TW202144406A - Sars-cov-2 antibody and the application thereof - Google Patents

Abstract

The application provides a SARS-CoV-2 antibody and the application thereof. The SARS-CoV-2 antibody of the application may have one or more of the following properties: blocking the binding of the RBD of the S protein or the mutant thereof of SARS-CoV-2 to human ACE2; blocking binding of the RBD of the S protein of SARS-CoV to human ACE2; specifically binding to the RBD of the S protein of SARS-CoV-2 or the mutant thereof with a very low KD value; having stronger affinity with the RBD of the S protein of SARS-CoV-2 than human ACE2-Fc; specifically binding to RBD of the S protein of SARS-CoV; having hydrophilicity; and being conducive to preparation and purification; having good thermal stability; having the activity of neutralizing SARS-CoV-2. The application also provides the preparation method thereof and the applications of the SARS-CoV-2 antibody.

Description

SARS-CoV-2 antibodies and their applications

The present application relates to the field of biomedicine, in particular to a SARS-CoV-2 antibody and its application.

The novel coronavirus (SARS-CoV-2) has a long incubation period, high invisibility and strong infectivity, and has rapidly spread around the world. As of May 27, 2020, 5.5 million people have been infected and 340,000 people have died; it has become a world health field public health emergencies. At the moment when the epidemic prevention and control situation is so severe, there is still no specific drug for the new coronavirus, so it is extremely urgent to develop specific antibody drugs for the prevention and treatment of the new coronavirus.

The new coronavirus is an enveloped virus with a positive RNA genome and belongs to the β genus of the Coronaviridae Nidovirales suborder. At present, there have been many cases of clinical treatment of critically ill patients with the use of convalescent specific plasma with remarkable results. However, after all, the source of plasma products is limited, and they must undergo strict blood biosafety testing before they can be used in clinical practice, which has not fully met the needs of the current epidemic prevention and control.

The present application provides a SARS-CoV-2 antibody and application thereof. The SARS-CoV-2 antibody of the present application may have one or more of the following properties: block the binding of the RBD of the S protein of SARS-CoV-2 or a mutant thereof to human ACE2; block the S protein of SARS-CoV-2 The RBD of SARS-CoV-2 binds to human ACE2; it specifically binds to the RBD of the S protein of SARS-CoV-2 or its mutant with a very low K _D value; the affinity to the RBD of the S protein of SARS-CoV-2 is stronger than that of human ACE2 -Fc; RBD that specifically binds to the S protein of SARS-CoV; has hydrophilicity; facilitates preparation and purification; good stability, especially thermal stability; has the activity of neutralizing SARS-CoV-2. The present application also provides preparation methods and applications related to the SARS-CoV-2 antibody.

In one aspect, the application provides an isolated antigen-binding protein having one or more of the following properties: blocking the binding of the RBD of the S protein of SARS-CoV-2 or a mutant thereof to human ACE2; blocking SARS-CoV-2 Binding of the RBD of the S protein of CoV to 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 about 5.0*10-8 M; in the Octet assay, with} The K _D value below about 6.0*10 ^-10 M specifically binds to the mutant of the RBD of the S protein of SARS-CoV-2; the affinity to the RBD of the S protein of SARS-CoV-2 is stronger than that of human ACE2-Fc; specific RBD of S protein that binds SARS-CoV; hydrophilic; main peak of charge heterogeneity analysis is about 45%-85%; Tm is at least about 75°C in Thermal shift assay; has neutralizing SARS-CoV-2 activity .

In certain embodiments, the isolated antigen binding protein comprises at least one CDR in the VL of the light chain variable region, the VL comprising the amino acid sequence set forth in SEQ ID NO:124 or SEQ ID NO:125.

In certain embodiments, the isolated antigen binding protein comprises at least one CDR in a heavy chain variable region VH comprising 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 The amino acid sequence set forth in any one of 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 .

In certain embodiments, the isolated antigen-binding protein comprises an antibody or antigen-binding fragment thereof.

In certain embodiments, the antigen-binding fragment comprises a Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and/or dAb.

In certain embodiments, the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR3 comprising the amino acid sequence set forth in any one of SEQ ID NO:122-SEQ ID NO:123.

In certain embodiments, the LCDR3 comprises 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.

In certain embodiments, the LCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:118-SEQ ID NO:119.

In certain embodiments, the LCDR1 comprises 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 the amino acid sequence shown in any one of SEQ ID NO: 111.

In certain embodiments, the LCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NO:120-SEQ ID NO:121.

In certain embodiments, the LCDR2 comprises 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 the amino acid sequence shown in any one of SEQ ID NO: 112.

In certain embodiments, the VH comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises SEQ ID NO:5, SEQ ID NO:22, SEQ ID NO:30, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:51 The amino acid sequence shown in any one of ID NO:55, SEQ ID NO:63, and SEQ ID NO:114.

In certain embodiments, the HCDR2 comprises 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. The amino acid sequence of any one of SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:67, and SEQ ID NO:115.

In certain embodiments, the HCDR3 comprises 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. Set forth in any one of 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 amino acid sequence.

In certain embodiments, the VL includes framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

In certain embodiments, the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, and the L-FR1 comprises an amine set forth in any one of SEQ ID NO:74-SEQ ID NO:75 base acid sequence.

In certain embodiments, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the set forth in any one of SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78 amino acid sequence.

In certain embodiments, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, and SEQ ID NO:82 Any of the amino acid sequences shown.

In certain embodiments, the N-terminus of the L-FR4 is directly or indirectly linked to the C-terminus of the LCDR3, and the L-FR4 comprises SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 85 The amino acid sequence shown in any of ID NO:86.

In certain embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO:124 or SEQ ID NO:125.

In certain embodiments, the VL comprises 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 the amino acid sequence shown in any one of SEQ ID NO: 113.

In certain embodiments, the isolated antigen binding protein comprises an antibody light chain constant region, and the antibody light chain constant region comprises a human Igκ constant region or a human Igλ constant region.

In certain embodiments, the VH includes framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

In certain embodiments, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 89, 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.

In certain embodiments, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98 Any of the amino acid sequences shown.

In certain embodiments, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, The amino acid sequence set forth in any one of SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 106.

In certain embodiments, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 109 The amino acid sequence shown in any of ID NO: 110.

In certain embodiments, the VH comprises 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. Set forth in any one of 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 amino acid sequence.

In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a human IgG constant region.

In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a human IgGl constant region.

In another aspect, 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.

In certain embodiments, the bispecific antigen binding protein comprises a first targeting moiety that specifically binds the RBD of the S protein of SARS-CoV-2, wherein the first targeting moiety comprises an isolated antigen binding agent of the present application protein.

In certain embodiments, 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 an isolated antigen binding protein of the present application.

In certain embodiments, the bispecific antigen binding protein comprises an antibody.

In certain embodiments, 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 and the first light chain The two light chains are the same.

In certain embodiments, the first light chain and the second light chain comprise the amino acid sequence set forth in SEQ ID NO:44.

In certain embodiments, the VH of the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO:48.

In certain embodiments, the VH of the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO:50.

In certain embodiments, the VH of the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO:48, and the VH of the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO:50.

In another aspect, the present application provides an isolated one or more nucleic acid molecules encoding the isolated antigen binding proteins of the present application, and/or the bispecific antigen binding proteins of the present application.

In another aspect, the present application provides a vector comprising the nucleic acid molecule of the present application.

In another aspect, the present application provides a cell comprising the nucleic acid molecule of the present application or the vector of the present application.

In another aspect, the present application provides a method for preparing the isolated antigen-binding protein of the present application, the bispecific antigen-binding protein of the present application, the method comprising making the isolated antigen-binding protein of the present application The cells of the present application are cultured under conditions in which the original binding protein and/or the bispecific antigen-binding protein of the present application is expressed.

In another aspect, the present application provides a pharmaceutical composition comprising the isolated antigen-binding protein of the present application, the bispecific antigen-binding protein of the present application, the nucleic acid molecule of the present application, the carrier of the present application and/or the cell of the present application , and optionally pharmaceutically acceptable adjuvants.

In another aspect, the present application provides an isolated antigen-binding protein of the present application, a bispecific antigen-binding protein of the present application, a nucleic acid molecule of the present application, a carrier of the present application, a cell of the present application and/or the pharmaceutical composition of the present application Use of the compound in the preparation of a medicament for preventing, relieving and/or treating coronavirus infection.

In certain embodiments, the coronavirus infection includes COVID-19.

On the other hand, the present application provides a method for blocking the binding of the RBD of the S protein of SARS-CoV-2 or its mutant to human ACE2, comprising the following steps, administering the isolated antigen-binding protein of the present application, the present application The bispecific antigen binding protein of the present application, the nucleic acid molecule of the present application, the vector of the present application, the cell of the present application and/or the pharmaceutical composition of the present application.

On the other hand, the present application provides a method for blocking the binding of the RBD of the S protein of SARS-CoV to human ACE2, comprising the steps of applying the isolated antigen-binding protein of the present application, the bispecific antigen-binding protein of the present application The protein, the nucleic acid molecule of the present application, the vector of the present application, the cell of the present application and/or the pharmaceutical composition of the present application.

Other aspects and advantages of the present application can be readily appreciated by those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to Changes may be made to the disclosed embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the drawings and descriptions in the specification of the present application are exemplary only and not restrictive.

The specific features of the invention to which this application relates are set forth in the appended claims. A better understanding of the features and advantages of the inventions involved in this application can be obtained by reference to the exemplary embodiments and drawings described in detail hereinafter. The picture is a brief description as follows:

Figures 1A to 1D show the detection results of the binding and dissociation rates of the antibodies of the present application to SARS-CoV-2 S1.

Figure 2 shows that the antibody of the present application blocks the binding of SARS-CoV-2 S1 to hACE2-Fc.

Figure 3 shows that the antibody of the present application blocks the binding of SARS-CoV S1 to hACE2.

Figure 4 shows the coefficient of hydrophilicity of the antibody of the present application.

Figure 5 shows the results of the antibody charge heterogeneity analysis of the present application.

Figure 6 shows the results of the antibody charge heterogeneity profiling of the present application.

Figure 7 shows the melting temperature of the antibody (Fab) of the present application.

FIG. 8A and FIG. 8B show the results of the virus neutralization activity experiment of the antibody of the present application.

FIG. 9A and FIG. 9B show the results of the virus neutralization activity experiment of the antibody of the present application.

Figure 10 shows the results of the half-reduced neutralization concentration assay of the antibody plaques of the present application.

Figure 11 shows the detection results of the binding and dissociation rates of the antibody of the present application to each mutant of SARS-CoV-2 S1 RBD.

12A to 12E show the neutralizing antibodies of the present application block S1 RBD 2 SARS-CoV-mutations and the IC ₅₀ value curve hACE2 body and binding.

The embodiments of the invention of the present application are described below with specific specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the contents disclosed in this specification.

Definition of Terms

In this application, the term "SARS-CoV" generally refers to SARS coronavirus, namely severe acute respiratory syndrome coronavirus (Severe acute respiratory syndrome coronavirus in English), which belongs to the genus Betacoronavirus of the family Coronaviridae ( Betacoronavirus ) subgenus Sarbecovirus (Sarbecovirus).

In this application, the term "SARS-CoV-2" generally 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 coronavirus family (Coronaviridae) betacoronavirus genus (Betacoronavirus) Sable virus subgenus (Sarbecovirus). SARS-CoV-2 is an enveloped, non-segmented positive-stranded single-stranded RNA virus. SARS-CoV-2 can cause novel coronavirus pneumonia (COVID-19). In the present application, the SARS-CoV-2 may include the S protein (spike protein).

In this application, the term "coronavirus S protein" generally refers to the spike protein of the coronavirus. The S protein can be assembled into trimers, which contain about 1300 amino acids. The S protein may belong to a Class I viral fusion protein. The S protein may generally contain two subunits, S1 and S2. S1 mainly contains the receptor binding domain (RBD), which can be responsible for recognizing receptors of cells. S2 contains the basic elements required for the membrane fusion process, including an intrinsic membrane fusion peptide (fusion peptide), two 7-peptide repeats (heptad repeats, HR), a membrane-adjacent region rich in aromatic amino acids (membrane proximal external region, MPER), and transmembrane region (transmembrane, TM). S1 protein can be further divided into two domains, namely N-terminal domain (NTD) and C-terminal domain (CTD). The S protein can determine the host range and specificity of viruses (such as the coronavirus SARS-CoV-2), and can also be an important site of action for host-neutralizing antibodies, and/or a key target for vaccine design. The S protein can be the S protein of SARS-CoV-2, for example, its structure can be found in Daniel Wrapp et al., Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation, Science .

In the present application, the term "ACE2" generally refers to angiotensin-converting enzyme II (Angiotensin-converting enzyme 2) or a functional fragment thereof. The angiotensin-converting enzyme II is an exopeptidase that catalyzes the conversion of angiotensin I to angiotensin-(1-9) or angiotensin II to angiotensin-(1-7). The ACE2 can include an N-terminal PD domain (peptidase domain, peptidase domain) and a C-terminal CLD domain (Collectrin-like domain). The angiotensin-converting enzyme II can be a receptor for SARS coronavirus (SARS-CoV) or severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), eg, the extracellular domain of ACE2 (eg, The PD region of the ACE2) can bind to the RBD of the S protein of the coronavirus. The accession number of human angiotensin-converting enzyme II in the UniProt database is Q9BYF1. The human ACE2 gene can contain 18 exons, see 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, Table 1 of 2000. In the present application, the functional fragment of the ACE2 protein can include truncations or variants of the complete ACE2 protein, as long as the functional fragment still has the ability to function as a coronavirus (eg, SARS-CoV and/or SARS-CoV-2) receptor function.

In the present application, the term "ACE2-Fc" generally refers to a fusion protein comprising the binding protein of the S protein of the present application or a functional fragment thereof and the Fc region of an IgG antibody. In the present application, the binding protein of the S protein or its functional fragment is directly or indirectly linked to the Fc region of the IgG antibody. For example, the Fc region of the IgG antibody can be located at the C-terminus of the ACE2-Fc fusion protein. In the present application, the IgG may be IgG1, for example, may be human IgG1.

In the present application, the term "coronavirus" generally refers to a virus belonging to the mesh mode (the Nidovirales) coronavirus family (Coronaviridae) Coronavirus (Coronavirus) virus. 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 can have a methylated cap structure at the 5' end and a poly(A) tail at the 3' end, and the full length of the genome is about 27-32 kb. In this application, the coronavirus includes severe acute respiratory syndrome-related coronavirus, namely Severe acute respiratory syndrome-related coronavirus, which is a species of the genus betacoronavirus of the family Coronaviridae. In this application, 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).

In this application, the term "mutant" generally refers to a mutated amino acid sequence by deletion, insertion or substitution of one or more selected amino acids. In the present application, the mutant may comprise at least about 90% (eg, at least about 95%, at least about 96%, at least about 97%) compared to 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. For example, 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 enhance the structure stability and infectivity of the spike protein, bioRxiv , 2020.

In this application, the term "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, and some can progress to viral pneumonia, multiple organ failure, or cytokine storm. The disease spreads primarily through close person-to-person contact, such as through small droplets produced by coughing, sneezing and talking. The World Health Organization declared the outbreak of COVID-19 a pandemic on March 11, 2020. There is currently no vaccine or specific treatment available for COVID-19.

In this application, the term "antigen-binding protein" generally refers to a protein comprising an antigen-binding moiety, as well as a scaffold or backbone moiety that optionally allows the antigen-binding moiety to adopt a conformation that facilitates binding of the antigen-binding protein to the antigen. Examples of 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 dAbs), immunoconjugates antibodies, multispecific antibodies (eg, bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.

In this application, the term "Fab" generally refers to a fragment containing the variable domain of the heavy chain and the variable domain of the light chain, and also containing the constant domain of the light chain and the first constant domain (CH1) of the heavy chain The term "Fab" generally refers to a fragment that differs from Fab by adding a small number of residues (including one or more cysteines from the antibody hinge region) to the carboxy terminus of the heavy chain CH1 domain; the term "F( ab') ₂ " generally refers to a dimer of Fab', an antibody fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region. The term "Fv" generally refers to a complete antigen recognition and binding site containing The smallest antibody fragment.In some cases, the fragment may consist of a heavy chain variable region and a light chain variable region in a tightly non-covalently bound dimer; the term "dsFv" generally refers to disulfide bonds A stable Fv fragment, the bond between its single light chain variable region and single heavy chain variable region is a disulfide bond. The term "dAb fragment" generally refers to an antibody fragment consisting of a VH domain. In this application, the term "scFv" generally refers to a monovalent molecules heavy chain variable domain and a light chain variable domain of the antibody by a flexible peptide linker pair to form a covalent linkage; such scFv molecules can have the general structure: NH ₂ -VL- linker -VH-COOH or NH ₂ -VH- linker -VL-COOH.

In this application, the term "antibody" is used in the broadest sense and specifically encompasses, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies comprising two light chains and two heavy chains), polyclonal antibodies, Multispecific antibodies (eg bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies and camelized single domain antibodies. An "antibody" may generally comprise a protein comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region. In certain naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region comprises three domains, CH1, CH2 and CH3. In certain naturally occurring antibodies, each light chain comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which alternate with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four framework regions (FRs), arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable domains of native heavy and light chains each comprise four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4) , mostly adopt a β-sheet configuration, connected by three CDRs, forming loop connections, and in some cases forming part of the β-sheet structure. The CDRs in each chain are brought together in close proximity by the FR regions and form together with the CDRs from the other chain the antigen-binding site of the antibody. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

In this application, the term "variable" generally refers to the fact that some portion of the sequence of the variable domains of an antibody varies strongly which contributes to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments in the light and heavy chain variable regions, called complementarity determining regions (CDRs) or hypervariable regions (HVRs). The more highly conserved portion of the variable domain is called the framework (FR). In the art, the CDRs of antibodies can be defined by a variety of methods, such as the Kabat definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Besse 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 and KABAT Definition Rules for IMGT Scientific Chart Rules.

IMGT refers to the International ImMunoGeneTics Information System, a global reference database for immunogenetics and immunoinformatics (http://www.imgt.org). IMGT specializes in immunoglobulins (IG) or antibodies from humans and other vertebrates, T cell receptors (TR), major histocompatibility (MH), and the immunoglobulin superfamily from vertebrates and invertebrates (IgSF), MH superfamily (MhSF), and immune system-related proteins (RPI).

In this application, the term "isolated" antigen binding protein generally refers to an antigen binding protein that has been identified, isolated and/or recovered from components of the environment in which it is produced (eg, native or recombinant). Contaminant components of its producing environment are often substances that interfere with its research, diagnostic or therapeutic use and can include Enzymes, hormones and other proteinaceous or non-proteinaceous solutes. An isolated antigen binding protein or antibody will generally be prepared by at least one purification step.

In this application, the term "monoclonal antibody" generally refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies in the population are identical except for minor natural mutations that may be present. Monoclonal antibodies are generally highly specific for a single antigenic site. Also, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by fusion tumor culture without contamination by other immunoglobulins. The modifier "monoclonal" denotes a characteristic of an antibody obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies used in the present application can be made in fusionoma cells, or can be made by recombinant DNA methods.

In this application, the term "chimeric antibody" generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species. Typically, the variable regions are derived from antibodies from experimental animals such as rodents ("parental antibodies"), and the constant regions are derived from human antibodies, such that the resulting chimeric antibody is less robust in humans than the parental (eg, mouse-derived) antibody There is a reduced likelihood of eliciting an adverse immune response in an individual.

In this application, the term "humanized antibody" generally refers to a non-human antibody (eg, a mouse antibody) in which some or all of the amino acids outside the CDR regions have been replaced by corresponding amino acids derived from human immunoglobulins Substituted antibody. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions may also be permissible as long as they still retain the ability of the antibody to bind to a particular antigen. Humanized antibodies optionally contain at least a portion of a human immunoglobulin constant region. A "humanized antibody" retains antigenic specificity similar to the original antibody. "Humanized" forms of non-human (eg, murine) antibodies may minimally comprise chimeric antibodies that contain sequences derived from non-human immunoglobulins. In some cases, human immunoglobulin (receptor The CDR region residues in the antibody) are CDR region residues of a non-human species (donor antibody) (such as mouse, rat, rabbit or non-human primate) having the desired properties, affinity and/or ability base replacement. In certain instances, FR region residues of the human immunoglobulin can be replaced with corresponding non-human residues. In addition, humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve antibody properties, such as binding affinity.

In the present application, the term "fully human antibody" generally refers to the antibody expressed by the human antibody gene-encoding gene transferred into a genetically engineered antibody gene-deficient animal. All parts of an 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 to the human body. Methods for obtaining fully human antibodies in the art include phage display technology, transgenic mouse technology, ribosome display technology and RNA-polypeptide technology.

In this application, the terms "binding", "specific binding" or "specific for" generally refer to a measurable and reproducible interaction, such as the binding between an antigen and an antibody, which can be determined at The presence of a target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody binds to an epitope via its antigen binding domain, and this binding requires some complementarity between the antigen binding domain and the epitope. For example, an antibody that specifically binds a target (which may be an epitope) is an antibody that binds to that target with greater affinity, avidity, easier, and/or for a greater duration than it binds to other targets. An antibody is said to "specifically bind" to an antigen when it binds an epitope more readily through its antigen-binding domain than it would bind a random, unrelated epitope. An "epitope" refers to a specific atomic group (eg, sugar side chain, phosphonium, sulfonyl) or amino acid on an antigen to which an antigen-binding protein (eg, an antibody) binds.

In the present application, the term "KD", "K _D" are used interchangeably, generally refers to the equilibrium dissociation constant, "KD" is the dissociation rate constant (Kdis, also referred to as "dissociation rate (off-rate) (koff) " or "kd") to the ratio of the association rate constant (kon, also known as "association rate (kon)" or "ka"). The association rate constant may be used (kon), dissociation rate constant (Kdis) and the equilibrium dissociation constant (K _D) represents the antigen binding protein (e.g. an antibody) binding affinity for the antigen. Methods for determining association and dissociation rate constants are well known in the art and include, but are 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 wafer techniques. The measured affinity for a particular protein-protein interaction can vary if measured under different conditions (eg, salt concentration, pH).

In the present application, the term "reference antibody" generally refers to an antibody with which the antigen-binding protein of the present application competes for binding to an antigen (eg, the RBD of the S protein of SARS-CoV-2).

In this application, the term "CDC" generally refers to the process initiated by the binding of complement factor C1q to the Fc portion of most IgG antibody subclasses. Binding of C1q to the antibody may result from defined protein-protein interactions of the binding site site of the Fc moiety. The binding site sites of these Fc moieties may comprise amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbered according to the EU index of Kabat). Antibodies of the IgGl, IgG2, and IgG3 subtypes can generally exhibit complement priming including C1q and C3 binding, whereas IgG4 does not prime the complement system and may not bind Clq and/or C3.

In this application, the term "ADCC" or "antibody-dependent cell-mediated cytotoxicity" generally refers to a form of cytotoxicity in which some secreted immunoglobulins bind to certain cytotoxic effector cells (eg, NK cells, Fc receptors (FcRs) on neutrophils and macrophages), enabling these cytotoxic effector cells to specifically bind antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Primary cells that mediate ADCC (eg, NK cells) express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII (see table in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991) p. 464) 3). In vitro and/or in vivo cytotoxicity assays can be performed To assess ADCC activity of a molecule of interest, for example, in vitro ADCC assays can be performed as described in US Pat. No. 5,500,362 or No. 5,821,337 or US Pat. No. 6,737,056 (Presta). Useful effector cells for such assays include PBMC and NK cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, eg, in animal models such as those disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998). For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcyR binding (and thus likely lacks ADCC activity), but retains FcRn binding ability.

ADCC activity can be reduced by modifying the Fc region. In certain instances, sites that affect binding to Fc receptors can be removed, eg, to remove sites that are not salvage receptor binding sites. In certain instances, the Fc region can be modified to remove the ADCC site. ADCC sites are known in the art, see eg, Sarmay et al. (1992) Molec. Immunol. 29(5):633-9 for ADCC sites of IgGl.

In this application, the term "between" generally 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 its N-terminus is connected to the first amino acid fragment. The C-terminus of the diamino acid fragment is linked directly or indirectly. In the light chain, for example, the N-terminus of the L-FR2 is directly or indirectly linked to the C-terminus of the LCDR1, and the C-terminus of the L-FR2 is directly or indirectly linked to the N-terminus of the LCDR2. For another example, the N-terminus of L-FR3 is directly or indirectly connected to the C-terminus of LCDR2, and the C-terminus of L-FR3 is directly or indirectly connected to the N-terminus of LCDR3. In the heavy chain, for example, the N-terminus of the H-FR2 is directly or indirectly linked to the C-terminus of the HCDR1, and the C-terminus of the H-FR2 is directly or indirectly linked to the N-terminus of the HCDR2. In another example, the N-terminus of the H-FR3 is directly or indirectly connected to the C-terminus of the HCDR2, and the C-terminus of the H-FR3 is directly or indirectly connected to the N-terminus of the HCDR3. In the present application, the "first amino acid fragment" and the "second amino acid fragment" may be the same or different amino acid fragments.

In this application, the term "isolated" antigen binding protein generally refers to an antigen binding protein that has been identified, isolated and/or recovered from components of the environment in which it is produced (eg, native or recombinant). Contaminant components of its producing environment are often substances that interfere with its research, diagnostic or therapeutic use, and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. An isolated antigen binding protein or antibody will generally be prepared by at least one purification step.

In this application, the term "isolated nucleic acid molecule" or "isolated polynucleotide" generally refers to DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, which is not related to the polynucleus found in nature All or a portion of the nucleotides are associated, or linked, to polynucleotides to which they are not linked in nature.

In this application, 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. Such vectors may include vectors primarily used for insertion of DNA or RNA into cells, vectors primarily used for replication of DNA or RNA, and vectors primarily for expression of transcription and/or translation of DNA or RNA. The vector also includes a vector having a variety of the above-mentioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce the desired expression product by culturing a suitable host cell containing the vector.

In the present application, the term "cell" generally refers to an individual cell, cell line or cell culture that can or has contained a plasmid or vector comprising a nucleic acid molecule of the present application, or capable of expressing an antibody or antigen-binding fragment thereof of the present application. The cells can include progeny of a single host cell. Due to natural, accidental or intentional mutations, the progeny cells may not necessarily be morphologically or genomically identical to the original parental cells, but are capable of expressing the antibodies of the present application or antigen-binding fragments thereof. The cells can be obtained by transfecting cells in vitro with the vector of the present application. The cell may be a prokaryotic cell (such as a colon bacteria), but also eukaryotic cells (eg, yeast cells such as COS cells, Chinese Hamster Ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells, or myeloma cells). In certain instances, the cell can be a mammalian cell. For example, the mammalian cells can be CHO-K1 cells. In this application, the term "recombinant cell" generally refers to a cell into which a recombinant expression vector has been introduced. The recombinant host cell includes not only a specific cell, but also progeny of these cells.

In this application, the term "pharmaceutically acceptable adjuvant" generally includes a pharmaceutically acceptable carrier, excipient or stabilizer that is non-toxic to the cells or mammals to which it is exposed at the doses and concentrations employed of. Typically, the physiologically acceptable carrier is a pH buffered aqueous solution. Examples of physiologically acceptable carriers can include buffers, antioxidants, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions and/or nonionics Surfactant.

As used herein, the terms "administering" and "treating" refer to the application of an exogenous drug, therapeutic agent, diagnostic agent or composition to an animal, human, subject, cell, tissue, organ or biological fluid. "Administering" and "treatment" can refer to therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of cells includes contact of reagents with cells, as well as contact of reagents with fluids, and contact of fluids with cells. "Administering" and "treating" also mean in vitro and ex vivo treatment by an agent, diagnostic, binding composition, or by another cell. "Treatment" when applied to humans, animals, or research subjects means therapeutic treatment, prophylactic or preventive measures, research and diagnosis; , contact of cells, tissues, physiological compartments or physiological fluids.

As used herein, the term "treatment" refers to the administration of an internal or external therapeutic agent, including any one of the SARS-CoV-2 antigen binding proteins and compositions thereof of the present application, to a patient with one or more disease symptoms for which the treatment is known Drugs have a therapeutic effect on these symptoms. Typically, a patient is administered to a patient in an amount of the therapeutic agent effective to alleviate one or more symptoms of the disease (therapeutically effective amount). Desired effects of treatment include reducing Low rate of disease progression, improvement or alleviation of disease state, and regression or improved prognosis. For example, if one or more symptoms associated with cancer are alleviated or eliminated, including, but not limited to, reducing (or destroying) cancer cell proliferation, reducing disease-derived symptoms, improving the quality of life of those suffering from the disease, reducing Dosages of other drugs needed to treat the disease, delay the progression of the disease, and/or prolong the survival of the individual, then the individual is successfully "treated."

In this application, the term "comprising" generally refers to the meaning of including, encompassing, containing or encompassing. In some cases, the meaning of "for" and "consisting of" is also expressed.

In this application, 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%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

[Detailed description of the invention]

antigen binding protein

In one aspect, the application provides an isolated antigen binding protein having one or more of the following properties:

Block the binding of the RBD of the S protein of SARS-CoV-2 or its mutants to human ACE2;

Block the binding of the RBD of the S protein of SARS-CoV to human ACE2;

In the Octet measurement, about 5.0* ^10-8 M or less (for example, it may 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 or approx. The K _D value of 1.0*10 ^-10 M or less) specifically binds to the RBD of the S protein of SARS-CoV-2;

In the Octet measurement, about 6.0* ^10-10 M or less (for example, it may 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 M or less ^-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 or A mutant of RBD that specifically binds to the S protein of SARS-CoV-2 with a K _D value of about 1.5*10 ^-10 M or less;

The affinity with the RBD of the S protein of SARS-CoV-2 is stronger than that of human ACE2-Fc; the RBD of the S protein of SARS-CoV specifically binds;

hydrophilic;

The charge heterogeneity analysis main peak is about 45%-85% (for example, it can be about 45%-about 85%, about 45%-about 84%, about 45%-about 83%, about 45%-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%);

In a Thermal shift assay, the Tm is at least about 75°C (eg, 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);

Has the activity of neutralizing SARS-CoV-2.

The isolated antigen-binding protein of the present 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, and the reference antibody The heavy chain variable region of the HCDR1 may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:5, and the HCDR2 may comprise the amine shown in SEQ ID NO:6 and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 7, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2 and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3.

The isolated antigen-binding protein of the present 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, and the reference antibody The variable region of the heavy chain may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 5, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 7, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, And the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO:3.

The isolated antigen-binding protein of the present 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, and the reference antibody The variable region of the heavy chain may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 5, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 15, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 16, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 11, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 12, And the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 13.

The isolated antigen-binding protein of the present 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, and the reference antibody The variable region of the heavy chain may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 22, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 23, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 24, the LCDR1 The LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 18, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 19, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 20.

The isolated antigen-binding protein of the present 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, and the reference antibody The variable region of the heavy chain may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 30, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 31, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 32, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 26, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 27, And the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 28.

The isolated antigen-binding protein of the present 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, and the reference antibody The variable region of the heavy chain may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 30, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 38, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 39, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 34, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 35, And the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO:36.

The isolated antigen-binding protein of the present 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, and the reference antibody The variable region of the heavy chain may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 45, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 46, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 47, the LCDR1 The LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:41, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:42, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:43.

The isolated antigen-binding protein of the present 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, and the reference antibody The variable region of the heavy chain may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 5, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 49, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 41, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 42, And the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO:43.

The isolated antigen-binding protein of the present 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, and the reference antibody The variable region of the heavy chain may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 51, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 52, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 53, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 41, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 42, And the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO:43.

The isolated antigen-binding protein of the present 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, and the reference antibody The variable region of the heavy chain may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 55, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 56, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 57, the LCDR1 The LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:41, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:42, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:43.

The isolated antigen-binding protein of the present 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, and the reference antibody The variable region of the heavy chain may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 63, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 65, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 59, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 60, And the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO:61.

The isolated antigen-binding protein of the present 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, and the reference antibody The variable region of the heavy chain may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 5, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 67, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 68, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 41, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 42, And the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO:43.

The isolated antigen-binding protein of the present 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, and the reference antibody The variable region of the heavy chain may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 55, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 56, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 70, the LCDR1 The LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:41, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:42, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:43.

The isolated antigen-binding protein of the present 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, and the reference antibody The variable region of the heavy chain may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 55, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 56, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 72, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 41, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 42, And the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO:43.

In the present application, the isolated antigen binding protein may comprise at least one CDR in the VL of the light chain variable region, and the VL may comprise the amino acid sequence shown in SEQ ID NO:124 or SEQ ID NO:125.

In the present application, the isolated antigen binding protein may comprise at least one CDR in the heavy chain variable region VH, the VH may comprise 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 , the amino acid sequence shown in any one of SEQ ID NO: 71 and SEQ ID NO: 73.

For example, the isolated antigen-binding protein can comprise an antibody or antigen-binding fragment thereof.

For example, the antigen-binding fragment can include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and/or dAb.

In the present application, the VL may comprise LCDR1, LCDR2 and LCDR3, and the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:122-SEQ ID NO:123.

For example, wherein the LCDR3 may comprise 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 . For example, the sequence may be a sequence determined according to the KABAT definition rules.

For example, wherein the LCDR3 can 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. For example, the sequence may be a sequence determined according to the KABAT definition rules.

For example, the LCDR3 may comprise 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 item.

For example, the LCDR1 can comprise the amino acid sequence set forth in any one of SEQ ID NO:118-SEQ ID NO:119.

For example, wherein the LCDR1 can 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. For example, the sequence may be a sequence determined according to the KABAT definition rules.

For example, wherein the LCDR1 can 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, 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 absent. For example, the sequence may be a sequence determined according to the KARAT definition rules.

For example, the LCDR1 may comprise 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 The amino acid sequence shown in any one of NO: 111.

For example, the LCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NO:120-SEQ ID NO:121.

For example, wherein 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. For example, the sequence may be a sequence determined according to the KABAT definition rules.

For example, wherein the LCDR2 can 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, and X ₆ is Ala or Glu. For example, the sequence may be a sequence determined according to the KABAT definition rules.

For example, the LCDR2 may comprise 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 The amino acid sequence shown in any one of NO: 112.

For example, the VH can comprise HCDRl, HCDR2, and HCDR3, and the HCDRl can comprise SEQ ID NO: 5, SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 45, SEQ ID NO: 51, SEQ ID NO: 55. The amino acid sequence set forth in any one of SEQ ID NO:63 and SEQ ID NO:114.

For example, the HCDR2 may comprise 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, The amino acid sequence set forth in any one of SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:67, and SEQ ID NO:115.

For example, the HCDR3 can comprise 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 The amino acid sequence shown in any one of 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 .

For example, the VL may include framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

For example, the C-terminus of the L-FR1 can be directly or indirectly linked to the N-terminus of the LCDR1, and the L-FR1 can comprise the amino acid sequence shown in any one of SEQ ID NO:74-SEQ ID NO:75 .

For example, the L-FR2 can be located between the LCDR1 and the LCDR2, and the L-FR2 can comprise the amino acid set forth in any one of SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78 sequence.

For example, the L-FR3 can be located between the LCDR2 and the LCDR3, and the L-FR3 can comprise any one of SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, and SEQ ID NO:82 The amino acid sequence shown.

For example, the N-terminus of the L-FR4 can be directly or indirectly linked to the C-terminus of the LCDR3, and the L-FR4 can comprise SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: The amino acid sequence shown in any one of 86.

In the present application, the VL may comprise the amino acid sequence shown in SEQ ID NO:124 or SEQ ID NO:125.

For example, where the VL can comprise SEQ ID NO: 124 shown in the amino acid _{sequence: 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 It 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, and X ₇₁ is Phe or Tyr, X ₉₆ is Pro or Ser, X ₉₇ is Ile or absent, X ₁₀₄ is Lys or Arg, and X ₁₀₅ is Leu or Val. For example, the sequence may be a sequence determined according to the KABAT definition rules.

For example, wherein the 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, Wherein, 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 ₁₇ is Asp or Glu, X ₁₉ is Ala or Val, X ₂₁ is Ile or Leu, X ₂₂ is Ser or Thr, X ₂₄ is Gln or Arg, X ₂₈ is Asp, Gly or Ser, X ₂₉ is Ile or Val, X ₃₀ is Asn or Ser, X ₃₁ is Gly, Asn or Ser, X ₃₂ is Ser or does not exist, X ₃₃ is Trp or Tyr, X ₃₅ is Ala or Asn, X ₄₃ is Lys or Gln, X ₄₆ is Lys or Arg, X ₅₁ is Ala, Asp or Gly, X ₅₄ is Asn, Ser or Thr, X ₅₅ is Leu or Arg, X ₅₆ is Ala or Glu, X ₅₉ is Ile or Val, X ₆₁ is Asp or Ser, X ₇₄ is Phe or Leu, X ₇₈ is Arg or Ser, X ₈₀ is Glu or Gln, X ₈₄ is Phe or Ile, X ₈₆ is Thr or Val, X ₉₃ is Asp or Gly, X ₉₄ is Asn or Ser, X ₉₅ is Leu or Ser, X ₉₇ is Ile, Leu, Gln or Val, X ₁₀₁ is Gly or Gln, X ₁₀₄ is Lys or Arg, and X ₁₀₅ is Leu or Val. For example, the sequence may be a sequence determined according to the KABAT definition rules.

For example, the VL may comprise 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 The amino acid sequence shown in any one of NO: 113.

For example, the isolated antigen binding protein can include an antibody light chain constant region, and the antibody light chain constant region includes a human Igκ constant region or a human Igλ constant region.

In the present application, the gene encoding the human Igκ constant region can be shown as GenBank accession number 50802 of the NCBI database; the gene encoding the human Igλ constant region can be shown as the GenBank accession number 3535 of the NCBI database.

For example, the VH may include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

For example, the C-terminus of the H-FR1 may be directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 may comprise SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90. The amino acid sequence of any one of SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, and SEQ ID NO:94.

For example, the H-FR2 can be located between the HCDR1 and the HCDR2, and the H-FR2 can comprise any one of SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98 The amino acid sequence shown.

For example, the H-FR3 can be located between the HCDR2 and the HCDR3, and the H-FR3 can comprise SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: : the amino acid sequence of any one of SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 106.

For example, the N-terminus of the H-FR4 can be linked directly or indirectly to the C-terminus of the HCDR3, and the H-FR4 can comprise 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.

For example, the VH may comprise 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 The amino acid sequence set forth in any one of 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 .

For example, the isolated antigen binding protein can include an antibody heavy chain constant region, and the antibody heavy chain constant region includes a human IgG constant region.

For example, the isolated antigen binding protein can include an antibody heavy chain constant region, and the antibody heavy chain constant region includes a human IgGl constant region.

In the present application, the gene encoding the human IgG1 constant region can be shown as GenBank accession number 3500 of the NCBI database.

In the present application, the isolated antigen-binding protein may comprise antibody light chain variable region CDRs---LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1, and the LCDR2 may comprise SEQ ID NO: 1 The amino acid sequence shown in ID NO: 2, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3.

In the present application, the isolated antigen-binding protein may comprise antibody light chain variable region CDRs---LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 11, and the LCDR2 may comprise SEQ ID NO: 11 The amino acid sequence shown in ID NO: 12, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 13.

In the present application, the isolated antigen-binding protein may comprise antibody light chain variable region CDRs---LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, and the LCDR2 may comprise SEQ ID NO: 18 The amino acid sequence shown in ID NO: 19, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 20.

In the present application, the isolated antigen-binding protein may comprise antibody light chain variable region CDRs---LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 26, and the LCDR2 may comprise SEQ ID NO: 26 The amino acid sequence shown in ID NO: 27, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 28.

In the present application, the isolated antigen binding protein may comprise antibody light chain variable region CDRs---LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 34, and the LCDR2 may comprise SEQ ID NO: 34 The amino acid sequence shown in ID NO: 35, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 36.

In the present application, the isolated antigen-binding protein may comprise antibody light chain variable region CDRs---LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 41, and the LCDR2 may comprise SEQ ID NO: 41 The amino acid sequence shown in ID NO: 42, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 43.

In the present application, the isolated antigen-binding protein may comprise antibody light chain variable region CDRs---LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 59, and the LCDR2 may comprise SEQ ID NO: 59 The amino acid sequence shown in ID NO: 60, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 61.

In the present application, the isolated antigen-binding protein may comprise antibody heavy chain variable region CDRs---HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise SEQ ID NO: 5 The amino acid sequence, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:7.

In the present application, the isolated antigen-binding protein may comprise antibody heavy chain variable region CDRs---HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 may comprise SEQ ID NO: 5 The amino acid sequence shown in ID NO:9, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:7.

In the present application, the isolated antigen-binding protein may comprise antibody heavy chain variable region CDRs---HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 may comprise SEQ ID NO: 5 The amino acid sequence shown in ID NO: 15, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 16.

In the present application, the isolated antigen-binding protein may comprise antibody heavy chain variable region CDRs---HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 22, and the HCDR2 may comprise SEQ ID NO: 22 The amino acid sequence shown in ID NO: 23, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 24.

In the present application, the isolated antigen-binding protein may comprise antibody heavy chain variable region CDRs---HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 30, and the HCDR2 may comprise SEQ ID NO: 30 The amino acid sequence shown in ID NO:31, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:32.

In the present application, the isolated antigen-binding protein may comprise antibody heavy chain variable region CDRs---HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 30, and the HCDR2 may comprise SEQ ID NO: 30 The amino acid sequence shown in ID NO:38, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:39.

In the present application, the isolated antigen-binding protein may comprise antibody heavy chain variable region CDRs---HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 45, and the HCDR2 may comprise SEQ ID NO: 45 The amino acid sequence shown in ID NO:46, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:47.

In the present application, the isolated antigen-binding protein may comprise antibody heavy chain variable region CDRs---HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 may comprise SEQ ID NO: 5 The amino acid sequence shown in ID NO:9, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:49.

In the present application, the isolated antigen-binding protein may comprise antibody heavy chain variable region CDRs---HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 51, and the HCDR2 may comprise SEQ ID NO: 51 The amino acid sequence shown in ID NO:52, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:53.

In the present application, the isolated antigen-binding protein may comprise antibody heavy chain variable region CDRs---HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 55, and the HCDR2 may comprise SEQ ID NO: 55 The amino acid sequence shown in ID NO:56, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:57.

In the present application, the isolated antigen-binding protein may comprise antibody heavy chain variable region CDRs---HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 63, and the HCDR2 may comprise SEQ ID NO: 63 The amino acid sequence shown in ID NO:64, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:65.

In the present application, the isolated antigen-binding protein may comprise antibody heavy chain variable region CDRs---HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise SEQ ID NO: 5 The amino acid sequence, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:67, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:68.

In the present application, the isolated antigen-binding protein may comprise antibody heavy chain variable region CDRs---HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 55, and the HCDR2 may comprise SEQ ID NO: 55 The amino acid sequence shown in ID NO:56, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:70.

In the present application, the isolated antigen-binding protein may comprise antibody heavy chain variable region CDRs---HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 55, and the HCDR2 may comprise SEQ ID NO: 55 The amino acid sequence shown in ID NO:56, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:72.

In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 may comprise SEQ ID NO: The amino acid sequence shown in 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 The amino acid sequence shown in NO:2, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3.

In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 may comprise SEQ ID NO: The amino acid sequence shown in 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 The amino acid sequence shown in NO:2, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3.

In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 may comprise 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, and the LCDR2 may include SEQ ID The amino acid sequence shown in NO: 12, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 13.

In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 22, and the HCDR2 may comprise 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 The amino acid sequence shown in NO: 19, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 20.

In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 30, and the HCDR2 may comprise 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, and the LCDR2 may include SEQ ID The amino acid sequence shown in NO: 27, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 28.

In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 30, and the HCDR2 may comprise SEQ ID NO: The amino acid sequence shown in 38, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 39, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 34 The LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:35, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:36.

In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 45, and the HCDR2 may comprise SEQ ID NO: The amino acid sequence shown in 46, and 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, and the LCDR2 may include SEQ ID The amino acid sequence shown in NO:42, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:43.

In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 may comprise 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, and the LCDR2 may include SEQ ID The amino acid sequence shown in NO:42, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:43.

In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 51, and the HCDR2 may comprise SEQ ID NO: The amino acid sequence shown in 52, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 53, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 41, and the LCDR2 may comprise SEQ ID The amino acid sequence shown in NO:42, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:43.

In the present application, the isolated antigen-binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1 may comprise SEQ ID NO: 55 The amino acid sequence, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 56, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 57, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 41 The LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:42, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:43.

In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 63, and the HCDR2 may comprise SEQ ID NO: The amino acid sequence shown in 64, and 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, and the LCDR2 may include SEQ ID The amino acid sequence shown in NO:60, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:61.

In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 5, and the HCDR2 may comprise SEQ ID NO: The amino acid sequence shown in 67, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 68, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 41, and the LCDR2 may comprise SEQ ID The amino acid sequence shown in NO:42, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:43.

In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 55, and the HCDR2 may comprise SEQ ID NO: The amino acid sequence shown in 56, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 70, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 41, and the LCDR2 may comprise SEQ ID The amino acid sequence shown in NO:42, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:43.

In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 55, and the HCDR2 may comprise SEQ ID NO: The amino acid sequence shown in 56, and 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, and the LCDR2 may include SEQ ID The amino acid sequence shown in NO:42, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:43.

For example, the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL, the VH can comprise the amino acid sequence set forth in SEQ ID NO:8, and the VL can comprise SEQ ID NO: 4 shows the amino acid sequence. This isolated antigen binding protein can be referred to as Ab2001.02.

For example, the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL, the VH can comprise the amino acid sequence set forth in SEQ ID NO: 10, and the VL can comprise SEQ ID NO: 4 shows the amino acid sequence. This isolated antigen binding protein can be referred to as Ab2001.03.

For example, the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL, the VH can comprise the amino acid sequence set forth in SEQ ID NO: 17, and the VL can comprise SEQ ID NO: The amino acid sequence shown in 14. This isolated antigen binding protein can be referred to as Ab2001.04.

For example, the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL, the VH can comprise the amino acid sequence set forth in SEQ ID NO: 25, and the VL can comprise SEQ ID NO: The amino acid sequence shown in 21. This isolated antigen binding protein can be referred to as Ab2001.08.

For example, the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL, the VH can comprise the amino acid sequence set forth in SEQ ID NO: 33, and the VL can comprise SEQ ID NO: The amino acid sequence shown in 29. This isolated antigen binding protein can be referred to as Ab2001.09.

For example, the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL, the VH can comprise the amino acid sequence set forth in SEQ ID NO:40, and the VL can comprise SEQ ID NO: The amino acid sequence shown in 37. This isolated antigen binding protein may be referred to as Ab2001.10.

For example, the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL, the VH can comprise the amino acid sequence set forth in SEQ ID NO:48, and the VL can comprise SEQ ID NO: The amino acid sequence shown in 44. This isolated antigen binding protein may be referred to as Ab2001.11.

For example, the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL, the VH can comprise the amino acid sequence set forth in SEQ ID NO:50, and the VL can comprise SEQ ID NO: The amino acid sequence shown in 44. This isolated antigen binding protein may be referred to as Ab2001.12.

For example, the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL, the VH can comprise the amino acid sequence set forth in SEQ ID NO: 54, and the VL can comprise SEQ ID NO: The amino acid sequence shown in 44. This isolated antigen binding protein can be referred to as Ab2001.13.

For example, the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL, the VH can comprise the amino acid sequence set forth in SEQ ID NO: 58, and the VL can comprise SEQ ID NO: The amino acid sequence shown in 44. This isolated antigen binding protein may be referred to as Ab2001.14.

For example, the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL, the VH can comprise the amino acid sequence set forth in SEQ ID NO:66, and the VL can comprise SEQ ID NO: The amino acid sequence shown in 62. This isolated antigen binding protein may be referred to as Ab2001.19.

For example, the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL, the VH can comprise the amino acid sequence set forth in SEQ ID NO:69, and the VL can comprise SEQ ID NO: The amino acid sequence shown in 44. This isolated antigen binding protein can be referred to as Ab2001.23.

For example, the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL, the VH can comprise the amino acid sequence set forth in SEQ ID NO:71, and the VL can comprise SEQ ID NO: The amino acid sequence shown in 44. This isolated antigen binding protein can be referred to as Ab2001.24.

For example, the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL, the VH can comprise the amino acid sequence set forth in SEQ ID NO:73, and the VL can comprise SEQ ID NO: The amino acid sequence shown in 44. This isolated antigen binding protein may be referred to as Ab2001.25.

In the present application, the VL of antibodies Ab2001.2, Ab2001.3, Ab2001.11, Ab2001.12, Ab2001.13, Ab2001.14, Ab2001.23, Ab2001.24 and Ab2001.25 may comprise SEQ ID NO: 124 The amino acid sequence shown.

In the present application, the VL of antibodies Ab2001.4, Ab2001.8, Ab2001.9, Ab2001.10 and Ab2001.19 may comprise the amino acid sequence shown in SEQ ID NO:125.

The antigen binding proteins of the present application (eg, SARS-CoV-2 antibodies) are capable of specifically binding to the RBD of the S protein of SARS-CoV-2. "Specific binding" SARS-CoV-2 antigens (e.g., RBD S protein of SARS-CoV-2) antigen binding proteins (e.g., antibody) may generally be about 5.0 * 10 ^-8 M in K _D value or more in Affinity (eg, below about 9.0* ^10-9 M, below about 8.0* ^10-9 M) binds to the RBD of the S protein of SARS-CoV-2, but not to other proteins lacking the SARS-CoV-2 sequence.

Whether an antigen binding protein (eg, an antibody) binds a SARS-CoV-2 antigen (eg, the RBD of the S protein of SARS-CoV-2) can be determined using any assay known in the art. Examples of assays known in the art to determine binding affinity include surface plasmon resonance (eg, BIACORE) or similar techniques (eg, KinExa or OCTET). In certain instances, the SARS-CoV-2 antibodies of the present application may also cross-react with SARS-CoV. For example, by flow cytometry and ELISA detected. As used herein, "cross-reactivity" refers to the ability of an antibody to react with homologous proteins from other species.

The antigen-binding protein (eg, SARS-CoV-2 antibody) of the present application is capable of blocking the binding of the RBD of the S protein of SARS-CoV-2 or a mutant thereof to human ACE2. Blocking assays can be detected using competitive methods, for example, combining the antigen-binding protein (eg, SARS-CoV-2 antibody) with the antigen (or, cells that can express the antigen) and the ligand of the antigen (or, the ligand expressing the ligand). cells), and the ability of the antigen-binding protein to compete with its ligand for binding to the antigen is reflected in the intensity (eg, fluorescence intensity or concentration) of the detectable label. In the present application, the antigen binding protein (eg, SARS-CoV-2 antibody) can also block the binding of the RBD of the S protein of SARS-CoV to human ACE2.

bispecific antigen binding protein

In another aspect, 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.

In the present application, 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 isolated antigen binding protein of the present application protein.

For example, antibodies Ab2001.2, Ab2001.3, Ab2001.11, Ab2001.12, Ab2001.13, Ab2001.14, Ab2001.23, Ab2001.24, and Ab2001.25 of the present application; and Ab2001.4 of the present application , Ab2001.8, Ab2001.9, Ab2001.10 and Ab2001.19, and the antigen-binding fragments thereof (for example, can be VL, VH), can specifically bind to the RBD of the S protein of SARS-CoV-2.

In the present application, the bispecific antigen binding protein may comprise a second targeting moiety that specifically binds to the RBD of the S protein of SARS-CoV, wherein the second targeting moiety may comprise the isolated antigen binding protein of the present application.

For example, the antibodies Ab2001.2, Ab2001.3, Ab2001.11, Ab2001.14, Ab2001.24, and Ab2001.25 of the present application, as well as antigen-binding fragments thereof (eg, may be VL, VH), may be specific RBD that binds to the S protein of SARS-CoV.

For example, the bispecific antigen binding protein may comprise an antibody.

For example, the first targeting moiety can comprise a first heavy chain and a first light chain, the second targeting moiety can comprise a second heavy chain and a second light chain, wherein the first light chain and the second light chain can be the same.

For example, the first light chain and the second light chain can comprise the amino acid sequence set forth in SEQ ID NO:44.

For example, the VH of the first heavy chain may comprise the amino acid sequence set forth in SEQ ID NO:48.

For example, the VH of the second heavy chain may comprise the amino acid sequence set forth in SEQ ID NO:50.

For example, the VH of the first heavy chain can comprise the amino acid sequence set forth in SEQ ID NO:48, and the VH of the second heavy chain can comprise the amino acid sequence set forth in SEQ ID NO:50.

In the present application, the bispecific antigen binding protein can be antibody Ab2001.16, wherein the first light chain and the second light chain can comprise the amino acid sequence shown in SEQ ID NO: 44; the first heavy chain The VH of the chain may comprise the amino acid sequence set forth in SEQ ID NO:48, and the VH of the second heavy chain may comprise the amino acid sequence set forth 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 scope: variants or homologues with the same or similar functions as the protein or polypeptide.

In the present application, the variant may be a protein in which one or more amino acids have been substituted, deleted, or added to the amino acid sequence of the protein and/or the polypeptide (eg, the antigen binding protein of the present application). or peptides. For example, the functional variant may comprise at least 1, such as 1-30, 1-20, or 1-10, also such as 1, 2, 3, 4, or 5 amine groups that have been Proteins or polypeptides with amino acid substitutions, deletions and/or insertions. The functional variant may substantially retain the biological properties of the protein or polypeptide prior to the alteration (eg, substitution, deletion, or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding capacity) of the protein or polypeptide prior to the alteration. For example, the substitution can be a conservative substitution.

In the present application, a portion 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 class. For example, both the variable and constant portions of an antibody can be derived from the variable and constant regions of an antibody of an animal species (eg, human). In the present application, the homologue may be at least about 85% (eg, having at least about 85%, 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.

In this application, the homology generally refers to the similarity, similarity or relatedness between two or more sequences. "Percent sequence homology" can be calculated by comparing the two sequences to be aligned in a comparison window to determine the presence of identical nucleic acid bases (eg, A, T, C, G) in the two sequences or identical amino acid residues (eg, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) the number of positions to get The number of matched positions, divided by the total number of positions in the comparison window (ie, the window size), and the result multiplied by 100 to yield percent sequence homology. Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length sequences being compared or within the region of the sequence of interest. The homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W. R. Pearson and D. J. Lipman, "Improved Tools for Biological Sequence Comparison," Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; J. Lipman and W. R. Pearson, "Fast and Sensitive Protein Similarity Search," Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E. W. Myers and D. Lipman, "A Basic Local Alignment Search Tool," Journal of Molecular Biology, 215: 403-410, 1990.

Nucleic acids, vectors, cells and preparation methods

In another aspect, the present application provides an isolated one or more nucleic acid molecules encoding the isolated antigen binding proteins of the present application, and/or the bispecific antigen binding proteins of the present application.

In another aspect, the present application provides a vector, which can contain the nucleic acid molecule of the present application.

In another aspect, the present application provides a cell, which may comprise the nucleic acid molecule of the present application or the vector of the present application.

The nucleic acid molecules of the present application may be isolated. For example, it can be produced or synthesized by: (i) amplified in vitro, for example by polymerase chain reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified, e.g. fractionated by digestion and gel electrophoresis isolated, or (iv) synthesized, eg, by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA techniques.

In the present application, nucleic acids encoding the antibodies, antigen-binding fragments thereof, can be prepared by a variety of methods known in the art, including, but not limited to, using restriction fragment manipulation or using overlap extension of synthetic oligonucleotides For PCR, see 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.

In another aspect, the application provides one or more vectors comprising one or more nucleic acid molecules of the application. One or more of the nucleic acid molecules may be included in each vector. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. In addition, the vector may contain expression control elements that allow for the correct expression of the coding region in an appropriate host. Such control elements are well known to those of skill in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like. In certain embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequence may vary depending on species or cell type function, but typically includes 5' untranslated sequences and 5' and 3' untranslated sequences involved in transcription and translation initiation, respectively, such as TATA boxes, capping sequence, CAAT sequence, etc. For example, a 5' non-transcribed expression control sequence may comprise a promoter region, which may comprise a promoter sequence for transcriptional control of a functionally linked nucleic acid. The expression control sequences may also include enhancer sequences or upstream activator sequences. In the present application, suitable promoters may include, for example, the promoters for SP6, T3 and T7 polymerases, the human U6 RNA promoter, the CMV promoter, and artificial hybrid promoters thereof (such as CMV), wherein the promoter's Some parts can be combined with other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) genes A portion of the promoter is fused, which may or may not contain additional introns. One or more nucleic acid molecules of the present application can be operably linked to the expression control element. The vector may include, for example, a plasmid, cosmid, virus, phage or other vectors commonly used, for example, in genetic engineering. For example, the vector is an expression vector.

In another aspect, the present application provides host cells that can comprise one or more nucleic acid molecules of the present application and/or one or more vectors of the present application. In certain embodiments, each or each host cell may comprise one or one nucleic acid molecule or vector of the present application. In certain embodiments, each or each host cell may comprise a plurality (eg, 2 or more) or more (eg, 2 or more) of the nucleic acid molecules or vectors of the present application. For example, the vectors of the present application can be introduced into such host cells, eg, eukaryotic cells, such as cells from plants, fungi or yeast cells, and the like. The vectors of the present application can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like.

In another aspect, the present application provides a method for preparing the isolated antigen-binding protein of the present application, the bispecific antigen-binding protein of the present application, the method comprising making the isolated antigen-binding protein of the present application and/or the bispecific antigen-binding protein of the present application The cells of the present application are cultured under conditions in which the specific antigen-binding protein is expressed.

The method can include culturing the host cell of the present application under conditions such that the antigen binding protein is expressed. For example, these methods can be understood by those of ordinary skill in the art by using an appropriate medium, appropriate temperature and incubation time, and the like.

Any method suitable for producing monoclonal antibodies can be used to produce the antigen binding proteins of the present application (eg, SARS-CoV-2 antibodies).

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) for the production of antibodies specific to SARS-CoV-2, screening the biology of the antibody active. The immunogens can be used alone or in combination with one or more immunogenicity enhancers known in the art. The DNA encoding the immunogen can be expressed using suitable genetic vectors including, but not limited to, adenoviral vectors, adeno-associated viral vectors, baculoviral vectors, plasmids, and non-viral vectors.

Humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA and IgE. In this application, the antibody is an IgG antibody, and the IgG1 subtype is used. Likewise, any type of light chain can be used in the compounds and methods herein. For example, kappa, lambda chains or variants thereof are suitable for use in this application.

The sequences of the DNA molecules of the antigen-binding proteins or fragments thereof of the present application can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences for the light and heavy chains can be fused together to form single chain antibodies.

Once the relevant sequences have been obtained, recombinant methods can be used to obtain the relevant sequences in bulk. This is usually by colonizing it into a vector and then transferring it into a cell, and then isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, synthetic methods can also be used to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation. The nucleic acid molecule can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.

The present application also relates to vectors comprising suitable nucleic acid molecules as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins quality. Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. For example, animal cells can include: CHO-S, CHO-K1 and/or HEK-293 cells.

The step of transforming a host cell with recombinant DNA in this application can be performed using techniques well known in the art. The obtained transformants can be cultured by conventional methods, and the transformants express the polypeptides encoded by the nucleic acid molecules of the present application. Depending on the host cell used, it is cultured with conventional media under appropriate conditions. Typically, the transformed host cells are cultured under conditions suitable for expression of the antigen binding proteins of the present application. Then use conventional 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. Those skilled in the art The antigen-binding protein of the present application can be obtained by well-known conventional separation and purification methods.

The obtained monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays such as flow cytometric sorting (FACS), radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).

Pharmaceutical composition

In another aspect, the present application provides a pharmaceutical composition, which can comprise the isolated antigen-binding protein of the present application, the bispecific antigen-binding protein of the present application, the nucleic acid molecule of the present application, the carrier of the present application and/or the cells, and optionally pharmaceutically acceptable adjuvants.

The pharmaceutical composition of the present application can be directly used to bind the RBD of the S protein of SARS-CoV-2, and thus can be used to prevent and treat diseases related to coronavirus infection (eg, COVID-19). In addition, other therapeutic agents may also be used concomitantly.

The pharmaceutical composition of the present application may contain a safe and effective amount (eg, 0.001-99 wt %, 0.01-90 wt %, or 0.1-80 wt %) of the antigen-binding protein of the present application and a pharmaceutically acceptable adjuvant (which can be including carriers or excipients). Such carriers can include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. The drug formulation should match the mode of administration. The pharmaceutical composition of the present application can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. In addition, the antigen binding proteins of the present application can also be used with other therapeutic agents.

The antigen binding proteins or pharmaceutical compositions herein can be formulated, administered and administered in a manner consistent with good medical practice. Considerations in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the etiology of the disorder, the site of drug delivery, the method of administration, and other factors known to medical practitioners. Therapeutic agents (eg, SARS-CoV-2 antibodies) need not be formulated and/or administered concurrently with one or more agents currently used to prevent or treat the condition in question, but as needed. The effective amount of such other agents depends on the amount of therapeutic agent (eg, SARS-CoV-2 antibody) present in the formulation, the type of disorder or treatment, and other factors discussed above. These agents can generally be administered in any dose and by any route as empirically/clinically determined to be appropriate. The dose of antibody administered in combination therapy can be reduced compared to single therapy. The progress of this therapy is readily monitored by conventional techniques.

use

In another aspect, the present application provides an isolated antigen-binding protein of the present application, a bispecific antigen-binding protein of the present application, a nucleic acid molecule of the present application, a carrier of the present application, a cell of the present application and/or the pharmaceutical composition of the present application Use of the compound in the preparation of a medicament for preventing, relieving and/or treating coronavirus infection.

The present application provides a method of preventing, alleviating and/or treating coronavirus infection, comprising administering to a subject in need thereof the isolated antigen-binding protein of the present application, the bispecific antigen-binding protein of the present application, the Nucleic acid molecules, vectors of the present application, cells of the present application and/or pharmaceutical compositions of the present application.

The present application provides isolated antigen binding proteins, bispecific antigen binding proteins of the present application, nucleic acid molecules of the present application, vectors of the present application, cells of the present application and/or pharmaceutical compositions of the present application, which can prevent, alleviate and/or treatment of coronavirus infection.

In this application, the coronavirus infection may include COVID-19. The coronavirus infection can also include SARS.

On the other hand, the present application provides a method for blocking the binding of the RBD of the S protein of SARS-CoV-2 or its mutant to human ACE2, comprising the following steps, administering the isolated antigen-binding protein of the present application, the present application The bispecific antigen binding protein of the present application, the nucleic acid molecule of the present application, the vector of the present application, the cell of the present application and/or the pharmaceutical composition of the present application.

On the other hand, the present application provides a method for blocking the binding of the RBD of the S protein of SARS-CoV to human ACE2, comprising the steps of applying the isolated antigen-binding protein of the present application, the bispecific antigen-binding protein of the present application The protein, the nucleic acid molecule of the present application, the vector of the present application, the cell of the present application and/or the pharmaceutical composition of the present application.

The antigen binding proteins of the present application can be used in detection applications, eg, for detection of samples, thereby providing diagnostic information. For example, the antibodies and/or methods of the present application can be used to treat a subject (eg, a patient suspected of being infected with SARS-CoV-2 or SARS-CoV, or having been infected with SARS-CoV-2 or SARS-CoV) Specimens (eg, throat swab test samples such as serum, whole blood, sputum, oral/nasopharyngeal secretions or washings, urine, feces, pleural effusion, cerebrospinal fluid, and tissue specimens) are tested as Indicators of efficacy observation and indicators of whether it is infectious and whether it needs to be isolated. For example, the antibodies and/or methods of the present application can provide monitoring protocols for therapeutic intervention.

In this application, the sample (sample) employed includes cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to those skilled in the art. Biopsy as used in this application may thus include tissue samples prepared, for example, by endoscopic methods or needle or needle biopsy of organs. For example, the sample can include a fixed or preserved cell or tissue sample.

The present application also provides a kit containing the antigen-binding protein of the present application. In some cases, the kit may also include a container, instructions for use, buffers, and the like. For example, the pro-binding protein of the present application can be immobilized on a detection plate.

Not to be limited by any theory, the following examples are only intended to illustrate the fusion protein, preparation method and use of the present application, and are not intended to limit the scope of the invention of the present application.

[Example]

Example 1 Antibody Screening

1.1 Yeast display library construction and screening

The common light chain phage library and the single-chain phage library were selected, and two rounds of panning were performed against SARS-CoV-2 S1 RBD (sino biological product number: 40592-V05H) to obtain positive enrichment. Using the plasmid after two rounds of phage panning as a template, primers were designed to amplify single-chain antibody (scFv) by polymerase chain reaction (PCR). EBY100 (purchased from ATCC) inserts the scFv gene into the yeast display plasmid by homologous recombination of Saccharomyces cerevisiae, thereby realizing the display of single-chain antibody on the surface of yeast cell wall. The yeast-displayed single-chain antibody library and the common light chain antibody library were named JYYDL056 and JYYDL057, respectively. After electroporation, libraries JYYDL056 and JYYDL057 were cultured in 100 mL of SD-Trp medium (Clontech, product number: 630308) at 30°C and 225 rpm overnight; 1.0×10 ⁸ bacteria were resuspended in 20 mL YPGP liquid medium (2% half Lactose, 2% protein gluten, 1% yeast extract, 0.54% Na ₂ HPO ₄ , 0.86% NaH ₂ PO ₄ .H ₂ O), 20 ℃, 225 r/min culture and induction for 24 hours, placed in a 4 ℃ refrigerator for later use .

_{After induction, the OD 600} of the bacterial solution was determined, and 1 OD was ^{calculated as 1.0 × 10 7} cells, 4.0 × 10 ⁷ cells were taken from each, and flow staining was carried out according to the following steps: 1. Use 2 mL of 1 × PBSA ( 1×PBS+1%BSA) washed once, centrifuged at 3000 rpm for 3 minutes (the following conditions are all centrifuged), discard the supernatant; 2. Each tube contains 100nM SARS-CoV-2 S1 RBD mFc (Sino biological, Item No.: 1×PBSA of 40592-V05H), incubate for 30 minutes at room temperature; 3. Add 1 mL of 1×PBSA to each, and centrifuge to discard the supernatant; 4. Add 100 μL of 300nM Human ACE2-biotin (Kactus, Item No.: ACE-HM401), incubate on ice for 20 minutes; 5. Add 1 mL of 1×PBSA to each, and centrifuge to discard the supernatant; 6. Add 500 μL of 1×PBSA containing fluorescent antibody to each (SA-PE manufacturer eBioscience, Item No.: 12) -4317-8, diluted 1:200; Goat anti mouse-Alexa Fluor 647 manufacturer thermo fisher, product number: A-21235, diluted 1:400), incubate on ice protected from light for 20 minutes; 7. Repeat step 5, add The cells were resuspended in 2 mL of 1×PBSA, and the cell population with strong fluorescence signal Alexa Fluor 647 signal and weak PE signal was collected for flow sorting. After sorting, the cells were cultured in 5 mL SD-Trp liquid medium at 30°C and 225 r/min overnight; 1 mL of bacterial broth was added to 4 mL of YPGP liquid medium, cultured at 20°C, 225 r/min for 24 hours, and placed in a 4°C refrigerator for later use. . The same as the first round of screening, the concentration of SARS-CoV-2 S1 RBD mFc was reduced to 50 nM, and the second round of sorting was performed. After sorting, the cells were plated on SD-Trp solid medium (Clontech, catalog number: 630309), and kept at 30°C. Incubate for 3 days.

1.2 Simple strain yeast colony sequencing and flow staining identification

The second round of screening products of JYYDL056 and JYYDL057, 400 simple strains were selected for sequencing. Finally, 34 unique single-chain antibody sequences were obtained from the second round of screening products of the JYYDL056 library; 42 unique single-chain antibody sequences were obtained from the second round of screening products of the JYYDL057 library. Common light chain antibody sequence. Flow staining analysis was performed on the corresponding yeast simple strain colonies. Referring to the staining steps in Example 2.1, 1×10 ⁶ cells were taken and stained according to the following scheme (Table 1).

The strength of the cell population of protocol 1 binding to SARS-CoV-2-RBD is reflected by the mean fluorescence signal intensity (MFI) of PE, and the strength of antibody blocking SARS-CoV-2-RBD and hACE2-Fc is reflected by the MFI value of Alexa Fluor 647 reflect;

Similarly, Scheme 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 the single-chain antibody, respectively.

Table 1 Identification scheme of simple strain yeast colony by flow staining

After each simplex was stained with three staining protocols, flow cytometry was performed using the GUAVA Microcapillary Cell Analysis Platform. According to the experimental results, pick the SARS-CoV-2-RBD in scheme 1. Pure strains with strong binding (high MFI value of PE fluorescence signal) and blocking SARS-CoV-2 RBD binding to hACE2-Fc (low MFI value of Alexa Fluor 647); at the same time, the binding signal with irrelevant antigens in Scheme 3 was excluded. High pure strain sequence.

Based on the results, 8 monoclonal antibodies (Y28A5, Y28B6, Y29B2, Y29F6, Y34H3, Y35F3, Y35G1, Y36F4) were obtained from the JYYDL056 library; 7 co-light chain monoclonal antibodies (Y28B4, Y30B4, Y34B6) were obtained from the JYYDL057 library , Y34G5, Y38A3, Y39B3, Y39G2) (the results are shown in Table 2). The common light chain simple strains (Y28B4 and Y34G5) were selected, and the common light chain bispecific antibody Ab2001.16 was constructed by Knob and Hole mutation in the Fc region (see US5731168A for mutation modification), as shown in Table 3. A total of 16 antibodies were handed over to a contract outsourcing company for mammalian cell expression, and antibody samples were prepared in small quantities.

Table 2 Flow cytometry analysis results of yeast simple strain colonies after the second round of library screening

Table 3 Expression combinations of co-light chain bispecific antibodies

Example 2 Antibody Expression

The candidate antibody sequences were coded and divided into CDR regions by the Kabat numbering method. Taizhou Baiying Biotechnology Co., Ltd. was entrusted to carry out plasmid construction and antibody expression and purification. The antibody sequences and corresponding expression information are shown in Table 4.

30 ml of HEK293 cells were transiently transfected, and candidate antibody proteins were obtained after expression and purification. The specific information is shown in Table 5.

Table 4 Antibody sequence information

Table 5 Antibody Expression Information

Example 3 In vitro activity screening

3.1 Octet Red detects the binding and dissociation rate of candidate antibodies to SARS-CoV-2 S1

The binding and dissociation rate of the candidate antibody to SARS-CoV-2 S1 was determined, and the candidate antibody was immobilized on the AHC sensor (Fortébio, cat. 40591-V08H) binding-dissociation rate. Each cycle consists of the following steps: 1) sensor regeneration; 2) immersion buffer (PBST, 10 μL Tween 20 in 50 mL PBS) for 60 seconds; 3) 5 μg/mL fully human antibody immobilized on the AHC sensor , the time was 40 seconds; 4) The sensor was immersed in the buffer for 180 seconds; 5) The binding of 100 nM SARS-CoV-2 S1 to the antibody, the time was 180 seconds; 6) The dissociation of the antigen-antibody, the time was 10 minutes. The results of affinity were analyzed by Octet Data Analysis Software (Fortébio), and the results are shown in Table 6.

Table 6

As can be seen from Table 6, except for Ab2001.07, the candidate antibodies all effectively bind to the antigen SARS-CoV-2 S1. Among them, Ab2001.08, Ab2001.10, and Ab2001.16 have higher affinity. The binding and dissociation rates of SARS-CoV-2 S1 at different concentrations were further determined. The concentration of SARS-CoV-2 S1 was 50 There are 5 concentrations of nM, 25nM, 12.5nM, 6.3nM and 3.2nM, which are compared with hACE2-Fc (Kaiqi Bio, Cat. No.: ACE-HM501) (see Table 7, Figure 1)

Table 7

It can be seen from Table 7 that the affinities of Ab2001.08, Ab2001.10, Ab2001.16, hACE2-Fc and SARS-CoV-2 S1 were 2.93nM (Figure 1A), 4.21nM (Figure 1B), and 2.39nM (Figure 1C), respectively. ), 11.1 nM (Fig. 1D), the affinity of the antibody to SARS-CoV-2 S1 was stronger than that of hACE2-Fc.

3.2 Candidate antibodies block the binding of SARS-CoV-2 S1 to hACE2-Fc

According to the affinity data of the candidate antibodies, Ab2001.02, Ab2001.03, Ab2001.08, Ab2001.09, Ab2001.10, Ab2001.11, and Ab2001.16 were selected to block the binding of SARS-CoV-2 S1 to hACE2; Since the Ab2001.16 bispecific antibody is composed of the sequences of Ab2001.11 and Ab2001.12, the same amount of Ab2001.11+Ab2001.12 combination was used for comparison.

Carry out the experiment as follows,

1. Dilute hACE2-Fc to 1 μg/mL coated microtiter plate with 1×PBS, 100 μl/well, stick the plate membrane, incubate at 4°C overnight, and then wash with washing solution (1×PBS+0.05% TWEEN) -20) Wash the plate 3 times. Then, the blocking solution (1×PBS+0.05% TWEEN-20+2%BSA) was prepared with washing solution, and 300 cells were added to each well. μl blocking solution, blocked at 37°C for 1 hour. 2. The final concentration of biotinylated SARS-CoV-2 S1 Fc (Kaiyi Bio, Cat. No.: COV-VM5S1) was prepared with blocking solution to a final concentration of 0.2 μg/mL; then the antibody was diluted with blocking solution, and the initial concentration of the antibody was 15 μg /mL, 5-fold serial dilution (dilution of 7 concentration points + 1 concentration point of 0 μg/mL), then anti-SARS-CoV-2 antibody and biotinylated SARS-CoV-2 S1 Fc were incubated at 37°C for 1 Hour. 3. Take out the sealed ELISA plate in step 1, wash the plate three times with washing solution, add 100 μl/well of the incubation solution in step 2, and incubate at 37°C for 1 hour. 4. Wash the plate 3 times with washing solution, dilute SA-HRP (sigma, S2438-250UG) with the blocking solution at a volume ratio of 1:5000, add 100 μl/well to the ELISA plate, and incubate at 37°C for 1 hour; 5. Use Wash the plate 3 times with washing solution, add TMB chromogenic solution (Biopanda, product number: TMB-S-003) at 100 μl/well, react at room temperature for 10 minutes in the dark, and then add stop solution (Solarbio, product number: C1058 at 50 μl/well) ) to terminate the reaction, and the absorbance was measured at a wavelength of 450 nm. The data were processed with GraphPad Prism software, as shown in Figure 2. It can be seen from Figure 2 that the blocking effect of Ab2001.08 and Ab2001.10 is good, about 15 times stronger than that of hACE2-Fc.

3.3 Identification of candidate antibodies and SARS-CoV-2 antigen-binding epitopes

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 interacts with the first antibody and the second antibody in turn, The second antibody binding signal is detected to determine whether the two antibodies recognize the same epitope. In this experiment, each cycle consisted of the following steps: 1) the sensor was first immersed in buffer (PBST, 10 μL of Tween 20 in 50 mL of PBS) for 30 s; 2) 1 μg/ml of Biotin human SARS-CoV-2 RBD his (Kaiqi Bio, Cat. No.: COV+VM4BD) was cured on the SA sensor (Fortébio, Cat. No.: 18-0009) for 65 seconds; 3) The sensor was immersed in the buffer for 30 seconds; 4) 100nM first Each antibody binds to the antigen on the sensor surface for 3 minutes to saturate the binding site of the first antibody on the antigen; 5) 100nM The second antibody binds to the antigen for 3 minutes. The results of epitope identification were analyzed by Octet Data Analysis Software (Fortébio), and the results are shown in Table 8.

Table 8 Identification of candidate antibodies and SARS-CoV-2 antigen-binding epitopes

Self-reaction signal <20%, the experimental data is valid. The criteria for determining the experimental results are: 1) 60%-100%: no competition at all; 2) 20%-60%: partial competition; 3) <20%: complete competition, if one direction meets this standard, it is considered that two antibodies compete. The data show that Ab2001.10, 12, 13 antibodies are one binding epitope; Ab2001.02, Ab2001.03, Ab2001.11 four antibodies are another binding epitope.

Ab2001.10 antibody was selected to determine whether the antigen-binding epitopes 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 were two different antigen-binding epitopes, and the Ab2001.16 double antibody contained two different antigen-binding epitopes, Ab2001.11 and Ab2001.12.

Table 9 Identification of candidate antibodies and SARS-CoV-2 antigen-binding epitopes

3.4 Candidate antibodies block the binding of SARS-CoV S1 to hACE2

It can be seen from Table 2 that after Y28B4 pure strain binds to SARS-CoV S1, the MFI of SARS-CoV S1 and hACE2-Fc is only 43.5, indicating that it may have the ability to block the binding of SARS-CoV S1 to hACE2. CR3022 is a neutralizing antibody against SARS-CoV S1 reported in the literature, which can block the binding of SARS-CoV S1 to hACE2 and can be used as a positive control. The antibody number corresponding to the pure Y28B4 strain 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. The ability of CoV S1 to bind to hACE2. The experimental scheme is the same as that in Example 3.2. The antibody concentration starts from 200 μg/mL and is diluted in 5 gradients, with a total of 7 concentrations, and an antibody concentration point of 0 μg/mL is added. The results are shown in Figure 3.

It can be seen from Figure 3 that the control antibody has a strong ability to block the binding of SARS-CoV S1 to hACE2, with an IC ₅₀ value of 6.21nM; Ab2001.11 has a weaker blocking ability, with an IC ₅₀ value of 303.87nM; the other antibodies have no blocking ability. Although the blocking ability of Ab2001.11 is weak, if the affinity is improved through affinity maturation, the blocking ability may be improved, and it is expected to become a broad-spectrum neutralizer that blocks both SARS-CoV-2 S1 and the binding of SARS-CoV S1 to hACE2 antibody.

Example 4 Physicochemical properties evaluation

Experimental Materials

Sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium hydroxide, guanidine hydrochloride, sodium chloride, ammonium sulfate, hydrochloric acid, glycine, sodium hydroxide, ethanol, and ammonium bicarbonate were purchased from Sinopharm Reagent; 1% methyl cellulose Element, CIEF Peptide Marker Kit, TTM solution, electrode solution, capillary cleaning solution, cIEF Cartridge FC-COATED were purchased from Protein Simple Company; TSKG3000SWxl (7.8×300mm, 5μm,) was purchased from TOSOH; Protein Thermal Shift ^TM Starter Kit, Propac WCX -10 (4 x 250 mm, 5 μm), MAbPacTMHIC-10 (4 x 250 mm, 5 μm, ) were purchased from Thermo.

4.1 Hydrophilic analysis of candidate antibodies

It can be seen from Figure 3 that the candidate antibodies Ab2001.02, Ab2001.03, Ab2001.08, Ab2001.09, Ab2001.10, Ab2001.11, Ab2001.12, and Ab2001.16 have the ability to block SARS-CoV-2 S1 and human receptors. The ability of the somatic protein ACE2 to bind. The hydrophilicity of these candidate antibodies was compared with that of omalizumab. The specific experimental steps are as follows, and the experimental results are shown in Figure 4.

(1) Dilute the sample to 1 mg/mL, mix well, centrifuge at 12,000 rpm for 5 minutes, take the supernatant and transfer it to a sample bottle and put it into the HPLC sample tray.

(2) Set the chromatographic conditions:

(3) Use 50mM phosphate buffer/1M ammonium sulfate, pH 7.0 as mobile phase A, use 50mM phosphate buffer, pH 7.0 as mobile phase B, carry out gradient elution analysis, perform data analysis with chromatographic software, and use hydrophilic The peak retention time of a good reference antibody is divided by the peak retention time of the candidate antibody, and the hydrophilic coefficient of each sample is calculated; according to past experience, the hydrophilic coefficient greater than 0.5 is defined as an acceptable standard line.

It can be seen from Figure 4 that the hydrophilic coefficients of the candidate antibodies are all greater than 0.5, which meets the internal druggability standard. The candidate antibodies AB2001.02, AB2001.03, AB2001.09, and AB2001.10 show excellent hydrophilicity.

4.2 Analysis of candidate antibody charge heterogeneity

Charge heterogeneity of candidate antibodies Ab2001.02, Ab2001.03, Ab2001.08, Ab2001.09, Ab2001.10, Ab2001.11, Ab2001.12, Ab2001.16 using imaging capillary isopoint focusing electrophoresis (iCIEF) Analysis, the specific steps are as follows:

(1) Take the sample solution and add it to the following system that has been thoroughly mixed: 1% methylcellulose (MC) 70 μL, urea 4M, 8 μL ampholyte Pharmalyte pH 3-10, pI marker 5.5 and 2 μL each of 9.5. Add an appropriate volume of ultrapure water to 200 μL and mix.

(2) Centrifuge all samples at 6000 rpm for 3 minutes to remove air bubbles, then transfer the supernatant to a sample bottle, place it on the sample tray, and record the sample position.

(3) Start the operation, and after the analysis, import the result file into Chrom Perfect software for spectrum integration processing and calculate the isoelectric point of each peak and the percentage of each peak.

The results of the analysis of the charge heterogeneity of the candidate antibodies are shown in Figure 5. It can be seen from Figure 5 that except for the low main peak of Ab2001.09, the other candidate antibodies have good charge heterogeneity, and the purity of the main peak is more than 70%. According to the analysis of charge heterogeneity, as shown in Figure 6, the Ab2001.16 bispecific antibody was purified by Protein A in one step, the purity of the main peak has reached 74.4%, and the proportion of heterodimers is close to 93%, or it can be compared with the monoclonal antibody. Shared production and purification processes.

4.3 Thermostability study of candidate antibodies

Thermal stability studies were performed on candidate antibodies Ab2001.02, Ab2001.03, Ab2001.08, Ab2001.09, Ab2001.10, Ab2001.11, Ab2001.12, Ab2001.16.

The specific steps are as follows: 1. Dilute the test product with sample buffer to 1 mg/mL; 2. According to the ^{instructions of the Protein Thermal Shift TM} Starter Kit, add 13 μL of the test solution to the PCR tube; 3. Add 5 μL Protein Thermal shift After TM buffer, add 2 μL of 10× staining solution to make the reaction volume 20 μL; 4. After mixing, centrifuge at 12,000 rpm for 5 minutes to remove air bubbles; 5. Put the test sample in the PCR machine, analyze the sample, and record the sample Fab melting temperature (Fab Tm), the results are shown in Figure 7.

In general, the candidate antibodies Ab2001.02, Ab2001.03, Ab2001.08, Ab2001.10, Ab2001.11, Ab2001.12, and Ab2001.16 have good physicochemical properties.

Example 5 Virus neutralization experiment

Based on the experimental results of Examples 3 and 4, candidate antibodies Ab2001.02, Ab2001.03, Ab2001.08, Ab2001.09, Ab2001.10, Ab2001.11, Ab2001.12, Ab2001.13, Ab2001.16; Ab2001 .07 was used as a negative control to evaluate the ability of candidate antibodies to neutralize 2019-nCoV infection in monkey kidney cells (Vero E6 cells). Due to the use of live viruses, the experiment was entrusted to the Wuhan Institute of Virology, Chinese Academy of Sciences, which has a Biosafety Level 3 Laboratory (BSL3). The specific experimental steps are as follows:

Reagents and Consumables:

Reagents: DMEM (Thermo Fisher), FBS (Gibco), methylcellulose (Millipore) Antibody diluent: PBS (Thermo Fisher)

Virus RNA extraction: QIAamp 96 Virus QIAcube HT Kit, Qiagen, 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.

Main instruments:

High-throughput nucleic acid automatic purification instrument: QIAcube HT 9001793 (Qiagen)

Fluorescence quantitative PCR instrument: CFX96 Touch Real-Time PCR Detection System (Bio-rad)

experimental design:

(1) SARS-CoV-2 virus (WIV04, GenBank: MN996528.1) amplification, titer: P6 generation of SARS-CoV-2 Vero-E6 cells were inoculated with virus at 37 ℃, 5% CO ₂ incubator After culturing for 48 hours, the cultured virus supernatant was collected, centrifuged, and aliquoted, and frozen at -80°C. Virus titers were determined in Vero-E6 cells using the classical plaque assay.

(2) Preliminary evaluation of antibodies: Vero-E6 cells were evenly plated in 24-well plates (10 ⁵ cells/well), and 16 hours later, culture medium containing different antibodies (final antibody concentration was 10 μg/mL) was added, and SARS cells with a MOI of 0.005 were added. -CoV-2 was infected, and the cell culture supernatant was collected after 24h and 72h for viral RNA extraction and detection.

(3) Effective antibody concentration gradient verification: Concentration gradient experiments (10 μg/mL, 2 μg/mL, 0.4 μg/mL) were performed on the effective antibodies screened in step 2, and the experimental protocol was the same.

(4) Plaque reduction neutralization test (PRNT)

The antibody to be tested was added to DMEM medium containing 2% FBS for 3-fold gradient dilution, and the diluted antibody was added to a 96-well plate, 100 μL per well; the virus was diluted to 2000 PFU/mL, and 100 μL were mixed with different concentrations of antibodies. ; (The final antibody concentrations are 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) , incubated at 37°C for 30 min, and then added the antibody and virus mixture to the cell culture. After 96 h of virus infection of the cells, a classical plaque experiment was performed to calculate the PRNT50 concentration.

Data processing and statistical analysis

PRNT50 was calculated using GraphPad prism software.

Experimental results

Preliminary evaluation of candidate antibody single concentration

Each candidate antibody (10 μg/mL) was added to the cell culture medium and then added with 0.005 MOI virus. After 24 hours, the viral RNA content of the supernatant was detected. The combined use of Ab2001.08 and Ab2001.10 (5 μg each) and the combined use of Ab2001.11 and Ab2001.12 (5 μg each) group decreased viral RNA content by about 2 orders of magnitude, as shown in Figure 8A; 72h detection of supernatant viral RNA Copy, Ab2001.10, Ab2001.08 and Ab2001.10 combined group decreased by more than 2 orders of magnitude, Ab2001.08 decreased by more than 1 order of magnitude, as shown in Figure 8B; 120h observation of cell status, Ab2001.08, Ab2001.10, The cells of the three groups were in good condition, and the other groups had obvious lesions.

Candidate Antibody Multi-Concentration Gradient Validation

Ab2001.08, Ab2001.10 and their combination were verified by multi-concentration gradients. 10μg/mL, 2μg/mL, and 0.4μg/mL antibodies were added to the cell culture medium, respectively, and then 0.005 MOI virus was added. After 24 hours, the viral RNA content in the supernatant was detected. Both decreased by about 2 orders of magnitude, as shown in Figure 9A; after 72 h, the viral RNA content of the supernatant was detected, only Ab2001.08 and the combination group decreased by about 2 orders of magnitude, as shown in Figure 9B.

Candidate Antibody Plaque Reduction Half Neutralization Concentration Assay

Ab2001.08, Ab2001.10 and the combination of the two groups were determined according to the experimental design scheme to reduce the neutralization concentration of the plaque by half, and the results are shown in Figure 10. As can be seen from Figure 10, the candidate antibodies Ab2001.08, Ab2001.10 and their combination all have the activity of neutralizing SARS-CoV-2, and their PRNT50 concentrations (the half-neutralizing concentration of plaque reduction) are: 0.012 μg/ mL, 0.062 μg/mL, and 0.022 μg/mL are expected to become effective drugs for the prevention and treatment of the new crown epidemic.

Example 6 Binding and blocking ability with different new coronavirus mutant strains

6.1 Binding and dissociation rates of neutralizing antibodies to SARS-CoV-2 S1 RBD and its mutants

Determination of binding of neutralizing antibodies Ab2001.08, Ab2001.10 to SARS-CoV-2 S1 RBD protein and mutant S1 RBD protein prevalent in different regions (doi: https://doi.org/10.1101/2020.03.15.991844) dissociation rate.

The neutralizing antibody was immobilized on the AHC sensor (Fortébio, Cat. No. 18-0015), and its interaction with SARS-CoV-2 S1 RBD and its mutants (ACRO, Cat. No.: SPD-S52H4, SPD-S52H5, SPD- S52H3, SPD-S52H8) binding and dissociation rates. Each cycle consists of the following steps: 1) sensor regeneration; 2) immersion buffer (PBST, 10 μL Tween 20 in 50 mL PBS) for 60 seconds; 3) 10 μg/mL fully human antibody immobilized on the AHC sensor , the time is 15 seconds; 4) The sensor is immersed in the buffer for 180 seconds; 5) 100nM SARS-CoV-2 S1 RBD binds to the antibody for 180 seconds; 6) Dissociation of antigen-antibody, time 5 minutes. Affinity results were analyzed by 1:1 fitting analysis by Octet Data Analysis Software (Fortébio), and the results are shown in Figure 11 .

As can be seen from the results in Figure 11, although the affinity of each mutant of SARS-CoV-2 S1 RBD to the human ACE2 receptor is higher than that of the wild type, the affinity of the neutralizing antibodies Ab2001.08 and Ab2001.10 to the RBD mutant also increases. The affinity is always higher than that of the RBD protein and the human ACE2 receptor, which means that even if the new coronavirus strain is mutated, the neutralizing antibody can maintain a good efficacy.

6.2 Neutralizing antibodies block the binding of SARS-CoV-2 S1 RBD and its mutants to hACE2-Fc

Blocking experiments were used to further demonstrate whether neutralizing antibodies could well block the binding of SARS-CoV-2 S1 RBD and its mutants to the human receptor ACE2.

The experimental method was carried out according to Example 3.2, the final concentration of SARS-CoV-2 S1 RBD and its mutants was 0.1 μg/mL, the initial concentration of antibody and hACE2-Fc was 100 nM, and the 5-fold gradient dilution (dilution 7 concentration points) was carried out. +1 concentration point of 0 μg/mL). The results are shown in Figures 12A to 12E, wherein Figures 12A to 12E respectively show that neutralizing antibodies blocked SARS-CoV-2 S1 RBD, SARS-CoV-2 S1 RBD V357F mutant, and SARS-CoV-2 S1 RBD N354D Binding of mutants, SARS-CoV-2 S1 RBD N354D and D304Y mutants, and SARS-CoV-2 S1 RBD R408I mutant to hACE2-Fc. From the results in Figure 12A to Figure 12E, it can be seen that the candidate antibody can well block the binding of the SARS-CoV-2 S1 RBD mutant and the human receptor ACE2, and the blocking effect is stronger than that of hACE2-Fc.

The embodiments of the present application are described in detail above. However, the present application is not limited to the specific details in the above-mentioned embodiments. Within the scope of the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications belong to the scope of protection of this application. In addition, it needs to be explained Yes, the specific technical features described in the above-mentioned embodiments can be combined in any suitable manner under the condition of no contradiction. In order to avoid unnecessary repetition, the present application does not separately describe the various possible combinations. instruction. In addition, the various embodiments of the present application can also be combined arbitrarily, as long as they do not violate the idea of the present application, they should also be regarded as the content disclosed in the present application.

<110> SHANGHAI JEMINCARE PHARM CO.,LTD. JIANGXI JEMINCARE GROUP CO.,LTD.

<120> SARS-CoV-2 antibody and its application

<130> 0134-PA-001TW

<160> 125

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<210> 84

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<212> PRT

<213> Artificial Sequence

<220>

<223> LFR4

<400> 84

<210> 85

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> LFR4

<400> 85

<210> 86

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> LFR4

<400> 86

<210> 87

<211> 30

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR1

<400> 87

<210> 88

<211> 30

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR1

<400> 88

<210> 89

<211> 30

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR1

<400> 89

<210> 90

<211> 30

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR1

<400> 90

<210> 91

<211> 30

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR1

<400> 91

<210> 92

<211> 30

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR1

<400> 92

<210> 93

<211> 30

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR1

<400> 93

<210> 94

<211> 30

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR1

<400> 94

<210> 95

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR2

<400> 95

<210> 96

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR2

<400> 96

<210> 97

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR2

<400> 97

<210> 98

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR2

<400> 98

<210> 99

<211> 32

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR3

<400> 99

<210> 100

<211> 32

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR3

<400> 100

<210> 101

<211> 32

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR3

<400> 101

<210> 102

<211> 32

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR3

<400> 102

<210> 103

<211> 32

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR3

<400> 103

<210> 104

<211> 32

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR3

<400> 104

<210> 105

<211> 32

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR3

<400> 105

<210> 106

<211> 32

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR3

<400> 106

<210> 107

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR4

<400> 107

<210> 108

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR4

<400> 108

<210> 109

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR4

<400> 109

<210> 110

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> HFR4

<400> 110

<210> 111

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR1

<400> 111

<210> 112

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR2

<400> 112

<210> 113

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> VL

<400> 113

<210> 114

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR1

<400> 114

<210> 115

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR2

<400> 115

<210> 116

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR3

<400> 116

<210> 117

<211> 127

<212> PRT

<213> Artificial Sequence

<220>

<223> VH

<400> 117

<210> 118

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Ab2001.02LCDR1; Ab2001.03LCDR1; Ab2001.11LCDR1; Ab2001.12LCDR1; Ab2001.13LCDR1;Ab2001.14LCDR1;Ab2001.23LCDR1;Ab2001.24LCDR1;Ab2001.25LCDR1; Ab2001.16LCDR1

<220>

<221> misc_feature

<222> (5)..(5)

<223> Xaa=Gly or Ser

<220>

<221> misc_feature

<222> (8)..(8)

<223> Xaa=Asn or Ser

<220>

<221> misc_feature

<222> (9)..(9)

<223> Xaa=Ser or Tyr

<220>

<221> misc_feature

<222> (11)..(11)

<223> Xaa=Ala or Asn

<400> 118

<210> 119

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Ab2001.04LCDR1; Ab2001.08LCDR1; Ab2001.09LCDR1; Ab2001.10LCDR1; Ab2001.19LCDR1

<220>

<221> misc_feature

<222> (1)..(1)

<223> Xaa=Gln or Arg

<220>

<221> misc_feature

<222> (5)..(5)

<223> Xaa=Asp, Gly or Ser

<220>

<221> misc_feature

<222> (6)..(6)

<223> Xaa=Ile or Val

<220>

<221> misc_feature

<222> (7)..(7)

<223> Xaa=Asn or Ser

<220>

<221> misc_feature

<222> (8)..(8)

<223> Xaa=Gly, Asn or Ser

<220>

<221> misc_feature

<222> (9)..(9)

<223> Xaa=Ser, Trp or Tyr

<220>

<221> misc_feature

<222> (10)..(10)

<223> Xaa=Leu or Tyr

<220>

<221> misc_feature

<222> (11)..(11)

<223> Xaa=Ala, Leu or Asn

<220>

<221> misc_feature

<222> (12)..(12)

<223> Xaa=Ala or does not exist

<400> 119

<210> 120

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Ab2001.02LCDR2; Ab2001.03LCDR2; Ab2001.11LCDR2; Ab2001.12LCDR2; Ab2001.13LCDR2;Ab2001.14LCDR2;Ab2001.23LCDR2;Ab2001.24LCDR2;Ab2001.25LCDR2; Ab2001.16LCDR2

<220>

<221> misc_feature

<222> (4)..(4)

<223> Xaa=Arg or Ser

<220>

<221> misc_feature

<222> (6)..(6)

<223> Xaa=Glu or Gln

<400> 120

<210> 121

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Ab2001.04LCDR2; Ab2001.08LCDR2; Ab2001.09LCDR2; Ab2001.10LCDR2; Ab2001.19LCDR2

<220>

<221> misc_feature

<222> (1)..(1)

<223> Xaa=Ala, Asp or Gly

<220>

<221> misc_feature

<222> (4)..(4)

<223> Xaa=Asn, Ser or Thr

<220>

<221> m.sc_feature

<222> (5)..(5)

<223> Xaa=Leu or Arg

<220>

<221> misc_feature

<222> (6)..(6)

<223> Xaa=Ala or Glu

<400> 121

<210> 122

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Ab2001.02LCDR3; Ab2001.03LCDR3; Ab2001.11LCDR3; Ab2001.12LCDR3; Ab2001.13LCDR3;Ab2001.14LCDR3;Ab2001.23LCDR3;Ab2001.24LCDR3;Ab2001.25LCDR3; Ab2001.16LCDR3

<220>

<221> misc_feature

<222> (8)..(8)

<223> Xaa=Pro or Ser

<220>

<221> mise_feature

<222> (9)..(9)

<223> Xaa=Ile or Thr

<220>

<221> misc_feature

<222> (10)..(10)

<223> Xaa=Thr or not present

<400> 122

<210> 123

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Ab2001.04LCDR3; Ab2001.08LCDR3; Ab2001.07LCDR3; Ab2001.09LCDR3; Ab2001.10LCDR3; Ab2001.19LCDR3

<220>

<221> misc_feature

<222> (4)..(4)

<223> Xaa=Asp or Gly

<220>

<221> misc_feature

<222> (5)..(5)

<223> Xaa=Asn or Ser

<220>

<221> misc_feature

<222> (6)..(6)

<223> Xaa=Leu or Ser

<220>

<221> misc_feature

<222> (8)..(8)

<223> Xaa=Ile, Leu, Gln or Val

<400> 123

<210> 124

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Ab2001.02VL; Ab2001.03VL; Ab2001.11VL; Ab2001.12VL; Ab2001.13VL; Ab2001.14VL; Ab2001.23VL; Ab2001.24VL; Ab2001.25VL; Ab2001.16VL

<220>

<221> misc_feature

<222> (28)..(28)

<223> Xaa=Gly or Ser

<220>

<221> misc_feature

<222> (31)..(31)

<223> Xaa=Asn or Ser

<220>

<221> misc_feature

<222> (32)..(32)

<223> Xaa=Ser or Tyr

<220>

<221> misc_feature

<222> (34)..(34)

<223> Xaa=Ala or Asn

<220>

<221> misc_feature

<222> (48)..(48)

<223> Xaa=Ile or Leu

<220>

<221> misc_feature

<222> (53)..(53)

<223> Xaa=Arg or Ser

<220>

<221> misc_feature

<222> (55)..(55)

<223> Xaa=Glu or Gln

<220>

<221> misc_feature

<222> (71)..(71)

<223> Xaa=Phe or Tyr

<220>

<221> misc_feature

<222> (96)..(96)

<223> Xaa=Pro or Ser

<220>

<221> misc_feature

<222> (97)..(97)

<223> Xaa=Ile or does not exist

<220>

<221> misc_feature

<222> (104)..(104)

<223> Xaa=Lys or Arg

<220>

<221> misc_feature

<222> (105)..(105)

<223> Xaa=Leu or Val

<400> 124

<210> 125

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Ab2001.04VL; Ab2001.08VL; Ab2001.09VL; Ab2001.10VL; Ab2001.19VL

<220>

<221> misc_feature

<222> (1)..(1)

<223> Xaa=Asp or Glu

<220>

<221> misc_feature

<222> (3)..(3)

<223> Xaa=Gln or Val

<220>

<221> misc_feature

<222> (4)..(4)

<223> Xaa=Leu or Met

<220>

<221> misc_feature

<222> (9)..(9)

<223> Xaa=Gly or Ser

<220>

<221> misc_feature

<222> (10)..(10)

<223> Xaa=Ser or Thr

<220>

<221> misc_feature

<222> (13)..(13)

<223> Xaa=Ala or Leu

<220>

<221> misc_feature

<222> (15)..(15)

<223> Xaa=Pre or Val

<220>

<221> misc_feature

<222> (17)..(17)

<223> Xaa=Asp or Glu

<220>

<221> misc_feature

<222> (19)..(19)

<223> Xaa=Ala or Val

<220>

<221> misc_feature

<222> (21)..(21)

<223> Xaa=Ile or Leu

<220>

<221> misc_feature

<222> (22)..(22)

<223> Xaa=Ser or Thr

<220>

<221> misc_feature

<222> (24)..(24)

<223> Xaa=Gln or Arg

<220>

<221> misc_feature

<222> (28)..(28)

<223> Xaa=Asp, Gly or Ser

<220>

<221> misc_feature

<222> (29)..(29)

<223> Xaa=

<220>

<221> misc_feature

<222> (30)..(30)

<223> Xaa=Asn or Ser

<220>

<221> misc_feature

<222> (31)..(31)

<223> Xaa=Gly, Asn or Ser

<220>

<221> misc_feature

<222> (32)..(32)

<223> Xaa=Ser or does not exist

<220>

<221> misc_feature

<222> (33)..(33)

<223> Xaa=Trp or Tyr

<220>

<221> misc_feature

<222> (35)..(35)

<223> Xaa=Ala or Asn

<220>

<221> misc_feature

<222> (43)..(43)

<223> Xaa=Lys or Gln

<220>

<221> misc_feature

<222> (46)..(46)

<223> Xaa=Lys or Arg

<220>

<221> misc_feature

<222> (51)..(51)

<223> Xaa=Ala, Asp or Gly

<220>

<221> misc_feature

<222> (54)..(54)

<223> Xaa=Asn, Ser or Thr

<220>

<221> misc_feature

<222> (55)..(55)

<223> Xaa=Leu or Arg

<220>

<221> misc_feature

<222> (56)..(56)

<223> Xaa=Ala or Glu

<220>

<221> misc_feature

<222> (59)..(59)

<223> Xaa=Ile or Val

<220>

<221> misc_feature

<222> (61)..(61)

<223> Xaa=Asp or Ser

<220>

<221> misc_feature

<222> (74)..(74)

<223> Xaa=Phe or Leu

<220>

<221> misc_feature

<222> (78)..(78)

<223> Xaa=Arg or Ser

<220>

<221> misc_feature

<222> (80)..(80)

<223> Xaa=Glu or Gln

<220>

<221> misc_feature

<222> (84).(84)

<223> Xaa=Phe or Ile

<220>

<221> misc_feature

<222> (86)..(86)

<223> Xaa=Thr or Val

<220>

<221> misc_feature

<222> (93)..(93)

<223> Xaa=Asp or Gly

<220>

<221> misc_feature

<222> (94)..(94)

<223> Xaa=Asn or Ser

<220>

<221> misc_feature

<222> (95)..(95)

<223> Xaa=Leu or Ser

<220>

<221> misc_feature

<222> (97)..(97)

<223> Xaa=Ile, Leu, Gln or Val

<220>

<221> misc_feature

<222> (101)..(101)

<223> Xaa=Gly or Gln

<220>

<221> misc_feature

<222> (104)..(104)

<223> Xaa=Lys or Arg

<220>

<221> misc_feature

<222> (105)..(105)

<223> Xaa=Leu or Val

<400> 125

Claims (49)

An isolated antigen binding protein having one or more of the following properties: 1) Block the binding of the RBD of the S protein of SARS-CoV-2 or its mutant to human ACE2; 2) Block the binding of the RBD of the S protein of SARS-CoV to human ACE2; 3) In the Octet assay, it specifically binds to the RBD of the S protein of SARS-CoV-2 with a K _D ^{value below about 5.0*10 -8 M;} 4) 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 about 6.0*10-10 M;} 5) The affinity with the RBD of the S protein of SARS-CoV-2 is stronger than that of human ACE2-Fc; 6) RBD that specifically binds to the S protein of SARS-CoV; 7) Hydrophilic; 8) The main peak of charge heterogeneity analysis is about 45%-85%; 9) Tm is at least about 75°C in Thermal shift assay; 10) It has the activity of neutralizing SARS-CoV-2. The isolated antigen-binding protein of claim 1, comprising at least one CDR in the VL of the light chain variable region, the VL comprising the amino acid sequence shown in SEQ ID NO:124 or SEQ ID NO:125. The isolated antigen-binding protein of claim 1 or 2, comprising at least one CDR in the VH of the heavy chain variable region, the VH comprising 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 The amino acid sequence shown in any one of NO:69, SEQ ID NO:71 and SEQ ID NO:73. The isolated antigen-binding protein of any one of claims 1 to 3, which comprises an antibody or antigen-binding fragment thereof. The isolated antigen-binding protein of claim 4, wherein the antigen-binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and/or dAb. The isolated antigen-binding protein of claim 4 or 5, wherein the antibody is a fully human antibody. The isolated antigen-binding protein of any one of claims 1-6, wherein the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR3 comprising any of SEQ ID NO: 122-SEQ ID NO: 123 amino acid sequence. The isolated antigen binding protein of claim 7, wherein the LCDR3 comprises SEQ ID NO: 3, SEQ ID NO: 13, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 36, SEQ ID NO : the amino acid sequence shown in any one of SEQ ID NO: 61. The isolated antigen-binding protein of claim 7 or 8, wherein the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:118-SEQ ID NO:119. The isolated antigen binding protein of any one of claims 7 to 9, wherein the LCDR1 comprises SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO : 34, the amino acid sequence shown in any one of SEQ ID NO: 41, SEQ ID NO: 59, and SEQ ID NO: 111. The isolated antigen-binding protein of any one of claims 7 to 10, wherein the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:120-SEQ ID NO:121. The isolated antigen binding protein of any one of claims 7 to 11, wherein the LCDR2 comprises SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 19, SEQ ID NO: 27. The amino acid sequence of any one of SEQ ID NO:35, SEQ ID NO:42, SEQ ID NO:60, and SEQ ID NO:112. The isolated antigen binding protein of any one of claims 3 to 12, wherein the VH comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprising SEQ ID NO: 5, SEQ ID NO: 22, SEQ ID NO: 30, The amino acid sequence set forth in any one of SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:63, and SEQ ID NO:114. The isolated antigen binding protein of claim 13, wherein the HCDR2 comprises SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO The amino acid sequence shown in any one of SEQ ID NO: 38, SEQ ID NO: 46, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 67, and SEQ ID NO: 115. The isolated antigen-binding protein of claim 13 or 14, wherein the HCDR3 comprises SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 39, SEQ ID NO: 39, 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 : the amino acid sequence shown in any one of 116. The isolated antigen binding protein of any one of claims 1 to 15, wherein the VL comprises framework regions L-FR1, L-FR2, L-FR3, and L-FR4. The isolated antigen-binding protein of claim 16, wherein the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, and the L-FR1 comprises SEQ ID NO:74-SEQ ID NO:75 Any of the amino acid sequences shown. The isolated antigen binding protein of claim 16 or 17, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO : the amino acid sequence shown in any one of 78. The isolated antigen binding protein of any one of claims 16 to 18, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises SEQ ID NO:79, SEQ ID NO:80 , the amino acid sequence shown in any one of SEQ ID NO: 81 and SEQ ID NO: 82. The isolated antigen-binding protein of any one of claims 16 to 19, wherein the N-terminus of the L-FR4 is directly or indirectly linked to the C-terminus of the LCDR3, and the L-FR4 comprises SEQ ID NO: 83, The amino acid sequence set forth in any one of SEQ ID NO:84, SEQ ID NO:85, and SEQ ID NO:86. The isolated antigen binding protein of any one of claims 2 to 20, wherein the VL comprises the amino acid sequence shown in SEQ ID NO:124 or SEQ ID NO:125. The isolated antigen binding protein of any one of claims 2 to 21, wherein the VL comprises SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 29, SEQ ID NO : the amino acid sequence shown in any one of SEQ ID NO: 44, SEQ ID NO: 62, and SEQ ID NO: 113. The isolated antigen-binding protein of any one of claims 4 to 22, which comprises an antibody light chain constant region, and the antibody light chain constant region comprises a human Igκ constant region or a human Igλ constant region. The isolated antigen binding protein of any one of claims 3 to 23, wherein the VH comprises framework regions H-FR1, H-FR2, H-FR3, and H-FR4. The isolated antigen-binding protein of claim 24, wherein the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises SEQ ID NO: 87, An amine group set forth in any one of SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, and SEQ ID NO:94 acid sequence. The isolated antigen binding protein of claim 24 or 25, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO : 97 and the amino acid sequence shown in any one of SEQ ID NO: 98. The isolated antigen binding protein of any one of claims 24 to 26, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises 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 amino acid sequence shown in any one. The isolated antigen-binding protein of any one of claims 24 to 27, wherein the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO: 107, The amino acid sequence set forth in any one of SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110. The isolated antigen binding protein of any one of claims 3 to 28, wherein the VH comprises 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 the amino acid sequence shown in any one of SEQ ID NO:73. The isolated antigen-binding protein of any one of claims 4 to 29, which comprises an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a human IgG constant region. The isolated antigen-binding protein of any one of claims 4 to 30, which comprises an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a human IgGl constant region. A bispecific antigen-binding protein that specifically binds 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 of claim 32, comprising a first targeting moiety that specifically binds to the RBD of the S protein of SARS-CoV-2, wherein the first targeting moiety comprises claims 1 to 31 The isolated antigen binding protein of any one. The bispecific antigen-binding protein of claim 32 or 33, comprising a second targeting moiety that specifically binds to the RBD of the S protein of SARS-CoV, wherein the second targeting moiety comprises claims 1 to 31 The isolated antigen binding protein of any one. The bispecific antigen binding protein of any one of claims 32 to 34, which comprises an antibody. The bispecific antigen binding protein of claim 34 or 35, wherein the first targeting moiety comprises a first heavy chain and a first light chain, and the second targeting moiety comprises a second heavy chain and a second light chain , wherein the first light chain and the second light chain are the same. The bispecific antigen-binding protein of claim 36, wherein the first light chain and the second light chain comprise the amino acid sequence shown in SEQ ID NO:44. The bispecific antigen-binding protein of claim 36 or 37, wherein the VH of the first heavy chain comprises the amino acid sequence shown in SEQ ID NO:48. The bispecific antigen binding protein of any one of claims 36 to 38, wherein the VH of the second heavy chain comprises the amino acid sequence shown in SEQ ID NO:50. The bispecific antigen-binding protein of any one of claims 36 to 39, wherein the VH of the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 48, and the VH of the second heavy chain Contains the amino acid sequence shown in SEQ ID NO:50. An isolated nucleic acid molecule encoding the isolated antigen binding protein of any one of claims 1 to 31, and/or the bispecific antigen binding protein of any one of claims 32 to 40. A vector comprising the nucleic acid molecule of claim 41. A cell comprising the nucleic acid molecule of claim 41 or the vector of claim 42. A method of preparing the isolated antigen-binding protein of any one of claims 1 to 31, the bispecific antigen-binding protein of any one of claims 32 to 40, the method comprising: The isolated antigen-binding protein according to any one of items 1 to 31 and/or the bispecific antigen-binding protein according to any one of claims 32 to 40 is expressed, cultured as described in claim 43 Cell. A pharmaceutical composition comprising the isolated antigen binding protein according to any one of claims 1 to 31, the bispecific antigen binding protein according to any one of claims 32 to 40, and the antigen binding protein according to any one of claims 41 Said nucleic acid molecule, the vector according to claim 42 and/or the cell according to claim 43, and optionally a pharmaceutically acceptable adjuvant. An isolated antigen binding protein according to any one of claims 1 to 31, a bispecific antigen binding protein according to any one of claims 32 to 40, a nucleic acid molecule according to claim 41, Use of the vector according to claim 42, the cell according to claim 43 and/or the pharmaceutical composition according to claim 45 in the preparation of a medicament for preventing, relieving and/or treating coronavirus infection. The use of claim 46, wherein the coronavirus infection comprises COVID-19. A method for blocking the binding of the RBD of the S protein of SARS-CoV-2 or a mutant thereof to human ACE2, comprising the steps of administering the isolated antigen-binding protein as described in any one of claims 1 to 31 , the bispecific antigen binding protein according to any one of claims 32 to 40, the nucleic acid molecule according to claim 41, the vector according to claim 42, the cell according to claim 43 and/ Or the pharmaceutical composition according to claim 45. A method for blocking the binding of the RBD of the S protein of SARS-CoV to human ACE2, comprising the steps of administering the isolated antigen-binding protein as described in any one of claims 1 to 31, as claimed in items 32 to 32 The bispecific antigen binding protein according to any one of 40, the nucleic acid molecule according to claim 41, the vector according to claim 42, the cell according to claim 43 and/or as claimed in claim 45 the described pharmaceutical composition.

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