WO2021237516A1 - Sars-cov-2抗体及其应用 - Google Patents

Sars-cov-2抗体及其应用 Download PDF

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WO2021237516A1
WO2021237516A1 PCT/CN2020/092634 CN2020092634W WO2021237516A1 WO 2021237516 A1 WO2021237516 A1 WO 2021237516A1 CN 2020092634 W CN2020092634 W CN 2020092634W WO 2021237516 A1 WO2021237516 A1 WO 2021237516A1
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seq
antigen binding
amino acid
binding protein
acid sequence
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PCT/CN2020/092634
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English (en)
French (fr)
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顾春银
邓俗俊
王宗达
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上海济煜医药科技有限公司
江西济民可信集团有限公司
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Priority to PCT/CN2020/092634 priority Critical patent/WO2021237516A1/zh
Priority to CN202080099668.XA priority patent/CN115315442B/zh
Priority to TW110119266A priority patent/TW202144406A/zh
Publication of WO2021237516A1 publication Critical patent/WO2021237516A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • This application relates to the field of biomedicine, and specifically relates to a SARS-CoV-2 antibody and its application.
  • the new coronavirus (SARS-CoV-2) has a long incubation period, high concealment, and strong contagion; it has become a public health emergency in the world health field. In such a severe situation of epidemic prevention and control, there are still no specific drugs for the new coronavirus. Therefore, it is extremely urgent to develop specific antibody drugs for the prevention and control of the new coronavirus epidemic.
  • the novel coronavirus is an enveloped virus with a positive RNA genome and belongs to the genus Nidovirales ⁇ of the coronavirus family.
  • Nidovirales ⁇ of the coronavirus family belongs to the genus Nidovirales ⁇ of the coronavirus family.
  • plasma products have limited sources and must undergo strict blood biosafety testing before they can be used in clinical practice, which has not yet fully met the needs of current epidemic prevention and control.
  • This application provides a SARS-CoV-2 antibody and its application.
  • the SARS-CoV-2 antibody described in the present application may have one or more of the following properties: block the binding of the RBD or its mutant of the S protein of SARS-CoV-2 to human ACE2; block SARS- Binding of the RBD of the S protein of CoV to human ACE2; specific binding to the RBD of the S protein of SARS-CoV-2 or its mutants with a very low K D value; affinity with the RBD of the S protein of SARS-CoV-2 Stronger than human ACE2-Fc; RBD that specifically binds to SARS-CoV S protein; Hydrophilic; Conducive to preparation and purification; Good stability, especially thermal stability; It has the activity of neutralizing SARS-CoV-2 .
  • the application also provides related preparation methods and applications of the SARS-CoV-2 antibody.
  • the present application provides an isolated antigen binding protein, which has one or more of the following properties: blocking the binding of the RBD of the S protein of SARS-CoV-2 or its mutants to human ACE2; blocking; The binding between the RBD of the S protein of SARS-CoV and human ACE2; in the Octet assay, it specifically binds to the RBD of the S protein of SARS-CoV-2 with a K D value below 5.0*10 -8 M; in the Octet assay A mutant of RBD that specifically binds to the S protein of SARS-CoV-2 with a K D value below 6.0*10 -10 M; the affinity of the RBD of the S protein of SARS-CoV-2 is stronger than that of human ACE2-Fc
  • the main peak of charge heterogeneity analysis is about 45%-85%; In Thermal shift determination, Tm is at least about 75°C; It neutralizes SARS-Co
  • the isolated antigen binding protein comprises at least one CDR in the light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO: 124 or SEQ ID NO: 125.
  • the isolated antigen binding protein comprises at least one CDR in a heavy chain variable region VH, and the VH comprises SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 17, and SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID NO : 69, the amino acid sequence shown in any one of SEQ ID NO: 71 and SEQ ID NO: 73.
  • the isolated antigen binding protein comprises an antibody or antigen binding fragment thereof.
  • the antigen-binding fragment includes Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and/or dAb.
  • the VL includes LCDR1, LCDR2, and LCDR3, and the LCDR3 includes the amino acid sequence shown in any one of SEQ ID NO: 122 to SEQ ID NO: 123.
  • the LCDR3 includes SEQ ID NO: 3, SEQ ID NO: 13, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 36, SEQ ID NO: 43, and SEQ ID NO : The amino acid sequence shown in any one of 61.
  • the LCDR1 includes the amino acid sequence shown in any one of SEQ ID NO: 118-SEQ ID NO: 119.
  • the LCDR1 includes SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 41, SEQ ID NO The amino acid sequence shown in any one of: 59 and SEQ ID NO: 111.
  • the LCDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 120 to SEQ ID NO: 121.
  • the LCDR2 includes SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 35, SEQ ID NO: 42, SEQ ID NO The amino acid sequence shown in any one of: 60 and SEQ ID NO: 112.
  • the VH includes HCDR1, HCDR2, and HCDR3, and the HCDR1 includes SEQ ID NO: 5, SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 45, SEQ ID NO: 51 , SEQ ID NO: 55, SEQ ID NO: 63, and SEQ ID NO: 114 as shown in the amino acid sequence.
  • the HCDR2 includes SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 38, SEQ ID NO The amino acid sequence shown in any one of: 46, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 67, and SEQ ID NO: 115.
  • the HCDR3 includes SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 39, SEQ ID NO: 47, SEQ ID NO : 49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, and SEQ ID NO: 116 The amino acid sequence.
  • the VL includes the framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
  • the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 includes any one of SEQ ID NO: 74-SEQ ID NO: 75 The amino acid sequence shown.
  • the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 includes any one of SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78 The amino acid sequence shown in the item.
  • the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 includes SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID The amino acid sequence shown in any one of NO:82.
  • the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3, and the L-FR4 includes SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: The amino acid sequence shown in any one of 85 and SEQ ID NO: 86.
  • the VL includes the amino acid sequence shown in SEQ ID NO: 124 or SEQ ID NO: 125.
  • the VL includes SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 44, SEQ ID NO The amino acid sequence shown in any one of: 62 and SEQ ID NO: 113.
  • the isolated antigen binding protein includes an antibody light chain constant region, and the antibody light chain constant region includes a human Ig ⁇ constant region or a human Ig ⁇ constant region.
  • the VH includes framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 includes SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89.
  • the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 includes SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID The amino acid sequence shown in any one of NO:98.
  • the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 includes SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID The amino acid sequence shown in any one of NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 106.
  • the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 includes SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: The amino acid sequence shown in any one of 109 and SEQ ID NO: 110.
  • the VH includes SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 71, and SEQ ID NO: 73
  • SEQ ID NO: 8 amino acid sequence.
  • the isolated antigen binding protein includes an antibody heavy chain constant region, and the antibody heavy chain constant region includes a human IgG constant region.
  • the isolated antigen binding protein includes an antibody heavy chain constant region, and the antibody heavy chain constant region includes a human IgG1 constant region.
  • the present application provides a bispecific antigen binding protein that specifically binds to the RBD of the S protein of SARS-CoV-2 and the RBD of the S protein of SARS-CoV.
  • the bispecific antigen binding protein comprises a first targeting moiety that specifically binds to the RBD of the S protein of SARS-CoV-2, wherein the first targeting moiety comprises the Isolated antigen binding protein.
  • the bispecific antigen binding protein comprises a second targeting moiety that specifically binds to the RBD of the S protein of SARS-CoV, wherein the second targeting moiety comprises the isolated Antigen binding protein.
  • the bispecific antigen binding protein includes an antibody.
  • the first targeting moiety comprises a first heavy chain and a first light chain
  • the second targeting moiety comprises a second heavy chain and a second light chain, wherein the first light chain Same as the second light chain.
  • the first light chain and the second light chain comprise the amino acid sequence shown in SEQ ID NO:44.
  • the VH of the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 48.
  • the VH of the second heavy chain comprises the amino acid sequence shown in SEQ ID NO:50.
  • the VH of the first heavy chain includes the amino acid sequence shown in SEQ ID NO: 48
  • the VH of the second heavy chain includes the amino acid sequence shown in SEQ ID NO: 50.
  • this application provides an isolated one or more nucleic acid molecules that encode the isolated antigen binding protein described in this application, and/or the bispecific antigen binding protein described in this application.
  • the present application provides a vector, which contains the nucleic acid molecule described in the present application.
  • this application provides a cell comprising the nucleic acid molecule described in this application or the vector described in this application.
  • the present application provides a method for preparing the isolated antigen binding protein described in this application and the bispecific antigen binding protein described in this application, and the method includes making the isolated antigen binding protein described in this application bind Culture the cells described in this application under the condition of expression of the protein and/or the bispecific antigen binding protein described in this application.
  • this application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated antigen binding protein described in this application, the bispecific antigen binding protein described in this application, the nucleic acid molecule described in this application, and the nucleic acid molecule described in this application.
  • this application provides an isolated antigen binding protein described in this application, a bispecific antigen binding protein described in this application, a nucleic acid molecule described in this application, a vector described in this application, and a carrier described in this application.
  • the coronavirus infection includes COVID-19.
  • the present application provides a method for blocking the binding of the RBD of the S protein of SARS-CoV-2 or its mutants to human ACE2, which includes the following steps, administering the isolated antigen binding protein described in the present application , The bispecific antigen binding protein described in this application, the nucleic acid molecule described in this application, the vector described in this application, the cell described in this application, and/or the pharmaceutical composition described in this application.
  • the present application provides a method for blocking the binding of the RBD of the S protein of SARS-CoV to human ACE2, which includes the following steps: administering the isolated antigen binding protein described in this application, The bispecific antigen binding protein, the nucleic acid molecule described in this application, the vector described in this application, the cell described in this application, and/or the pharmaceutical composition described in this application.
  • Figures 1A-1D show the detection results of the binding and dissociation rate of the antibody described in this application with SARS-CoV-2 S1.
  • Figure 2 shows that the antibody described in this application blocks the binding of SARS-CoV-2 S1 to hACE2-Fc.
  • Figure 3 shows how the antibody described in this application blocks the binding of SARS-CoV S1 to hACE2.
  • Figure 4 shows the hydrophilic coefficient of the antibody described in this application.
  • FIG. 5 shows the results of the analysis of the charge heterogeneity of the antibodies described in this application.
  • FIG. 6 shows the result of the analysis of the antibody charge heterogeneity profile described in this application.
  • FIG. 7 shows the melting temperature of the antibody (Fab) described in this application.
  • FIGS 8A-8B show the results of the antibody virus neutralization activity experiment described in this application.
  • FIGS 9A-9B show the results of the antibody virus neutralization activity experiment described in this application.
  • Figure 10 shows the results of the neutralization concentration of the antibody plaque reduced by half.
  • Figure 11 shows the detection results of the binding and dissociation rates of the antibodies of the present application and SARS-CoV-2 S1 RBD mutants.
  • FIGS 12A-12E show that the present application and in the antibody blocks S1 RBD 2 SARS-CoV-mutations and the IC 50 value curve hACE2 body and binding.
  • SARS-CoV usually refers to SARS coronavirus, that is, Severe Acute Respiratory Syndrome Coronavirus (Severe Acute Respiratory Syndrome Coronavirus), which belongs to the Coronavirus family (Coronaviridae) type B coronavirus genus (Betacoronavirus) Sarbecovirus.
  • SARS-CoV-2 usually refers to Severe Acute Respiratory Syndrome Coronavirus Type 2.
  • the full English name is Severe Acute Respiratory Syndrome Coronavirus 2.
  • SARS-CoV-2 belongs to the Coronaviridae (Coronaviridae) genus Betacoronavirus (Sarbecovirus).
  • SARS-CoV-2 is an enveloped, unsegmented positive-stranded single-stranded RNA virus. SARS-CoV-2 can cause a new type of coronavirus pneumonia (COVID-19).
  • the SARS-CoV-2 may include S protein (spike protein).
  • coronavirus S protein generally refers to the spike protein (spike protein) of the coronavirus.
  • the S protein can be combined into a trimer, which contains approximately 1300 amino acids.
  • the S protein may belong to the first class of membrane fusion protein (Class I viral fusion protein).
  • the S protein can generally contain two subunits, S1 and S2.
  • S1 mainly contains receptor binding domain (RBD), which can be responsible for recognizing cell receptors.
  • S2 contains the basic elements required for the membrane fusion process, including an intrinsic membrane fusion peptide (fusion peptide), two 7 peptide repeats (heptad repeat, HR), and a membrane proximal region rich in aromatic amino acids (membrane proximal external) region, MPER), and transmembrane (TM).
  • the S1 protein can be further divided into two domains, namely the N-terminal domain (N-terminal domain, NTD) and the C-terminal domain (C-terminal domain, CTD).
  • the S protein can determine the host range and specificity of a virus (such as the coronavirus SARS-CoV-2), and can also be an important site for host neutralization antibodies and/or a key target for vaccine design.
  • the S protein may be the S protein of SARS-CoV-2, for example, for its structure, see Daniel Wrapp, etc., Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation, Science.
  • ACE2 generally refers to angiotensin-converting enzyme II (Angiotensin-converting enzyme 2) or a functional fragment thereof.
  • the angiotensin converting enzyme II can catalyze the conversion of angiotensin I into angiotensin-(1-9) or angiotensin II into an exopeptidase of angiotensin-(1-7).
  • the ACE2 may include an N-terminal PD region (peptidase domain) and a C-terminal CLD region (Collectrin-like domain).
  • the angiotensin converting enzyme II may be a receptor for SARS coronavirus (SARS-CoV) or severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), for example, the extracellular domain of ACE2 ( For example, the PD region of ACE2 can bind to the RBD of the S protein of the coronavirus.
  • SARS-CoV SARS coronavirus
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus type 2
  • ACE2 the extracellular domain of ACE2
  • the accession number of human angiotensin converting enzyme II in UniProt database is Q9BYF1.
  • the human ACE2 gene can contain 18 exons, see Tipnis, SR, Hooper, NM, Hyde, R., Karran, E., Christie, G., Turner, AJA human homolog of angiotensin-converting enzyme: cloning and functional expression as a captopril-insensitive carboxypeptidase. J. Biol. Chem. 275: 33238-33243, Table 1 of 2000.
  • the functional fragment of the ACE2 protein may include a truncation or variant of the complete ACE2 protein, as long as the functional fragment still has the function of a coronavirus (such as SARS-CoV and/or SARS-CoV). -2) The function of the receptor.
  • ACE2-Fc generally refers to a fusion protein comprising the binding protein of the S protein described in this application or a functional fragment thereof and the Fc region of an IgG antibody.
  • the binding protein of the S protein or its functional fragment is directly or indirectly connected to the Fc region of the IgG antibody.
  • the Fc region of the IgG antibody may be located at the C-terminus of the ACE2-Fc fusion protein.
  • the IgG may be IgG1, for example, it may be human IgG1.
  • coronavirus generally refers to a virus belonging to the Coronavirus genus (Coronavirus) of the order Nidovirales (Coronaviridae).
  • the coronavirus is a linear single-stranded positive-stranded RNA virus.
  • the coronavirus may include an envelope with spinous processes.
  • the genome of the coronavirus may have a methylated cap structure at the 5'end and a poly(A) tail at the 3'end, and the total length of the genome is about 27-32 kb.
  • the coronaviruses include severe acute respiratory syndrome-related coronaviruses, namely Severe acute respiratory syndrome-related coronavirus, which is a species of the genus B coronavirus of the Coronavirus family.
  • the coronavirus can cause colds as well as Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS) and/or Novel Coronavirus Pneumonia (COVID-19).
  • MERS Middle East Respiratory Syndrome
  • SARS Severe Acute Respiratory Syndrome
  • COVID-19 Novel Coronavirus Pneumonia
  • the term "mutant" generally refers to a mutated amino acid sequence by deletion, insertion or substitution of one or more selected amino acids.
  • the mutant may comprise at least about 90% (for example, at least about 95%, at least about 96%, at least about 97%) compared with the amino acid sequence of the RBD of the S protein of SARS-CoV-2. , At least about 98%, at least about 99% or more) identical amino acid sequences.
  • the amino acid sequence of the RBD mutant of the S protein of SARS-CoV-2 can be found in RBD mutations from circulating SARS-CoV-2 strains enhancement of the structure stability and infectivity of the spike protein, bioRxiv, 2020.
  • COVID-19 generally refers to Coronavirus Disease 2019, which is a respiratory disease caused by the SARS-CoV-2 virus.
  • Common symptoms of COVID-19 include fever, cough, fatigue, shortness of breath, and loss of smell and taste. Some symptoms can develop into viral pneumonia, multiple organ failure or cytokine storm. The disease is mainly spread during close contact between people, for example, through small droplets produced by coughing, sneezing, and talking.
  • the World Health Organization declared the COVID-19 outbreak to be a pandemic on March 11, 2020. There is currently no vaccine or specific treatment for COVID-19 available.
  • the term "antigen-binding protein” generally refers to a protein comprising a portion that binds to an antigen, and optionally a scaffold or framework portion that allows the portion that binds to the antigen to adopt a conformation that promotes the binding of the antigen-binding protein to the antigen.
  • antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab) 2 , Fv fragments, F(ab') 2 , scFv, di-scFv and/or dAb), immunoconjugates Compounds, multispecific antibodies (such as bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • Fab antigen-binding fragments
  • Fv fragments F(ab') 2
  • scFv fragments
  • di-scFv and/or dAb immunoconjugates
  • immunoconjugates Compounds, multispecific antibodies (such as bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • Fab generally refers to a fragment containing the variable domain of the heavy chain and the variable domain of the light chain, and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab' usually refers to a fragment that is different from Fab by adding a small number of residues (including one or more cysteine from the hinge region of an antibody) to the carboxyl end of the CH1 domain of the heavy chain
  • F(ab ') 2 generally refers to Fab' dimer antibody fragments comprising two Fab fragments by a disulfide bridge at the hinge region.
  • Fv generally refers to the smallest antibody fragment that contains a complete antigen recognition and binding site.
  • the fragment may consist of a dimer in which a heavy chain variable region and a light chain variable region are tightly non-covalently bound;
  • dsFv usually refers to a disulfide bond-stabilized Fv fragment, The bond between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.
  • dAb fragment generally refers to an antibody fragment composed of a VH domain.
  • scFv generally refers to a monovalent molecule formed by covalently connecting and pairing a heavy chain variable domain and a light chain variable domain of an antibody through a flexible peptide linker; such scFv molecules may have general Structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • an “antibody” is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies comprising two light chains and two heavy chains), polyclonal antibodies , Multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies and camelized single domain antibodies.
  • An “antibody” can generally comprise a protein of at least two heavy chains (HC) and two light chains (LC) connected to each other by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region.
  • VH heavy chain variable region
  • the heavy chain constant region contains three domains, CH1, CH2, and CH3.
  • each light chain contains a light chain variable region (VL) and a light chain constant region.
  • the constant region of the light chain contains one domain, CL.
  • the VH and VL regions can be further subdivided into hyperdenatured regions, called complementarity determining regions (CDR), which alternate with more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL contains three CDRs and four framework regions (FR), which are arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • variable domains of the natural heavy and light chains each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4) Most of them adopt the ⁇ -sheet configuration, connected by three CDRs to form a loop connection, and in some cases form part of the ⁇ -sheet structure.
  • the CDRs in each chain are closely brought together by the FR region, and together with the CDRs from the other chain, form the antigen binding site of the antibody.
  • the constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (for example, effector cells) and the first component (Clq) of the classical complement system.
  • variable generally refers to the fact that certain parts of the sequence of the variable domain of an antibody change strongly, which forms the binding and specificity of various specific antibodies to their specific antigens.
  • variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the light chain and heavy chain variable regions, which are called complementarity determining regions (CDR) or hypervariable regions (HVR).
  • CDR complementarity determining regions
  • HVR hypervariable regions
  • the more highly conserved parts of variable domains are called the framework (FR).
  • the CDR of an antibody can be defined by a variety of methods, such as the Kabat definition rule based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Bethe Star, Maryland (1991)), Chothia definition rules based on the location of structural loop regions (see, A1-Lazikani et al., JMol Biol 273:927-48, 1997) and concepts based on IMGT-ONTOLOGY (IMGT-ONTOLOGY) And the KABAT definition rule of IMGT Scientific chart rule.
  • IMGT refers to the International ImmunoGeneTics Information System, a global reference database for immunogenetics and immunoinformatics (http://www.imgt.org). IMGT specializes in immunoglobulin (IG) or antibodies from humans and other vertebrates, T cell receptor (TR), major histocompatibility (MH), and immunoglobulin superfamily from vertebrates and invertebrates (IgSF), MH superfamily (MhSF) and immune system related protein (RPI).
  • IG immunoglobulin
  • TR T cell receptor
  • MH major histocompatibility
  • IgSF immunoglobulin superfamily from vertebrates and invertebrates
  • MhSF MH superfamily
  • RPI immune system related protein
  • isolated antigen binding protein generally refers to an antigen binding protein that has been identified, separated and/or recovered from a component of its production environment (for example, natural or recombinant).
  • the pollutant components of the environment are usually substances that interfere with its research, diagnostic or therapeutic uses, and can include enzymes, hormones and other protein or non-protein solutes.
  • the isolated antigen binding protein or antibody will usually be prepared through at least one purification step.
  • the term "monoclonal antibody” generally refers to an antibody obtained from a group of substantially homogeneous antibodies, that is, the individual antibodies in the group are the same, except for a small number of natural mutations that may exist.
  • Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which usually have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen.
  • the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without being contaminated by other immunoglobulins.
  • the modifier "monoclonal” refers to the characteristics of an antibody obtained from a substantially homogeneous antibody population, and is not construed as requiring the production of the antibody by any specific method.
  • the monoclonal antibodies used in this application can be prepared in hybridoma cells, or can be prepared by recombinant DNA methods.
  • chimeric antibody generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species.
  • the variable region is derived from an antibody of an experimental animal such as a rodent ("parent antibody”), and the constant region is derived from a human antibody, so that the resulting chimeric antibody is compared with the parental (e.g., mouse-derived) antibody in a human individual The possibility of triggering an adverse immune response is reduced.
  • humanized antibody generally refers to an antibody in which some or all of the amino acids other than the CDR region of a non-human antibody (such as a mouse antibody) are replaced with corresponding amino acids derived from human immunoglobulin. In the CDR regions, small additions, deletions, insertions, substitutions or modifications of amino acids may also be allowed, as long as they still retain the ability of the antibody to bind to a specific antigen.
  • the humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region. "Humanized antibodies” retain antigen specificity similar to the original antibodies.
  • “Humanized” forms of non-human (e.g., murine) antibodies can contain, at a minimum, chimeric antibodies derived from non-human immunoglobulin sequences.
  • the CDR region residues in human immunoglobulins can be equipped with non-human species (donor antibodies) (such as mice, rats) with desired properties, affinity, and/or capabilities.
  • donor antibodies such as mice, rats
  • Rabbits or non-human primates residues in the CDR region In some cases, the FR region residues of the human immunoglobulin can be replaced with corresponding non-human residues.
  • humanized antibodies may contain amino acid modifications that are not in the recipient antibody or in the donor antibody. These modifications can be made to further improve antibody performance, such as binding affinity.
  • Fully human antibody generally refers to the antibody expressed by the human antibody-encoding gene transferred to the genetically engineered antibody gene-deficient animal. All parts of the antibody (including the variable and constant regions of the antibody) are encoded by genes of human origin. Fully human antibodies can greatly reduce the immune side effects caused by heterologous antibodies on the human body. Methods for obtaining fully human antibodies in the art include phage display technology, transgenic mouse technology, ribosome display technology, RNA-polypeptide technology, and the like.
  • binding generally refer to a measurable and reproducible interaction, such as the binding between an antigen and an antibody, which can determine the presence of a molecule
  • targets in the context of heterogeneous populations (including biological molecules).
  • an antibody binds to an epitope through its antigen binding domain, and this binding requires some complementarity between the antigen binding domain and the epitope.
  • an antibody that specifically binds a target is an antibody that binds to this target with greater affinity, avidity, easier, and/or for a greater duration than it binds to other targets.
  • epitope When an antibody binds to an epitope through its antigen binding domain more easily than it will bind to a random, unrelated epitope, the antibody is said to "specifically bind” to that antigen.
  • epitope refers to a specific atomic group (for example, sugar side chain, phosphoryl group, sulfonyl group) or amino acid that binds to an antigen-binding protein (such as an antibody) on an antigen.
  • KD and “K D” are used interchangeably, usually referring to the equilibrium dissociation constant
  • KD is the dissociation rate constant (kdis, also known as the “off-rate ) (koff)” or “kd") and the ratio of the association rate constant (kon, also known as “combination rate (kon)” or “ka”).
  • the binding rate constant (kon), the dissociation rate constant (kdis), and the equilibrium dissociation constant (K D ) can be used to express the binding affinity of an antigen-binding protein (for example, an antibody) to an antigen.
  • Methods for determining the rate constants of association and dissociation are well known in the art, including but not limited to Biofilm Interferometry (BLI), Radioimmunoassay (RIA), Equilibrium Dialysis, Surface Plasmon Resonance (SPR), Fluorescence Resonance Energy Transfer (FRET) , Co-immunoprecipitation (Co-IP) and protein chip technology. If measured under different conditions (such as salt concentration, pH), the measured affinity of a specific protein-protein interaction can be different.
  • reference antibody generally refers to an antibody with which the antigen binding protein described in this application competes for binding to an antigen (for example, the RBD of the S protein of SARS-CoV-2).
  • CDC generally refers to the process initiated by the binding of complement factor C1q to the Fc part of most IgG antibody subclasses.
  • the binding of C1q to the antibody can be caused by the defined protein-protein interaction of the binding site of the Fc part.
  • the binding sites of these Fc parts may contain amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbered according to Kabat's EU index).
  • Antibodies of IgG1, IgG2, and IgG3 subtypes can generally show complement activation including the binding of C1q and C3, while IgG4 does not activate the complement system and may not bind C1q and/or C3.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • cytotoxic effector cells eg, NK cells
  • FcR Fc receptor
  • the main cells that mediate ADCC (such as NK cells) only express Fc ⁇ RIII, while monocytes express Fc ⁇ RI, Fc ⁇ RII and Fe ⁇ RIII (see Ravetch and Kinet, Annu. Rev. Immunol.
  • in vitro and/or in vivo cytotoxicity assays can be performed to evaluate the ADCC activity of the molecule of interest.
  • in vitro ADCC assays can be performed, see U.S. Patent No. 5,500,362 or No. 5,821,337 or U.S. Patent No. 6,737,056 (Presta).
  • Useful effector cells for such assays include PBMC and NK cells.
  • the ADCC activity of the molecule of interest can be assessed in vivo, for example in animal models such as those disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).
  • an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks Fc ⁇ R binding (and therefore may lack ADCC activity), but retains FcRn binding ability.
  • ADCC activity can be reduced by modifying the Fc region.
  • sites that affect binding to Fc receptors can be removed, for example, sites that are not salvage receptor binding sites are removed.
  • the Fc region can be modified to remove ADCC sites.
  • ADCC sites are known in the art. For ADCC sites of IgG1, see, for example, Sarmay et al. (1992) Molec. Immunol. 29(5):633-9.
  • the term "between” usually means that the C-terminus of a certain amino acid fragment is directly or indirectly connected to the N-terminus of the first amino acid fragment, and the N-terminus is directly or indirectly connected to the C-terminus of the second amino acid fragment. Indirect connection.
  • the N-terminus of the L-FR2 is directly or indirectly connected to the C-terminus of the LCDR1
  • the C-terminus of the L-FR2 is directly or indirectly connected to the N-terminus of the LCDR2.
  • the N-terminus of the L-FR3 is directly or indirectly connected to the C-terminus of the LCDR2
  • the C-terminus of the L-FR3 is directly or indirectly connected to the N-terminus of the LCDR3.
  • the N-terminus of the H-FR2 is directly or indirectly connected to the C-terminus of the HCDR1
  • the C-terminus of the H-FR2 is directly or indirectly connected to the N-terminus of the HCDR2.
  • the N-terminus of the H-FR3 is directly or indirectly connected to the C-terminus of the HCDR2
  • the C-terminus of the H-FR3 is directly or indirectly connected to the N-terminus of the HCDR3.
  • the "first amino acid fragment" and the "second amino acid fragment” can be any amino acid fragment that is the same or different.
  • isolated antigen binding protein generally refers to an antigen binding protein that has been identified, separated and/or recovered from a component of its production environment (for example, natural or recombinant).
  • the pollutant components of the environment are usually substances that interfere with its research, diagnostic or therapeutic uses, and can include enzymes, hormones and other protein or non-protein solutes.
  • the isolated antigen binding protein or antibody will usually be prepared through at least one purification step.
  • isolated nucleic acid molecule or isolated polynucleotide
  • isolated polynucleotide generally refers to genome, mRNA, cDNA, or synthetically derived DNA or RNA or a certain combination thereof, which is not compatible with the polynucleus found in nature. All or part of the nucleotides are associated or linked to polynucleotides to which they are not linked in nature.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells.
  • the vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of DNA or RNA transcription and/or translation.
  • the carrier also includes a carrier having a variety of the above-mentioned functions.
  • the vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the vector, the vector can produce the desired expression product.
  • the term "cell” generally refers to an individual cell, cell line, or cell line that can or already contains a plasmid or vector containing the nucleic acid molecule described in this application, or capable of expressing the antibody or antigen-binding fragment thereof described in this application.
  • the cell may include the progeny of a single host cell. Due to natural, accidental or deliberate mutations, the progeny cells and the original parent cells may not necessarily be identical in morphology or genome, but they can express the antibodies or antigen-binding fragments described in this application.
  • the cells can be obtained by transfecting cells in vitro using the vectors described in this application.
  • the cell may be a prokaryotic cell (such as Escherichia coli), or a eukaryotic cell (such as yeast cells, such as COS cells, Chinese Hamster Ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells or Myeloma cells).
  • the cell may be a mammalian cell.
  • the mammalian cell may be a CHO-K1 cell.
  • the term "recombinant cell” generally refers to a cell into which a recombinant expression vector is introduced.
  • the recombinant host cell includes not only certain specific cells, but also the progeny of these cells.
  • the term "pharmaceutically acceptable adjuvant” generally includes pharmaceutically acceptable carriers, excipients or stabilizers, which are not effective for the cells or mammals exposed to them at the dose and concentration used. Poisonous.
  • the physiologically acceptable carrier is a pH buffered aqueous solution.
  • physiologically acceptable carriers may include buffers, antioxidants, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions and/or nonionic surface activity Agent.
  • administering refers to the application of exogenous drugs, therapeutic agents, diagnostic agents or compositions to animals, humans, subjects, cells, tissues, organs, or biological fluids.
  • administering can refer to treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact between reagents and cells, contact between reagents and fluids, and contact between fluids and cells.
  • administering also mean treatment by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to humans, animals or research subjects, refers to treatment, preventive or preventive measures, research and diagnosis; including coronavirus (such as SARS-CoV-2) and humans or animals, subjects , Cells, tissues, physiological compartments or physiological fluid contact.
  • coronavirus such as SARS-CoV-2
  • subjects Cells, tissues, physiological compartments or physiological fluid contact.
  • treatment refers to the administration of an internal or external therapeutic agent, including any SARS-CoV-2 antigen binding protein and composition thereof in the present application, to a patient who has one or more disease symptoms,
  • the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • the patient is administered in an amount (therapeutically effective amount) of a therapeutic agent that is effective to alleviate one or more disease symptoms.
  • the desired effects of treatment include reducing the rate of disease progression, improving or alleviating the disease state, and regression or improved prognosis.
  • one or more symptoms related to cancer are alleviated or eliminated, including, but not limited to, reducing (or destroying) the proliferation of cancer cells, reducing symptoms derived from the disease, and improving the quality of life of individuals suffering from the disease .
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the application provides an isolated antigen binding protein, which has one or more of the following properties:
  • the value is about 5.0*10 -8 M or less (for example, it can be about 5.0*10 -8 M or less, about 2.0*10 -8 M or less, about 1.0*10 -8 M or less, about 9.0*10 -9 M or less, about 8.0 * 10 -9 M or less, about 7.0 * 10 -9 M or less, about 6.0 * 10 - 9 M or less, about 5.0 * 10 -9 M or less, about 4.0 * 10 -9 M or less, about 3.0 * 10 -9 M or less, about 2.0 * 10 -9 M or less, about 1.0 * 10 -10 M or less, about 9.0 * 10 -10 M or less, about 8.0 * 10 -10 M or less, about 7.0 * 10 - 10 M or less, about 6.0*10 -10 M or less, about 5.0*10 -10 M or less, about 4.0*10 -10 M or less, about 3.0*10 -10 M or less, about 2.0*10 -10 M or less
  • the Octet measurement it is about 6.0*10 -10 M or less (for example, it can be about 6.0*10 -10 M or less, about 5.5*10 -10 M or less, about 5.0*10 -10 M or less, about 4.5*10 -10 M or less, about 4.0*10 -10 M or less, about 3.5*10 -10 M or less, about 3.0*10 -10 M or less, about 2.5*10 -10 M or less, about 2.0*10 -10 M or less
  • the K D value of 1.5*10 -10 M or less specifically binds to the RBD mutant of the S protein of SARS-CoV-2;
  • the affinity with the RBD of SARS-CoV-2 S protein is stronger than that of human ACE2-Fc; it specifically binds to the RBD of SARS-CoV S protein;
  • the main peak of charge heterogeneity analysis is about 45%-85% (for example, it can be about 45% to about 85%, about 45% to about 84%, about 45% to about 83%, about 45% to about 81%, About 45% to about 80%, about 45% to about 79%, about 45% to about 78%, about 45% to about 77%, about 45% to about 76%, about 45% to about 75%, or about 45 %-About 74%);
  • Tm is at least about 75°C (for example, it can be at least about 75°C, at least about 76°C, at least about 77°C, at least about 78°C, at least about 79°C, at least about 80°C, at least about 81°C. °C, at least about 82 °C, at least about 83 °C, or at least about 84 °C);
  • the isolated antigen-binding protein described in this application can compete with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, so
  • the heavy chain variable region of the reference antibody may include HCDR1, HCDR2 and HCDR3,
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 6
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 7
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 1
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 2
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 3.
  • the isolated antigen-binding protein described in this application can compete with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, so
  • the heavy chain variable region of the reference antibody may include HCDR1, HCDR2, and HCDR3,
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 9
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 7
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 1
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 2
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 3.
  • the isolated antigen-binding protein described in this application can compete with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, so
  • the heavy chain variable region of the reference antibody may include HCDR1, HCDR2, and HCDR3,
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 15
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 16
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 11
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 12
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 13.
  • the isolated antigen-binding protein described in this application can compete with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, so
  • the heavy chain variable region of the reference antibody may include HCDR1, HCDR2 and HCDR3,
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 22,
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 23
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 24,
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 18, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 19, and
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO:20.
  • the isolated antigen-binding protein described in this application can compete with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, so
  • the heavy chain variable region of the reference antibody may include HCDR1, HCDR2 and HCDR3,
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 30, and the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 31,
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 32
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 26
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 27, and
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO:28.
  • the isolated antigen-binding protein described in this application can compete with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, so
  • the heavy chain variable region of the reference antibody may include HCDR1, HCDR2, and HCDR3,
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 30, and the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 38
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 39
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 34
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 35
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 36.
  • the isolated antigen-binding protein described in this application can compete with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, so
  • the heavy chain variable region of the reference antibody may include HCDR1, HCDR2 and HCDR3,
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 45
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 46
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 47
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 41
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 42
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 43.
  • the isolated antigen-binding protein described in this application can compete with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, so
  • the heavy chain variable region of the reference antibody may include HCDR1, HCDR2, and HCDR3,
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 9
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 49
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 41
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 42
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 43.
  • the isolated antigen-binding protein described in this application can compete with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, so
  • the heavy chain variable region of the reference antibody may include HCDR1, HCDR2, and HCDR3,
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 51
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 52
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 53
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 41
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 42
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 43.
  • the isolated antigen-binding protein described in this application can compete with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, so
  • the heavy chain variable region of the reference antibody may include HCDR1, HCDR2 and HCDR3,
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 55
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 57
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 41
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 42
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 43.
  • the isolated antigen-binding protein described in this application can compete with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, so
  • the heavy chain variable region of the reference antibody may include HCDR1, HCDR2, and HCDR3,
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 63
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 64
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 65
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 59
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 60
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 61.
  • the isolated antigen-binding protein described in this application can compete with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, so
  • the heavy chain variable region of the reference antibody may include HCDR1, HCDR2 and HCDR3, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 67,
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 68
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 41
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 42, and
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 43.
  • the isolated antigen-binding protein described in this application can compete with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, so
  • the heavy chain variable region of the reference antibody may include HCDR1, HCDR2 and HCDR3,
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 55
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 70
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 41
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 42
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 43.
  • the isolated antigen-binding protein described in this application can compete with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, so
  • the heavy chain variable region of the reference antibody may include HCDR1, HCDR2 and HCDR3,
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 55
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 72
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 41
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 42
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 43.
  • the isolated antigen binding protein may include at least one CDR in the light chain variable region VL, and the VL may include the amino acid sequence shown in SEQ ID NO: 124 or SEQ ID NO: 125.
  • the isolated antigen binding protein may include at least one CDR in the heavy chain variable region VH, and the VH may include SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 17, and SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID NO : 69, the amino acid sequence shown in any one of SEQ ID NO: 71 and SEQ ID NO: 73.
  • the isolated antigen binding protein may comprise an antibody or antigen binding fragment thereof.
  • the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and/or dAb.
  • the VL may include LCDR1, LCDR2, and LCDR3, and the LCDR3 may include the amino acid sequence shown in any one of SEQ ID NO: 122 to SEQ ID NO: 123.
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 122: QQSYSTPX 8 X 9 X 10 , wherein X 8 is Pro or Ser, X 9 is Ile or Thr, and X 10 is Thr or absent.
  • the sequence may be a sequence determined according to KABAT definition rules.
  • the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 123; QQYX 4 X 5 X 6 PX 8 T, wherein X 4 is Asp or Gly, X 5 is Asn or Ser, and X 6 is Leu or Ser, X 8 is Ile, Leu, Gln or Val.
  • the sequence may be a sequence determined according to KABAT definition rules.
  • the LCDR3 may include any of SEQ ID NO: 3, SEQ ID NO: 13, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 36, SEQ ID NO: 43, and SEQ ID NO: 61 The amino acid sequence shown in one item.
  • the LCDR1 may include the amino acid sequence shown in any one of SEQ ID NO: 118-SEQ ID NO: 119.
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 118: RASQX 5 ISX 8 X 9 LX 11 , wherein X 5 is Gly or Ser, X 8 is Asn or Ser, and X 9 is Ser or Tyr , X 11 is Ala or Asn.
  • the sequence may be a sequence determined according to KABAT definition rules.
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 119; X 1 ASQX 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 , wherein X 1 is Gln or Arg, and X 5 is Asp, Gly or Ser, X 6 is Ile or Val, X 7 is Asn or Ser, X 8 is Gly, Asn or Ser, X 9 is Ser, Trp or Tyr, X 10 is Leu or Tyr, X 11 is Ala, Leu or Asn, X 12 is Ala or does not exist.
  • the sequence may be a sequence determined according to KABAT definition rules.
  • the LCDR1 may include SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 41, SEQ ID NO: 59, and SEQ ID NO: the amino acid sequence shown in any one of 111.
  • the LCDR2 may include the amino acid sequence shown in any one of SEQ ID NO: 120-SEQ ID NO: 121.
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 120: AASX 4 LX 6 S, wherein X 4 is Arg or Ser, and X 6 is Glu or Gln.
  • the sequence may be a sequence determined according to KABAT definition rules.
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 121; X 1 ASX 4 X 5 X 6 T, wherein X 1 is Ala, Asp or Gly, X 4 is Asn, Ser or Thr, X 5 is Leu or Arg, X 6 is Ala or Glu.
  • the sequence may be a sequence determined according to KABAT definition rules.
  • the LCDR2 may include SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 35, SEQ ID NO: 42, SEQ ID NO: 60, and SEQ ID NO: the amino acid sequence shown in any one of 112.
  • the VH may include HCDR1, HCDR2, and HCDR3, and the HCDR1 may include SEQ ID NO: 5, SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 45, SEQ ID NO: 51, SEQ ID The amino acid sequence shown in any one of NO: 55, SEQ ID NO: 63, and SEQ ID NO: 114.
  • the HCDR2 may include SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 38, SEQ ID NO: 46, SEQ The amino acid sequence shown in any one of ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 67, and SEQ ID NO: 115.
  • the HCDR3 may include SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 39, SEQ ID NO: 47, SEQ ID NO: 49, SEQ The amino acid sequence shown in any one of ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, and SEQ ID NO: 116.
  • the VL may include the framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
  • the C-terminus of the L-FR1 may be directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 may include the amino acid shown in any one of SEQ ID NO: 74-SEQ ID NO: 75 sequence.
  • the L-FR2 may be located between the LCDR1 and the LCDR2, and the L-FR2 may include any one of SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78.
  • the amino acid sequence may be located between the LCDR1 and the LCDR2, and the L-FR2 may include any one of SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78. The amino acid sequence.
  • the L-FR3 may be located between the LCDR2 and the LCDR3, and the L-FR3 may include SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82 The amino acid sequence shown in any one of them.
  • the N-terminus of the L-FR4 may be directly or indirectly connected to the C-terminus of the LCDR3, and the L-FR4 may include SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 and SEQ The amino acid sequence shown in any one of ID NO: 86.
  • the VL may include the amino acid sequence shown in SEQ ID NO: 124 or SEQ ID NO: 125.
  • the VL can comprise SEQ ID NO: amino acid sequence of 124: DIQMTQSPSSLSASVGDRVTITCRASQX 28 ISX 31 X 32 LX 34 WYQQKPGKAPKLLX 48 YAASX 53 LX 55 SGVPSRFSGSGSGTDX 71 TLTISSLQPEDFATYYCQQSYSTPX 96 X 97 TFGQGTX 104 X 105 EIK, wherein, X 28 is Gly or Ser, X 31 is Asn or Ser, X 32 is Ser or Tyr, X 34 is Ala or Asn, X 48 is Ile or Leu, X 53 is Arg or Ser, X 55 is Glu or Gln, X 71 is Phe Or Tyr, X 96 is Pro or Ser, X 97 is Ile or not present, X 104 is Lys or Arg, and X 105 is Leu or Val.
  • the sequence may be a sequence determined according to KABAT definition rules.
  • VL may comprise the amino acid sequence shown in SEQ ID NO: 125; X 1 IX 3 X 4 TQSPX 9 X 10 LSX 13 SX 15 GX 17 RX 19 TX 21 X 22 CX 24 ASQX 28 X 29 X 30 X 31 X 32 X 33 LX 35 WYQQKPGX 43 APX 46 LLIYX 51 ASX 54 X 55 X 56 TGX 59 PX 61 RFSGSGSGTDFTX 74 TISX 78 LX 80 PEDX 84 AX 86 YYCQQYX 93 X 94 X 95 PX 97 TFGX 101 GTX 104 X 105 EIK, of which , X 1 is Asp or Glu, X 3 is Gln or Val, X 4 is Leu or Met, X 9 is Gly or Ser, X 10 is Ser or Thr, X 13 is Ala or Leu, X 15 is Pro or Val, X 1 is Asp or
  • the VL may include SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 44, SEQ ID NO: 62, and SEQ ID NO: the amino acid sequence shown in any one of 113.
  • the isolated antigen binding protein may include an antibody light chain constant region, and the antibody light chain constant region includes a human Ig ⁇ constant region or a human Ig ⁇ constant region.
  • the gene encoding the human Ig ⁇ constant region can be shown in the GenBank accession number 50802 of the NCBI database; the gene encoding the human Ig ⁇ constant region can be shown as the GenBank accession number 3535 of the NCBI database.
  • the VH may include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
  • the C-terminus of the H-FR1 may be directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 may include SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ The amino acid sequence shown in any one of ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, and SEQ ID NO: 94.
  • the H-FR2 may be located between the HCDR1 and the HCDR2, and the H-FR2 may include SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO: 98 The amino acid sequence shown in any one of them.
  • the H-FR3 may be located between the HCDR2 and the HCDR3, and the H-FR3 may include SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102 , SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 106.
  • the N-terminus of the H-FR4 may be directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 may include SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: the amino acid sequence shown in any one of 110.
  • the VH may include SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID NO: 40, SEQ ID NO: 48, SEQ The amino acid sequence shown in any one of ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 71, and SEQ ID NO: 73.
  • the isolated antigen binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region includes a human IgG constant region.
  • the isolated antigen binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region includes a human IgG1 constant region.
  • the gene encoding the human IgG1 constant region can be as shown in the GenBank accession number 3500 of the NCBI database.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody light chain-LCDR1, LCDR2 and LCDR3, the LCDR1 may include the amino acid sequence shown in SEQ ID NO:1, and the LCDR2 may include The amino acid sequence shown in SEQ ID NO: 2, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 3.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody light chain-LCDR1, LCDR2 and LCDR3, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 11, and the LCDR2 may include The amino acid sequence shown in SEQ ID NO: 12, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 13.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody light chain-LCDR1, LCDR2 and LCDR3, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 18, and the LCDR2 may include The amino acid sequence shown in SEQ ID NO: 19, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 20.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody light chain-LCDR1, LCDR2 and LCDR3, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 26, and the LCDR2 may include The amino acid sequence shown in SEQ ID NO: 27, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 28.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody light chain-LCDR1, LCDR2, and LCDR3, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 34, and the LCDR2 may include The amino acid sequence shown in SEQ ID NO: 35, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 36.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody light chain-LCDR1, LCDR2 and LCDR3, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 41, and the LCDR2 may include The amino acid sequence shown in SEQ ID NO: 42, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 43.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody light chain-LCDR1, LCDR2 and LCDR3, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 59, and the LCDR2 may include The amino acid sequence shown in SEQ ID NO: 60, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 61.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody heavy chain—HCDR1, HCDR2, and HCDR3, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 may include The amino acid sequence shown in SEQ ID NO: 6, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 7.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody heavy chain—HCDR1, HCDR2, and HCDR3, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 may include The amino acid sequence shown in SEQ ID NO:9, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:7.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody heavy chain—HCDR1, HCDR2, and HCDR3, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 may include The amino acid sequence shown in SEQ ID NO: 15, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 16.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody heavy chain—HCDR1, HCDR2, and HCDR3, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 22, and the HCDR2 may include The amino acid sequence shown in SEQ ID NO: 23, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 24.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody heavy chain—HCDR1, HCDR2, and HCDR3, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 30, and the HCDR2 may include The amino acid sequence shown in SEQ ID NO: 31, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 32.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody heavy chain—HCDR1, HCDR2, and HCDR3, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 30, and the HCDR2 may include The amino acid sequence shown in SEQ ID NO: 38, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 39.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody heavy chain—HCDR1, HCDR2, and HCDR3, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 45, and the HCDR2 may include The amino acid sequence shown in SEQ ID NO: 46, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 47.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody heavy chain—HCDR1, HCDR2, and HCDR3, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 may include The amino acid sequence shown in SEQ ID NO:9, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:49.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody heavy chain—HCDR1, HCDR2, and HCDR3, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 51, and the HCDR2 may include The amino acid sequence shown in SEQ ID NO: 52, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 53.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody heavy chain—HCDR1, HCDR2, and HCDR3, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 55, and the HCDR2 may include The amino acid sequence shown in SEQ ID NO:56, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:57.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody heavy chain—HCDR1, HCDR2 and HCDR3, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 63, and the HCDR2 may include The amino acid sequence shown in SEQ ID NO: 64, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 65.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody heavy chain—HCDR1, HCDR2, and HCDR3, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 may include The amino acid sequence shown in SEQ ID NO: 67, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 68.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody heavy chain—HCDR1, HCDR2, and HCDR3, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 55, and the HCDR2 may include The amino acid sequence shown in SEQ ID NO:56, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:70.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody heavy chain—HCDR1, HCDR2, and HCDR3, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 55, and the HCDR2 may include The amino acid sequence shown in SEQ ID NO:56, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:72.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 may include SEQ ID NO. :6, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 7, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 1, and the LCDR2 may include SEQ ID NO: The amino acid sequence shown in 2, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 3.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 may include SEQ ID NO. :9, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 7, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 1, and the LCDR2 may include SEQ ID NO: The amino acid sequence shown in 2, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 3.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 may include SEQ ID NO.
  • the amino acid sequence shown in: 15 and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 16
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 11
  • the LCDR2 may include SEQ ID NO:
  • the amino acid sequence shown in 12 and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 13.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 22, and the HCDR2 may include SEQ ID NO.
  • the amino acid sequence shown in: 23, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 24, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 18, and the LCDR2 may include SEQ ID NO:
  • the amino acid sequence shown in 19, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 20.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 30, and the HCDR2 may include SEQ ID NO.
  • the amino acid sequence shown in: 31, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 32
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 26
  • the LCDR2 may include SEQ ID NO:
  • the amino acid sequence shown in 27, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 28.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 30, and the HCDR2 may include SEQ ID NO.
  • the amino acid sequence shown in: 38, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 39
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 34
  • the LCDR2 may include SEQ ID NO:
  • the amino acid sequence shown in 35, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 36.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 45
  • the HCDR2 may include SEQ ID NO.
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 47
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 41
  • the LCDR2 may include SEQ ID NO:
  • the amino acid sequence shown in 42, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 43.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 may include SEQ ID NO.
  • the amino acid sequence shown in: 9 and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 49
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 41
  • the LCDR2 may include SEQ ID NO:
  • the amino acid sequence shown in 42, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 43.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 51
  • the HCDR2 may include SEQ ID NO.
  • the amino acid sequence shown in SEQ ID NO: 52, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 53
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 41
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO:
  • the amino acid sequence shown in 42, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 43.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 55
  • the HCDR2 may include SEQ ID NO.
  • the amino acid sequence shown in SEQ ID NO: 57, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 57
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 41
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO:
  • the amino acid sequence shown in 42, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 43.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 63
  • the HCDR2 may include SEQ ID NO.
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 65
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 59
  • the LCDR2 may include SEQ ID NO:
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 61.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 may include SEQ ID NO. : 67, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 68, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 41, and the LCDR2 may include SEQ ID NO: The amino acid sequence shown in 42, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 43.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 55
  • the HCDR2 may include SEQ ID NO.
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 70
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 41
  • the LCDR2 may include SEQ ID NO:
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 43.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 55
  • the HCDR2 may include SEQ ID NO.
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 72
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 41
  • the LCDR2 may include SEQ ID NO:
  • the amino acid sequence shown in 42, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 43.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 8, and the VL may include SEQ ID NO : The amino acid sequence shown in 4.
  • the isolated antigen binding protein can be called Ab2001.02.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 10, and the VL may include SEQ ID NO : The amino acid sequence shown in 4.
  • the isolated antigen binding protein can be called Ab2001.03.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 17, and the VL may include SEQ ID NO : The amino acid sequence shown in 14.
  • the isolated antigen binding protein can be called Ab2001.04.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 25, and the VL may include SEQ ID NO : The amino acid sequence shown in 21.
  • the isolated antigen binding protein can be called Ab2001.08.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 33, and the VL may include SEQ ID NO : The amino acid sequence shown in 29.
  • the isolated antigen binding protein can be called Ab2001.09.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 40, and the VL may include SEQ ID NO : The amino acid sequence shown in 37.
  • the isolated antigen binding protein can be called Ab2001.10.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 48, and the VL may include SEQ ID NO : The amino acid sequence shown in 44.
  • the isolated antigen binding protein can be called Ab2001.11.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 50, and the VL may include SEQ ID NO : The amino acid sequence shown in 44.
  • the isolated antigen binding protein can be called Ab2001.12.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 54, and the VL may include SEQ ID NO : The amino acid sequence shown in 44.
  • the isolated antigen binding protein can be called Ab2001.13.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 58, and the VL may include SEQ ID NO : The amino acid sequence shown in 44.
  • the isolated antigen binding protein can be called Ab2001.14.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 66, and the VL may include SEQ ID NO : The amino acid sequence shown in 62.
  • the isolated antigen binding protein can be called Ab2001.19.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 69, and the VL may include SEQ ID NO : The amino acid sequence shown in 44.
  • the isolated antigen binding protein can be called Ab2001.23.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 71, and the VL may include SEQ ID NO : The amino acid sequence shown in 44.
  • the isolated antigen binding protein can be called Ab2001.24.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 73, and the VL may include SEQ ID NO : The amino acid sequence shown in 44.
  • the isolated antigen binding protein can be called Ab2001.25.
  • the VL of antibodies Ab2001.2, Ab2001.3, Ab2001.11, Ab2001.12, Ab2001.13, Ab2001.14, Ab2001.23, Ab2001.24, and Ab2001.25 may include SEQ ID NO: 124 The amino acid sequence shown.
  • the VL of antibodies Ab2001.4, Ab2001.8, Ab2001.9, Ab2001.10, and Ab2001.19 may include the amino acid sequence shown in SEQ ID NO: 125.
  • the antigen binding protein described in this application can specifically bind to the RBD of the S protein of SARS-CoV-2.
  • the antigen binding protein (eg, antibody) that "specifically binds" SARS-CoV-2 antigen eg, the RBD of SARS-CoV-2 S protein
  • SARS-CoV-2 antigen eg, the RBD of SARS-CoV-2 S protein
  • an antigen binding protein binds to SARS-CoV-2 antigen (e.g., the RBD of the S protein of SARS-CoV-2)
  • SARS-CoV-2 antigen e.g., the RBD of the S protein of SARS-CoV-2
  • assays known in the art to determine binding affinity include surface plasmon resonance (e.g., BIACORE) or similar techniques (e.g., KinExa or OCTET).
  • the SARS-CoV-2 antibody described in this application can also cross-react with SARS-CoV. For example, it can be detected by flow cytometry technology and enzyme-linked immunoassay.
  • cross-reactivity refers to the ability of an antibody to react with homologous proteins from other species.
  • the antigen binding protein described in this application can block the binding of the RBD of the S protein of SARS-CoV-2 or its mutants with human ACE2.
  • the blocking experiment can be detected by a competition method, for example, the antigen binding protein (for example, SARS-CoV-2 antibody) is combined with the antigen (or, the cell that can express the antigen) and the ligand of the antigen (or, the expression ligand). Somatic cells) are mixed to reflect the ability of the antigen-binding protein to competitively bind the antigen with the ligand of the antigen based on the intensity (for example, fluorescence intensity or concentration) of the detectable label.
  • the antigen binding protein for example, SARS-CoV-2 antibody
  • the present application provides a bispecific antigen binding protein that specifically binds to the RBD of the S protein of SARS-CoV-2 and the RBD of the S protein of SARS-CoV.
  • the bispecific antigen binding protein may comprise a first targeting moiety that specifically binds to the RBD of the S protein of SARS-CoV-2, wherein the first targeting moiety may comprise the first targeting moiety described in this application Isolated antigen binding protein.
  • the bispecific antigen binding protein may include a second targeting moiety that specifically binds to the RBD of the S protein of SARS-CoV, wherein the second targeting moiety may include the isolated Antigen binding protein.
  • the antibodies Ab2001.2, Ab2001.3, Ab2001.11, Ab2001.14, Ab2001.24, and Ab2001.25 described in this application, as well as the antigen-binding fragments thereof may be VL, VH
  • the antigen-binding fragments thereof may be specific sexually binds the RBD of the S protein of SARS-CoV.
  • the bispecific antigen binding protein may include an antibody.
  • the first targeting moiety may include a first heavy chain and a first light chain
  • the second targeting moiety may include a second heavy chain and a second light chain, wherein the first light chain and the The second light chain can be the same.
  • the first light chain and the second light chain may comprise the amino acid sequence shown in SEQ ID NO:44.
  • the VH of the first heavy chain may include the amino acid sequence shown in SEQ ID NO: 48.
  • the VH of the second heavy chain may include the amino acid sequence shown in SEQ ID NO:50.
  • the VH of the first heavy chain may include the amino acid sequence shown in SEQ ID NO: 48
  • the VH of the second heavy chain may include the amino acid sequence shown in SEQ ID NO: 50.
  • the bispecific antigen binding protein may be the antibody Ab2001.16, wherein the first light chain and the second light chain may comprise the amino acid sequence shown in SEQ ID NO: 44;
  • the VH of one heavy chain may include the amino acid sequence shown in SEQ ID NO: 48, and the VH of the second heavy chain may include the amino acid sequence shown in SEQ ID NO: 50.
  • the protein, polypeptide, and/or amino acid sequence involved in this application should also be understood to include at least the following range: a variant or homologue that has the same or similar functions as the protein or polypeptide.
  • the variant may be one or more amino acids in the amino acid sequence of the protein and/or the polypeptide (for example, the antigen binding protein described in the present application).
  • Protein or peptide may contain at least one, such as 1-30, 1-20, or 1-10, and another example, 1, 2, 3, 4, or 5 amino acid substitutions.
  • the functional variant may substantially maintain the biological properties of the protein or the polypeptide before the change (e.g., substitution, deletion or addition).
  • the functional variant can maintain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding ability) of the protein or polypeptide before the change.
  • the substitution may be a conservative substitution.
  • a part of the amino acid sequence of the antigen binding protein may be homologous to the corresponding amino acid sequence in an antibody from a specific species, or belong to a specific category.
  • both the variable region and constant part of an antibody can be derived from the variable region and constant region of an antibody of an animal species (such as human).
  • the homologue may be at least about 85% (for example, having at least about 85% of the amino acid sequence of the protein and/or the polypeptide (for example, the antigen binding protein described in this application) %, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology Protein or peptide.
  • the homology generally refers to the similarity, similarity or association between two or more sequences.
  • the "percentage of sequence homology" can be calculated in the following way: the two sequences to be aligned are compared in the comparison window, and it is determined that the same nucleic acid base (for example, A, T, C, G) or Positions of the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) To get the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (ie, the window size), and multiply the result by 100 to produce the sequence homology percentage.
  • the same nucleic acid base for example, A, T, C, G
  • Positions of the same amino acid residues e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu
  • the alignment to determine the percentage of sequence homology can be achieved in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for sequence alignment, including any algorithm required to achieve the maximum alignment within the full-length sequence being compared or within the target sequence region.
  • the homology can also be determined by the following methods: FASTA and BLAST. For a description of the FASTA algorithm, see "Improved Tools for Biological Sequence Comparison" by WRPearson and DJ Lipman, Proc. Natl. Acad.
  • this application provides an isolated one or more nucleic acid molecules that encode the isolated antigen binding protein described in this application, and/or the bispecific antigen binding protein described in this application.
  • the present application provides a vector, which may contain the nucleic acid molecule described in the present application.
  • this application provides a cell, which may contain the nucleic acid molecule described in this application or the vector described in this application.
  • the nucleic acid molecules described in this application may be isolated. For example, it can be produced or synthesized by the following methods: (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified , For example, fractionation by restriction enzyme digestion and gel electrophoresis, or (iv) synthesis, for example, by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • the nucleic acid encoding the antibody and its antigen-binding fragment can be prepared by a variety of methods known in the art. These methods include, but are not limited to, the use of restriction fragment operations or the use of synthetic oligonucleotides. Overlapping extension PCR. For specific operations, please refer to Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York NY, 1993.
  • this application provides one or more vectors, which comprise one or more nucleic acid molecules described in this application.
  • Each vector may contain one or more of the nucleic acid molecules.
  • the vector may also contain other genes, such as a marker gene that allows the vector to be selected in a suitable host cell and under suitable conditions.
  • the vector may also contain expression control elements that allow the coding region to be correctly expressed in a suitable host.
  • control elements are well known to those skilled in the art. For example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation.
  • the expression control sequence is a tunable element.
  • the specific structure of the expression control sequence may vary according to the function of the species or cell type, but usually includes 5'non-transcribed sequences and 5'and 3'non-translated sequences involved in transcription and translation initiation, such as TATA box, plus Cap sequence, CAAT sequence, etc.
  • the 5' non-transcriptional expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcriptional control functionally linked to the nucleic acid.
  • the expression control sequence may also include an enhancer sequence or an upstream activator sequence.
  • suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerases, human U6 RNA promoters, CMV promoters and artificial hybrid promoters (such as CMV), wherein A certain part may be fused with a certain part of the promoter of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), and it may or may not contain additional introns.
  • One or more nucleic acid molecules described in this application can be operably linked to the expression control element.
  • the vector may include, for example, a plasmid, a cosmid, a virus, a phage, or other vectors commonly used in, for example, genetic engineering.
  • the vector is an expression vector.
  • the application provides a host cell, which may comprise one or more nucleic acid molecules described in this application and/or one or more vectors described in this application.
  • each or each host cell may contain one or one of the nucleic acid molecules or vectors described in this application.
  • each or each host cell may contain multiple (e.g., two or more) or multiple (e.g., two or more) nucleic acid molecules or vectors described in the present application.
  • the vector described in the present application can be introduced into the host cell, such as a eukaryotic cell, such as a plant-derived cell, fungus, or yeast cell.
  • the vector described in the present application can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like.
  • the present application provides a method for preparing the isolated antigen binding protein described in this application and the bispecific antigen binding protein described in this application, and the method includes making the isolated antigen binding protein described in this application bind Culture the cells described in this application under the condition of expression of the protein and/or the bispecific antigen binding protein described in this application.
  • the method may include culturing the host cell described in the present application under conditions that allow the expression of the antigen binding protein. For example, it is possible to use an appropriate culture medium, an appropriate temperature, a culture time, etc., and these methods are understood by those of ordinary skill in the art.
  • Any method suitable for producing monoclonal antibodies can be used to produce the antigen binding protein of the present application (for example, SARS-CoV-2 antibody).
  • SARS-CoV-2 any suitable form of SARS-CoV-2 (such as the RBD of the S protein of SARS-CoV-2) can be used as an immunogen (antigen) to produce antibodies specific to SARS-CoV-2, and to screen the organisms of the antibodies Study activity.
  • the immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art.
  • Suitable genetic vectors can be used to express DNA encoding immunogens, including but not limited to adenovirus vectors, adeno-associated virus vectors, baculovirus vectors, materials, and non-viral vectors.
  • the humanized antibody can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE.
  • the antibody is an IgG antibody, and the IgG1 subtype is used.
  • any type of light chain can be used in the compounds and methods herein. For example, kappa, lambda chains or variants thereof are applicable in this application.
  • sequence of the DNA molecule of the antigen-binding protein or its fragments of the present application can be obtained by conventional techniques, such as PCR amplification or genomic library screening.
  • the coding sequences of the light chain and the heavy chain can be fused together to form a single chain antibody.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences. The nucleic acid molecule can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • This application also relates to vectors containing the above-mentioned appropriate nucleic acid molecules and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • animal cells may include: CHO-S, CHO-K1 and/or HEK-293 cells.
  • the steps of transforming host cells with recombinant DNA described in this application can be performed by techniques well known in the art.
  • the obtained transformant can be cultured by conventional methods, and the transformant expresses the polypeptide encoded by the nucleic acid molecule of the present application.
  • the host cell used it is cultured in a conventional medium under suitable conditions.
  • the transformed host cells are cultured under conditions suitable for the expression of the antigen binding protein of the present application.
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc.
  • the antigen-binding protein of the present application is purified by conventional separation and purification methods well-known to the person.
  • the obtained monoclonal antibody can be identified by conventional means.
  • the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays (such as flow cytometry (FACS), radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA)).
  • FACS flow cytometry
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • this application provides a pharmaceutical composition, which may comprise the isolated antigen binding protein described in this application, the bispecific antigen binding protein described in this application, the nucleic acid molecule described in this application, and the nucleic acid molecule described in this application.
  • the pharmaceutical composition described in the present application can be directly used for the RBD that binds to the S protein of SARS-CoV-2, and thus can be used for the prevention and treatment of diseases related to coronavirus infection (for example, COVID-19).
  • diseases related to coronavirus infection for example, COVID-19
  • other therapeutic agents can also be used at the same time.
  • the pharmaceutical composition of the present application may contain a safe and effective amount (such as 0.001-99 wt%, 0.01-90 wt%, or 0.1-80 wt%) of the antigen binding protein described in the present application and a pharmaceutically acceptable adjuvant (which may include a carrier Or excipients).
  • a pharmaceutically acceptable adjuvant which may include a carrier Or excipients.
  • Such carriers may include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition described in this application can be prepared in the form of injections, for example, with physiological saline or an aqueous solution containing glucose and other adjuvants for preparation by conventional methods. Pharmaceutical compositions such as injections and solutions should be manufactured under aseptic conditions.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • the antigen binding protein described in this application can also be used together with other therapeutic agents
  • the antigen binding protein or pharmaceutical composition described herein can be formulated, administered, and administered in a manner consistent with good medical practice.
  • the considerations in this situation include the specific condition being treated, the specific mammal being treated, the clinical condition of a single patient, the cause of the condition, the location of drug delivery, the method of administration, and other factors known to medical practitioners.
  • the therapeutic agent e.g., SARS-CoV-2 antibody
  • the effective amount of such other agents depends on the amount of therapeutic agent (e.g., SARS-CoV-2 antibody) present in the formulation, the type of disorder or treatment, and other factors discussed above.
  • These agents can generally be used in any dose empirically/clinically determined to be appropriate and through any route empirically/clinically determined to be appropriate. Compared with a single treatment, the dose of the antibody administered in the combination treatment can be reduced. It is easy to monitor the progress of this therapy by conventional techniques.
  • this application provides an isolated antigen binding protein described in this application, a bispecific antigen binding protein described in this application, a nucleic acid molecule described in this application, a vector described in this application, and a carrier described in this application.
  • This application provides a method for preventing, alleviating and/or treating coronavirus infection, which comprises administering the isolated antigen binding protein described in this application, the bispecific antigen binding protein described in this application to a subject in need
  • the protein, the nucleic acid molecule described in this application, the vector described in this application, the cell described in this application, and/or the pharmaceutical composition described in this application comprises administering the isolated antigen binding protein described in this application, the bispecific antigen binding protein described in this application to a subject in need.
  • This application provides isolated antigen binding proteins, bispecific antigen binding proteins described in this application, nucleic acid molecules described in this application, vectors described in this application, cells described in this application, and/or described in this application
  • the pharmaceutical composition can prevent, alleviate and/or treat coronavirus infection.
  • the coronavirus infection may include COVID-19.
  • the coronavirus infection may also include SARS.
  • the present application provides a method for blocking the binding of the RBD of the S protein of SARS-CoV-2 or its mutants to human ACE2, which includes the following steps, administering the isolated antigen binding protein described in the present application , The bispecific antigen binding protein described in this application, the nucleic acid molecule described in this application, the vector described in this application, the cell described in this application, and/or the pharmaceutical composition described in this application.
  • the present application provides a method for blocking the binding of the RBD of the S protein of SARS-CoV to human ACE2, which includes the following steps: administering the isolated antigen binding protein described in this application, The bispecific antigen binding protein, the nucleic acid molecule described in this application, the vector described in this application, the cell described in this application, and/or the pharmaceutical composition described in this application.
  • the antigen-binding protein of the present application can be used in detection applications, for example, to detect samples, so as to provide diagnostic information.
  • the antibodies and/or methods described in this application can be used to treat subjects (such as patients suspected of being infected by SARS-CoV-2 or SARS-CoV, or patients who have been infected by SARS-CoV-2 or SARS-CoV).
  • Specimens for example, throat swab test samples, such as serum, whole blood, sputum, oral/nasopharyngeal secretions or lotions, urine, feces, pleural effusion, cerebrospinal fluid and tissue specimens
  • Specimens for example, throat swab test samples, such as serum, whole blood, sputum, oral/nasopharyngeal secretions or lotions, urine, feces, pleural effusion, cerebrospinal fluid and tissue specimens
  • Curative effect observation indicators and indicators of whether they are infectious and whether they need to be isolated for example, the antibodies and/or methods described in this application can provide a monitoring program for therapeutic intervention.
  • samples include cells, tissue samples and biopsy specimens.
  • biopsy used in this application shall include all types of biopsy known to those skilled in the art. Therefore, the biopsy used in this application may include, for example, tissue samples prepared by endoscopic methods or organ puncture or needle biopsy.
  • the sample may include a fixed or preserved cell or tissue sample.
  • the application also provides a kit containing the antigen binding protein of the application.
  • the kit may also include a container, instructions for use, buffers, and the like.
  • the original binding protein of the present application can be immobilized on a detection plate.
  • a common light chain phage library and a single chain phage library were selected, and two rounds of panning were performed against SARS-CoV-2 S1 RBD (sino biological catalog number: 40592-V05H) to obtain positive enrichment.
  • primers were designed for polymerase chain reaction (PCR) to amplify single-chain antibody (scFv); the scFv gene fragment amplified by PCR was recovered and transferred to the yeast display plasmid into the Saccharomyces cerevisiae strain.
  • Yeast display single chain antibody library and common light chain antibody library are named JYYDL056 and JYYDL057, respectively.
  • the libraries JYYDL056 and JYYDL057 were cultured overnight in 100 mL SD-Trp medium (Clontech, catalog number: 630308) at 30°C and 225 revolutions per minute; each 1.0 ⁇ 10 8 bacteria was resuspended in 20 mL YPGP liquid medium (2 % Galactose, 2% peptone, 1% yeast extract, 0.54% Na 2 HPO 4 , 0.86% NaH 2 PO 4 ⁇ H 2 O), 20°C, 225 revolutions/min, incubate for 24 hours, place in a 4°C refrigerator stand-by.
  • the OD 600 of the bacterial solution was measured, and the number of cells was calculated based on 1OD of 1.0 ⁇ 10 7 cells.
  • Each 4.0 ⁇ 10 7 cells were taken, and the flow staining sorting was carried out according to the following steps: 1. Use 2mL 1 ⁇ PBSA(1 ⁇ PBS+1%BSA) wash once, centrifuge at 3000 rpm for 3 minutes (the following centrifugation is under this condition) discard the supernatant; 2. Each tube contains 100nM SARS-CoV-2 S1 RBD mFc (Sino biological, catalog number) : 40592-V05H) 1 ⁇ PBSA, incubate at room temperature for 30 minutes; 3.
  • step 5 Repeat step 5 and add 2mL 1 ⁇ Resuspend the cells in PBSA and collect the cell populations with strong Alexa Fluor 647 signal and weak PE signal for flow sorting.
  • the cells were cultured overnight in 5mL SD-Trp liquid medium at 30°C, 225 rpm; add 1 mL of bacterial solution to 4mL YPGP liquid medium, culture at 20°C, 225 rpm for 24 hours, and place in a 4°C refrigerator stand-by.
  • the SARS-CoV-2 S1 RBD mFc concentration was reduced to 50nM, and the second round of sorting was performed.
  • the cells were spread on SD-Trp solid medium (Clontech, catalog number: 630309), 30 Cultivation at °C for 3 days.
  • the second round of screening products of JYYDL056 and JYYDL057, 400 monoclonals were selected for sequencing, and finally 34 unique single-chain antibody sequences were obtained from the second round of screening products of JYYDL056 library; 42 unique single-chain antibody sequences were obtained from the second round of screening products of JYYDL057 library Common light chain antibody sequence.
  • the corresponding yeast monoclonal colonies were analyzed by flow cytometry, referring to the staining procedure in Example 2.1, and each 1 ⁇ 10 6 cells were stained according to the following scheme (Table 1).
  • Option 2 can evaluate the binding and blocking ability of antibodies to SARS S1;
  • the MFI values of PE and Alexa Fluor 647 in Scheme 3 reflect the display level and non-specific binding level of single-chain antibodies, respectively.
  • the GUAVA microcapillary cell analysis platform was used for flow cytometric analysis. According to the experimental results, select the strong binding to SARS-CoV-2-RBD (high MFI value of PE fluorescence signal) and block the strong binding of SARS-CoV-2 RBD to hACE2-Fc (Alexa Fluor 647 MFI) according to the experimental results. Value is low) clones; at the same time, the clone sequences with high binding signal to irrelevant antigens in Scheme 3 are excluded.
  • the co-light chain monoclonals (Y28B4 and Y34G5) were selected, and the common light chain bispecific antibody Ab2001.16 was constructed through the Knob and Hole mutations in the Fc region (for mutation modification see US5731168A), as shown in Table 3. A total of 16 antibodies will be handed over to a contracted outsourcing company for mammalian cell expression, and a small amount of antibody samples will be prepared.
  • the candidate antibody sequence is coded by Kabat numbering and the CDR regions are divided. Taizhou Baiying Biotechnology Co., Ltd. is entrusted to carry out plasmid construction and antibody expression and purification.
  • the antibody sequence and expression information are shown in Table 4.
  • Each cycle includes the following steps: 1) sensor regeneration; 2) immersion in buffer (PBST, 50mL PBS with 10 ⁇ L Tween 20) for 60 seconds; 3) 5 ⁇ g/mL fully human antibody immobilized on the AHC sensor for 40 Seconds; 4) The sensor is immersed in the buffer for 180 seconds; 5) 100nM SARS-CoV-2 S1 binds to the antibody, time is 180 seconds; 6) Antigen and antibody dissociation, time is 10 minutes. The results of affinity were analyzed by Octet Data Analysis Software (Fortébio), and the results are shown in Table 6.
  • biotinylated SARS-CoV-2 S1 Fc with blocking solution (Kaiqi Biology, item number: COV-VM5S1) to a final concentration of 0.2 ⁇ g/mL; then dilute the antibody with blocking solution, and the initial concentration of antibody is 15 ⁇ g /mL, 5-fold gradient dilution (dilution of 7 concentration points + 1 concentration point of 0 ⁇ g/mL), and then incubate the anti-SARS-CoV-2 antibody and biotinylated SARS-CoV-2 S1 Fc at 37°C 1 Hour. 3.
  • step 1 Take out the ELISA plate sealed in step 1, wash the plate 3 times with washing solution, add the incubation solution in step 2 at 100 ⁇ l/well, and incubate at 37°C for 1 hour. 4. Wash the plate 3 times with washing solution, dilute SA-HRP (sigma, S2438-250UG) with a volume ratio of 1:5000 in the blocking solution, add 100 ⁇ l/well to the microtiter plate, and incubate at 37°C for 1 hour; 5.
  • SA-HRP Sigma, S2438-250UG
  • the candidate antibody and SARS-CoV-2 S1 antigen binding epitope adopts the In-tandem method, that is, the SARS-CoV-2 S1 antigen is immobilized on the sensor, and then it interacts with the first antibody and the second antibody in sequence to detect the second antibody.
  • the two antibodies bind the signal to determine whether the two antibodies recognize the same epitope.
  • each cycle includes the following steps: 1) The sensor is first immersed in buffer (PBST, 50mL PBS with 10 ⁇ L Tween 20) for 30s; 2) 1 ⁇ g/ml Biotin human SARS-CoV-2 RBD his (Kay) ⁇ bio, item number: COV-VM4BD) is cured on the SA sensor (Fortébio, item number: 18-0009), time is 65 seconds; 3) the sensor is immersed in buffer for 30 seconds; 4) the first antibody of 100nM is on the surface of the sensor The antigen binding time is 3 minutes to saturate the binding site of the first antibody on the antigen; 5) The second antibody of 100 nM binds to the antigen, time 3 minutes.
  • the results of epitope identification were analyzed by Octet Data Analysis Software (Fortébio), and the results are shown in Table 8.
  • the criteria for determining the results of the experiment are: 1) 60%-100%: no competition at all; 2) 20%-60%: partial competition; 3) ⁇ 20%: perfect competition.
  • Each antibody competes.
  • the data show that Ab2001.10, 12, and 13 antibodies are one binding epitope; the four antibodies of Ab2001.02, Ab2001.03, and Ab2001.11 are another binding epitope.
  • the Ab2001.10 antibody was selected to determine whether the antigen binding epitope of Ab2001.08 and Ab2001.16 were the same. The results are shown in Table 9. The results showed that Ab2001.08 and Ab2001.10 are two different antigen-binding epitopes, and the Ab2001.16 double antibody contains two different antigen-binding epitopes, Ab2001.11 and Ab2001.12.
  • the antibody number corresponding to the Y28B4 clone is Ab2001.11, and the bispecific antibody Ab2001.16 is composed of Ab2001.11 and Ab2001.12, so the candidate antibodies Ab2001.11, Ab2001.12, and Ab2001.16 are further evaluated to block SARS CoV.
  • the experimental protocol is the same as in Example 3.2.
  • the antibody concentration starts from 200 ⁇ g/mL with 5 gradient dilutions, a total of 7 concentrations, and an antibody concentration point of 0 ⁇ g/mL is added. The result is shown in Figure 3.
  • the control antibody has a strong ability to block the binding of SARS-CoV S1 and hACE2, with an IC 50 value of 6.21 nM;
  • Ab2001.11 has a weak blocking ability with an IC 50 value of 303.87 nM; none of the other antibodies Blocking ability.
  • the blocking ability of Ab2001.11 is weak, if the affinity is matured to increase the affinity, the blocking ability may be improved, and it is expected to be a broad-spectrum neutralization that blocks both SARS-CoV-2 S1 and the binding of SARS-CoV S1 to hACE2 Antibody.
  • Sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium hydroxide, guanidine hydrochloride, sodium chloride, ammonium sulfate, hydrochloric acid, glycine, sodium hydroxide, ethanol, ammonium bicarbonate were purchased from Sinopharm reagents; 1% methyl cellulose, CIEF Peptide Marker Kit, TTM solution, electrode solution, capillary cleaning solution, cIEF Cartridge FC-COATED were purchased from Protein Simple; TSKG3000SWxl (7.8 ⁇ 300mm, 5 ⁇ m,) was purchased from TOSOH; Protein Thermal Shift TM Starter Kit, Propac WCX-10 (4 ⁇ 250mm, 5 ⁇ m), MAbPacTM HIC-10 (4x250mm, 5 ⁇ m,) were purchased from Thermo.
  • the hydrophilic coefficients of the candidate antibodies are all greater than 0.5, which meets the internal druggability standards.
  • the candidate antibodies AB2001.02, AB2001.03, AB2001.09, and AB2001.10 show excellent hydrophilicity.
  • the candidate antibodies Ab2001.02, Ab2001.03, Ab2001.08, Ab2001.09, Ab2001.10, Ab2001.11, Ab2001.12, Ab2001.16 are charged heterogeneity
  • the specific steps are as follows:
  • the candidate antibodies Ab2001.02, Ab2001.03, Ab2001.08, Ab2001.10, Ab2001.11, Ab2001.12, Ab2001.16 have good physicochemical properties.
  • DMEM Thermo Fisher
  • FBS Gabco
  • Methyl Cellulose Methyl Cellulose (Millipore)
  • PBS Thermo Fisher
  • Viral RNA extraction QIAamp 96 Virus QIAcube HT Kit, Qiagen Company, CAT#57731;
  • Viral Nucleic Acid Detection (Fluorescence Quantitative PCR) Kit Developed by the Diagnostic Microbiology Group of Wuhan Institute of Virology, targeting the RBD2 gene.
  • Fluorescence quantitative PCR instrument CFX96 Touch Real-Time PCR Detection System (Bio-rad)
  • SARS-CoV-2 virus (WIV04, GenBank: MN996528.1) amplification and titer determination: P6 generation of SARS-CoV-2 virus inoculates Vero-E6 cells, in a 37°C, 5% CO 2 incubator Cultivate for 48 hours, collect the cultured virus supernatant, centrifuge and aliquot, and store in a refrigerator at -80°C. The classical plaque method was used to determine the virus titer in Vero-E6 cells.
  • Vero-E6 cells are evenly spread on a 24-well plate (10 5 cells/well), and 16 hours later, medium containing different antibodies is added (the final concentration of antibody is 10 ⁇ g/mL), with 0.005 MOI SARS-CoV-2 was infected, and the cell culture supernatant was collected after 24h and 72h for viral RNA extraction and detection.
  • the antibody to be tested was added to the DMEM medium containing 2% FBS, and the diluted antibody was added to a 96-well plate with 100 ⁇ L per well; the virus was diluted to 2000PFU/mL, and 100 ⁇ L and different concentrations of antibodies were taken respectively.
  • Mix (Final antibody concentrations are 30 ⁇ g/mL, 10 ⁇ g/mL, 3.33 ⁇ g/mL, 1.11 ⁇ g/mL, 0.37 ⁇ g/mL, 0.12 ⁇ g/mL, 0.04 ⁇ g/mL, 0.014 ⁇ g/mL, 0.005 ⁇ g/mL ), incubate at 37°C for 30 minutes, and then add the antibody and virus mixture to the cell culture. After the virus infects the cells for 96 hours, perform the classical plaque experiment to calculate the PRNT50 concentration.
  • PRNT50 is calculated using GraphPad prism software.
  • Each candidate antibody (10 ⁇ g/mL) was added to the cell culture medium and 0.005 MOI virus was added. After 24 hours, the supernatant viral RNA content was detected, Ab2001.08, Ab2001.09, Ab2001.10, Ab2001.12, Ab2001.13, Ab2001.16 And the combined use of Ab2001.08 and Ab2001.10 (5 ⁇ g each), the combination of Ab2001.11 and Ab2001.12 (5 ⁇ g each) group decreased the viral RNA content by about 2 orders of magnitude, as shown in Figure 8A; the supernatant virus was detected at 72h RNA copy, Ab2001.10, Ab2001.08 and Ab2001.10 combined group reduced by more than 2 orders of magnitude, Ab2001.08 reduced by more than 1 order of magnitude, as shown in Figure 8B; 120h observe the cell status, Ab2001.08, Ab2001.10 The three groups of cells were in good condition, and the other groups had obvious pathological changes.
  • Cure the neutralizing antibody onto the AHC sensor (Fortébio, item number: 18-0015), and determine its relationship with SARS-CoV-2 S1 RBD and its mutants (ACRO, item number: SPD-S52H4, SPD-S52H5, SPD-S52H3, SPD-S52H8) binding and dissociation rate.
  • Each cycle includes the following steps: 1) sensor regeneration; 2) immersion in buffer (PBST, 50mL PBS with 10 ⁇ L Tween 20) for 60 seconds; 3) 10 ⁇ g/mL fully human antibody immobilized on the AHC sensor for 15 Seconds; 4) The sensor is immersed in the buffer for 180 seconds; 5) 100nM SARS-CoV-2 S1 RBD binds to the antibody, time is 180 seconds; 6) Antigen and antibody dissociation, time is 5 minutes.
  • the results of affinity were analyzed by Octet Data Analysis Software (Fortébio) with 1:1 fitting analysis, and the results are shown in Figure 11.
  • the neutralizing antibody can block the binding of SARS-CoV-2 S1 RBD and its mutants to the human receptor ACE2.
  • the experimental method was carried out in accordance with Example 3.2.
  • the final concentration of SARS-CoV-2 S1 RBD and its mutants was 0.1 ⁇ g/mL, and the starting concentration of antibody and hACE2-Fc was 100 nM, with 5-fold dilution (7 concentration points) +1 concentration point of 0 ⁇ g/mL).
  • Figures 12A-12E The results are shown in Figures 12A-12E, where Figures 12A-12E respectively show that neutralizing antibodies block SARS-CoV-2 S1 RBD, SARS-CoV-2 S1 RBD V357F mutant, SARS-CoV-2 S1 RBD N354D mutant , SARS-CoV-2 S1 RBD N354D and D304Y mutants and SARS-CoV-2 S1 RBD R408I mutants binding to hACE2-Fc. From the results of Figures 12A-12E, it can be seen that the candidate antibody can block the binding of the SARS-CoV-2 S1 RBD mutant to the human receptor ACE2, and both have a stronger blocking effect than hACE2-Fc.

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Abstract

一种SARS-CoV-2抗体及其应用。所述的SARS-CoV-2抗体可阻断SARS-CoV-2的S蛋白的RBD或其突变体与人ACE2的结合；阻断SARS-CoV的S蛋白的RBD与人ACE2的结合；以极低的K D值特异性结合SARS-CoV-2的S蛋白的RBD或其突变体；与SARS-CoV-2的S蛋白的RBD的亲和力强于人ACE2-Fc；特异性结合SARS-CoV的S蛋白的RBD；具有亲水性；并且利于制备和纯化；热稳定性佳；具有中和SARS-CoV-2的活性。还提供了所述SARS-CoV-2抗体相关的制备方法和应用。

Description

SARS-CoV-2抗体及其应用 技术领域
本申请涉及生物医药领域，具体的涉及一种SARS-CoV-2抗体及其应用。
背景技术
[根据细则9.2改正08.06.2020]　
新型冠状病毒(SARS-CoV-2)潜伏期长、隐蔽性高、传染性强；成为世界卫生领域突发公共卫生事件。在疫情防治形势如此严峻的当下，仍然没有针对新型冠状病毒的特效药物出现，因此开发出用于新型冠状病毒疫情防治的特效抗体药物显得极为迫切。
新型冠状病毒为阳性RNA基因组的含包膜病毒，属于冠状病毒科Nidovirales亚目β属。目前，已有多个利用康复者特异血浆临床治疗危重病人且效果显著的案例。但毕竟血浆制品来源有限，且必须经过严格的血液生物安全性检测方能用于临床，尚未充分满足当前疫情防治的需求。
发明内容
本申请提供了一种SARS-CoV-2抗体及其应用。本申请所述的SARS-CoV-2抗体可具有下述性质中的一种或多种：阻断SARS-CoV-2的S蛋白的RBD或其突变体与人ACE2的结合；阻断SARS-CoV的S蛋白的RBD与人ACE2的结合；以极低的K D值特异性结合SARS-CoV-2的S蛋白的RBD或其突变体；与SARS-CoV-2的S蛋白的RBD的亲和力强于人ACE2-Fc；特异性结合SARS-CoV的S蛋白的RBD；具有亲水性；有利于制备和纯化；稳定性，尤其是热稳定性佳；具有中和SARS-CoV-2的活性。本申请还提供了所述SARS-CoV-2抗体相关的制备方法和应用。
一方面，本申请提供了分离的抗原结合蛋白，其具有下述性质中的一种或多种：阻断SARS-CoV-2的S蛋白的RBD或其突变体与人ACE2的结合；阻断SARS-CoV的S蛋白的RBD与人ACE2的结合；在Octet测定中，以约5.0*10 -8M以下的K D值特异性结合SARS-CoV-2的S蛋白的RBD；在Octet测定中，以约6.0*10 -10M以下的K D值特异性结合SARS- CoV-2的S蛋白的RBD的突变体；与SARS-CoV-2的S蛋白的RBD的亲和力强于人ACE2-Fc；特异性结合SARS-CoV的S蛋白的RBD；亲水性；电荷异质性分析主峰约为45％-85％；在Thermal shift测定中，Tm至少为约75℃；具有中和SARS-CoV-2的活性。
在某些实施方式中，所述分离的抗原结合蛋白包含轻链可变区VL中的至少一个CDR，所述VL包含SEQ ID NO:124或SEQ ID NO:125所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:17、SEQ ID NO:25、SEQ ID NO:33、SEQ ID NO:40、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:54、SEQ ID NO:58、SEQ ID NO:66、SEQ ID NO:69、SEQ ID NO:71和SEQ ID NO:73中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv和/或dAb。
在某些实施方式中，所述VL包含LCDR1，LCDR2和LCDR3，所述LCDR3包含SEQ ID NO:122-SEQ ID NO:123中任一项所示的氨基酸序列。
在某些实施方式中，所述LCDR3包含SEQ ID NO:3、SEQ ID NO:13、SEQ ID NO:20、SEQ ID NO:28、SEQ ID NO:36、SEQ ID NO:43和SEQ ID NO:61中任一项所示的氨基酸序列。
在某些实施方式中，所述LCDR1包含SEQ ID NO:118-SEQ ID NO:119中任一项所示的氨基酸序列。
在某些实施方式中，所述LCDR1包含SEQ ID NO:1、SEQ ID NO:11、SEQ ID NO:18、SEQ ID NO:26、SEQ ID NO:34、SEQ ID NO:41、SEQ ID NO:59和SEQ ID NO:111中任一项所示的氨基酸序列。
在某些实施方式中，所述LCDR2包含SEQ ID NO:120-SEQ ID NO:121中任一项所示的氨基酸序列。
在某些实施方式中，所述LCDR2包含SEQ ID NO:2、SEQ ID NO:12、SEQ ID NO:19、SEQ ID NO:27、SEQ ID NO:35、SEQ ID NO:42、SEQ ID NO:60和SEQ ID NO:112中任一项所示的氨基酸序列。
在某些实施方式中，所述VH包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:5、SEQ ID NO:22、SEQ ID NO:30、SEQ ID NO:45、SEQ ID NO:51、SEQ ID NO:55、SEQ ID NO:63和SEQ ID NO:114中任一项所示的氨基酸序列。
在某些实施方式中，所述HCDR2包含SEQ ID NO:6、SEQ ID NO:9、SEQ ID NO:15、 SEQ ID NO:23、SEQ ID NO:31、SEQ ID NO:38、SEQ ID NO:46、SEQ ID NO:52、SEQ ID NO:56、SEQ ID NO:64、SEQ ID NO:67和SEQ ID NO:115中任一项所示的氨基酸序列。
在某些实施方式中，所述HCDR3包含SEQ ID NO:7、SEQ ID NO:16、SEQ ID NO:24、SEQ ID NO:32、SEQ ID NO:39、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:53、SEQ ID NO:57、SEQ ID NO:65、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72和SEQ ID NO:116中任一项所示的氨基酸序列。
在某些实施方式中，所述VL包括框架区L-FR1，L-FR2，L-FR3，和L-FR4。
在某些实施方式中，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:74-SEQ ID NO:75中任一项所示的氨基酸序列。
在某些实施方式中，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:76、SEQ ID NO:77和SEQ ID NO:78中任一项所示的氨基酸序列。
在某些实施方式中，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:79、SEQ ID NO:80、SEQ ID NO:81和SEQ ID NO:82中任一项所示的氨基酸序列。
在某些实施方式中，所述L-FR4的N末端与所述LCDR3的C末端直接或间接相连，且所述L-FR4包含SEQ ID NO:83、SEQ ID NO:84、SEQ ID NO:85和SEQ ID NO:86中任一项所示的氨基酸序列。
在某些实施方式中，所述VL包含SEQ ID NO:124或SEQ ID NO:125所示的氨基酸序列。
在某些实施方式中，所述VL包含SEQ ID NO:4、SEQ ID NO:14、SEQ ID NO:21、SEQ ID NO:29、SEQ ID NO:37、SEQ ID NO:44、SEQ ID NO:62和SEQ ID NO:113中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体轻链恒定区，且所述抗体轻链恒定区包括人Igκ恒定区或人Igλ恒定区。
在某些实施方式中，所述VH包括框架区H-FR1，H-FR2，H-FR3，和H-FR4。
在某些实施方式中，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91、SEQ ID NO:92、SEQ ID NO:93和SEQ ID NO:94中任一项所示的氨基酸序列。
在某些实施方式中，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:95、SEQ ID NO:96、SEQ ID NO:97和SEQ ID NO:98中任一项所示的氨基酸序列。
在某些实施方式中，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104、SEQ ID NO:105和SEQ ID NO:106中任一项所示的氨基酸序列。
在某些实施方式中，所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO:107、SEQ ID NO:108、SEQ ID NO:109和SEQ ID NO:110中任一项所示的氨基酸序列。
在某些实施方式中，所述VH包含SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:17、SEQ ID NO:25、SEQ ID NO:33、SEQ ID NO:40、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:54、SEQ ID NO:58、SEQ ID NO:66、SEQ ID NO:69、SEQ ID NO:71和SEQ ID NO:73中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体重链恒定区，且所述抗体重链恒定区包括人IgG恒定区。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体重链恒定区，且所述抗体重链恒定区包括人IgG1恒定区。
另一方面，本申请提供一种双特异性抗原结合蛋白，其特异性结合SARS-CoV-2的S蛋白的RBD和SARS-CoV的S蛋白的RBD。
在某些实施方式中，所述双特异性抗原结合蛋白包含特异性结合SARS-CoV-2的S蛋白的RBD的第一靶向部分，其中所述第一靶向部分包含本申请所述的分离的抗原结合蛋白。
在某些实施方式中，所述双特异性抗原结合蛋白包含特异性结合SARS-CoV的S蛋白的RBD的第二靶向部分，其中所述第二靶向部分包含本申请所述的分离的抗原结合蛋白。
在某些实施方式中，所述双特异性抗原结合蛋白包括抗体。
在某些实施方式中，所述第一靶向部分包含第一重链和第一轻链，所述第二靶向部分包含第二重链和第二轻链，其中所述第一轻链和所述第二轻链相同。
在某些实施方式中，所述第一轻链和所述第二轻链包含SEQ ID NO:44所示的氨基酸序列。
在某些实施方式中，所述第一重链的VH包含SEQ ID NO:48所示的氨基酸序列。
在某些实施方式中，所述第二重链的VH包含SEQ ID NO:50所示的氨基酸序列。
在某些实施方式中，所述第一重链的VH包含SEQ ID NO:48所示的氨基酸序列，且所述第二重链的VH包含SEQ ID NO:50所示的氨基酸序列。
另一方面，本申请提供一种分离的一种或多种核酸分子，其编码本申请所述的分离的抗原结合蛋白，和/或本申请所述的双特异性抗原结合蛋白。
另一方面，本申请提供一种载体，其包含本申请所述的核酸分子。
另一方面，本申请提供一种细胞，其包含本申请所述的核酸分子或本申请所述的载体。
另一方面，本申请提供一种制备本申请所述的分离的抗原结合蛋白、本申请所述的双特异性抗原结合蛋白的方法，所述方法包括在使得本申请所述的分离的抗原结合蛋白和/或本申请所述的双特异性抗原结合蛋白表达的条件下，培养本申请所述的细胞。
另一方面，本申请提供一种药物组合物，其包含本申请所述的分离的抗原结合蛋白、本申请所述的双特异性抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的佐剂。
另一方面，本申请提供一种本申请所述的分离的抗原结合蛋白、本申请所述的双特异性抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗冠状病毒的感染。
在某些实施方式中，所述冠状病毒的感染包括COVID-19。
另一方面，本申请提供一种阻断SARS-CoV-2的S蛋白的RBD或其突变体与人ACE2的结合的方法，其包括以下的步骤，施用本申请所述的分离的抗原结合蛋白、本申请所述的双特异性抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物。
另一方面，本申请提供一种阻断SARS-CoV的S蛋白的RBD与人ACE2的结合的方法，其包括以下的步骤，施用本申请所述的分离的抗原结合蛋白、本申请所述的双特异性抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1A-1D显示的是本申请所述抗体与SARS-CoV-2 S1结合解离速率的检测结果。
图2显示的是本申请所述抗体阻断SARS-CoV-2 S1与hACE2-Fc结合的情况。
图3显示的是本申请所述抗体阻断SARS-CoV S1与hACE2结合的情况。
图4显示的是本申请所述抗体的亲水系数。
图5显示的是本申请所述抗体电荷异质性分析结果。
图6显示的是本申请所述抗体电荷异质性图谱分析的结果。
图7显示的是本申请所述抗体(Fab)熔解温度。
图8A-8B显示的是本申请所述抗体病毒中和活性实验的结果。
图9A-9B显示的是本申请所述抗体病毒中和活性实验的结果。
图10显示的是本申请所述抗体噬菌斑减少半数中和浓度测定的结果。
图11显示的是本申请所述抗体与SARS-CoV-2 S1 RBD的各突变体结合解离速率的检测结果。
图12A-12E显示的是本申请所述中和抗体阻断SARS-CoV-2 S1 RBD及其突变体与hACE2结合的曲线与IC 50值。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“SARS-CoV”通常是指SARS冠状病毒，即严重急性呼吸道综合征冠状病毒(英文全称为Severe acute respiratory syndrome coronavirus)，其属于冠状病毒科(Coronaviridae)乙型冠状病毒属(Betacoronavirus)沙贝病毒亚属(Sarbecovirus)。
在本申请中，术语“SARS-CoV-2”通常是指严重急性呼吸道综合征冠状病毒2型，英文全称为Severe Acute Respiratory Syndrome Coronavirus 2。SARS-CoV-2属于冠状病毒科(Coronaviridae)乙型冠状病毒属(Betacoronavirus)沙贝病毒亚属(Sarbecovirus)。SARS-CoV-2是一种具有包膜的、不分节段的正链单股RNA病毒。SARS-CoV-2可以引发新型冠状病毒肺炎(COVID-19)。在本申请中，所述SARS-CoV-2可以包括S蛋白(刺突蛋白，spike蛋白)。
在本申请中，术语“冠状病毒的S蛋白”通常是指冠状蛋白的刺突蛋白(spike蛋白)。所述S蛋白可以组合成三聚体，其约含有1300个氨基酸。所述S蛋白可以属于第一类膜融合蛋白(Class I viral fusion protein)。所述S蛋白通常可以含有两个亚基(subunit)，S1和S2。S1主要包含有受体结合区(receptor binding domain RBD)，其可以负责识别细胞的受体。S2含有膜融合过程所需的基本元件，包括一个内在的膜融合肽(fusion peptide)，两个7肽重复序列(heptad repeat，HR)，一个富含芳香族氨基酸的膜临近区域(membrane proximal external region，MPER)，以及跨膜区(transmembrane，TM)。S1蛋白可进一步分成两个区域(domain)，即N-端区域(N-terminal domain，NTD)和C-端区域(C-terminal domain，CTD)。S蛋白可以决定病毒(例如冠状病毒SARS-CoV-2)的宿主范围和特异性，也可以为宿主中和抗体的而重要作用位点，和/或疫苗设计的关键靶点。所述S蛋白可以为SARS-CoV-2的S蛋白，例如，其结构可以参见Daniel Wrapp等，Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation,Science。
在本申请中，术语“ACE2”通常是指血管紧张素转化酶II(Angiotensin-converting enzyme 2)或其功能片段。所述血管紧张素转化酶II可以催化血管紧张素I转化为血管紧张素-(1-9)或血管紧张素II转化为血管紧张素-(1-7)的外肽酶。所述ACE2可以包括N端的PD区(peptidase domain，肽酶结构域)和C端CLD区(Collectrin-like domain)。所述血管紧张素转化酶II可以为SARS冠状病毒(SARS-CoV)或严重急性呼吸道综合征冠状病毒2型(SARS-CoV-2)的受体，例如，所述ACE2的胞外结构域(例如，所述ACE2的PD区)可以结合冠状病毒的S蛋白的RBD。人血管紧张素转化酶II在UniProt数据库的登录号为Q9BYF1。人ACE2基因可以包含18个外显子，参见Tipnis,S.R.,Hooper,N.M.,Hyde,R.,Karran,E.,Christie,G.,Turner,A.J.A human homolog of angiotensin-converting enzyme:cloning and functional expression as a captopril-insensitive carboxypeptidase.J.Biol.Chem.275:33238-33243,2000的表1。在本申请中，所述ACE2蛋白的功能性片段可以包括所述完整ACE2蛋白的截短体或变体，只要所述功能性片段仍具备作为冠状病毒(例如SARS-CoV和/或SARS-CoV-2)受体的功能。
在本申请中，术语“ACE2-Fc”通常是指包含本申请所述S蛋白的结合蛋白或其功能性片段以及IgG抗体的Fc区域的融合蛋白。在本申请中，所述S蛋白的结合蛋白或其功能性片段与所述IgG抗体的Fc区域直接或间接连接。例如，所述IgG抗体的Fc区域可以位于所述ACE2-Fc融合蛋白的C端。在本申请中，所述IgG可以为IgG1，例如可以为人IgG1。
在本申请中，术语“冠状病毒”通常是指属于套式病毒目(Nidovirales)冠状病毒科(Coronaviridae)冠状病毒属(Coronavirus)中的病毒。所述冠状病毒为线性单股正链的RNA病毒。所述冠状病毒可以包括具有棘突的包膜。所述冠状病毒的基因组可以5’端具有甲基化的帽状结构，3’端具有poly(A)尾，基因组全长约为27-32kb。在本申请中，所述冠状病毒包括严重急性呼吸道综合征相关冠状病毒，即Severe acute respiratory syndrome-related coronavirus，其是冠状病毒科乙型冠状病毒属的一个种。在本申请中，所述冠状病毒可引起感冒以及中东呼吸综合征(MERS)、严重急性呼吸综合征(SARS)和/或新型冠状病毒肺炎(COVID-19)。
在本申请中，术语“突变体”通常是指通过一种或多种选择的氨基酸的缺失、插入或替换的突变的氨基酸序列。在本申请中，所述突变体可以包含与SARS-CoV-2的S蛋白的RBD的氨基酸序列相比，具有至少约90％(例如，至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或更多)的同一性的氨基酸序列。例如，SARS-CoV-2的S蛋白的RBD突变体的氨基酸序列可以参见RBD mutations from circulating SARS-CoV-2 strains enhance the structure stability and infectivity of the spike protein，bioRxiv，2020。
在本申请中，术语“COVID-19”通常是指冠状病毒病2019，其是由SARS-CoV-2病毒引起的呼吸道疾病。COVID-19的常见症状包括发烧，咳嗽，疲劳，呼吸急促以及气味和味觉丧失，某些症状会发展为病毒性肺炎，多器官功能衰竭或细胞因子风暴。该疾病主要在人与人之间密切接触时传播，例如可以通过咳嗽，打喷嚏和说话产生的小液滴传播。世界卫生组织于2020年3月11日宣布COVID-19的爆发是大流行病(pandemic)。目前没有针对COVID-19的可用的疫苗或特异性的治疗方法。
在本申请中，术语“抗原结合蛋白”通常是指包含结合抗原的部分的蛋白质，以及任选地允许结合抗原的部分采用促进抗原结合蛋白与抗原结合的构象的支架或骨架部分。抗原结合蛋白的实例包括但不限于抗体、抗原结合片段(Fab，Fab’，F(ab) 2，Fv片段，F(ab’) 2，scFv，di-scFv和/或dAb)、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物或融合蛋白等，只要它们显示出所需的抗原结合活性即可。
在本申请中，术语“Fab”通常是指含有重链可变结构域和轻链可变结构域的片段，并且还含有轻链的恒定结构域和重链的第一恒定结构域(CH1)；术语“Fab’”通常是指在重链CH1结构域的羧基端添加少量残基(包括一个或多个来自抗体铰链区的半胱氨酸)而不同于Fab的片段；术语“F(ab') 2”通常是指Fab’的二聚体，包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。术语“Fv”通常是指含有完整抗原识别与结合位点的最小抗体片段。在某 些情形中，该片段可以由一个重链可变区和一个轻链可变区以紧密非共价结合的二聚体组成；术语“dsFv”通常是指二硫键稳定的Fv片段，其单个轻链可变区与单个重链可变区之间的键是二硫键。术语“dAb片段”通常是指由VH结构域组成的抗体片段。在本申请中，术语“scFv”通常是指抗体的一个重链可变结构域和一个轻链可变结构域通过柔性肽连接子共价连接配对形成的单价分子；此类scFv分子可具有一般结构：NH 2-VL-连接子-VH-COOH或NH 2-VH-连接子-VL-COOH。
在本申请中，术语“抗体”其以最广泛意义使用，且具体涵盖，但不限于，单克隆抗体(包括包含两条轻链和两条重链的全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、完全人类抗体、嵌合抗体和骆驼化单结构域抗体。“抗体”通常可以包含通过二硫键互相连接的至少两条重链(HC)和两条轻链(LC)的蛋白，或其抗原结合片段。每条重链包含重链可变区(VH)和重链恒定区。在某些天然存在的IgG、IgD和IgA抗体中，重链恒定区包含三个结构域，CH1、CH2和CH3。在某些天然存在的抗体中，各轻链包含轻链可变区(VL)和轻链恒定区。轻链恒定区包含一个结构域，CL。VH和VL区可进一步细分为超变性的区域，称为互补决定区(CDR)，其与称为框架区(FR)的较保守的区域交替。各VH和VL包含三个CDR和四个框架区(FR)，从氨基端至羧基端按以下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3和FR4。天然重链和轻链的可变结构域各自包含四个FR区(H-FR1，H-FR2，H-FR3，H-FR4，L-FR1，L-FR2，L-FR3，L-FR4)，大部分采用β-折叠构型，通过三个CDRs连接，形成环连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDRs通过FR区紧密靠近在一起，并与来自另一条链的CDR一起形成抗体的抗原结合位点。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(Clq)结合。
在本申请中，术语“可变”通常是指这样的事实，即抗体的可变结构域的序列的某些部分变化强烈，它形成各种特定抗体对其特定抗原的结合和特异性。然而，变异性并非均匀地分布在抗体的整个可变区中。它集中在轻链和重链可变区中的三个区段，被称为互补决定区(CDR)或高变区(HVR)。可变域中更高度保守的部分被称为框架(FR)。在本领域中，可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))、基于结构环区域位置的Chothia定义规则(参见，A1-Lazikani等人，JMol Biol 273:927-48,1997)和基于IMGT本体论(IMGT-ONTOLOGY)的概念和IMGT Scientific图表规则的KABAT定义规则。
IMGT指国际ImMunoGeneTics信息系统，一种免疫遗传学和免疫信息学的全球参考数据库(http://www.imgt.org)。IMGT专门研究来自人类和其他脊椎动物的免疫球蛋白(IG)或抗体、T细胞受体(TR)、主要组织相容性(MH)，以及来自脊椎动物和非脊椎动物的免疫球蛋白超家族(IgSF)、MH超家族(MhSF)和免疫系统相关蛋白(RPI)。
在本申请中，术语“分离的”抗原结合蛋白通常是指已经从其产生环境(例如，天然的或重组的)的组分中识别，分离和/或回收的抗原结合蛋白。其产生环境的污染组分通常是干扰其研究、诊断或治疗用途的物质，可以包括酶、激素和其他蛋白质或非蛋白质溶质。分离的抗原结合蛋白或抗体通常将通过至少一个纯化步骤来制备。
在本申请中，术语“单克隆抗体”通常是指从一群基本上同质的抗体获得的抗体，即集群中的个别抗体是相同的，除了可能存在的少量的自然突变。单克隆抗体通常针对单个抗原位点具有高度特异性。而且，与常规多克隆抗体制剂(通常具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性之外，单克隆抗体的优点在于它们可以通过杂交瘤培养合成，不受其他免疫球蛋白污染。修饰语“单克隆”表示从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。例如，本申请使用的单克隆抗体可以在杂交瘤细胞中制备，或者可以通过重组DNA方法制备。
在本申请中，术语“嵌合抗体”通常是指其中可变区源自一个物种，而恒定区源自另一个物种的抗体。通常，可变区源自实验动物诸如啮齿动物的抗体(“亲本抗体”)，且恒定区源自人类抗体，使得所得嵌合抗体与亲本(例如小鼠来源)抗体相比，在人类个体中引发不良免疫反应的可能性降低。
在本申请中，术语“人源化抗体”通常是指非人抗体(例如小鼠抗体)的CDR区以外的部分或全部有的氨基酸被源自人免疫球蛋白的相应的氨基酸置换的抗体。在CDR区中，氨基酸的小的添加、缺失、插入、置换或修饰也可以是允许的，只要它们仍保留抗体结合特定抗原的能力。人源化抗体可任选地包含人类免疫球蛋白恒定区的至少一部分。“人源化抗体”保留类似于原始抗体的抗原特异性。非人(例如鼠)抗体的“人源化”形式可以最低限度地包含衍生自非人免疫球蛋白的序列的嵌合抗体。在某些情形中，可以将人免疫球蛋白(受体抗体)中的CDR区残基用具有所期望性质、亲和力和/或能力的非人物种(供体抗体)(诸如小鼠，大鼠，家兔或非人灵长类动物)的CDR区残基替换。在某些情形中，可以将人免疫球蛋白的FR区残基用相应的非人残基替换。此外，人源化抗体可包含在受体抗体中或在供体抗体中没有的氨基酸修饰。进行这些修饰可以是为了进一步改进抗体的性能，诸如结合亲和力。
在本申请中，术语“全人源抗体”通常是指将人类编码抗体的基因转移至基因工程改造的抗体基因缺失动物中，使动物表达的抗体。抗体所有部分(包括抗体的可变区和恒定区)均由人类来源的基因所编码。全人源抗体可以大大减少异源抗体对人体造成的免疫副反应。本领域获得全人源抗体的方法可以有噬菌体展示技术、转基因小鼠技术、核糖体展示技术和RNA-多肽技术等。
在本申请中，术语“结合”、“特异性结合”或“对…特异性的”通常是指可测量且可再现的相互作用，诸如抗原和抗体之间的结合，其可以确定在存在分子(包括生物学分子)的异质群体的情况中靶物的存在。例如，抗体通过其抗原结合域与表位结合，并且该结合需要抗原结合域和表位之间的一些互补性。例如，特异性结合靶物(其可以是表位)的抗体是以比其结合其它靶物更大的亲和力、亲合力、更容易和/或以更大的持续时间结合此靶物的抗体。当抗体相比于其将结合随机的、不相关的表位而言更容易通过其抗原结合域与表位结合时，抗体被称为“特异性结合”该抗原。“表位”是指抗原上与抗原结合蛋白(如抗体)结合的特定的原子基团(例如，糖侧链、磷酰基、磺酰基)或氨基酸。
在本申请中，术语“KD”、“K D”可互换地使用，通常是指平衡解离常数，“KD”是解离速率常数(kdis，也称为“解离率(off-rate)(koff)”或“kd”)与结合速率常数(kon，也称为“结合率(kon)”或“ka”)的比值。可使用结合速率常数(kon)、解离速率常数(kdis)和平衡解离常数(K D)表示抗原结合蛋白(例如抗体)对抗原的结合亲和力。确定结合和解离速率常数的方法为本领域熟知，包括但不限于生物膜干涉技术(BLI)、放射免疫法(RIA)、平衡透析法、表面等离子共振(SPR)、荧光共振能量迁移(FRET)、免疫共沉淀(Co-IP)以及蛋白质芯片技术。如果在不同的条件(例如盐浓度、pH)下测量，则所测得的某种特定蛋白-蛋白相互作用的亲和力可不同。
在本申请中，术语“参比抗体”通常是指本申请所述抗原结合蛋白与之竞争结合抗原(例如SARS-CoV-2的S蛋白的RBD)的抗体。
在本申请中，术语“CDC”通常是指由补体因子C1q与大多数IgG抗体亚类的Fc部分结合而起始的过程。C1q与抗体的结合可以由Fc部分的结合位点的限定的蛋白-蛋白相互作用所导致。这些Fc部分的结合位点可以包含氨基酸L234，L235，D270，N297，E318，K320，K322，P331，和P329(根据Kabat的EU索引编号)。IgG1，IgG2，和IgG3亚型的抗体通常可以显示包括C1q和C3结合的补体激活，而IgG4不激活补体系统并且可以不结合C1q和/或C3。
在本申请中，术语“ADCC”或“抗体依赖性细胞介导的细胞毒性”通常是指这样一种细 胞毒性形式，一些分泌型免疫球蛋白结合到某些细胞毒性效应细胞(例如，NK细胞、嗜中性粒细胞和巨噬细胞)上的Fc受体(FcR)上，使得这些细胞毒性效应细胞能够特异性结合携带抗原的靶细胞，随后用细胞毒素杀死靶细胞。介导ADCC的主要细胞(例如NK细胞)只表达FcγRIII，而单核细胞表达FcγRI、FcγRII和FeγRIII(可参见Ravetch and Kinet,Annu.Rev.Immunol.9:457-92(1991)第464页表3)。可以进行体外和/或体内细胞毒性测定法以评估目的分子的ADCC活性，例如，可进行体外ADCC测定法，可参见美国专利No.5,500,362或No.5,821,337或美国专利No.6,737,056(Presta)中所记载的。可用于此类测定法的效应细胞包括PBMC和NK细胞。或者/另外，可在体内评估目的分子的ADCC活性，例如在动物模型中，诸如Clynes et al.，PNAS(USA)95:652-656(1998)中所披露的。例如，可以进行Fc受体(FcR)结合测定法以确保抗体缺乏FcγR结合(因此有可能缺乏ADCC活性)，但是保留FcRn结合能力。
ADCC活性可通过修饰Fc区而减少。在某些情形中，影响与Fc受体结合的位点可被除去，例如，除去并非补救受体结合位点的位点。在某些情形中，Fc区可经修饰以除去ADCC位点。ADCC位点是本领域已知的，关于IgG1的ADCC位点，参见例如，Sarmay et al.(1992)Molec.Immunol.29(5):633-9。
在本申请中，术语“在……之间”通常是指某种氨基酸片段的C端与第一氨基酸片段的N端直接或间接连接，并且其N端与第二氨基酸片段的C端直接或间接连接。在轻链中，例如，所述L-FR2的N末端与所述LCDR1的C末端直接或间接相连，且所述L-FR2的C末端与所述LCDR2的N末端直接或间接相连。又例如，所述L-FR3的N末端与所述LCDR2的C末端直接或间接相连，且所述L-FR3的C末端与所述LCDR3的N末端直接或间接相连。在重链中，例如，所述H-FR2的N末端与所述HCDR1的C末端直接或间接相连，且所述H-FR2的C末端与所述HCDR2的N末端直接或间接相连。又例如，所述H-FR3的N末端与所述HCDR2的C末端直接或间接相连，且所述H-FR3的C末端与所述HCDR3的N末端直接或间接相连。在本申请中，“第一氨基酸片段”和“第二氨基酸片段”可以为相同或不同的任意一段氨基酸片段。
在本申请中，术语“分离的”抗原结合蛋白通常是指已经从其产生环境(例如，天然的或重组的)的组分中识别，分离和/或回收的抗原结合蛋白。其产生环境的污染组分通常是干扰其研究、诊断或治疗用途的物质，可以包括酶、激素和其他蛋白质或非蛋白质溶质。分离的抗原结合蛋白或抗体通常将通过至少一个纯化步骤来制备。
在本申请中，术语“分离的核酸分子”或“分离的多核苷酸”通产是指基因组、mRNA、 cDNA或合成来源的DNA或RNA或其一定组合，其不与在自然界中发现的多核苷酸的全部或一部分缔合，或连接至其在自然界中不连接的多核苷酸。
在本申请中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。
在本申请中，术语“细胞”通常是指可以或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述的抗体或其抗原结合片段的个体细胞、细胞系或细胞培养物。所述细胞可以包括单个宿主细胞的子代。由于天然的、意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的抗体或其抗原结合片段即可。所述细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。在某些情形中，所述细胞可以是哺乳动物细胞。例如，所述哺乳动物细胞可以是CHO-K1细胞。在本申请中，术语“重组细胞”通常是指在其中引入了重组表达载体的细胞。所述重组宿主细胞不仅包括某种特定的细胞，还包括这些细胞的后代。
在本申请中，术语“药学上可接受的佐剂”通常包括药剂学可接受的载体、赋形剂或稳定剂，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒的。通常，生理学可接受的载体是pH缓冲水溶液。生理学可接受载体的例子可包括缓冲剂，抗氧化剂，亲水性聚合物，氨基酸，单糖，二糖和其它碳水化合物，螯合剂，糖醇，成盐反荷离子和/或非离子表面活性剂。
如本文所用，术语“给予”和“处理”是指外源性药物、治疗剂、诊断剂或组合物应用于动物、人、受试者、细胞、组织、器官或生物流体。“给予”和“处理”可以指治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触、以及试剂与流体的接触、流体与细胞的接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理。“处理”当应用于人、动物或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断；包括冠状病毒(例如SARS-CoV-2)与人或动物、受试者、细胞、组织、生理区室或生理流体的接触。
如本文所用，术语“治疗”指给予患者内用或外用治疗剂，包含本申请的任何一种SARS-CoV-2抗原结合蛋白及其组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，以有效缓解一种或多种疾病症状的治疗剂的量(治疗有效量)给予患者。治疗的期望效果包括降低疾病进展速率，改善或减轻疾病状态，和消退或改善的预后。例如，若一种或多种与癌症有关的症状是减轻或消除的，包括但不限于，降低(或破坏)癌细胞增殖，减少源自疾病的症状，提高那些患有疾病的个体的生命质量，降低治疗疾病需要的其它药物的剂量，延迟疾病的进展，和/或延长个体存活，则个体得到成功“治疗”。
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
抗原结合蛋白
一方面，本申请提供了分离的抗原结合蛋白，其具有下述性质中的一种或多种：
阻断SARS-CoV-2的S蛋白的RBD或其突变体与人ACE2的结合；
阻断SARS-CoV的S蛋白的RBD与人ACE2的结合；
在Octet测定中，以约5.0*10 -8M以下(例如，可以为约5.0*10 -8M以下、约2.0*10 -8M以下、约1.0*10 -8M以下、约9.0*10 -9M以下、约8.0*10 -9M以下、约7.0*10 -9M以下、约6.0*10 - 9M以下、约5.0*10 -9M以下、约4.0*10 -9M以下、约3.0*10 -9M以下、约2.0*10 -9M以下、约1.0*10 -10M以下、约9.0*10 -10M以下、约8.0*10 -10M以下、约7.0*10 -10M以下、约6.0*10 -10M以下、约5.0*10 -10M以下、约4.0*10 -10M以下、约3.0*10 -10M以下、约2.0*10 -10M以下或约1.0*10 -10M以下)的K D值特异性结合SARS-CoV-2的S蛋白的RBD；
在Octet测定中，以约6.0*10 -10M以下(例如，可以为约6.0*10 -10M以下、约5.5*10 -10M以下、约5.0*10 -10M以下、约4.5*10 -10M以下、约4.0*10 -10M以下、约3.5*10 -10M以下、约3.0*10 -10M以下、约2.5*10 -10M以下、约2.0*10 -10M以下或约1.5*10 -10M以下)的K D值特异性结合SARS-CoV-2的S蛋白的RBD的突变体；
与SARS-CoV-2的S蛋白的RBD的亲和力强于人ACE2-Fc；特异性结合SARS-CoV的S蛋白的RBD；
具有亲水性；
电荷异质性分析主峰约为45％-85％(例如，可以为约45％-约85％、约45％-约84％、约45％-约83％、约45％-约81％、约45％-约80％、约45％-约79％、约45％-约78％、约45％-约77％、约45％-约76％、约45％-约75％或约45％-约74％)；
在Thermal shift测定中，Tm至少为约75℃(例如，可以为至少约75℃、至少约76℃、至少约77℃、至少约78℃、至少约79℃、至少约80℃、至少约81℃、至少约82℃、至少约83℃或至少约84℃)；
具有中和SARS-CoV-2的活性。
本申请所述的分离的抗原结合蛋白，能够与参比抗体竞争结合SARS-CoV-2的S蛋白的RBD，其中所述参比抗体可包含重链可变区和轻链可变区，所述参比抗体的重链可变区可以包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:6所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:7所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:1所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:2所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:3所示的氨基酸序列。
本申请所述的分离的抗原结合蛋白，能够与参比抗体竞争结合SARS-CoV-2的S蛋白的RBD，其中所述参比抗体可包含重链可变区和轻链可变区，所述参比抗体的重链可变区可以包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:7所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:1所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:2所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:3所示的氨基酸序列。
本申请所述的分离的抗原结合蛋白，能够与参比抗体竞争结合SARS-CoV-2的S蛋白的RBD，其中所述参比抗体可包含重链可变区和轻链可变区，所述参比抗体的重链可变区可以包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:15所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:16所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:11所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:12所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:13所示的氨基酸序列。
本申请所述的分离的抗原结合蛋白，能够与参比抗体竞争结合SARS-CoV-2的S蛋白的RBD，其中所述参比抗体可包含重链可变区和轻链可变区，所述参比抗体的重链可变区可以 包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:22所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:23所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:24所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:19所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:20所示的氨基酸序列。
本申请所述的分离的抗原结合蛋白，能够与参比抗体竞争结合SARS-CoV-2的S蛋白的RBD，其中所述参比抗体可包含重链可变区和轻链可变区，所述参比抗体的重链可变区可以包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:30所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:31所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:32所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:26所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:27所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:28所示的氨基酸序列。
本申请所述的分离的抗原结合蛋白，能够与参比抗体竞争结合SARS-CoV-2的S蛋白的RBD，其中所述参比抗体可包含重链可变区和轻链可变区，所述参比抗体的重链可变区可以包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:30所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:38所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:39所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:34所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:35所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:36所示的氨基酸序列。
本申请所述的分离的抗原结合蛋白，能够与参比抗体竞争结合SARS-CoV-2的S蛋白的RBD，其中所述参比抗体可包含重链可变区和轻链可变区，所述参比抗体的重链可变区可以包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:45所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:47所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:41所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:42所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:43所示的氨基酸序列。
本申请所述的分离的抗原结合蛋白，能够与参比抗体竞争结合SARS-CoV-2的S蛋白的RBD，其中所述参比抗体可包含重链可变区和轻链可变区，所述参比抗体的重链可变区可以包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:49所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:41所示的氨基酸序列，所述LCDR2可 包含SEQ ID NO:42所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:43所示的氨基酸序列。
本申请所述的分离的抗原结合蛋白，能够与参比抗体竞争结合SARS-CoV-2的S蛋白的RBD，其中所述参比抗体可包含重链可变区和轻链可变区，所述参比抗体的重链可变区可以包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:51所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:52所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:53所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:41所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:42所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:43所示的氨基酸序列。
本申请所述的分离的抗原结合蛋白，能够与参比抗体竞争结合SARS-CoV-2的S蛋白的RBD，其中所述参比抗体可包含重链可变区和轻链可变区，所述参比抗体的重链可变区可以包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:55所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:56所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:57所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:41所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:42所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:43所示的氨基酸序列。
本申请所述的分离的抗原结合蛋白，能够与参比抗体竞争结合SARS-CoV-2的S蛋白的RBD，其中所述参比抗体可包含重链可变区和轻链可变区，所述参比抗体的重链可变区可以包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:63所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:65所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:59所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:60所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:61所示的氨基酸序列。
本申请所述的分离的抗原结合蛋白，能够与参比抗体竞争结合SARS-CoV-2的S蛋白的RBD，其中所述参比抗体可包含重链可变区和轻链可变区，所述参比抗体的重链可变区可以包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:67所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:68所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:41所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:42所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:43所示的氨基酸序列。
本申请所述的分离的抗原结合蛋白，能够与参比抗体竞争结合SARS-CoV-2的S蛋白的 RBD，其中所述参比抗体可包含重链可变区和轻链可变区，所述参比抗体的重链可变区可以包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:55所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:56所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:70所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:41所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:42所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:43所示的氨基酸序列。
本申请所述的分离的抗原结合蛋白，能够与参比抗体竞争结合SARS-CoV-2的S蛋白的RBD，其中所述参比抗体可包含重链可变区和轻链可变区，所述参比抗体的重链可变区可以包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:55所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:56所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:72所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:41所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:42所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:43所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含轻链可变区VL中的至少一个CDR，所述VL可以包含SEQ ID NO:124或SEQ ID NO:125所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含重链可变区VH中的至少一个CDR，所述VH可以包含SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:17、SEQ ID NO:25、SEQ ID NO:33、SEQ ID NO:40、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:54、SEQ ID NO:58、SEQ ID NO:66、SEQ ID NO:69、SEQ ID NO:71和SEQ ID NO:73中任一项所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可以包含抗体或其抗原结合片段。
例如，所述抗原结合片段可以包括Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv和/或dAb。
在本申请中，所述VL可以包含LCDR1，LCDR2和LCDR3，所述LCDR3可以包含SEQ ID NO:122-SEQ ID NO:123中任一项所示的氨基酸序列。
例如，其中所述LCDR3可以包含SEQ ID NO:122所示的氨基酸序列：QQSYSTPX 8X 9X 10，其中，X 8为Pro或Ser，X 9为Ile或Thr，X 10为Thr或不存在。例如，该序列可以是根据KABAT定义规则确定的序列。
例如，其中所述LCDR3可以包含SEQ ID NO:123所示的氨基酸序列；QQYX 4X 5X 6PX 8T，其中，X 4为Asp或Gly，X 5为Asn或Ser，X 6为Leu或Ser，X 8为Ile、Leu、Gln或Val。例如，该序列可以是根据KABAT定义规则确定的序列。
例如，所述LCDR3可以包含SEQ ID NO:3、SEQ ID NO:13、SEQ ID NO:20、SEQ ID NO:28、SEQ ID NO:36、SEQ ID NO:43和SEQ ID NO:61中任一项所示的氨基酸序列。
例如，所述LCDR1可以包含SEQ ID NO:118-SEQ ID NO:119中任一项所示的氨基酸序列。
例如，其中所述LCDR1可以包含SEQ ID NO:118所示的氨基酸序列：RASQX 5ISX 8X 9LX 11，其中，X 5为Gly或Ser，X 8为Asn或Ser，X 9为Ser或Tyr，X 11为Ala或Asn。例如，该序列可以是根据KABAT定义规则确定的序列。
例如，其中所述LCDR1可以包含SEQ ID NO:119所示的氨基酸序列；X 1ASQX 5X 6X 7X 8X 9X 10X 11X 12，其中，X 1为Gln或Arg，X 5为Asp、Gly或Ser，X 6为Ile或Val，X 7为Asn或Ser，X 8为Gly、Asn或Ser，X 9为Ser、Trp或Tyr，X 10为Leu或Tyr，X 11为Ala、Leu或Asn，X 12为Ala或不存在。例如，该序列可以是根据KABAT定义规则确定的序列。
例如，所述LCDR1可以包含SEQ ID NO:1、SEQ ID NO:11、SEQ ID NO:18、SEQ ID NO:26、SEQ ID NO:34、SEQ ID NO:41、SEQ ID NO:59和SEQ ID NO:111中任一项所示的氨基酸序列。
例如，所述LCDR2可以包含SEQ ID NO:120-SEQ ID NO:121中任一项所示的氨基酸序列。
例如，其中所述LCDR2可以包含SEQ ID NO:120所示的氨基酸序列：AASX 4LX 6S，其中，X 4为Arg或Ser，X 6为Glu或Gln。例如，该序列可以是根据KABAT定义规则确定的序列。
例如，其中所述LCDR2可以包含SEQ ID NO:121所示的氨基酸序列；X 1ASX 4X 5X 6T，其中，X 1为Ala、Asp或Gly，X 4为Asn、Ser或Thr，X 5为Leu或Arg，X 6为Ala或Glu。例如，该序列可以是根据KABAT定义规则确定的序列。
例如，所述LCDR2可以包含SEQ ID NO:2、SEQ ID NO:12、SEQ ID NO:19、SEQ ID NO:27、SEQ ID NO:35、SEQ ID NO:42、SEQ ID NO:60和SEQ ID NO:112中任一项所示的氨基酸序列。
例如，所述VH可以包含HCDR1，HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:5、SEQ ID NO:22、SEQ ID NO:30、SEQ ID NO:45、SEQ ID NO:51、SEQ ID NO:55、SEQ ID NO:63和SEQ ID NO:114中任一项所示的氨基酸序列。
例如，所述HCDR2可以包含SEQ ID NO:6、SEQ ID NO:9、SEQ ID NO:15、SEQ ID NO:23、SEQ ID NO:31、SEQ ID NO:38、SEQ ID NO:46、SEQ ID NO:52、SEQ ID NO:56、 SEQ ID NO:64、SEQ ID NO:67和SEQ ID NO:115中任一项所示的氨基酸序列。
例如，所述HCDR3可以包含SEQ ID NO:7、SEQ ID NO:16、SEQ ID NO:24、SEQ ID NO:32、SEQ ID NO:39、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:53、SEQ ID NO:57、SEQ ID NO:65、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72和SEQ ID NO:116中任一项所示的氨基酸序列。
例如，所述VL可以包括框架区L-FR1，L-FR2，L-FR3，和L-FR4。
例如，所述L-FR1的C末端可以与所述LCDR1的N末端直接或间接相连，且所述L-FR1可以包含SEQ ID NO:74-SEQ ID NO:75中任一项所示的氨基酸序列。
例如，所述L-FR2可以位于所述LCDR1与所述LCDR2之间，且所述L-FR2可以包含SEQ ID NO:76、SEQ ID NO:77和SEQ ID NO:78中任一项所示的氨基酸序列。
例如，所述L-FR3可以位于所述LCDR2与所述LCDR3之间，且所述L-FR3可以包含SEQ ID NO:79、SEQ ID NO:80、SEQ ID NO:81和SEQ ID NO:82中任一项所示的氨基酸序列。
例如，所述L-FR4的N末端可以与所述LCDR3的C末端直接或间接相连，且所述L-FR4可以包含SEQ ID NO:83、SEQ ID NO:84、SEQ ID NO:85和SEQ ID NO:86中任一项所示的氨基酸序列。
在本申请中，所述VL可以包含SEQ ID NO:124或SEQ ID NO:125所示的氨基酸序列。
例如，其中所述VL可以包含SEQ ID NO:124所示的氨基酸序列：DIQMTQSPSSLSASVGDRVTITCRASQX 28ISX 31X 32LX 34WYQQKPGKAPKLLX 48YAASX 53LX 55SGVPSRFSGSGSGTDX 71TLTISSLQPEDFATYYCQQSYSTPX 96X 97TFGQGTX 104X 105EIK，其中，X 28为Gly或Ser，X 31为Asn或Ser，X 32为Ser或Tyr，X 34为Ala或Asn，X 48为Ile或Leu，X 53为Arg或Ser，X 55为Glu或Gln，X 71为Phe或Tyr，X 96为Pro或Ser，X 97为Ile或不存在，X 104为Lys或Arg，X 105为Leu或Val。例如，该序列可以是根据KABAT定义规则确定的序列。
例如，其中所述VL可以包含SEQ ID NO:125所示的氨基酸序列；X 1IX 3X 4TQSPX 9X 10LSX 13SX 15GX 17RX 19TX 21X 22CX 24ASQX 28X 29X 30X 31X 32X 33LX 35WYQQKPGX 43APX 46LLIYX 51ASX 54X 55X 56TGX 59PX 61RFSGSGSGTDFTX 74TISX 78LX 80PEDX 84AX 86YYCQQYX 93X 94X 95PX 97TFGX 101GTX 104X 105EIK，其中，X 1为Asp或Glu，X 3为Gln或Val，X 4为Leu或Met，X 9为Gly或Ser，X 10为Ser或Thr，X 13为Ala或Leu，X 15为Pro或Val，X 17为Asp或Glu，X 19为Ala或Val，X 21为Ile或Leu，X 22为Ser或Thr，X 24为Gln或Arg，X 28为Asp、Gly或Ser，X 29为Ile或Val，X 30为Asn或Ser，X 31为Gly、Asn或Ser，X 32为Ser或 不存在，X 33为Trp或Tyr，X 35为Ala或Asn，X 43为Lys或Gln，X 46为Lys或Arg，X 51为Ala、Asp或Gly，X 54为Asn、Ser或Thr，X 55为Leu或Arg，X 56为Ala或Glu，X 59为Ile或Val，X 61为Asp或Ser，X 74为Phe或Leu，X 78为Arg或Ser，X 80为Glu或Gln，X 84为Phe或Ile，X 86为Thr或Val，X 93为Asp或Gly，X 94为Asn或Ser，X 95为Leu或Ser，X 97为Ile、Leu、Gln或Val，X 101为Gly或Gln，X 104为Lys或Arg，X 105为Leu或Val。例如，该序列可以是根据KABAT定义规则确定的序列。
例如，所述VL可以包含SEQ ID NO:4、SEQ ID NO:14、SEQ ID NO:21、SEQ ID NO:29、SEQ ID NO:37、SEQ ID NO:44、SEQ ID NO:62和SEQ ID NO:113中任一项所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可以包括抗体轻链恒定区，且所述抗体轻链恒定区包括人Igκ恒定区或人Igλ恒定区。
在本申请中，编码所述人Igκ恒定区的基因可以如NCBI数据库的GenBank登录号50802所示；编码所述人Igλ恒定区的基因可以如NCBI数据库的GenBank登录号3535所示。
例如，所述VH可以包括框架区H-FR1，H-FR2，H-FR3，和H-FR4。
例如，所述H-FR1的C末端可以与所述HCDR1的N末端直接或间接相连，且所述H-FR1可以包含SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91、SEQ ID NO:92、SEQ ID NO:93和SEQ ID NO:94中任一项所示的氨基酸序列。
例如，所述H-FR2可以位于所述HCDR1与所述HCDR2之间，且所述H-FR2可以包含SEQ ID NO:95、SEQ ID NO:96、SEQ ID NO:97和SEQ ID NO:98中任一项所示的氨基酸序列。
例如，所述H-FR3可以位于所述HCDR2与所述HCDR3之间，且所述H-FR3可以包含SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104、SEQ ID NO:105和SEQ ID NO:106中任一项所示的氨基酸序列。
例如，所述H-FR4的N末端可以与所述HCDR3的C末端直接或间接相连，且所述H-FR4可以包含SEQ ID NO:107、SEQ ID NO:108、SEQ ID NO:109和SEQ ID NO:110中任一项所示的氨基酸序列。
例如，所述VH可以包含SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:17、SEQ ID NO:25、SEQ ID NO:33、SEQ ID NO:40、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:54、SEQ ID NO:58、SEQ ID NO:66、SEQ ID NO:69、SEQ ID NO:71和SEQ ID NO:73中任一项所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可以包括抗体重链恒定区，且所述抗体重链恒定区包括 人IgG恒定区。
例如，所述分离的抗原结合蛋白可以包括抗体重链恒定区，且所述抗体重链恒定区包括人IgG1恒定区。
在本申请中，编码所述人IgG1恒定区的基因可以如NCBI数据库的GenBank登录号3500所示。
在本申请中，所述分离的抗原结合蛋白可包含抗体轻链可变区CDR——LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:1所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:2所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:3所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体轻链可变区CDR——LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:11所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:12所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:13所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体轻链可变区CDR——LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:19所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:20所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体轻链可变区CDR——LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:26所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:27所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:28所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体轻链可变区CDR——LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:34所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:35所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:36所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体轻链可变区CDR——LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:41所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:42所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:43所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体轻链可变区CDR——LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:59所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:60所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:61所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:6所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:7所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2可包含SEQ  ID NO:9所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:7所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:15所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:16所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:22所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:23所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:24所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:30所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:31所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:32所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:30所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:38所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:39所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:45所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:47所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:49所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:51所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:52所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:53所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:55所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:56所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:57所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:63所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:65所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:67所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:68所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:55所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:56所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:70所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:55所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:56所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:72所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:6所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:7所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:1所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:2所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:3所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:7所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:1所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:2所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:3所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:15所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:16所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:11所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:12所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:13所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:22所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:23所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:24所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:19所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:20所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:30所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:31所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:32所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:26所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:27所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:28所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:30所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:38所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:39所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:34所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:35所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:36所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:45所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:47所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:41所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:42所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:43所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:9所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:49所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:41所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:42所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:43所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:51所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:52所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:53所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:41所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:42所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:43所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:55所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:56所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:57所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:41所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:42所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:43所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:63所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:65所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:59所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:60所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:61所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2 和LCDR3，所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:67所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:68所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:41所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:42所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:43所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:55所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:56所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:70所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:41所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:42所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:43所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:55所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:56所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:72所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:41所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:42所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:43所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:8所示的氨基酸序列，且所述VL可包含SEQ ID NO:4所示的氨基酸序列。该分离的抗原结合蛋白可称为Ab2001.02。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:10所示的氨基酸序列，且所述VL可包含SEQ ID NO:4所示的氨基酸序列。该分离的抗原结合蛋白可称为Ab2001.03。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:17所示的氨基酸序列，且所述VL可包含SEQ ID NO:14所示的氨基酸序列。该分离的抗原结合蛋白可称为Ab2001.04。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:25所示的氨基酸序列，且所述VL可包含SEQ ID NO:21所示的氨基酸序列。该分离的抗原结合蛋白可称为Ab2001.08。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:33所示的氨基酸序列，且所述VL可包含SEQ ID NO:29所示的氨基酸序列。该分离的抗原结合蛋白可称为Ab2001.09。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:40所示的氨基酸序列，且所述VL可包含SEQ ID NO:37所示的氨基酸序列。 该分离的抗原结合蛋白可称为Ab2001.10。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:48所示的氨基酸序列，且所述VL可包含SEQ ID NO:44所示的氨基酸序列。该分离的抗原结合蛋白可称为Ab2001.11。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:50所示的氨基酸序列，且所述VL可包含SEQ ID NO:44所示的氨基酸序列。该分离的抗原结合蛋白可称为Ab2001.12。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:54所示的氨基酸序列，且所述VL可包含SEQ ID NO:44所示的氨基酸序列。该分离的抗原结合蛋白可称为Ab2001.13。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:58所示的氨基酸序列，且所述VL可包含SEQ ID NO:44所示的氨基酸序列。该分离的抗原结合蛋白可称为Ab2001.14。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:66所示的氨基酸序列，且所述VL可包含SEQ ID NO:62所示的氨基酸序列。该分离的抗原结合蛋白可称为Ab2001.19。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:69所示的氨基酸序列，且所述VL可包含SEQ ID NO:44所示的氨基酸序列。该分离的抗原结合蛋白可称为Ab2001.23。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:71所示的氨基酸序列，且所述VL可包含SEQ ID NO:44所示的氨基酸序列。该分离的抗原结合蛋白可称为Ab2001.24。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:73所示的氨基酸序列，且所述VL可包含SEQ ID NO:44所示的氨基酸序列。该分离的抗原结合蛋白可称为Ab2001.25。
在本申请中，抗体Ab2001.2、Ab2001.3、Ab2001.11、Ab2001.12、Ab2001.13、Ab2001.14、Ab2001.23、Ab2001.24和Ab2001.25的VL可以包含SEQ ID NO:124所示的氨基酸序列。
在本申请中，抗体Ab2001.4、Ab2001.8、Ab2001.9、Ab2001.10和Ab2001.19的VL可以包含SEQ ID NO:125所示的氨基酸序列。
本申请所述的抗原结合蛋白(例如，SARS-CoV-2抗体)能够特异性结合SARS-CoV-2的S蛋白的RBD。“特异性结合”SARS-CoV-2抗原(例如SARS-CoV-2的S蛋白的RBD)的抗 原结合蛋白(例如，抗体)通常可以以约5.0*10 -8M的K D值或更高亲和力(例如，约9.0*10 - 9M以下、约8.0*10 -9M以下)结合SARS-CoV-2的S蛋白的RBD，但不结合缺乏SARS-CoV-2序列的其它蛋白。
抗原结合蛋白(例如，抗体)是否结合SARS-CoV-2抗原(例如SARS-CoV-2的S蛋白的RBD)可使用本领域中已知的任何测定法确定。本领域中已知测定结合亲和力的分析的实例包括表面等离子共振(例如，BIACORE)或类似技术(例如，KinExa或OCTET)。在某些情形中，本申请所述的SARS-CoV-2抗体还可以与SARS-CoV交叉反应。例如，通过流式分析技术和酶联免疫反应所检测的。如本文所用，“交叉反应性”是指抗体与来自其它物种的同源蛋白反应的能力。
本申请所述的抗原结合蛋白(例如，SARS-CoV-2抗体)能够阻断SARS-CoV-2的S蛋白的RBD或其突变体与人ACE2的结合。阻断实验可以使用竞争法进行检测，例如，将所述的抗原结合蛋白(例如，SARS-CoV-2抗体)与抗原(或，可表达抗原的细胞)和抗原的配体(或，表达配体的细胞)混合，根据可检测标记的强度(例如，荧光强度或浓度)反应抗原结合蛋白与抗原的配体竞争性结合抗原的能力。在本申请中，所述的抗原结合蛋白(例如，SARS-CoV-2抗体)还可以阻断SARS-CoV的S蛋白的RBD与人ACE2的结合。
双特异性抗原结合蛋白
另一方面，本申请提供一种双特异性抗原结合蛋白，其特异性结合SARS-CoV-2的S蛋白的RBD和SARS-CoV的S蛋白的RBD。
在本申请中，所述双特异性抗原结合蛋白可以包含特异性结合SARS-CoV-2的S蛋白的RBD的第一靶向部分，其中所述第一靶向部分可以包含本申请所述的分离的抗原结合蛋白。
例如，本申请所述抗体Ab2001.2、Ab2001.3、Ab2001.11、Ab2001.12、Ab2001.13、Ab2001.14、Ab2001.23、Ab2001.24和Ab2001.25；以及本申请所述抗体Ab2001.4、Ab2001.8、Ab2001.9、Ab2001.10和Ab2001.19，以及其中的抗原结合片段(例如，可以为其VL、VH)，可以特异性结合SARS-CoV-2的S蛋白的RBD。
在本申请中，所述双特异性抗原结合蛋白可以包含特异性结合SARS-CoV的S蛋白的RBD的第二靶向部分，其中所述第二靶向部分可以包含本申请所述的分离的抗原结合蛋白。
例如，本申请所述抗体Ab2001.2、Ab2001.3、Ab2001.11、Ab2001.14、Ab2001.24和Ab2001.25，以及其中的抗原结合片段(例如，可以为其VL、VH)，可以特异性结合SARS-CoV的S蛋白的RBD。
例如，所述双特异性抗原结合蛋白可以包括抗体。
例如，所述第一靶向部分可以包含第一重链和第一轻链，所述第二靶向部分可以包含第 二重链和第二轻链，其中所述第一轻链和所述第二轻链可以是相同的。
例如，所述第一轻链和所述第二轻链可以包含SEQ ID NO:44所示的氨基酸序列。
例如，所述第一重链的VH可以包含SEQ ID NO:48所示的氨基酸序列。
例如，所述第二重链的VH可以包含SEQ ID NO:50所示的氨基酸序列。
例如，所述第一重链的VH可以包含SEQ ID NO:48所示的氨基酸序列，且所述第二重链的VH可以包含SEQ ID NO:50所示的氨基酸序列。
在本申请中，所述双特异性抗原结合蛋白可以为抗体Ab2001.16，其中所述第一轻链和所述第二轻链可以包含SEQ ID NO:44所示的氨基酸序列；所述第一重链的VH可以包含SEQ ID NO:48所示的氨基酸序列，且所述第二重链的VH可以包含SEQ ID NO:50所示的氨基酸序列。
本申请中涉及的蛋白质、多肽和/或氨基酸序列，还应理解为至少包含以下的范围：与该所述蛋白质或多肽具备相同或类似功能的变体或同源物。
在本申请中，所述变体可以为，在所述蛋白质和/或所述多肽(例如，本申请所述的抗原结合蛋白)的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽。例如，所述功能性变体可包含已经通过至少1个，例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的蛋白质或多肽。所述功能性变体可基本上保持改变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如，所述功能性变体可保持改变之前的所述蛋白质或所述多肽的至少60％，70％，80％，90％，或100％的生物学活性(例如抗原结合能力)。例如，所述取代可以为保守取代。
在本申请中，所述抗原结合蛋白的氨基酸序列的一部分可以与来自特定物种的抗体中相应的氨基酸序列同源，或者属于特定的类别。例如，抗体的可变区及恒定部分均可以来自一个动物物种(如人)的抗体的可变区及恒定区。在本申请中，所述同源物可以为，与所述蛋白质和/或所述多肽(例如，本申请所述的抗原结合蛋白)的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的蛋白质或多肽。
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。可以通过以下方式计算“序列同源性百分比”：将两条待比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同源性百分比。为了确定序列同源性百分数而进行 的比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435-1441，1989。对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。
核酸、载体、细胞和制备方法
另一方面，本申请提供一种分离的一种或多种核酸分子，其编码本申请所述的分离的抗原结合蛋白，和/或本申请所述的双特异性抗原结合蛋白。
另一方面，本申请提供一种载体，其可以包含本申请所述的核酸分子。
另一方面，本申请提供一种细胞，其可以包含本申请所述的核酸分子或本申请所述的载体。
本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。
在本申请中，可以通过本领域已知的多种方法来制备编码所述抗体、其抗原结合片段的核酸，这些方法包括但不限于，采用限制性片段操作或采用合成性寡核苷酸的重叠延伸PCR，具体操作可参见Sambrook等人，Molecular Cloning，A Laboratory Manual，Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1989；和Ausube等人Current Protocols in Molecular Biology，Greene Publishing and Wiley-Interscience，New York N.Y.，1993。
在另一个方面，本申请提供了一种或多种载体，其包含本申请所述的一种或多种核酸分子。每种载体中可包含一种或多种所述核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。在某些实施方式中，所述表达控制序列为可调的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化，但通常包含分别参与转录和翻译起始的5’ 非转录序列和5’及3’非翻译序列，例如TATA盒、加帽序列、CAAT序列等。例如，5’非转录表达控制序列可包含启动子区，启动子区可包含用于转录控制功能性连接核酸的启动子序列。所述表达控制序列还可包括增强子序列或上游活化子序列。在本申请中，适当的启动子可包括，例如用于SP6、T3和T7聚合酶的启动子、人U6RNA启动子、CMV启动子及其人工杂合启动子(如CMV)，其中启动子的某部分可与其他细胞蛋白(如人GAPDH，甘油醛-3-磷酸脱氢酶)基因启动子的某部分融合，其可包含或不包含另外的内含子。本申请所述的一种或多种核酸分子可以与所述表达控制元件可操作地连接。所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体为表达载体。
在另一个方面，本申请提供了宿主细胞，所述宿主细胞可包含本申请所述的一种或多种核酸分子和/或本申请所述的一种或多种载体。在某些实施方式中，每种或每个宿主细胞可包含一个或一种本申请所述的核酸分子或载体。在某些实施方式中，每种或每个宿主细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述宿主细胞中，例如真核细胞，如来自植物的细胞、真菌或酵母细胞等。可通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。
另一方面，本申请提供一种制备本申请所述的分离的抗原结合蛋白、本申请所述的双特异性抗原结合蛋白的方法，所述方法包括在使得本申请所述的分离的抗原结合蛋白和/或本申请所述的双特异性抗原结合蛋白表达的条件下，培养本申请所述的细胞。
所述方法可包括，在使得所述的抗原结合蛋白表达的条件下，培养所述本申请所述的宿主细胞。例如，可通过使用适当的培养基、适当的温度和培养时间等，这些方法是本领域普通技术人员所了解的。
任何适于产生单克隆抗体的方法都可用于产生本申请的抗原结合蛋白(例如，SARS-CoV-2抗体)。
任何合适形式的SARS-CoV-2(例如SARS-CoV-2的S蛋白的RBD)都可以作为免疫原(抗原)，用于产生对SARS-CoV-2特异的抗体，筛选所述抗体的生物学活性。免疫原可以单独使用，或与本领域已知的一种或多种免疫原性增强剂组合使用。可以使用合适的遗传载体表达编码免疫原的DNA，所述载体包括但不限于腺病毒载体、腺相关病毒载体、杆状病毒载体、质料和非病毒载体。
人源化抗体可以选自任何种类的免疫球蛋白，包括IgM、IgD、IgG、IgA和IgE。在本申请中，抗体是IgG抗体，使用IgG1亚型。同样，任一类轻链都可以在本文的化合物和方法中使用。例如，κ、λ链或其变体在本申请中是适用的。
本申请的抗原结合蛋白或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。然后可将该核酸分子引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本申请还涉及包含上述的适当核酸分子以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。例如，动物细胞可以包括：CHO-S、CHO-K1和/或HEK-293细胞。
本申请中所述的用重组DNA转化宿主细胞的步骤可用本领域熟知的技术进行。获得的转化子可用常规方法培养，转化子表达本申请的核酸分子所编码的多肽。根据所用的宿主细胞，用常规培养基在合适的条件下培养。通常，在适合本申请抗原结合蛋白表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本申请的抗原结合蛋白。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如流式细胞分选技术(FACS)、放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。
药物组合物
另一方面，本申请提供一种药物组合物，其可以包含本申请所述的分离的抗原结合蛋白、本申请所述的双特异性抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的佐剂。
本申请所述的药物组合物可直接用于结合SARS-CoV-2的S蛋白的RBD，因而可用于预防和治疗冠状病毒感染相关的疾病(例如，COVID-19)。此外，还可同时使用其他治疗剂。
本申请的药物组合物可以含有安全有效量(如0.001-99wt％，0.01-90wt％，或0.1-80wt％)的本申请所述的抗原结合蛋白以及药学上可接受的佐剂(可包括载体或赋形剂)。这类载体可以包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本申请所述的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡 萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量。此外，本申请所述的抗原结合蛋白还可与其他治疗剂一起使用。
本文所述的抗原结合蛋白或药物组合物可以符合良好医疗实践的方式配制、给药和施用。在此情形下的考虑因素包括所治疗的特定病症、所治疗的特定哺乳动物、单个患者的临床病状、病症的病因、药剂递送部位、施用方法和医学从业者已知的其他因素。治疗剂(例如，SARS-CoV-2抗体)无需但任选地与一种或多种当前用来预防或治疗所考虑的病症的药剂一起配制和/或同时施用。此类其他药剂的有效量取决于制剂中存在的治疗剂(例如，SARS-CoV-2抗体)的量、病症或治疗的类型以及以上论述的其他因素。这些药剂通常可以凭经验/临床上确定为适当的任何剂量且通过凭经验/临床上确定为适当的任何途径加以使用。与单个治疗相比，可减少组合治疗中施用的抗体的剂量。通过常规技术易于监测此疗法的进展。
用途
另一方面，本申请提供一种本申请所述的分离的抗原结合蛋白、本申请所述的双特异性抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗冠状病毒的感染。
本申请提供一种预防、缓解和/或治疗冠状病毒的感染的方法，其包括向有需要的受试者施用本申请所述的分离的抗原结合蛋白、本申请所述的双特异性抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物。
本申请提供了分离的抗原结合蛋白、本申请所述的双特异性抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物，其可以预防、缓解和/或治疗冠状病毒的感染。
在本申请中，所述冠状病毒的感染可以包括COVID-19。所述冠状病毒的感染还可以包括SARS。
另一方面，本申请提供一种阻断SARS-CoV-2的S蛋白的RBD或其突变体与人ACE2的结合的方法，其包括以下的步骤，施用本申请所述的分离的抗原结合蛋白、本申请所述的双特异性抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物。
另一方面，本申请提供一种阻断SARS-CoV的S蛋白的RBD与人ACE2的结合的方法，其包括以下的步骤，施用本申请所述的分离的抗原结合蛋白、本申请所述的双特异性抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物。
本申请的抗原结合蛋白可用于检测应用，例如用于检测样本，从而提供诊断信息。例如，本申请所述的抗体和/或方法，可以用于对受试者(例如疑似被SARS-CoV-2或SARS-CoV感染，或已经被SARS-CoV-2或SARS-CoV感染的患者)的标本(例如，咽拭子检测样品，例如血清、全血、痰液、口腔/鼻咽分泌物或洗液、尿液、粪便、胸腹腔积液、脑脊液和组织标本)进行检测，作为疗效观察的指标及是否具有传染性和是否需要隔离的指标。例如，本申请所述的抗体和/或方法，可以为治疗性干预提供监测方案。
在本申请中，所采用的样本(样品)包括细胞、组织样本和活检标本。本申请使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本申请中使用的活检可以包括例如通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。例如，所述样本可以包括固定的或保存的细胞或组织样本。
本申请还提供了一种指含有本申请的抗原结合蛋白的试剂盒。在某些情形中，所述的试剂盒还可以包括容器、使用说明书、缓冲剂等。例如，本申请的原结合蛋白可以固定于检测板。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的融合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1抗体筛选
1.1酵母展示文库构建与筛选
选用共同轻链噬菌体文库与单链噬菌体文库，针对SARS-CoV-2 S1 RBD(sino biological货号：40592-V05H)进行两轮淘选，获得阳性富集。以噬菌体两轮淘选后质粒为模板，设计引物进行聚合酶链式反应(PCR)扩增单链抗体(scFv)；PCR扩增的scFv基因片段回收后与酵母展示质粒共转入酿酒酵母菌株EBY100(购自ATCC)，通过酿酒酵母的同源重组使scFv基因插入至酵母展示质粒中，进而实现在酵母细胞壁表面展示单链抗体。酵母展示单链抗体库与共同轻链抗体库分别命名为JYYDL056、JYYDL057。文库JYYDL056、JYYDL057电转后在100mL的SD-Trp培养基(Clontech，货号：630308)，30℃、225转/分钟培养过夜；各取1.0×10 8菌量重悬于20mL YPGP液体培养基(2％半乳糖，2％蛋白胨，1％酵母提取物，0.54％Na 2HPO 4，0.86％NaH 2PO 4·H 2O)，20℃、225转/分钟培养诱导24小时，置于4℃冰箱待用。
诱导后菌液，测定菌液的OD 600，按1OD为1.0×10 7细胞数计算，各取4.0×10 7细胞， 按如下步骤进行流式染色分选：1.用2mL 1×PBSA(1×PBS+1％BSA)洗涤一次，3000转/分钟离心3分钟(以下离心均为此条件)弃上清；2.每管与500μL含100nM SARS-CoV-2 S1 RBD mFc(Sino biological，货号：40592-V05H)的1×PBSA，室温孵育30分钟；3.各加入1mL 1×PBSA，离心弃上清；4.各加入100μL经1×PBSA稀释的300nM Human ACE2-biotin(Kactus，货号：ACE-HM401)，冰上孵育20分钟；5.各加入1mL 1×PBSA，离心弃上清；6.各加入500μL含荧光抗体的1×PBSA(SA-PE厂家eBioscience，货号：12-4317-8，按1:200稀释；Goat anti mouse-Alexa Fluor 647厂家thermo fisher，货号：A-21235，按1:400稀释)，避光冰上孵育20分钟；7.重复步骤5，加入2mL 1×PBSA重悬细胞，收集荧光信号Alexa Fluor 647信号强且PE信号弱的细胞群进行流式分选。分选后细胞在5mL SD-Trp液体培养基中30℃、225转/分钟培养过夜；取1mL菌液加入4mL YPGP液体培养基，20℃、225转/分钟培养24小时，置于4℃冰箱待用。同第一轮筛选方案，将SARS-CoV-2 S1 RBD mFc浓度降为50nM，进行第二轮分选，分选后细胞涂布于SD-Trp固体培养基(Clontech，货号：630309)，30℃静置培养3天。
1.2单克隆酵母菌落测序与流式染色鉴定
JYYDL056、JYYDL057第二轮筛选产物，各挑400个单克隆进行测序，最终JYYDL056文库第二轮筛选产物获得34个独一的单链抗体序列；JYYDL057文库第二轮筛选产物获得42个独一的共同轻链抗体序列。对相应的酵母单克隆菌落进行流式染色分析，参考实施例2.1的染色步骤，各取1×10 6个细胞按如下的方案(表1)进行染色。
方案1与SARS-CoV-2-RBD结合细胞群的强弱由PE平均荧光信号强度(MFI)反映，抗体阻断SARS-CoV-2-RBD与hACE2-Fc的强弱由Alexa Fluor 647的MFI值反映；
同理方案2可以评估抗体与SARS S1的结合与阻断能力；
方案3的PE与Alexa Fluor 647的MFI值分别反映了单链抗体的展示水平与非特异性结合水平。
表1单克隆酵母菌落流式染色鉴定方案
Figure PCTCN2020092634-appb-000001
Figure PCTCN2020092634-appb-000002
每个单克隆用三种染色方案染色后，使用GUAVA微毛细管细胞分析平台进行流式分析。根据实验结果，挑取方案1中与SARS-CoV-2-RBD结合强(PE荧光信号的MFI值高)，且阻断SARS-CoV-2 RBD与hACE2-Fc结合强(Alexa Fluor 647的MFI值低)的克隆；同时排除方案3中与无关抗原结合信号高的克隆序列。
综合所得，从JYYDL056文库中获得8个单克隆抗体(Y28A5、Y28B6、Y29B2、Y29F6、Y34H3、Y35F3、Y35G1、Y36F4)；从JYYDL057文库中获得7个共轻链单克隆抗体(Y28B4、Y30B4、Y34B6、Y34G5、Y38A3、Y39B3、Y39G2)(结果如表2所示)。选取其中的共轻链单克隆(Y28B4与Y34G5)，通过Fc区的Knob与Hole突变(其突变修饰参见US5731168A)构建共同轻链双特异性抗体Ab2001.16，如表3。总计16个抗体，交由合同外包公司进行哺乳动物细胞表达，小量制备抗体样品。
表2文库二轮筛选后酵母单克隆菌落流式染色分析结果
Figure PCTCN2020092634-appb-000003
表3共轻链双特异性抗体的表达组合
抗体名称 Knob克隆号 Hole克隆号 轻链
Ab2001.16 Y28B4 Y34G5 同轻链germline1-39
实施例2抗体表达
候选抗体序列以Kabat编号法编码及划分CDR区，委托泰州市百英生物科技有限公司进 行质粒构建与抗体的表达、纯化，抗体序列及表达相应信息见表4。
瞬转30毫升HEK293细胞，表达、纯化后获得候选抗体蛋白，具体信息见表5。
表4抗体序列信息
Figure PCTCN2020092634-appb-000004
表5抗体表达信息
Figure PCTCN2020092634-appb-000005
Figure PCTCN2020092634-appb-000006
实施例3体外活性筛选
3.1 Octet Red检测候选抗体与SARS-CoV-2 S1的结合解离速率
候选抗体测定与SARS-CoV-2 S1的结合解离速率，将候选抗体固化到AHC传感器上(Fortébio,货号：18-0015)，测定其与SARS-CoV-2 S1(Sino Biological,货号：40591-V08H)的结合解离速率。每个循环包含以下步骤：1)传感器再生；2)浸入缓冲液(PBST，50mL PBS中加入10μL吐温20)60秒；3)5μg/mL的全人抗体固化在AHC传感器上，时间为40秒；4)传感器浸入缓冲液180秒；5)100nM SARS-CoV-2 S1与抗体结合，时间180秒；6)抗原抗体的解离，时间10分钟。亲和力的结果通过Octet Data Analysis Software(Fortébio)进行分析，结果如表6所示。
表6
Figure PCTCN2020092634-appb-000007
Figure PCTCN2020092634-appb-000008
由表6可知，除Ab2001.07以外，候选抗体均与抗原SARS-CoV-2 S1有效结合。其中Ab2001.08、Ab2001.10、Ab2001.16亲和力较高，进一步测定与不同浓度SARS-CoV-2 S1的结合解离速率，SARS-CoV-2 S1浓度选用50nM、25nM、12.5nM、6.3nM、3.2nM共5个浓度，同时与hACE2-Fc(恺佧生物,货号：ACE-HM501)比较(见表7，图1)
表7
Figure PCTCN2020092634-appb-000009
由表7可知，Ab2001.08、Ab2001.10、Ab2001.16、hACE2-Fc与SARS-CoV-2 S1的亲和力分别为2.93nM(图1A)、4.21nM(图1B)、2.39nM(图1C)、11.1nM(图1D)，抗体与SARS-CoV-2 S1的亲和力均强于hACE2-Fc。
3.2候选抗体阻断SARS-CoV-2 S1与hACE2-Fc结合
根据候选抗体的亲和力数据，选取Ab2001.02、Ab2001.03、Ab2001.08、Ab2001.09、Ab2001.10、Ab2001.11、Ab2001.16进行SARS-CoV-2 S1与hACE2结合的阻断实验；由于Ab2001.16双特异性抗体由Ab2001.11与Ab2001.12的序列组合而成，使用同量的Ab2001.11+Ab2001.12组合进行对比。
按如下步骤开展实验，
1.用1×PBS将hACE2-Fc稀释到1μg/mL的包被酶标板，100μl/孔，贴封板膜，置于4℃孵育过夜，然后用洗涤液(1×PBS+0.05％TWEEN-20)洗板3次。继而用洗涤液配制封闭液(1×PBS+0.05％TWEEN-20+2％BSA)，每孔加入300μl封闭液，37℃封闭1小时。2.用封闭液配制生物素化的SARS-CoV-2 S1 Fc(恺佧生物，货号：COV-VM5S1)终浓度为0.2μg/mL；随后用封闭液稀释抗体，抗体的起始终浓度为15μg/mL，5倍梯度稀释(稀释7个浓度点+1个0μg/mL的浓度点)，然后将抗SARS-CoV-2抗体和生物素化的SARS-CoV-2 S1 Fc 37℃共孵育1小时。3.取出步骤1中封闭好的酶标板，用洗涤液洗板3次，以100μl/孔加入步骤2 中的孵育液，37℃孵育1小时。4.用洗涤液洗板3次，封闭液按体积比1：5000稀释SA-HRP(sigma，S2438-250UG)，以100μl/孔加至酶标板内，37℃孵育1小时；5.用洗涤液洗板3次，以100μl/孔加入TMB显色液(Biopanda，货号：TMB-S-003)，室温避光反应10分钟，然后以50μl/孔加入终止液(Solarbio，货号：C1058)终止反应，在450nm波长处测定吸光值。数据用GraphPad Prism软件来进行处理，如图2所示。由图2可知，Ab2001.08、Ab2001.10的阻断效果好，比hACE2-Fc的阻断效果强约15倍。
3.3候选抗体与SARS-CoV-2抗原结合表位鉴定
候选抗体与SARS-CoV-2 S1抗原结合表位采用In-tandem的方法，即将SARS-CoV-2 S1抗原固化在传感器上，然后依次与第一个抗体和第二个抗体相互作用，检测第二个抗体结合信号以判定两个抗体是否识别同一表位。在本实验中，每个循环包含以下步骤：1)传感器先浸入缓冲液(PBST，50mL PBS中加入10μL吐温20)30s；2)1μg/ml的Biotin人SARS-CoV-2 RBD his(恺佧生物，货号：COV-VM4BD)固化在SA传感器上(Fortébio,货号：18-0009)，时间为65秒；3)传感器浸入缓冲液30秒；4)100nM的第一个抗体与传感器表面的抗原结合，时间3分钟，使抗原上第一个抗体的结合位点饱和；5)100nM的第二个抗体与抗原结合，时间3分钟。抗原表位鉴定的结果通过Octet Data Analysis Software(Fortébio)进行分析，结果如表8所示。
表8候选抗体与SARS-CoV-2抗原结合表位鉴定
Figure PCTCN2020092634-appb-000010
自反应信号<20％，该实验数据有效。实验结果的判定标准为：1)60％-100％：完全不竞争；2)20％-60％：部分竞争；3)<20％：完全竞争，如果有一个方向达到这标准，则认为两个抗体有竞争。数据显示Ab2001.10，12，13号抗体为一个结合表位；Ab2001.02、Ab2001.03、Ab2001.11四个抗体为另一个结合表位。
挑选Ab2001.10抗体测定与Ab2001.08和Ab2001.16的抗原结合表位是否相同，结果见表9。结果显示，Ab2001.08，Ab2001.10为2个不同的抗原结合表位,且Ab2001.16双抗含有Ab2001.11与Ab2001.12两个不同的抗原结合表位。
表9候选抗体与SARS-CoV-2抗原结合表位鉴定
Figure PCTCN2020092634-appb-000011
3.4候选抗体阻断SARS-CoV S1与hACE2结合
从表2中可知，Y28B4克隆结合SARS-CoV S1后，SARS-CoV S1再结合hACE2-Fc的MFI只有43.5，说明其可能具有阻断SARS-CoV S1与hACE2结合的能力。CR3022为文献报道针对SARS-CoV S1的中和抗体，可阻断SARS-CoV S1与hACE2结合，可作为阳性对照。Y28B4克隆对应的抗体编号为Ab2001.11，同时双特异性抗体Ab2001.16由Ab2001.11、Ab2001.12号组成，所以进一步评估候选抗体Ab2001.11、Ab2001.12、Ab2001.16阻断SARS CoV S1与hACE2结合的能力。实验方案同实施例3.2，抗体浓度从200μg/mL开始5梯度稀释，共7个浓度，增加一个抗体为0μg/mL的浓度点，结果如图3所示。
由图3可知，对照抗体具有较强阻断SARS-CoV S1与hACE2结合的能力，IC 50值在6.21nM；Ab2001.11有较弱阻断能力，IC 50值在303.87nM；其余抗体均无阻断能力。虽然Ab2001.11阻断能力较弱，但若经过亲和力成熟提高亲和力或许能提高阻断能力，有望成为既阻断SARS-CoV-2 S1又阻断SARS-CoV S1与hACE2结合的广谱中和抗体。
实施例4理化成药性评价
实验材料
磷酸二氢钠、磷酸氢二钠、氢氧化钠、盐酸胍、氯化钠、硫酸铵、盐酸、甘氨酸、氢氧化钠、乙醇、碳酸氢铵购自国药试剂；1％的甲基纤维素、CIEF Peptide Marker Kit、TTM溶液、电极液、毛细管清洗液、cIEF Cartridge FC-COATED购自Protein Simple公司；TSKG3000SWxl(7.8×300mm，5μm，)购自TOSOH；Protein Thermal Shift TM Starter Kit、Propac WCX-10(4×250mm，5μm)、MAbPacTMHIC-10(4x250mm，5μm，)购自Thermo。
4.1候选抗体亲水性分析
由图3可知，候选抗体Ab2001.02、Ab2001.03、Ab2001.08、Ab2001.09、Ab2001.10、Ab2001.11、Ab2001.12、Ab2001.16具有阻断SARS-CoV-2 S1与人受体蛋白ACE2结合的能 力。对这些候选抗体进行亲水性与奥马珠单抗进行对比，具体实验步骤如下，实验结果见图4。
(1)将样品稀释至1mg/mL，混匀，12000转离心5分钟，取上清转移至样品瓶内，放入HPLC样品盘。
(2)设置色谱条件：
Figure PCTCN2020092634-appb-000012
(3)以50mM磷酸盐缓冲液/1M硫酸铵，pH 7.0作为流动相A，以50mM磷酸盐缓冲液，pH 7.0作为流动相B，进行梯度洗脱分析，色谱软件进行数据分析，用亲水性好的参比抗体出峰保留时间除以候选抗体的出峰保留时间，计算各样品亲水系数；根据以往经验内部定义亲水系数大于0.5为可接受标准线。
由图4可知，候选抗体的亲水系数都大于0.5，符合内部成药性标准，其中候选抗体AB2001.02、AB2001.03、AB2001.09、AB2001.10表现出优异的亲水性。
4.2候选抗体电荷异质性分析
应用成像毛细管等点聚焦电泳技术(iCIEF)，对候选抗体Ab2001.02、Ab2001.03、Ab2001.08、Ab2001.09、Ab2001.10、Ab2001.11、Ab2001.12、Ab2001.16进行电荷异质性分析，具体步骤如下：
(1)取样品溶液加入到已经充分混匀的以下体系中：1％的甲基纤维素(MC)70μL，尿素4M，8μL两性电解质Pharmalyte pH 3-10，pI marker 5.5和9.5各2μL。补加适当体积超纯水至200μL，混匀。
(2)所有样品6000转离心3分钟除气泡，然后转移上清到样品瓶中，置于样品盘上，记录样品位置。
(3)开机运行，分析结束后，将结果文件导入Chrom Perfect软件进行图谱积分处理并计算各峰的等电点以及各峰百分比。
候选抗体电荷异质性分析结果如图5所示，由图5可知，除Ab2001.09主峰偏低外，其余候选抗体电荷异质性良好，主峰纯度均大于70％以上。通过电荷异质性图谱分析，如图6所示，Ab2001.16双特异性抗体经Protein A一步纯化，主峰纯度已达到74.4％，异源二聚体 比例接近93％，或可与单克隆抗体共用生产、纯化工艺。
4.3候选抗体热稳定性研究
对候选抗体Ab2001.02、Ab2001.03、Ab2001.08、Ab2001.09、Ab2001.10、Ab2001.11、Ab2001.12、Ab2001.16进行热稳定性研究。
具体步骤如下：1.将供试品用样品缓冲液稀释至1mg/mL；2.按Protein Thermal Shift TM Starter Kit说明书，取供试品溶液13μL加入至PCR管内；3.加入5μL Protein Thermal shift TM缓冲液后，再加入2μL 10×染色液，使反应体积为20μL；4.混匀后，12000转离心5分钟去除气泡；5.将检测样品置于PCR仪内，进行样品分析，记录样品的Fab熔解温度(Fab Tm)，结果如图7所示。
综合而言，候选抗体Ab2001.02、Ab2001.03、Ab2001.08、Ab2001.10、Ab2001.11、Ab2001.12、Ab2001.16具有良好的理化成药性质。
实施例5病毒中和实验
综合实施例3与4的实验结果，挑选候选抗体Ab2001.02、Ab2001.03、Ab2001.08、Ab2001.09、Ab2001.10、Ab2001.11、Ab2001.12、Ab2001.13、Ab2001.16；Ab2001.07作为阴性对照，评估候选抗体中和新型冠状病毒侵染猴肾细胞(Vero E6细胞)的能力。由于使用活病毒，实验委托有生物安全三级实验室(BSL3)的中国科学院武汉病毒所进行，具体实验步骤如下：
试剂与耗材：
试剂：DMEM(Thermo Fisher)，FBS(Gibco)，甲基纤维素(Millipore)抗体稀释剂：PBS(Thermo Fisher)
病毒RNA提取：QIAamp 96 Virus QIAcube HT Kit，Qiagen公司，CAT#57731；
病毒核酸检测(荧光定量PCR)试剂盒：武汉病毒研究所诊断微生物学科组研制，针对RBD2基因。
主要仪器：
高通量核酸自动纯化仪：QIAcube HT 9001793(Qiagen)
荧光定量PCR仪：CFX96 Touch Real-Time PCR Detection System(Bio-rad)
实验设计：
(1)SARS-CoV-2病毒(WIV04，GenBank:MN996528.1)扩增、滴度测定：P6代SARS-CoV-2病毒接种Vero-E6细胞，于37℃、5％CO 2培养箱中培养48h，收取培养的病毒上清，离心后分装，-80℃冰箱冻存。在Vero-E6细胞中采用经典噬菌斑法测定病毒滴度。
(2)抗体初步评价：Vero-E6细胞均匀铺于24孔板(10 5个/孔)，16小时后进行加入含不同抗体的培养基(抗体终浓度为10μg/mL)，以0.005MOI的SARS-CoV-2进行感染，24h、72h后收取细胞培养上清，进行病毒RNA提取和检测。
(3)有效抗体浓度梯度验证：对步骤2筛选中有效的抗体进行浓度梯度实验(10μg/mL、2μg/mL、0.4μg/mL)，实验方案相同。
(4)噬菌斑减少中和实验(Plaque reduction neutralization test,PRNT)
待测抗体加入含2％FBS的DMEM培养基中，进行3倍梯度稀释，稀释后的抗体加入96孔板中，每孔100μL；病毒稀释为2000PFU/mL，分别取100μL与不同浓度的抗体进行混合；(抗体终浓度分别为30μg/mL、10μg/mL、3.33μg/mL、1.11μg/mL、0.37μg/mL、0.12μg/mL、0.04μg/mL、0.014μg/mL、0.005μg/mL)，37℃孵育30min，再将抗体、病毒混合液加入细胞培养，病毒感染细胞96h后进行经典噬菌斑实验计算PRNT50浓度。
数据处理及统计分析
PRNT50利用GraphPad prism软件计算。
实验结果
候选抗体单浓度初步评价
各候选抗体(10μg/mL)加入细胞培养基后加入0.005MOI病毒，24h后检测上清病毒RNA含量，Ab2001.08、Ab2001.09、Ab2001.10、Ab2001.12、Ab2001.13、Ab2001.16以及Ab2001.08与Ab2001.10联用(各5μg)、Ab2001.11与Ab2001.12联用组(各5μg)组病毒RNA含量下降约2个数量级，如图8A所示；72h检测上清病毒RNA拷贝，Ab2001.10、Ab2001.08与Ab2001.10联用组降低超2个数量级，Ab2001.08降低超1个数量级，如图8B所示；120h观察细胞状态，Ab2001.08、Ab2001.10、联用等三组细胞状态良好，其他组均病变明显。
候选抗体多浓度梯度验证
Ab2001.08、Ab2001.10及两者联用多浓度梯度验证，分别以10μg/mL、2μg/mL、0.4μg/mL抗体加入细胞培养基后加入0.005MOI病毒，24h后检测上清病毒RNA含量，均下降约2个数量级，如图9A所示；72h后检测上清病毒RNA含量，只有Ab2001.08、联用组下降约2个数量级，如图9B所示。
候选抗体噬菌斑减少半数中和浓度测定
Ab2001.08、Ab2001.10及两者联用组按实验设计的方案进行噬菌斑减少半数中和浓度的测定，结果如图10所示。由图10可知，候选抗体Ab2001.08、Ab2001.10及两者联用均具有 中和SARS-CoV-2的活性，其PRNT50浓度(噬菌斑减少半数中和浓度)分别为：0.012μg/mL、0.062μg/mL、0.022μg/mL，有望成为新冠疫情防治的特效药物。
实施例6与不同新冠突变毒株的结合与阻断能力
6.1中和抗体与SARS-CoV-2 S1 RBD及其突变体的结合解离速率
测定中和抗体Ab2001.08、Ab2001.10与SARS-CoV-2 S1 RBD蛋白及在不同地区流行的突变体S1 RBD蛋白(doi:https://doi.org/10.1101/2020.03.15.991844)的结合解离速率。
将中和抗体固化到AHC传感器上(Fortébio,货号：18-0015)，测定其与SARS-CoV-2 S1 RBD及其突变体(ACRO，货号：SPD-S52H4，SPD-S52H5，SPD-S52H3，SPD-S52H8)的结合解离速率。每个循环包含以下步骤：1)传感器再生；2)浸入缓冲液(PBST，50mL PBS中加入10μL吐温20)60秒；3)10μg/mL的全人抗体固化在AHC传感器上，时间为15秒；4)传感器浸入缓冲液180秒；5)100nM SARS-CoV-2 S1 RBD与抗体结合，时间180秒；6)抗原抗体的解离，时间5分钟。亲和力的结果通过Octet Data Analysis Software(Fortébio)进行1：1拟合分析，结果如图11所示。
由图11的结果可知，虽然SARS-CoV-2 S1 RBD的各突变体与人ACE2受体的亲和力比野生型高，但是中和抗体Ab2001.08和Ab2001.10与RBD突变体的亲和力也随之提高，且亲和力始终比RBD蛋白与人ACE2受体的亲和力高，这就说明即使新冠毒株发生突变，中和抗体也能保持很好的药效。
6.2中和抗体阻断SARS-CoV-2 S1 RBD及其突变体与hACE2-Fc结合
通过阻断实验来进一步证明中和抗体能否很好地阻断SARS-CoV-2 S1 RBD及其突变体和人受体ACE2的结合。
实验方法按照实施例3.2进行，SARS-CoV-2 S1 RBD及其突变体的终浓度为0.1μg/mL，抗体和hACE2-Fc的起始终浓度为100nM，5倍梯度稀释(稀释7个浓度点+1个0μg/mL的浓度点)。结果如图12A-12E所示，其中图12A-12E分别表示中和抗体阻断SARS-CoV-2 S1 RBD、SARS-CoV-2 S1 RBD V357F突变体、SARS-CoV-2 S1 RBD N354D突变体、SARS-CoV-2 S1 RBD N354D和D304Y突变体以及SARS-CoV-2 S1 RBD R408I突变体与hACE2-Fc结合的情况。由图12A-12E的结果可知，候选抗体可以很好地阻断SARS-CoV-2 S1 RBD突变体和人受体ACE2的结合，并且都比hACE2-Fc的阻断效果强。
以上详细描述了本申请的实施方式，但是，本申请并不限于上述实施方式中的具体细节， 在本申请的技术构思范围内，可以对本申请的技术方案进行多种简单变型，这些简单变型均属于本申请的保护范围。另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本申请对各种可能的组合方式不再另行说明。此外，本申请的各种不同的实施方式之间也可以进行任意组合，只要其不违背本申请的思想，其同样应当视为本申请所公开的内容。

Claims (49)

  1. 分离的抗原结合蛋白，其具有下述性质中的一种或多种：
    1)阻断SARS-CoV-2的S蛋白的RBD或其突变体与人ACE2的结合；
    2)阻断SARS-CoV的S蛋白的RBD与人ACE2的结合；
    3)在Octet测定中，以约5.0*10 -8M以下的K D值特异性结合SARS-CoV-2的S蛋白的RBD；
    4)在Octet测定中，以约6.0*10 -10M以下的K D值特异性结合SARS-CoV-2的S蛋白的RBD的突变体；
    5)与SARS-CoV-2的S蛋白的RBD的亲和力强于人ACE2-Fc；
    6)特异性结合SARS-CoV的S蛋白的RBD；
    7)亲水性；
    8)电荷异质性分析主峰约为45％-85％；
    9)在Thermal shift测定中，Tm至少为约75℃；
    10)具有中和SARS-CoV-2的活性。
  2. 根据权利要求1所述的分离的抗原结合蛋白，其包含轻链可变区VL中的至少一个CDR，所述VL包含SEQ ID NO:124或SEQ ID NO:125所示的氨基酸序列。
  3. 根据权利要求1-2中任一项所述的分离的抗原结合蛋白，其包含重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:17、SEQ ID NO:25、SEQ ID NO:33、SEQ ID NO:40、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:54、SEQ ID NO:58、SEQ ID NO:66、SEQ ID NO:69、SEQ ID NO:71和SEQ ID NO:73中任一项所示的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的分离的抗原结合蛋白，其包括抗体或其抗原结合片段。
  5. 根据权利要求4所述的分离的抗原结合蛋白，其中所述抗原结合片段包括Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv和/或dAb。
  6. 根据权利要求4-5中任一项所述的分离的抗原结合蛋白，其中所述抗体为全人源抗体。
  7. 根据权利要求1-6中任一项所述的分离的抗原结合蛋白，其中所述VL包含LCDR1，LCDR2和LCDR3，所述LCDR3包含SEQ ID NO:122-SEQ ID NO:123中任一项所示的氨基酸序列。
  8. 根据权利要求7所述的分离的抗原结合蛋白，其中所述LCDR3包含SEQ ID NO:3、SEQ ID NO:13、SEQ ID NO:20、SEQ ID NO:28、SEQ ID NO:36、SEQ ID NO:43和SEQ ID NO:61中任一项所示的氨基酸序列。
  9. 根据权利要求7-8中任一项所述的分离的抗原结合蛋白，其中所述LCDR1包含SEQ ID NO:118-SEQ ID NO:119中任一项所示的氨基酸序列。
  10. 根据权利要求7-9中任一项所述的分离的抗原结合蛋白，其中所述LCDR1包含SEQ ID NO:1、SEQ ID NO:11、SEQ ID NO:18、SEQ ID NO:26、SEQ ID NO:34、SEQ ID NO:41、SEQ ID NO:59和SEQ ID NO:111中任一项所示的氨基酸序列。
  11. 根据权利要求7-10中任一项所述的分离的抗原结合蛋白，其中所述LCDR2包含SEQ ID NO:120-SEQ ID NO:121中任一项所示的氨基酸序列。
  12. 根据权利要求7-11中任一项所述的分离的抗原结合蛋白，其中所述LCDR2包含SEQ ID NO:2、SEQ ID NO:12、SEQ ID NO:19、SEQ ID NO:27、SEQ ID NO:35、SEQ ID NO:42、SEQ ID NO:60和SEQ ID NO:112中任一项所示的氨基酸序列。
  13. 根据权利要求3-12中任一项所述的分离的抗原结合蛋白，其中所述VH包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:5、SEQ ID NO:22、SEQ ID NO:30、SEQ ID NO:45、SEQ ID NO:51、SEQ ID NO:55、SEQ ID NO:63和SEQ ID NO:114中任一项所示的氨基酸序列。
  14. 根据权利要求13所述的分离的抗原结合蛋白，其中所述HCDR2包含SEQ ID NO:6、SEQ ID NO:9、SEQ ID NO:15、SEQ ID NO:23、SEQ ID NO:31、SEQ ID NO:38、SEQ ID NO:46、SEQ ID NO:52、SEQ ID NO:56、SEQ ID NO:64、SEQ ID NO:67和SEQ ID NO:115中任一项所示的氨基酸序列。
  15. 根据权利要求13-14中任一项所述的分离的抗原结合蛋白，其中所述HCDR3包含SEQ ID NO:7、SEQ ID NO:16、SEQ ID NO:24、SEQ ID NO:32、SEQ ID NO:39、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:53、SEQ ID NO:57、SEQ ID NO:65、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72和SEQ ID NO:116中任一项所示的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的分离的抗原结合蛋白，其中所述VL包括框架区L-FR1，L-FR2，L-FR3，和L-FR4。
  17. 根据权利要求16所述的分离的抗原结合蛋白，其中所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:74-SEQ ID NO:75中任一项所示的氨基酸序列。
  18. 根据权利要求16-17中任一项所述的分离的抗原结合蛋白，其中所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:76、SEQ ID NO:77和SEQ ID NO:78中任一项所示的氨基酸序列。
  19. 根据权利要求16-18中任一项所述的分离的抗原结合蛋白，其中所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:79、SEQ ID NO:80、SEQ ID NO:81和SEQ ID NO:82中任一项所示的氨基酸序列。
  20. 根据权利要求16-19中任一项所述的分离的抗原结合蛋白，其中所述L-FR4的N末端与所述LCDR3的C末端直接或间接相连，且所述L-FR4包含SEQ ID NO:83、SEQ ID NO:84、SEQ ID NO:85和SEQ ID NO:86中任一项所示的氨基酸序列。
  21. 根据权利要求2-20中任一项所述的分离的抗原结合蛋白，其中所述VL包含SEQ ID NO:124或SEQ ID NO:125所示的氨基酸序列。
  22. 根据权利要求2-21中任一项所述的分离的抗原结合蛋白，其中所述VL包含SEQ ID NO:4、SEQ ID NO:14、SEQ ID NO:21、SEQ ID NO:29、SEQ ID NO:37、SEQ ID NO:44、SEQ ID NO:62和SEQ ID NO:113中任一项所示的氨基酸序列。
  23. 根据权利要求4-22中任一项所述的分离的抗原结合蛋白，其包括抗体轻链恒定区，且所述抗体轻链恒定区包括人Igκ恒定区或人Igλ恒定区。
  24. 根据权利要求3-23中任一项所述的分离的抗原结合蛋白，其中所述VH包括框架区H-FR1，H-FR2，H-FR3，和H-FR4。
  25. 根据权利要求24所述的分离的抗原结合蛋白，其中所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91、SEQ ID NO:92、SEQ ID NO:93和SEQ ID NO:94中任一项所示的氨基酸序列。
  26. 根据权利要求24-25中任一项所述的分离的抗原结合蛋白，其中所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:95、SEQ ID NO:96、SEQ ID NO:97和SEQ ID NO:98中任一项所示的氨基酸序列。
  27. 根据权利要求24-26中任一项所述的分离的抗原结合蛋白，其中所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104、SEQ ID NO:105和SEQ ID NO:106中任一项所示的氨基酸序列。
  28. 根据权利要求24-27中任一项所述的分离的抗原结合蛋白，其中所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO:107、SEQ ID NO:108、SEQ ID NO:109和SEQ ID NO:110中任一项所示的氨基酸序列。
  29. 根据权利要求3-28中任一项所述的分离的抗原结合蛋白，其中所述VH包含SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:17、SEQ ID NO:25、SEQ ID NO:33、SEQ ID NO: 40、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:54、SEQ ID NO:58、SEQ ID NO:66、SEQ ID NO:69、SEQ ID NO:71和SEQ ID NO:73中任一项所示的氨基酸序列。
  30. 根据权利要求4-29中任一项所述的分离的抗原结合蛋白，其包括抗体重链恒定区，且所述抗体重链恒定区包括人IgG恒定区。
  31. 根据权利要求4-30中任一项所述的分离的抗原结合蛋白，其包括抗体重链恒定区，且所述抗体重链恒定区包括人IgG1恒定区。
  32. 双特异性抗原结合蛋白，其特异性结合SARS-CoV-2的S蛋白的RBD和SARS-CoV的S蛋白的RBD。
  33. 根据权利要求32所述的双特异性抗原结合蛋白，其包含特异性结合SARS-CoV-2的S蛋白的RBD的第一靶向部分，其中所述第一靶向部分包含权利要求1-31中任一项所述的分离的抗原结合蛋白。
  34. 根据权利要求32-33中任一项所述的双特异性抗原结合蛋白，其包含特异性结合SARS-CoV的S蛋白的RBD的第二靶向部分，其中所述第二靶向部分包含权利要求1-31中任一项所述的分离的抗原结合蛋白。
  35. 根据权利要求32-34中任一项所述的双特异性抗原结合蛋白，其包括抗体。
  36. 根据权利要求34-35所述的双特异性抗原结合蛋白，其中所述第一靶向部分包含第一重链和第一轻链，所述第二靶向部分包含第二重链和第二轻链，其中所述第一轻链和所述第二轻链相同。
  37. 根据权利要求36所述的双特异性抗原结合蛋白，其中所述第一轻链和所述第二轻链包含SEQ ID NO:44所示的氨基酸序列。
  38. 根据权利要求36-37中任一项所述的双特异性抗原结合蛋白，其中所述第一重链的VH包含SEQ ID NO:48所示的氨基酸序列。
  39. 根据权利要求36-38中任一项所述的双特异性抗原结合蛋白，其中所述第二重链的VH包含SEQ ID NO:50所示的氨基酸序列。
  40. 根据权利要求36-39中任一项所述的双特异性抗原结合蛋白，其中所述第一重链的VH包含SEQ ID NO:48所示的氨基酸序列，且所述第二重链的VH包含SEQ ID NO:50所示的氨基酸序列。
  41. 分离的一种或多种核酸分子，其编码权利要求1-31中任一项所述的分离的抗原结合蛋白，和/或权利要求32-40中任一项所述的双特异性抗原结合蛋白。
  42. 载体，其包含根据权利要求41所述的核酸分子。
  43. 细胞，其包含根据权利要求41所述的核酸分子或根据权利要求42所述的载体。
  44. 制备权利要求1-31中任一项所述的分离的抗原结合蛋白、权利要求32-40中任一项所述的双特异性抗原结合蛋白的方法，所述方法包括在使得权利要求1-31中任一项所述的分离的抗原结合蛋白和/或根据权利要求32-40中任一项所述的双特异性抗原结合蛋白表达的条件下，培养根据权利要求43所述的细胞。
  45. 药物组合物，其包含权利要求1-31中任一项所述的分离的抗原结合蛋白、权利要求32-40中任一项所述的双特异性抗原结合蛋白、权利要求41所述的核酸分子、权利要求42所述的载体和/或权利要求43所述的细胞，以及任选地药学上可接受的佐剂。
  46. 权利要求1-31中任一项所述的分离的抗原结合蛋白、权利要求32-40中任一项所述的双特异性抗原结合蛋白、权利要求41所述的核酸分子、权利要求42所述的载体、权利要求43所述的细胞和/或权利要求45所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗冠状病毒的感染。
  47. 根据权利要求46所述的用途，其中所述冠状病毒的感染包括COVID-19。
  48. 阻断SARS-CoV-2的S蛋白的RBD或其突变体与人ACE2的结合的方法，其包括以下的步骤，施用权利要求1-31中任一项所述的分离的抗原结合蛋白、权利要求32-40中任一项所述的双特异性抗原结合蛋白、权利要求41所述的核酸分子、权利要求42所述的载体、权利要求43所述的细胞和/或权利要求45所述的药物组合物。
  49. 阻断SARS-CoV的S蛋白的RBD与人ACE2的结合的方法，其包括以下的步骤，施用权利要求1-31中任一项所述的分离的抗原结合蛋白、权利要求32-40中任一项所述的双特异性抗原结合蛋白、权利要求41所述的核酸分子、权利要求42所述的载体、权利要求43所述的细胞和/或权利要求45所述的药物组合物。
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