding the antibody product or fragment thereof in a subject, such that the polynucleotide can be expressed in the subject to produce the antibody in vivo. The polynucleotide provided herein can be administered to a subject by, for example, transfection techniques such as electroporation and hydrodynamic injection, which are suitable for administration of naked polynucleotides. For polynucleotides in the form of viral vectors such as AAV, it can be administered via local injection (e.g. intramuscular, intranasal, intradermal, subcutaneous, etc. ) or systematic administration (e.g. intravenous administration) .
In certain embodiments, the methods comprises administering to the subject a therapeutically effective amount or a prophylactically effective amount of a combination of two or more of the antibodies (or the antigen-binding fragment thereof) provided herein. In certain embodiments, the two or more antibodies comprises a first antibody comprising heavy chain CDR sequences and light chain CDR sequences derived from P2C-1F11, and a second antibody comprising heavy chain CDR sequences and light chain CDR sequences derived from antibody P2B-2F6. In certain embodiments, the two or more antibodies or the antigen binding fragments thereof bind to different epitopes in RBD of spike protein of SARS-CoV-2. In certain embodiments, the two or more antibodies comprise a first antibody comprising P2C-1F11, and a second antibody which is selected from the group consisting of P2C-1A3, P2C-1C10, P2B-2F6, and P2A-1B3. In certain embodiments, the two or more antibodies comprise a first antibody comprising P2C-1A3 and a second antibody which is selected from the group consisting of P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof. In certain embodiments, the two or more antibodies comprise a first antibody comprising P2B-2F6 and a second antibody which is selected from the group consisting of P2C-1C10, P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof. In certain embodiments, the two or more antibodies comprises a first antibody comprising P2A-1B3 and a second antibody which selected from the group consisting of P2C-1A3, P2C-1C10, P2C-1F11, P2B-2F6, and P2A-1A10, or an antigen binding fragment thereof. In some embodiments, the two or more antibodies comprise a first antibody which comprises P2C-1C10 or an antigen binding fragment thereof, and a second antibody selected from the group consisting of P2C-1A3, P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof.
The antibodies or antigen-binding fragments thereof provided herein may be administered by any route known in the art, such as for example parenteral (e.g. subcutaneous,  intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g. oral, intranasal, intraocular, sublingual, rectal, or topical) routes.
In some embodiments, the antibodies or antigen-binding fragments thereof provided herein may be administered alone or in combination a therapeutically effective amount of a second active agent. The second active agent can be a therapeutic agent or a prophylactic agent.
In some embodiments, the second therapeutic agent is an anti-viral agent. In some embodiments, the anti-viral agent comprises an antiviral peptide, an anti-viral antibody, an anti-viral compound, an anti-viral cytokine, or an anti-viral oligonucleotide. In some embodiments, the anti-viral agent is an RNA dependent RNA polymerase inhibitor, a non-nucleoside reverse transcriptase inhibitor (NNRTI) , nucleoside reverse transcriptase inhibitor (NRTI) , purine nucleoside, antiviral cytokines such as interferon, adamantine antiviral compound, anti-RBD antibody, anti-S1 antibody, anti-S2 antibody, siRNAs Targeting mRNA of coronavirus proteins M, N, or E (Chinese patent applications CN101173275 and CN1648249) , siRNAs targeting replicase and RNA polymerase region (US patent application US20050004063) , RNA Aptamers (Korean patent applications KR2009128837 and KR 2012139512) , ribozymes (Japanese patent application JP2007043942) , antisense oligonucleotides (PCT patent application WO2005023083) , or any other suitable antiviral agent. In certain embodiments, the anti-viral compound is selected from the group consisting of remdesivir, chloroquine, hydroxychloroquine, lopinavir, ritonavir, APN01, favilavir, mesalazine, toremifene, eplerenone, paroxetine, sirolimus, dactinomycin, irbesartan, emodin, mercaptopurine, melatonin, quinacrine, carvedilol, colchicine, camphor, equilin, oxymetholone, nafamosta, camostat, baricitinib, darunavir, ribavirin, galidesivir, BCX-4430, Arbidol, nitazoxanide, derivatives thereof, or any combination thereof. More examples of potentially useful anti-viral agents for SARS-CoV-2 reviewed by C. Liu et al, ACS Cent. Sci. 2020, 6, 3, 315-331, which is incorporate herein to its entirety.
In certain embodiments, the second active agent (e.g. prophylactic agent) can be a SARS-CoV-2 vaccine (e.g. mRNA-1273 by Moderna, an AAV-based vaccine capable of expressing an SARS-CoV-2 immunogen) , an antibody (e.g. directed to SARS-CoV-2) , lymphokines, hematopoietic growth factors (such as IL-2, IL-3, IL-7, and IL-15) , which can  for example serve to increase the number or activity of effector cells which interact with the antibodies.
In certain embodiments, the second active agent can comprise hormonal therapy, immunotherapy, and anti-inflammatory agents.
In certain of these embodiments, an antibody or antigen-binding fragment thereof provided herein may be administered simultaneously with the one or more additional active agents, and in certain of these embodiments the antibody or antigen-binding fragment thereof and the additional therapeutic agent (s) may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment thereof administered “in combination” with another active agent does not have to be administered simultaneously with or in the same composition as the agent. An antibody or antigen-binding fragment thereof administered prior to or after another agent is considered to be administered “in combination” with that agent as the phrase is used herein, even if the antibody or antigen-binding fragment and the second agent are administered via different routes. Where possible, additional active agents administered in combination with the antibodies or antigen-binding fragments thereof disclosed herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to the Physicians'Desk Reference 2003 (Physicians'Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002) ) or protocols well known in the art.
Methods of virus detection
In another aspect, the present disclosure provides a method of detecting presence or amount of SARS-CoV-2 virus antigen in a sample. In some embodiments, the SARS-CoV-2 virus antigen comprises spike protein, or comprises the SARS-CoV-2 virus particle. In some embodiments, the method comprises contacting the sample with the antibody or antigen binding fragment disclosed herein, and determining the presence or the amount of the SARS-CoV-2 virus antigen in the sample.
In certain embodiments, the anti-SARS-CoV-2 antibody disclosed herein is used in a method of diagnosing a subject suffering from a disorder (e.g., SARS-CoV-2 infection) , the method comprising: determining the presence or amount of SARS-CoV-2 virus antigen in a sample obtained from the subject by contacting the sample with an anti-SARS-CoV-2 antibody of the disclosure and detecting the presence of the bound antibody.
Any sample suspected of containing SARS-CoV-2 virus can be used. In some embodiments, a suitable sample can be obtained from respiratory tract of the subject, for example, an upper respiratory nasopharyngeal swab (NP) , oropharyngeal swabs (OP) , sputum, a lower respiratory tract aspirate, bronchoalveolar lavage sample, nasopharyngeal wash, nasopharyngeal aspirate, nasal aspirate, a nasal swap, a throat swap, a bronchoalveolar lavage fluid (BALF) , a cell or tissue sample from respiratory tract or from lung, and the like. In some embodiments, a suitable sample can be a body fluid sample such as a whole blood sample, a serum sample, or a plasma sample. In some embodiments, a suitable sample can be a urine sample or a stool sample.
The presence or level of SARS-CoV-2 virus antigen in a sample can be determined based on the detection of the presence or level of the complex of the virus antigen bound by the antibody or the antigen binding fragment thereof disclosed herein. Any suitable methods can be used for such detection, for example, by immunoassays such as immunohistochemistry (IHC) , immunofluorescence (IF) , immunoblotting (e.g., Western blotting) , flow cytometry (e.g., FACS TM) , Enzyme-linked Immunosorbant Assay (ELISA) , enzyme immunoassay (EIA) , and radioimmunoassay (RIA) .
For a review of immunological and immunoassay procedures, see Basic and Clinical Immunology (Stites &Terr eds., 7 th ed. 1991) . Moreover, the immunoassays can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980) ; and Harlow &Lane, supra. For a review of the general immunoassays, see also Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993) ; Basic and Clinical Immunology (Stites &Terr, eds., 7 th ed. 1991) .
In certain embodiments, the antibodies or the antigen binding fragments thereof disclosed herein are detectably labeled, or are not labeled but can react with a second molecule which is detectably labeled (e.g. a detectably labeled secondary antibody) .
In certain embodiments, the antibodies or the antigen binding fragments thereof disclosed herein may be immobilized on a solid substrate. The immobilization can be via covalent linking or non-covalent attachment (e.g. coating) . Examples of solid substrate include porous and non-porous materials, latex particles, magnetic particles, microparticles, strips, beads, membranes, microtiter wells and plastic tubes. The choice of solid phase material and method of detectably labeling can be determined based upon desired assay format performance characteristics.
The level of the SARS-CoV-2 antigen can be determined, for example, by normalizing to a control value or to a standard curve. The control value can be predetermined, or determined concurrently.
The assays and methods provided herein for the measurement of the level of the SARS-CoV-2 antigen can be adapted or optimized for use in automated and semi-automated systems, or point of care assay systems.
Methods of antibody detection
In another aspect, the present disclosure provides a method of detecting presence or amount of an antibody capable of specifically binding to RBD of the spike protein of SARS-CoV-2 in a sample, comprising contacting the sample with a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 128, and determining the presence or the level of the antibody in the sample. In some embodiments, the absence of the antibody in the sample or the level of the antibody in the sample being below a threshold indicates that the subject is more likely to suffer from disease progression.
In another aspect, the present disclosure provides a method of determining or predicting the likelihood of disease progression in a subject infected with SARS-CoV-2, the method comprising: contacting a sample obtained from the subject with a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 128, and detecting the presence or the level of an antibody in the sample wherein the antibody is capable of specifically binding to RBD of the spike protein of the SARS-CoV-2, wherein the subject is likely to experience disease progression when the antibody in the sample is absent or is below a threshold.
A subject infected with SARS-CoV-2 can produce antibodies against the SARS-CoV-2 antigens. Such antibodies produced by human immune system are polyclonal, and can bind to different antigens or epitopes of SARS-CoV-2. Without wishing to be bound by any theory, it is unexpectedly found by the inventors that the presence or level of the antibodies specific to the RBD of the spike protein of the SARS-CoV-2 can be indicative of likelihood of disease progression in the subject. Antibodies capable of specifically binding to the RBD of the spike protein of the SARS-CoV-2 ( “RBD-specific antibodies” ) are found by the inventors to be capable of effectively competing with ACE2 receptor for binding to the RBD, and also provide for SARS-CoV-2 virus neutralizing activity. The presence of such a RBD-specific antibody can be associated with an effective immune  response to the SARS-CoV-2, and the titer of such RBD-specific antibody in the body may correlate to the prognosis of the SARS-CoV-2 infection or a disease, disorder or condition associated with SARs-CoV-2 infection.
A threshold of the level of the RBD-specific antibodies can be predetermined. The threshold refers to a level of the RBD-specific antibodies above which the sample is scored as being positive for RBD-specific antibodies. For example, the threshold can be a level above which the sample is scored as having sufficient neutralizing activity against the SARS-CoV-2. If the level of the RBD-specific antibodies is below the threshold, it could indicate insufficient protective immunity in the subject, and hence likelihood of disease progression. In contrast, if the level of the RBD-specific antibodies in the sample reaches or is above the threshold, it could indicate protective immunity in the subject, and hence less likely to suffer from disease progression.
Any sample suspected of containing antibodies can be used. In some embodiments, a suitable sample can be obtained from blood, for example, a whole blood sample, a serum sample, or a plasma sample. In some embodiments, said sample is obtained from a subject suspected of having, inflicted with, or under treatment for SARS-CoV-2 infection, or a disease, disorder or condition associated with SARs-CoV-2 infection.
Polypeptides comprising the RBD of the spike protein of SARS-CoV-2 can be used in the methods provided to herein to detect presence or level of the RBD-specific antibodies in the subject. In certain embodiments, the RBD of the spike protein of SARS-CoV-2 comprises an amino acid sequence comprising SEQ ID NO: 128. In certain embodiments, the polypeptides can further comprise a tag. Exemplary tag include, without limitation, 6xHis tag or its fusion such SEQ ID NO: 132 or SEQ ID NO: 133. The polypeptides comprising RBD may be produced by recombinant methods (e.g., by prokaryotic expression system or eukaryotic expression system) , or chemically synthesized (e.g. by solid phase synthesis, or solution synthesis method) . Solid phase synthesis method is described by Merrifield in J.A.C.S. 85: 2149-2154 (1963) or the standard solution synthesis method described in "Peptide Synthesis" by Bodanszky, et al, second edition, John Wiley and Sons, 1976. The polypeptides can be purified by methods known in the art. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymology, 182 (1990) ; Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982) . The purification  step (s) selected will depend, for example, on the nature of the production process used and the particular polypeptide of the present application produced.
The presence or level of RBD-specific antibodies in a sample can be determined based on the detection of the presence or level of the complex of the RBD bound by the RBD-specific antibodies. Any suitable methods can be used for such detection, for example, by immunoassays such as immunohistochemistry (IHC) , immunofluorescence (IF) , immunoblotting (e.g., Western blotting) , flow cytometry (e.g., FACS TM) , Enzyme-linked Immunosorbant Assay (ELISA) , enzyme immunoassay (EIA) , and radioimmunoassay (RIA) , as described above.
In certain embodiments, the polypeptide comprising RBD of the spike protein of the SARS-CoV-2 may be immobilized on a solid substrate. The immobilization can be via covalent linking or non-covalent attachment (e.g. coating) . The sample suspected of containing the RBD-specific antibodies can be brought into contact with the bound polypeptide. After a suitable period of incubation, for a period of time sufficient to allow capture of the RBD-specific antibodies via formation of antibody-antigen complex. After washing away any unreacted materials, a detection antibody specific to the captured antibody can be added, which can produce a detectable signal to allow detection of the captured antibody. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of the detectable signal.
In another aspect, the present disclosure provides a method of monitoring treatment response in a subject infected with SARS-CoV-2 and received a treatment, the method comprising: (i) contacting a sample from the subject with a peptide comprising an amino acid sequence comprising SEQ ID NO: 128; (ii) detecting a first level of an antibody in the sample wherein the antibody is capable of specifically binding to RBD of the spike protein of the SARS-CoV-2; and (iii) comparing the first level of the antibody with a second level of the antibody detected in the subject prior to the treatment; wherein the first level being higher than the second level indicates that the subject is responsive to the treatment.
In one embodiment, a sample is obtained from a subject or patient prior to any treatment. In another embodiment, a test sample is obtained during or after treatment such as anti-viral treatment.
In one aspect, the present disclosure provides a kit for detecting an antibody capable of specifically binding to receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, comprising a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 128. In some embodiments, the polypeptide is immobilized on a substrate. In some embodiments, the kit further comprises a set of reagents for detecting complex of the antibody bound to the polypeptide.
Kits
In certain embodiments, the present disclosure provides a kit comprising one or more of the antibody or an antigen-binding fragment thereof provided herein. In certain embodiments, the kit disclosed herein is a therapeutic kit. In certain embodiments, the kit disclosed herein is a diagnostic kit.
Such kits can further include, if desired, one or more of various conventional kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers etc., as will be readily apparent to a person skilled in the art. Instructions, either as inserts or a label, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.
In certain embodiments, where the antibody is labeled with an enzyme, the kit will include substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides the detectable chromophore or fluorophore) . In addition, other additives may be included such as stabilizers, buffers (e.g., a block buffer or lysis buffer) and the like. The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay. Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.
Also provided are diagnostic or detection reagents and kits comprising one or more such reagents for use in a variety of detection assays, including for example, immunoassays such as ELISA (sandwich-type or competitive format) . The kit's components may be pre-attached to a solid support, or may be applied to the surface of a solid support when the kit is used. In some embodiments, the signal generating means may come pre-associated with an antibody of the invention or may require combination with one or  more components, e.g., buffers, antibody-enzyme conjugates, enzyme substrates, or the like, prior to use. Kits may also include additional reagents, e.g., blocking reagents for reducing nonspecific binding to the solid phase surface, washing reagents, enzyme substrates, and the like. The solid phase surface may be in the form of a tube, a bead, a microtiter plate, a microsphere, or other materials suitable for immobilizing proteins, peptides, or polypeptides. In particular aspects, an enzyme that catalyzes the formation of a chemiluminescent or chromogenic product or the reduction of a chemiluminescent or chromogenic substrate is a component of the signal generating means. Such enzymes are well known in the art. Kits may comprise any of the capture agents and detection reagents described herein. Optionally the kit may also comprise instructions for carrying out the methods of the invention.
The detection kits disclosed herein may also be prepared that comprise at least one of the antibodies or antigen-binding fragments disclosed herein and instructions for using the composition as a detection reagent. Containers for use in such kits may typically comprise at least one vial, test tube, flask, bottle, syringe or other suitable container, into which one or more of the detection composition (s) may be placed, and preferably suitably aliquoted. The kits disclosed herein will also typically include a means for containing the vial (s) in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vial (s) are retained. Where a radiolabel, chromogenic, fluorigenic, or other type of detectable label or detecting means is included within the kit, the labeling agent may be provided either in the same container as the detection composition itself, or may alternatively be placed in a second distinct container means into which this second composition may be placed and suitably aliquoted. Alternatively, the detection reagent may be prepared in a single container means, and in most cases, the kit will also typically include a means for containing the vial (s) in close confinement for commercial sale and/or convenient packaging and delivery.
A device or apparatus for carrying out the detection or monitoring methods described herein is also provided. Such an apparatus may include a chamber or tube into which sample can be input, a fluid handling system optionally including valves or pumps to direct flow of the sample through the device, optionally filters to separate plasma or serum from blood, mixing chambers for the addition of capture agents or detection reagents, and optionally a detection device for detecting the amount of detectable label bound to the capture agent immunocomplex. The flow of sample may be passive (e.g., by capillary, hydrostatic, or other forces that do not require further manipulation of the device once sample  is applied) or active (e.g., by application of force generated via mechanical pumps, electroosmotic pumps, centrifugal force, or increased air pressure) , or by a combination of active and passive forces.
The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the present invention. It is the intention of the inventors that such variations are included within the scope of the invention.
EXAMPLE 1
Materials and Methods
Patients and blood samples. A total of eight patients aged 10 to 66 years old infected with SARS-CoV-2 were enrolled (Table 5) . A plasma sample from a healthy control was also included. Of these eight patients, six (P#1 through P#4, P#8, and P#16) had Wuhan exposure history through personal visit and two had direct contact with individuals from Wuhan. Four subjects (P#1 through P#4) were part of a family cluster (P#1 through P#5) infected while visiting Wuhan and subsequently transmitted infection to P#5 after returning to Shenzhen. All patients were hospitalized at Shenzhen Third People’s Hospital, the designated city hospital for treatment of COVID-19 infected patients, three to nine days after symptom onset. All patients presented with fever, fatigue, and dry cough and three (P#1, P#2 and P#5) developed severe pneumonia. Four patients (P#1, P#2, P#5, and P#22) were 60 years or older, of which three (P#1, P#2, and P#22) had underlying disease such as hypertension. SARS-CoV-2 infection status was verified by RT-PCR of nasopharyngeal swab and throat swab specimens. No patient had detectable influenza A, B, respiratory syncytial virus (RSV) , or adenovirus co-infections. Chest computed tomographic scans showed varying degrees of bilateral lung patchy shadows or opacity. All patients received antiviral and corticosteroid treatments, recovered and were discharged except for P#1, who succumbed to disease in hospital. Single (P#1, P#3, P#5, P#8, P#16, and P#22) or sequential  (P#2 and P#4) blood samples were collected during hospitalization and follow-up visits and separated into plasma and peripheral blood mononuclear cells (PBMCs) by Ficoll-Hypaque gradient (GE Healthcare) centrifugation. All plasma samples were heat-inactivated at 56 ℃ for 1h before being stored at -80 ℃. PBMCs were maintained in freezing media and stored in liquid nitrogen until use.
Recombinant RBDs and trimeric Spike from SARS-CoV-2, SARS-CoV, and  MERS-CoV and receptor ACE2. Recombinant RBDs and trimeric Spike for MERS-CoV, SARS-CoV, and SARS-CoV-2 and the N-terminal peptidase domain of human ACE2 (residues Ser19-Asp615) were expressed using the Bac-to-Bac baculovirus system (Invitrogen) as previously described (Gui, M. et al. Cell Res 27, 119-129 (2017) ; Song, W. et al. PLoS Pathog 14, e1007236-e1007236 (2018) ; Wang, N. et al. Cell Res 23, 986-993 (2013) ; Jiang, L. et al. Sci Transl Med 6, 234ra259-234ra259 (2014) ; Zhang, S. et al. Cell Rep 24, 441-452 (2018) ) . Amino acid sequence for RBD of spike protein for MERS-CoV is shown in SEQ ID NO: 126, and the polynucleotide sequence is shown in SEQ ID NO: 127. Amino acid sequence for extracellular domain of the spike protein for MERS-CoV is shown in SEQ ID NO: 123. Amino acid sequence for RBD of spike protein for SARS-CoV is shown in SEQ ID NO: 124, and the polynucleotide sequence is shown in SEQ ID NO: 125. Amino acid sequence for extracellular domain of the spike protein for SARS-CoV is shown in SEQ ID NO: 122. Amino acid sequence for RBD of spike protein for SARS-CoV-2 is shown in SEQ ID NO: 128, and the polynucleotide sequence is shown in SEQ ID NO: 129. Amino acid sequence for extracellular domain of the spike protein for SARS-CoV-2 is shown in SEQ ID NO: 121. Extracellular domains of the spike protein were fused to an artificial sequence to enable formation of a trimeric Spike structure in vitro.
SARS-CoV-2 RBD (residues Arg319-Phe541) containing the gp67 secretion signal peptide (SEQ ID NO: 130) and a C-terminal 6×His tag (SEQ ID NO: 132) was inserted into pFastBac-Dual vectors (Invitrogen) and transformed into DH10Bac component cells. The bacmid was extracted and further transfected into Sf9 cells using Cellfectin II Reagents (Invitrogen) . The recombinant viruses were harvested from the transfected supernatant and amplified to generate high-titer virus stock. Viruses were then used to infect Hi5 cells for RBD and trimeric Spike expression. Secreted RBD and trimeric Spike were harvested from the supernatant and purified by gel filtration chromatography as previously reported (Gui, M. et al. Cell Res 27, 119-129 (2017) ; Song, W. et al. PLoS Pathog 14,  e1007236-e1007236 (2018) ; Wang, N. et al. Cell Res 23, 986-993 (2013) ; Jiang, L. et al. Sci Transl Med 6, 234ra259-234ra259 (2014) ; Zhang, S. et al. Cell Rep 24, 441-452 (2018) ) .
ELISA analysis of plasma and antibody binding to RBD, trimeric Spike, and  NP proteins. The recombinant RBDs and trimeric Spike derived from SARS-CoV-2, SARS-CoV and MERS-CoV and the SARS-CoV-2 NP protein (Sino Biological, Beijing) were diluted to final concentrations of 0.5 μg/ml or 2μg/ml, then coated onto 96-well plates and incubated at 4℃ overnight. Samples were washed with PBS-T (PBS containing 0.05%Tween 20) and blocked with blocking buffer (PBS containing 5%skim milk and 2%BSA) at RT for 1h. Either serially diluted plasma samples or isolated mAbs were added the plates and incubated at 37℃ for 1h. Wells were then incubated with secondary anti-human IgG labeled with HRP (ZSGB-BIO, Beijing) and TMB substrate (Kinghawk, Beijing) and optical density (OD) was measured by a spectrophotometer at 450nm and 630nm. The serially diluted plasma from healthy individuals or mAbs against SARS-CoV, MERS-CoV or HIV-1 were used as controls.
Isolation of RBD-specific single B cells by FACS. RBD-specific single B cells were sorted as previously described (Kong, L. et al. Immunity 44, 939-950 (2016) ; Wu, X. et al. Science 329, 856-861 (2010) ) . In brief, PBMCs from infected and convalescent individuals were collected and incubated with an antibody and RBD cocktail for identification of RBD-specific B cells. The cocktail consisted of CD19-PE-Cy7, CD3-Pacific Blue, CD8-Pacific Blue, CD14-Pacific Blue, CD27-APC-H7, IgG-FITC (BD Biosciences) and the recombinant RBD-Strep or RBD-His described above. Three consecutive staining steps were conducted. The first was a LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen) in 50μl phosphate-buffered saline (PBS) applied at RT for 20 minutes to exclude dead cells. The second utilized an antibody and RBD cocktail for an additional 30 min at 4 ℃. The third staining at 4 ℃ for 30min involved either: Streptavidin-APC (eBioscience) and/or Streptavidin-PE (BD Biosciences) to target the Strep tag of RBD, or anti-his-APC and anti-his-PE antibodies (Abcam) to target the His tag of RBD. The stained cells were washed and resuspended in PBS before being strained through a 70μm cell mesh (BD Biosciences) . RBD-specific single B cells were gated as CD19+CD3-CD8-CD14-IgG+RBD+ and sorted into 96-well PCR plates containing 20μl of lysis buffer (5 μl of 5 x first strand buffer, 0.5 μl of RNase out, 1.25 μl of 0.1 M DTT (Invitrogen) per well and 0.0625 μl of Igepal (Sigma) . Plates were then snap-frozen on dry ice and stored at -80 ℃ until RT reaction.
Single B cell PCR, cloning and expression of monoclonal antibodies (mAbs) . The IgG heavy and light chain variable genes were amplified by nested PCR and cloned into linear expression cassettes or expression vectors to produce full IgG1 antibodies as previously described (Liao, H. -X. et al. J Virol Methods, 2009; Tiller, T. et al. J. Immunol Methods, 2008) . Specifically, all second round PCR primers containing tag sequences were used to produce the linear Ig expression cassettes by overlapping PCR. Separate primer pairs containing the specific restriction enzyme cutting sites (heavy chain, 5’-AgeI/3’-SalI; kappa chain, 5’-AgeI/3’-BsiWI; and lambda chain, 5’-AgeI/3’-XhoI) were used to amplify the cloned PCR products. The PCR products were purified and cloned into the backbone of antibody expression vectors containing the constant regions of human IgG1. The DNA sequence for the heavy chain constant region of human IgG1 is set forth in SEQ ID NO: 118, and the amino acid sequence for the heavy chain constant region of human IgG1 is shown in SEQ ID NO: 115. Overlapping PCR products of paired heavy and light chain expression cassettes were co-transfected into 293T cells (ATCC) grown in 24-well plates. Antigen-specific ELISA was used to detect the binding capacity of transfected culture supernatants to SARS-CoV-2 RBD. Monoclonal antibodies were produced by transient transfection of 293F cells (Life Technologies) with equal amounts of paired heavy and light chain plasmids.
Specifically, Table 4 shows the encoding DNA sequences and the amino acid sequences for the heavy chain and light chain variable regions of the monoclonal antibodies including P2A-1A8, P2A-1A9, P2A-1A10, P2A-1B3, P2B-2F6, P2B-2G4, P2B-2G11, P2C-1A3, P2C-1C8, P2C-1C10, P2C-1D5, and P2C-1F11. Antibodies P2A-1A8, P2A-1A9, P2B-2F6, P2B-2G4, P2B-2G11, and P2C-1D5 have lambda light chains, and the encoding DNA sequences and the amino acid sequences for the lambda constant region is shown in SEQ ID NO: 116 and SEQ ID NO: 119, respectively. Antibodies P2A-1A10, P2A-1B3, P2C-1A3, P2C-1C8, P2C-1C10, and P2C-1F11 have kappa light chains, and the encoding DNA sequences and the amino acid sequences for the kappa constant region is shown in SEQ ID NO: 117 and SEQ ID NO: 120, respectively.
Antibodies in the culture supernatant was purified by affinity chromatography using Protein A beads columns (National Engineering Research Center for Biotechnology, Beijing) according to the manufacturer’s protocol. Concentrations were determined by BCA Protein Assay Kits (Thermo Scientific) . SARS-CoV, MERS-CoV, and HIV-1 mAbs were also included as controls. SARS-CoV antibodies (S230 and m396) previously isolated by  others (Zhu, Z. et al. Proc Natl Acad Sci USA 104, 12123-12128 (2007) ) were synthesized and sequences verified before expression in 293T cells and purification by protein A chromatography. MERS-CoV antibodies (Mab-GD33) were derived from previously reported (Niu, P. et al. J Infect Dis 218, 1249-1260 (2018) ) . HIV-1 antibody VRC01 was a broadly neutralizing antibody directly isolated from a patient targeting the CD4 binding site of envelope glycoprotein 40.
Antibody binding kinetics, epitope mapping, and competition with receptor  ACE2 measured by SPR. The binding kinetics and affinity of mAbs to SARS-CoV-2 RBD were analyzed by SPR (Biacore T200, GE Healthcare) . Specifically, purified RBDs were covalently immobilized to a CM5 sensor chip via amine groups in 10mM sodium acetate buffer (pH 5.0) for a final RU around 250. SPR assays were run at a flow rate of 30ml/min in HEPE buffer. The sensograms were fit in a 1: 1 binding model with BIA Evaluation software (GE Healthcare) . For epitope mapping, two different antibodies were sequentially injected and monitored for binding activity to determine whether the two mAbs recognized separate or closely-situated epitopes. To determine competition with the human ACE2 peptidase domain, SARS-CoV-2 RBD was immobilized to a CM5 sensor chip via amine group for a final RU around 250. Antibodies (1 μM) were injected onto the chip until binding steady-state was reached. ACE2 (2 μM) , which was produced and purified as above, was then injected for 60 seconds. Blocking efficacy was determined by comparison of response units with and without prior antibody incubation.
Analysis of plasma and antibody binding to cell surface expressed trimeric  Spike protein. HEK 293T cells were transfected with expression plasmid encoding the full length spike of SARS-CoV-2, SARS-CoV or MERS-CoV and incubated at 37 ℃ for 36 h. The cells were digested with trypsin and distributed into 96 well plates for the individual staining. Cells were washed twice with 200μl staining buffer (PBS with 2%heated-inactivated FBS) between each following steps. The cells were stained at room temperature for 30 minutes in 100 μl staining buffer with 1: 100 dilutions of plasma or 20 μg/ml monoclonal antibodies. The cells were then stained with PE labeled anti-human IgG Fc secondary antibody (Biolegend) at a 1: 20 dilution in 50 μl staining buffer at room temperature for 30 minutes. Finally, the cells were re-suspended and analyzed with FACS Calibur instrument (BD Biosciences, USA) and FlowJo 10 software (FlowJo, USA) . HEK 293T cells without transfection were also stained as background control. S230 and m396 targeting the RBD of SARS-CoV spike (Zhu, Z. et al. Proc Natl Acad Sci USA 104,  12123-12128 (2007) ) and Mab-GD33 targeting the RBD of MERS-CoV spike (Niu, P. et al. J Infect Dis 218, 1249-1260 (2018) ) were used as positive primary antibody controls, while VRC01 targeting HIV-1 env (Wu, X. et al. Science 329, 856-861 (2010) ) was used as an irrelevant primary antibody control.
Neutralization activity of mAbs against pseudovirus and live SARS-CoV-2. SARS-CoV-2, SARS-CoV and MERS-CoV pseudovirus were generated by co-transfection of human immunodeficiency virus backbones expressing firefly luciferase (pNL43R-E-luciferase) and pcDNA3.1 (Invitrogen) expression vectors encoding the respective full length S proteins into 293T cells (ATCC) (Wang, N. et al. Cell Res 23, 986-993 (2013) ; Jiang, L. et al. Sci Transl Med 6, 234ra259-234ra259 (2014) ; Jia, W. et al. Emerg Microbes Infect 8, 760-772 (2019) ; Zhang, L. et al. J Med Virol 78, 1-8 (2006) ) . Viral supernatants were collected 48 h later. Viral titers were measured as luciferase activity in relative light units (Bright-Glo Luciferase Assay Vector System, Promega Biosciences) . Control envelope glycoproteins derived from human immunodeficiency virus (HIV) -1 and their corresponding pseudoviruses were produced in the same manner. Control mAbs included VRC01 against HIV-1 40; S230 and m396 against SARS-CoV (Zhu, Z. et al. Proc Natl Acad Sci USA 104, 12123-12128 (2007) ) ; and Merb-GD33 against MERS-CoV 43. Neutralization assays were performed by incubating pseudoviruses with serial dilutions of purified mAbs at 37℃ for 1h. Huh7 cells (ATCC) (approximately 1.5 × 10 4 per well) were added in duplicate to the virus-antibody mixture. Half-maximal inhibitory concentrations (IC 50) of the evaluated mAbs were determined by luciferase activity 48h after exposure to virus-antibody mixture using GraphPad Prism 6 (GraphPad Software Inc. ) , data were shown in Table 6.
All experiments involving live SARS-CoV-2 followed approved Biosafety Level 3 laboratory standard operating procedures. Neutralization assays against live SARS-CoV-2 were conducted using a clinical isolate (Beta/Shenzhen/SZTH-003/2020, EPI_ISL_406594 at GISAID) previously obtained from a nasopharyngeal swab of P#3. The isolate was amplified in Vero cell lines to make working stocks of the virus (1 × 10 5 PFU/ml) . To analyze the mAb neutralizing activities, Vero E6 cells were seeded at 10 4/well in 96-well culture plates and cultured at 37 ℃ to form a monolayer. Serial dilutions of mAbs were mixed separately with 100 PFU of SARS-CoV-2, incubated at 37 ℃ for 1 h, and added to the monolayer of Vero E6 cells in duplicates. Cells either unexposed to the virus or mixed with 100 PFU SARS-CoV-2 were used as negative (uninfected) and positive (infected) controls,  respectively. Cytopathic effects (CPE) were observed daily and recorded on Day 2 post-exposure.
Gene family usage and phylogenetic analysis of mAbs. The program IMGT/V-QUEST (http: //www. imgt. org/IMGT_vquest/vquest) was used to analyze germline gene, germline divergence or degree of somatic hypermutation (SHM) , the framework region (FR) and the loop length of the complementarity determining region 3 (CDR3) for each antibody clone. The IgG heavy and light chain variable genes were aligned using Clustal W in the BioEdit sequence analysis package (https: //bioedit. software. informer. com/7.2/) . Phylogenetic analyses were performed by the Maximum Likelihood method using MEGA X (Molecular Evolutionary Genetics Analysis across computing platforms) . Several forms of the phylogenetic trees are presented for clarity.
Antibody production. The production of antibodies was conducted as previously described (Jiang, L. et al. Sci Transl Med 6, 234ra259-234ra259 (2014) ; Zhang, Q. et al. Sci Rep 6, 25856-25856 (2016) ) . The genes encoding the heavy and light chains of isolated antibodies were separately cloned into expression vectors containing IgG1 constant regions and the vectors were transiently transfected into HEK293T or 293F cells using polyethylenimine (PEI) (Sigma) . After 72h, the antibodies secreted into the supernatant were collected and captured by protein A Sepharose (GE Healthcare) . The bound antibodies were eluted and further purified by gel-filtration chromatography using a Superdex 200 High Performance column (GE Healthcare) . The purified antibodies were either used in binding and neutralizing assays.
EXAMPLE 2
This example illustrates the identification of human plasma and B cell that responses specific to SARS-CoV-2 RBD.
Cross-sectional and longitudinal blood samples from eight SARS-CoV-2-infected and convalescent subjects were collected during the early outbreak in Shenzhen (see Table 5) . Samples were named by patient number and either A, B, or C depending on collection sequence. Six patients (P#1 through P#4, P#8, and P#16) had Wuhan travel history and the remaining two (P#5 and P#22) had direct contact with those from Wuhan. P#1 through P#5 is a family cluster with the first documented case of human-to-human transmission of SARS-CoV-2 in Shenzhen. All subjects recovered and were discharged from the hospital except for P#1 who succumbed to disease despite intensive  intervention. To analyze antibody binding, serial plasma dilutions were applied to enzyme-linked immunosorbent assay (ELISA) plates coated with either recombinant RBD or trimeric Spike derived from SARS-CoV-2, SARS-CoV, and MERS-CoV or recombinant NP from SARS-CoV-2. Binding activity was visualized using anti-human IgG secondary antibodies at an optical density (OD) of 450nm. Varying degrees of binding were found across individuals and among samples from the same individual. Samples from P#1, P#2, P#5, and P#16 demonstrated higher binding to both SARS-CoV-2 RBD and NP than the rest (Figure 1 (A) ) . Three sequential plasma samples collected from P#2 over nine days during early infection showed similar binding to SARS-CoV-2 RBD and NP and remained relative stable over the course of the infection. Surprisingly, virtually no cross-reactivity between SARS-CoV RBD and MERS-CoV RBD was detected (Figure 1 (A) ) , despite strong recognition by the positive control antibodies. However, strong cross-reactivity was detected against trimeric Spikes from SARS-CoV and MERS-CoV in both ELISA (Figure 1 (B) ) and cell-surface staining (Figure 5) . All samples except P#4A demonstrated significant levels of cross-binding to SARS-CoV trimeric Spike while only those from P#1, P#2 and P#4B cross recognized MERS-CoV trimeric Spike (Figure 1 (B) ) . None of the plasma samples were reactive to HIV-1 envelope trimer derived from strain BG505 27. The same plasma samples were also evaluated for neutralization of pseudoviruses bearing the Spike proteins of either SARS-CoV-2, SARS-CoV, or MERS-CoV. Consistent with the antibody binding results, varying degrees of neutralizing activities against SARS-CoV-2 were found across individuals (Figure 1 (C) ) . However, cross-neutralizing against SARS-CoV and MERS-CoV is rather minimal as all plasma samples tested, including healthy control plasma, had negligible levels of neutralization (Figure 1 (C) ) . No detectable neutralization was found for any plasma sample against the pseudovirus control bearing the HIV-1 envelope MG04 (Figure 1 (C) ) . Taken together, these results suggest that RBDs from SARS-CoV-2, SARS-CoV, and MERS-CoV are likely to be immunologically distinct despite substantial sequence and structural similarities. Thus, regions beyond RBDs likely contribute to the observed cross-reactivity against SARS-CoV and MERS-CoV Spike protein.
Flow cytometry with a range of gating strategies was used to study SARS-CoV-2-specific B cell responses and identity B cells recognizing fluorescent-labeled RBD probes (Figure 1 (D) and Figure 6) . As shown in Figure 1 (E) , the RBD-specific B cells constitute about 0.005-0.065%among the total B cell population and 0.023-0.329%among the memory subpopulations. The number of RBD-specific B cells are relatively higher  in P#2, P#5, P#16, and P#22 (Figure 1 (E) ) , which appeared to correlate well with binding activity of corresponding plasma samples to SARS-CoV-2 RBD and trimeric Spike protein (Figure 1 (A) and 1 (B) ) . However, sample P#1A demonstrated the lowest RBD-specific B cell response despite high-level plasma binding. As P#1 was the only patient succumb to disease, it is possible that this dichotomy of high plasma binding activity and low levels of RBD-specific B cells is a surrogate marker of rapid disease progression.
EXAMPLE 3
This example illustrates the cloning and analysis of single B cell antibody against SARS-CoV-2 RBD.
The RBD-binding B cells identified in EXAMPLE 2 were isolated into single cell suspension for cloning and evaluation of the mAb response (Figure 1 (D) and Figure 6) . Immunoglobulin heavy and light chains were amplified by RT-PCR using nested primers. The amplified products were cloned into linear expression cassettes to produce full IgG1 antibodies as previously described (Kong, L. et al. Immunity 44, 939-950 (2016) ; Liao, H. -X. et al. J Virol Methods 158, 171-179) . The number of B cell clones varied from 10 to 10 6 among the subjects and each clone has been differentially represented (Figure 7) . Individual IgGs were produced by transfection of linear expression cassettes and tested for SARS-CoV-2 RBD reactivity by ELISA. On average, fifty-eight percent of the antibody clones were reactive, although great variability was found among different individuals (Figure 7) . Out of 358 antibodies, 206 antibodies were found to specifically bind to SARS-CoV-2 RBD, and by B cell cloning and sequencing, 165 distinct sequences were obtained (Table 9) . These 206 antibodies demonstrated significant differences in binding activity. For example, a large number of antibodies from samples P#2B, P#2C, P#4A, P4#B, P#5A, P#16A, and P#22A had OD 450 values well over 4.0, while none of those from sample P#1A exceeded 4.0. There were too few antibodies from P#3A and P#8A to make meaningful evaluations (Figure 7) . Furthermore, samples from different study subjects also demonstrated substantial differences in heavy chain variable gene (VH) usage (Figure 2 (A) ) . For instance, P#1 samples are dominated by VH3-53, 3-13, and 1-69 which constituted approximately 21.4%, 14.3%, and 14.3%of the entire VH repertoire, respectively. Samples from P#2 and P#5 are more diverse in distribution and frequency of their VH usage. However, no single or group of VH families stood out among study subjects, suggesting patients have immunologically distinct responses to SARS-CoV-2 infection. This hypothesis is supported by the phylogenetic analysis of all 206 VH sequences superimposed with their corresponding  binding activities as presented in Figure 2B. The high-binding clusters (80%of clusters with OD 450 > 3) were widely distributed across multiple heavy chain families. In fact, majority of the high-binding antibodies were derived by clonal expansion of specific VH families in P#2, P#4, and P#5. Similarly, the middle- (60-80%of clusters with OD 450 > 3) and low- (<60%cluster with OD 450 > 3) binding clusters were also widely distributed and each consisted of disproportionally represented VH gene families.
As P#2 showed a large number of RBD-binding antibodies and was the only patient with three sequential blood samples, more detailed characterization of P#2 antibodies were conducted. Among a total of 69 antibodies from P#2, the majority (59%) were scattered across various branches and the remaining (41%) were clonally expanded into three major clusters (Figure 3 (A) ) . Antibodies from the three time points (A, B, C) do not appear to group together but rather interdigitate among themselves, suggesting they are highly related during early infection. Three clones were significantly enriched and each constituted between 12-14%of the entire tested repertoire (Figure 3 (A) ) . Their heavy-chain variable regions belong to the VH1-2*06, VH3-48*02, and VH3-9*01 families. The corresponding light-chain kappa (Igk) belongs to 2-40*01/2D-40*01, 3-20*01, and light-chain lambda (Igl) to 2-14*02 with the respective joining segment kappa 4 (Jk4) , Jk5 and joining segment lambda 1 (Jl1) (Table 9) . More importantly, these clonally expanded antibodies were identified in all three samples indicating that they are strongly selected for during infection. When comparing their representation within each cluster, VH1-2*06 and VH3-9*01 appeared to increase from approximately 33 to 45%, whereas VH3-48*02 decreased from 33 to 9%over the three time points, although the number of clones was too small for statistical significance. Interestingly, the somatic hypermutation (SHM) or germline divergence for VH1-2*06 was 0%and this cluster persisted during the study period. However, the SHM for VH3-48*02 reached as high as 9.6%and for VH3-9*01 reached 3.8%compared to the overall average of 2.2%± 3.3 %among the 69 VH sequences. Furthermore, the CDR3 length for VH1-2*06, VH3-48*02, and VH3-9*01 was 19aa, 16aa, and 23aa, respectively, compared with the overall average of 16 ±4aa among the 69 VH sequences. Close examination of the longest CDR3 from the VH3-9*01 cluster revealed richness in tyrosine, indicating potential hydrogen bonding and hydrophobic interactions with the surrounding residues. These results shed light on the clonal expansion and broad diversity of RBD-specific antibodies during early infection and their potential role in controlling SARS-CoV-2 infection.
EXAMPLE 4
This example illustrates the binding properties of the antibodies against SARS-CoV-2 RBD.
Based on their representation and distribution on the phylogenetic tree, 13 of the 69 P#2 antibodies sequences were selected for further analysis (Figure 3 (A) , starred) . Five P#1A antibody clones were used as controls. Surface plasmon resonance (SPR) with SARS-CoV-2 RBD showed that P#2 antibodies had dissociation constants (Kd) ranging from 10 -8 to 10 -9 M while those from P#1 ranged from not detectable to 10 -9 M (Table 7 and Figure 8) . SHM did not appear to correlate with Kd; some germline clones with 0%divergence in both VH and VL genes (P2A-1A10, P2B-2G4, P2C-1A3, and P2C-1E1) had Kd values comparable to clones with higher levels of SHM. The Kd of representative clones (P2A-1A8, P2A-1A10, and P2A-1B3) from the three clonally expanded clusters fell into a similar range, suggesting that their expansion may not be driven by affinity maturation. Next, each antibody for competition with ACE2 for binding to the SARS-CoV-2 RBD were measured. Specifically, the RBD was covalently immobilized on a CM5 sensor chip and first saturated by antibody and then flowed through with soluble ACE2. Competing capacity of each antibody was measured as percent reduction in ACE2 binding with the RBD (Table 7 and Figure 9) . As shown in Table 7, the evaluated antibodies demonstrated various competing capacity with ACE2. The most powerful were P2C-1F11 and P2B-2F6, which reduced ACE2 binding about 99.2%and 98.5%, respectively. Two of the three representative antibodies from the clonal expanded clusters (P2A-1A10 and P2A-1B3) had slightly over 80%and 90%reduction, respectively. The third representative (P2A-1A8) only showed 57%reduction. Many antibodies had only limited competing power with ACE2 despite impressive Kd values, suggesting binding affinity is not predictive of ACE2 competing capacity. Control antibodies from P#1 demonstrated even lower competing power with ACE2. Surprisingly, none of the antibodies tested demonstrated cross-binding with SARS-CoV and MERS-CoV RBD except P1A-1C7 (Kd=4.85μM) , for which only limited cross reactivity with SARS-CoV RBD was detected (Figure 8) .
EXAMPLE 5
This example illustrates the neutralizing properties of the antibodies against pseudoviruses bearing the Spike protein of SARS-CoV-2.
Consistent with the competing capacity findings, neutralizing activity varied considerably with IC 50 values ranging from 0.03 to > 50 μg/ml (Figure 4 (A) , Figure 4 (B)  and Table 7) . P2C-1F11 and P2B-2F6 were the most potent, followed by P2C-1A3 and P2C-1C10. Overall, ACE2 competing capacity correlated well with the neutralizing activities, although this correlation was not exact in some instances. Notably, no cross-neutralization was found either against pseudoviruses bearing the full length Spike of SARS-CoV or MERS-CoV or with cell-surface staining of trimeric SARS-CoV and MERS-CoV Spike (Figure 10) . Furthermore, we selected P2C-1F11, P2B-2F6, and P2C-1A3 for neutralizing activity analyses against live SARS-CoV-2. Consistent with their respective pseudovirus assay findings, P2C-1F11 and P2B-2F6 demonstrated potent neutralization activity while that of P2C-1A3 was somewhat lower, although it needs to be noted that CPE assay is not particularly quantitative (Figure 4 (C) ) .
To determine whether these antibodies compete for similar epitopes on the SARS-CoV-2 RBD, a total of six antibodies with ACE2 competitive capacities of at least 70%were selected and analyzed in a pairwise competition fashion using SPR. As shown in Table 8 and Figure 11, variable degrees of competition were found among the pairs of antibodies. P2C-1A3, for instance, was competitive against all antibodies tested with reduction capacity ranging from 52 to 76. P2C-1F11, on the other hand, was less competitive with other antibodies and in particular, only minimally competitive with P2C-1C10. P2B-2F6, another potent neutralizing antibody, was broadly competitive with all antibodies tested. These results indicate that the antibodies analyzed recognized both overlapping and distinct epitopes. Different mAbs may therefore exert their neutralizing activity through different mechanisms.
                               *************
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Figure PCTCN2020084097-appb-000014
Table6. Neutralizing activity of the 12 antibodies from patient #2
Figure PCTCN2020084097-appb-000015
Figure PCTCN2020084097-appb-000016
Figure PCTCN2020084097-appb-000017
*Antibodies having an IC50＜50μg/ml are defined as specific neutralizing antibody.
Figure PCTCN2020084097-appb-000018
Table 8. Epitope mapping of mAbs through competitive binding to SARS-CoV-2 RBD
Figure PCTCN2020084097-appb-000019
n.a.: not applicable
Table 9. Gene family analysis of monoclonal antibodies.
Figure PCTCN2020084097-appb-000020
Figure PCTCN2020084097-appb-000021
Figure PCTCN2020084097-appb-000022
The program IMGT/V-QUEST was applied to analyze gene germline, complementarity determining region (CDR) 3 length, and somatic hypermutation (SHM) . The CDR3 length was calculated from amino acids sequences. The SHM frequency was calculated from the mutated nucleotides.
Table 10. All sequences mentioned or used in the present application
Figure PCTCN2020084097-appb-000023
Figure PCTCN2020084097-appb-000024
Figure PCTCN2020084097-appb-000025
Figure PCTCN2020084097-appb-000026
Figure PCTCN2020084097-appb-000027
Figure PCTCN2020084097-appb-000028
Figure PCTCN2020084097-appb-000029
Figure PCTCN2020084097-appb-000030
Figure PCTCN2020084097-appb-000031
Figure PCTCN2020084097-appb-000032
Figure PCTCN2020084097-appb-000033
Figure PCTCN2020084097-appb-000034
Figure PCTCN2020084097-appb-000035
Figure PCTCN2020084097-appb-000036
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Claims (59)

  1. An isolated or recombinant antibody or an antigen-binding fragment thereof, which is capable of specifically binding to SARS-CoV-2, and exhibiting at least 50%less binding or non-detectable binding to SARS-CoV or MERS-CoV.
  2. An isolated or recombinant antibody or an antigen-binding fragment thereof, having one or more features selected from the group consisting of:
    a) capable of specifically binding to spike protein of SARS-CoV-2 and exhibiting at least 50%less binding to spike protein of SARS-CoV or spike protein of MERS-CoV;
    b) capable of specifically binding to receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 comprising the amino acid sequence of SEQ ID NO: 128;
    c) exhibiting binding to RBD of spike protein of SARS-CoV comprising the amino acid sequence of SEQ ID NO: 124 at a level that is non-detectable or that is no more than 50%of the binding to the RBD of spike protein of SARS-CoV-2;
    d) exhibiting binding to RBD of spike protein of MERS-CoV comprising the amino acid sequence of SEQ ID NO: 126 at a level that is non-detectable or that is no more than 50%of the binding to RBD of the spike protein of SARS-CoV-2;
    e) capable of binding to the RBD of spike protein of SARS-CoV-2 at a K d value of no more than 1x10 -7M as measured by Surface Plasmon Resonance (SPR) ;
    f) exhibiting binding to the RBD of spike protein of SARS-CoV or the RBD of spike protein of MERS-CoV at a K d value of at least 1x10 -6M as measured by SPR;
    g) capable of exhibiting at least 30%competition at 1μM, with 2 μM angiotensin converting enzyme 2 (ACE2) receptor, for binding to the RBD of spike protein of SARS-CoV-2 immobilized at a resonance units (RU) of 250, as measured by SPR; and
    h) capable of binding to the RBD of spike protein of SARS-CoV-2 at a neutralizing activity at an IC 50 value of no more than 100μg/ml, as measured by pseudovirus neutralization assay.
  3. An isolated or recombinant antibody or an antigen-binding fragment thereof capable of specifically binding to RBD of spike protein of SARS-CoV-2, comprising:
    a) 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3;
    b) 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13;
    c) 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23;
    d) 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33;
    e) 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43;
    f) 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53;
    g) 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67;
    h) 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77;
    i) 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87;
    j) 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97; or
    k) 1, 2, or 3 heavy chain CDR sequences selected from SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107.
  4. The antibody or antigen binding fragment of any of the preceding claims, comprising:
    a) 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6;
    b) 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16;
    c) 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26;
    d) 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36;
    e) 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46;
    f) 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56;
    g) 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70;
    h) 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80; and
    i) 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90.
    j) 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 98, SEQ ID NO: 99, and SEQ ID NO: 100; or
    k) 1, 2, or 3 light chain CDR sequences selected from SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110.
  5. The antibody or antigen binding fragment of any of the preceding claims, comprising:
    a) a heavy chain CDR1 (HCDR1) comprising the sequence of SEQ ID NO: 1, a heavy chain CDR2 (HCDR2) comprising the sequence of SEQ ID NO: 2, a heavy chain CDR3 (HCDR3) comprising the sequence of SEQ ID NO: 3; a light chain CDR1 (LCDR1) comprising the sequence of SEQ ID NO: 4, a light chain CDR2 (LCDR2) comprising the sequence of SEQ ID NO: 5, and a light chain CDR3 (LCDR3) comprising the sequence of SEQ ID NO: 6;
    b) a HCDR1 comprising the sequence of SEQ ID NO: 11, a HCDR2 comprising the sequence of SEQ ID NO: 12, a HCDR3 comprising the sequence of SEQ ID NO: 13, a LCDR1 comprising the sequence of SEQ ID NO: 14, a LCDR2 comprising the sequence of SEQ ID NO: 15, and a LCDR3 comprising the sequence of SEQ ID NO: 16;
    c) a HCDR1 comprising the sequence of SEQ ID NO: 21, a HCDR2 comprising the sequence of SEQ ID NO: 22, a HCDR3 comprising the sequence of SEQ ID NO: 23, a LCDR1 comprising the sequence of SEQ ID NO: 24, a LCDR2 comprising the sequence of SEQ ID NO: 25, and a LCDR3 comprising the sequence of SEQ ID NO: 26;
    d) a HCDR1 comprising the sequence of SEQ ID NO: 31, a HCDR2 comprising the sequence of SEQ ID NO: 32, a HCDR3 comprising the sequence of SEQ ID NO: 33, a LCDR1 comprising the sequence of SEQ ID NO: 34, a LCDR2 comprising the sequence of SEQ ID NO: 35, and a LCDR3 comprising the sequence of SEQ ID NO: 36;
    e) a HCDR1 comprising the sequence of SEQ ID NO: 41, a HCDR2 comprising the sequence of SEQ ID NO: 42, a HCDR3 comprising the sequence of SEQ ID NO: 43,  a LCDR1 comprising the sequence of SEQ ID NO: 44, a LCDR2 comprising the sequence of SEQ ID NO: 45, and a LCDR3 comprising the sequence of SEQ ID NO: 46;
    f) a HCDR1 comprising the sequence of SEQ ID NO: 51, a HCDR2 comprising the sequence of SEQ ID NO: 52, a HCDR3 comprising the sequence of SEQ ID NO: 53, a LCDR1 comprising the sequence of SEQ ID NO: 54, a LCDR2 comprising the sequence of SEQ ID NO: 55, and a LCDR3 comprising the sequence of SEQ ID NO: 56;
    g) a HCDR1 comprising the sequence of SEQ ID NO: 65, a HCDR2 comprising the sequence of SEQ ID NO: 66, a HCDR3 comprising the sequence of SEQ ID NO: 67, a LCDR1 comprising the sequence of SEQ ID NO: 68, a LCDR2 comprising the sequence of SEQ ID NO: 69, and a LCDR3 comprising the sequence of SEQ ID NO: 70;
    h) a HCDR1 comprising the sequence of SEQ ID NO: 75, a HCDR2 comprising the sequence of SEQ ID NO: 76, a HCDR3 comprising the sequence of SEQ ID NO: 77, a LCDR1 comprising the sequence of SEQ ID NO: 78, a LCDR2 comprising the sequence of SEQ ID NO: 79, and a LCDR3 comprising the sequence of SEQ ID NO: 80;
    i) a HCDR1 comprising the sequence of SEQ ID NO: 85, a HCDR2 comprising the sequence of SEQ ID NO: 86, a HCDR3 comprising the sequence of SEQ ID NO: 87, a LCDR1 comprising the sequence of SEQ ID NO: 88, a LCDR2 comprising the sequence of SEQ ID NO: 89, and a LCDR3 comprising the sequence of SEQ ID NO: 90;
    j) a HCDR1 comprising the sequence of SEQ ID NO: 95, a HCDR2 comprising the sequence of SEQ ID NO: 96, a HCDR3 comprising the sequence of SEQ ID NO: 97, a LCDR1 comprising the sequence of SEQ ID NO: 98, a LCDR2 comprising the sequence of SEQ ID NO: 99, and a LCDR3 comprising the sequence of SEQ ID NO: 100; or
    k) a HCDR1 comprising the sequence of SEQ ID NO: 105, a HCDR2 comprising the sequence of SEQ ID NO: 106, a HCDR3 comprising the sequence of SEQ ID NO: 107, a LCDR1 comprising the sequence of SEQ ID NO: 108, a LCDR2 comprising the sequence of SEQ ID NO: 109, and a LCDR3 comprising the sequence of SEQ ID NO: 110.
  6. The antibody or antigen binding fragment of any of the preceding claims, comprising a pair of heavy chain variable region and light chain variable region sequences selected from the group consisting of: SEQ ID NOs: 7/8, 17/18, 27/28, 37/38, 47/48, 57/58, 61/62, 71/72, 81/82, 91/92, 101/102, and 111/112, or a pair of homologous sequences thereof having at least 80%sequence identity yet retaining specific binding affinity to RBD of spike protein of SARS-CoV-2.
  7. The antibody or antigen binding fragment of any of the preceding claims, further comprising an immunoglobulin constant region, optionally a constant region of human immunoglobulin, or optionally a constant region of human IgG.
  8. The antibody or antigen binding fragment of any of the preceding claims, further comprising one or more amino acid residue substitutions or modifications yet retains specific binding affinity to RBD of spike protein of SARS-CoV-2.
  9. The antibody or antigen binding fragment of claim 8, which is an affinity variant, a glycosylation variant, a cysteine-engineered variant, or an Fc variant.
  10. The antibody or antigen binding fragment of claim 9, wherein the Fc variant comprises one or more amino acid residue modifications or substitutions resulting in increased effector functions relative to a wildtype Fc.
  11. The antibody or antigen binding fragment of claim 10, wherein the Fc variant comprises one or more amino acid substitution (s) at one or more of the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 246, 247, 248, 249, 252, 254, 255, 256, 258, 260, 262, 263, 264, 265, 267, 268, 269, 270, 272, 274, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 300, 301, 303, 304, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 345, 360, 373, 376, 378, 382, 388, 389, 396, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438, 439 and 440 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat.
  12. The antibody or antigen binding fragment of claim 11, wherein the Fc variant comprises one or more amino acid substitution selected from the group consisting of 234Y, 235Q, 236A, 236W, 239D, 239E, 239M, 243L, 247I, 267E, 268D, 268E, 268F, 270E, 280H, 290S, 292P, 298A, 298D, 298V, 300L, 305I, 324T, 326A, 326D, 326W, 330L, 330M, 333S, 332D, 332E, 298A, 333A, 334A, 334E, 339D, 339Q, 345R, 396L, 430G, 440Y, or any combination thereof.
  13. The antibody or antigen binding fragment of claim 9, wherein the Fc variant comprises one or more amino acid residue modifications or substitutions resulting in reduced effector functions relative to a wildtype Fc.
  14. The antibody or antigen binding fragment of claim 13, wherein the Fc variant comprises one or more amino acid substitution (s) at a position selected from the group consisting of: 220, 226, 229, 233, 234, 235, 236, 237, 238, 267, 268, 269, 270, 297, 309, 318, 320, 322, 325, 328, 329, 330, and 331 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat.
  15. The antibody or antigen binding fragment of claim 13, wherein the Fc variant comprises one or more amino acid substitution (s) selected from the group consisting of 220S, 226S, 228P, 229S, 233P, 234V, 234G, 234A, 234F, 234A, 235A, 235G, 235E, 236E, 236R, 237A, 237K, 238S, 267R, 268A, 268Q, 269R, 297A, 297Q, 297G, 309L, 318A, 322A, 325L, 328R, 330S, 331S, and any combination thereof.
  16. The antibody or antigen binding fragment of claim 9, wherein the Fc variant comprises one or more amino acid residue modifications or substitutions resulting in improved serum half-life or improved binding affinity to neonatal Fc receptor (FcRn) at pH 6.0 while retaining minimal binding at pH 7.4.
  17. The antibody or antigen binding fragment of claim 16, wherein the Fc variant comprises one or more amino acid substitution (s) at a position selected from the group consisting of: 234, 235, 238, 250, 252, 254, 256; 259; 272, 305, 307, 308, 311, 312, 322, 328, 331, 378, 380, 382, 428, 432, 433, 434, 435, 436 and 437 (all positions by EU numbering) .
  18. The antibody or antigen binding fragment of claim 17, wherein the Fc variant comprises one or more amino acid substitution (s) selected from the group consisting of 234F, 235Q, 238D, 250Q, 252T, 252Y, 254T, 256E, 259I, 272A, 305A, 307A, 308F, 311A, 322Q, 328E, 331S, 380A, 428L, 432C, 433K, 433S, 434S, 434Y, 434F, 434W, 434A, 435H, 436L, 437C and any combination thereof.
  19. The antibody or antigen binding fragment of claim 8, wherein at least one of the substitutions or modifications is in one or more of the CDR sequences, and/or in one or more of the non-CDR sequences of the heavy chain variable region or light chain variable region.
  20. The antibody or an antigen-binding fragment thereof of any one of the preceding claims, which is a monoclonal antibody, a bispecific antibody, a multi-specific antibody, a recombinant antibody, a chimeric antibody, a labeled antibody, a bivalent antibody, an anti-idiotypic antibody, a fusion protein, a dimerized or polymerized antibody, or a modified antibody (e.g. glycosylated antibody) .
  21. The antibody or antigen binding fragment of any of the preceding claims, which is a diabody, a Fab, a Fab', a F (ab')  2, a Fd, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv)  2, a bispecific dsFv (dsFv-dsFv') , a disulfide stabilized diabody (ds diabody) , a single-chain antibody molecule (scFv) , an scFv dimer (bivalent diabody) , a bispecific scFv dimer, a multispecific antibody, a heavy chain antibody, a camelized single domain antibody, a nanobody, a domain antibody, or a bivalent domain antibody.
  22. The antibody or antigen binding fragment of any of the preceding claims, which is bispecific and comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first and the second antigen-binding domains are derived:
    a) from P2C-1F11 and P2B-2F6, respectively;
    b) from P2C-1F11 and P2A-1A8, respectively;
    c) from P2C-1F11 and P2A-1A9, respectively;
    d) from P2C-1F11 and P2B-2G11, respectively;
    e) from P2C-1F11 and P2A-1A10, respectively;
    f) from P2C-1F11 and P2A-1B3, respectively;
    g) from P2C-1F11 and P2B-2G4, respectively;
    h) from P2C-1F11 and P2C-1A3, respectively;
    i) from P2C-1F11 and P2C-1C8, respectively;
    j) from P2C-1F11 and P2C-1C10, respectively;
    k) from P2C-1F11 and P2C-1D5, respectively;
    l) from P2C-1F11 and P2C-1F11, respectively;
    m) from P2A-1A8 and P2A-1A9, respectively;
    n) from P2A-1A8 and P2B-2G11, respectively;
    o) from P2A-1A8 and P2A-1A10, respectively;
    p) from P2A-1A8 and P2A-1B3, respectively;
    q) from P2A-1A8 and P2B-2F6, respectively;
    r) from P2A-1A8 and P2B-2G4, respectively;
    s) from P2A-1A8 and P2C-1A3, respectively;
    t) from P2A-1A8 and P2C-1C8, respectively;
    u) from P2A-1A8 and P2C-1C10, respectively;
    v) from P2A-1A8 and P2C-1D5, respectively;
    w) from P2A-1A9 and 2B-2G11, respectively;
    x) from P2A-1A9 and P2A-1A10, respectively;
    y) from P2A-1A9 and P2A-1B3, respectively;
    z) from P2A-1A9 and P2B-2F6, respectively;
    aa) from P2A-1A9 and P2B-2G4, respectively;
    bb) from P2A-1A9 and P2C-1A3, respectively;
    cc) from P2A-1A9 and P2C-1C8, respectively;
    dd) from P2A-1A9 and P2C-1C10, respectively;
    ee) from P2A-1A9 and P2C-1D5, respectively;
    ff) from P2B-2G11 and P2A-1A10, respectively;
    gg) from P2B-2G11 and P2A-1B3, respectively;
    hh) from P2B-2G11 and P2B-2F6, respectively;
    ii) from P2B-2G11 and P2B-2G4, respectively;
    jj) from P2B-2G11 and P2C-1A3, respectively;
    kk) from P2B-2G11 and P2C-1C8, respectively;
    ll) from P2B-2G11 and P2C-1C10, respectively;
    mm) from P2B-2G11 and P2C-1D5, respectively;
    nn) from P2A-1A10 and P2A-1B3, respectively;
    oo) from P2A-1A10 and P2B-2F6, respectively;
    pp) from P2A-1A10 and P2B-2G4, respectively;
    qq) from P2A-1A10 and P2C-1A3, respectively;
    rr) from P2A-1A10 and P2C-1C8, respectively;
    ss) from P2A-1A10 and P2C-1C10, respectively;
    tt) from P2A-1A10 and P2C-1D5, respectively;
    uu) from P2A-1B3 and P2B-2F6, respectively;
    vv) from P2A-1B3 and P2B-2G4, respectively;
    ww) from P2A-1B3 and P2C-1A3, respectively;
    xx) from P2A-1B3 and P2C-1C8, respectively;
    yy) from P2A-1B3 and P2C-1C10, respectively;
    zz) from P2A-1B3 and P2C-1D5, respectively;
    aaa) from P2B-2F6 and P2B-2G4, respectively;
    bbb) from P2B-2F6 and P2C-1A3, respectively;
    ccc) from P2B-2F6 and P2C-1C8, respectively;
    ddd) from P2B-2F6 and P2C-1C10, respectively;
    eee) from P2B-2F6 and P2C-1D5, respectively;
    fff) from P2B-2G4 and P2C-1A3, respectively;
    ggg) from P2B-2G4 and P2C-1C8, respectively;
    hhh) from P2B-2G4 and P2C-1C10, respectively;
    iii) from P2B-2G4 and P2C-1D5, respectively;
    jjj) from P2C-1A3 and P2C-1C8, respectively;
    kkk) from P2C-1A3 and P2C-1C10, respectively;
    lll) from P2C-1A3 and P2C-1D5, respectively;
    mmm) from P2C-1C8 and P2C-1C10, respectively;
    nnn) from P2C-1C8 and P2C-1D5, respectively; or
    ooo) from P2C-1C10 and P2C-1D5, respectively.
  23. The antibody or antigen binding fragment of any of the preceding claims, which is a full human antibody.
  24. The antibody or antigen binding fragment of any of the preceding claims, linked to one or more conjugate moieties.
  25. The antibody or antigen binding fragment of claim 24, wherein the conjugate moiety comprises a therapeutic agent, a radioactive isotope, a detectable label, a pharmacokinetic  modifying moiety, or a purifying moiety, and optionally the conjugate moiety is covalently attached either directly or via a linker.
  26. An antibody or an antigen-binding fragment thereof, which competes for binding to RBD of spike protein of SARS-CoV-2 with the antibody or an antigen-binding fragment thereof of any one of claims 1-25.
  27. A pharmaceutical composition comprising the antibody or antigen binding fragment of any of the preceding claims, and a pharmaceutically acceptable carrier, or comprising the polynucleotide of claim 34, and a pharmaceutically acceptable carrier.
  28. The pharmaceutical composition of claim 27, comprising a combination of two or more antibodies or antigen binding fragments of any of the preceding claims, and a pharmaceutically acceptable carrier.
  29. The pharmaceutical composition of claim 28, wherein the two or more antibodies or the antigen binding fragments thereof bind to different epitopes in RBD of spike protein of SARS-CoV-2.
  30. The pharmaceutical composition of claim 29, wherein the two or more antibodies comprise a first antibody which comprises P2C-1F11 or an antigen binding fragment thereof, and a second antibody which is selected from the group consisting of P2C-1A3, P2C-1C10, P2B-2F6, and P2A-1B3, or an antigen binding fragment thereof, optionally, the pharmaceutical composition comprises a first antibody comprising heavy chain CDR sequences and light chain CDR sequences derived from P2C-1F11, and a second antibody comprising heavy chain CDR sequences and light chain CDR sequences derived from antibody P2B-2F6.
  31. The pharmaceutical composition of claim 29, wherein the two or more antibodies comprise a first antibody which comprises P2C-1A3 or an antigen binding fragment thereof, and a second antibody which is selected from the group consisting of P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof.
  32. The pharmaceutical composition of claim 29, wherein the two or more antibodies comprise a first antibody which comprises P2B-2F6 or an antigen binding fragment thereof, and a second antibody selected from the group consisting of P2C-1C10, P2C-1F11, and P2A-1B3, or an antigen binding fragment thereof.
  33. The pharmaceutical composition of claim 29, wherein the two or more antibodies comprise a first antibody which comprises P2A-1B3 or an antigen binding fragment thereof, and a second antibody selected from the group consisting of P2C-1A3, P2C-1C10, P2C-1F11, P2B-2F6, and P2A-1A10, or an antigen binding fragment thereof.
  34. An isolated polynucleotide encoding the antibody or antigen binding fragment of any of the claims 1-26.
  35. The isolated polynucleotide of claim 34, comprising a nucleotide sequence selected from a group consisting of: SEQ ID NOs: 9-10, 19-20, 29-30, 39-40, 49-50, 59-60, 63-64, 73-74, 83-84, 93-94, 103-104, and 113-114, or a homologous sequence thereof having at least 80%sequence identity.
  36. The isolated polynucleotide of claim 35, wherein the homologue sequence encodes the same protein as encoded by any nucleotide sequence selected from the group consisting of SEQ ID NOs: 9-10, 19-20, 29-30, 39-40, 49-50, 59-60, 63-64, 73-74, 83-84, 93-94, 103-104, and 113-114.
  37. A vector comprising the isolated polynucleotide of any of claims 34-36, optionally the vector is an expression vector.
  38. A host cell comprising the vector of claim 37.
  39. A method of producing the antibody or antigen binding fragment of any of claims 1-26 comprising culturing the host cell of claim 38 under the condition at which the vector of claim 37 is expressed.
  40. The method of claim 39, further comprising purifying the antibody produced by the host cell.
  41. A kit for detecting a SARS-CoV-2 antigen, comprising the antibody or antigen binding fragment of any of claims 1-26.
  42. The kit of claim 41, further comprising a control reagent comprising RBD of spike protein of the SARS-CoV-2, optionally, the kit further comprises a set of reagents for detecting complex of the antibody or the antigen-binding fragment bound to the SARS-CoV-2 antigen.
  43. A method of treating SARS-CoV-2 infection or a disease, disorder or condition associated with SARs-CoV-2 infection in a subject, comprising administering a therapeutically effective amount of one or more of the antibody or antigen binding fragment of any of claims 1-26, or of one or more of the polynucleotides of any claims 34-36, or of one or more of the vectors of claim 37, or of the pharmaceutical composition of any of claims 27-33 to the subject.
  44. A method of preventing SARS-CoV-2 infection or a disease, disorder or condition associated with SARs-CoV-2 infection in a subject, comprising administering a therapeutically effective amount of one or more of the antibody or antigen binding fragment of any of claims 1-26, or of one or more of the polynucleotides of any claims 34-36, or of one or more of the vectors of claim 37, or of the pharmaceutical composition of any of claims 27-33 to the subject.
  45. The method of claim 43 or 44, wherein the administration is via oral, nasal, intravenous, subcutaneous, or intramuscular administration.
  46. The method of claim 45, wherein the subject is human.
  47. The method of any of claims 43-46, further comprising administering a therapeutically effective amount of a second active agent, optionally the second active agent is a therapeutic agent or a prophylactic agent.
  48. The method of claim 47, wherein the therapeutic agent is an anti-viral agent, optinally, the anti-viral agent comprises an antiviral peptide, an anti-viral antibody, an anti-viral compound, an anti-viral cytokine, or an anti-viral oligonucleotide.
  49. A method of detecting presence or amount of SARS-CoV-2 virus antigen in a sample, comprising contacting the sample with one or more of the antibody or antigen binding fragment of any of claims 1-26, and determining the presence or the amount of the SARS-CoV-2 virus antigen in the sample.
  50. Use of one or more of the antibody or antigen binding fragment of any of claims 1-26 in the manufacture of a medicament for treating SARS-CoV-2 infection or a disease, disorder or condition associated with SARs-CoV-2 infection.
  51. Use of one or more of the antibody or antigen binding fragment of any of claims 1-26 in the manufacture of a diagnostic reagent for detecting SARS-CoV-2 infection.
  52. A kit for detecting an antibody capable of specifically binding to receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, comprising a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 128.
  53. The kit of claim 52, wherein the polypeptide is immobilized on a substrate.
  54. The kit of claim 53, further comprising a set of reagents for detecting complex of the antibody bound to the polypeptide.
  55. A method of detecting presence or amount of an antibody capable of specifically binding to RBD of the spike protein of SARS-CoV-2 in a sample, comprising contacting the sample with a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 128, and determining the presence or the level of the antibody in the sample.
  56. The method of claim 55, wherein the absence of the antibody in the sample or the level of the antibody in the sample being below a threshold indicates that the subject is more likely to suffer from disease progression.
  57. A method of determining the likelihood of disease progression in a subject infected with SARS-CoV-2, the method comprising: contacting a sample obtained from the subject with a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 128, and detecting the presence or the level of an antibody in the sample wherein the antibody is capable of specifically binding to RBD of the spike protein of the SARS-CoV-2,
    wherein the subject is likely to experience disease progression when the antibody in the sample is absent or is below a threshold.
  58. A method of monitoring treatment response in a subject infected with SARS-CoV-2 and received a treatment, the method comprising:
    (i) contacting a sample from the subject with a peptide comprising an amino acid sequence comprising SEQ ID NO: 128;
    (ii) detecting a first level of an antibody in the sample wherein the antibody is capable of specifically binding to RBD of the spike protein of the SARS-CoV-2; and
    (iii) comparing the first level of the antibody with a second level of the antibody detected in the subject prior to the treatment;
    wherein the first level being higher than the second level indicates that the subject is responsive to the treatment.
  59. A method of neutralizing SARS-CoV-2 in a subject or in a sample in vitro, comprising administering a therapeutically effective amount of one or more of the antibody or antigen binding fragment of any of claims 1-26, or the pharmaceutical composition of any of claims 27-33 to the subject or to the sample.
PCT/CN2020/084097 2020-03-20 2020-04-10 Anti-sars-cov-2 antibodies and uses thereof WO2021203397A1 (en)

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