WO2023131262A1

WO2023131262A1 - Antigen-binding protein specifically bound to sars-cov-2

Info

Publication number: WO2023131262A1
Application number: PCT/CN2023/070864
Authority: WO
Inventors: 朱建伟; 韩雷; 谢跃庆; 江华; 王澍生; 王振玉; 常云松
Original assignee: 杰库(上海)生物医药研究有限公司; 美国杰科实验室有限公司; 杰科（天津）生物医药有限公司
Priority date: 2022-01-10
Filing date: 2023-01-06
Publication date: 2023-07-13

Abstract

An isolated antigen-binding protein specifically bound to SARS-CoV-2, comprising at least one CDR in a variable region of light chain (VL), wherein the VL comprises an amino acid sequence as shown in SEQ ID NO.2. Also provided are a preparation method for the antigen-binding protein and the pharmaceutical use thereof.

Description

Antigen-binding proteins that specifically bind SARS-CoV-2

technical field

This application relates to the field of biomedicine, in particular to an antigen-binding protein that specifically binds to SARS-CoV-2.

Background technique

The outbreak of COVID-19 caused by SARS-Cov-2 has become a major global public health event. Prevention and treatment strategies for COVID-19 are being developed in preclinical and clinical research, and there are currently no very potent drugs for the treatment of COVID-19.

Antibody-based therapy is a viable treatment option. Neutralizing antibodies are an important part of the host's immune response to pathogens, and neutralizing monoclonal antibodies have been developed for the treatment of viral infections such as RSV, influenza, Ebola, HIV, HCMV, and rabies. At present, monoclonal antibody preparation technologies include hybridoma technology, EBV transformed B lymphocyte technology, phage display technology, transgenic mouse technology, and single B cell antibody preparation technology.

Contents of the invention

The application provides an isolated antigen-binding protein that specifically binds SARS-CoV-2. The isolated antigen-binding protein described in the application has at least the following beneficial effects: 1) specifically binds to SARS-CoV-2; 2) has the activity of neutralizing SARS-CoV-2; 3) is effective against SARS-CoV-2 Infections have good prophylactic, therapeutic and/or palliative effects. The present application also provides a preparation method of the isolated antigen-binding protein specifically binding to SARS-CoV-2, and a pharmaceutical application of the isolated antigen-binding protein specifically binding to SARS-CoV-2.

In one aspect, the application provides an isolated antigen-binding protein that specifically binds to SARS-CoV-2, 1. an isolated antigen-binding protein that specifically binds to SARS-CoV-2, which comprises a light chain variable region VL At least one CDR of, wherein said VL comprises the amino acid sequence shown in SEQ ID NO.2.

In some embodiments, the VL comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.6.

In some embodiments, the VL comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO.7.

In some embodiments, the VL comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.NO.8.

In some embodiments, the VL comprises LCDR1 and LCDR2, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.6, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO.7.

In some embodiments, the VL comprises LCDR1 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.6, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.8.

In some embodiments, the VL comprises LCDR2 and LCDR3, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO.7, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.8.

In some embodiments, the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.6, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO.7; the LCDR3 Comprising the amino acid sequence shown in SEQ ID NO.8.

In certain embodiments, the VL includes framework regions L-FR1, L-FR2, L-FR3 and L-FR4, wherein the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, And the L-FR1 comprises the amino acid sequence shown in SEQ ID NO.9.

In some embodiments, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO.10.

In some embodiments, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO.11.

In some embodiments, the N-terminal of the L-FR4 is directly or indirectly connected to the C-terminal of the LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO.12.

In some embodiments, the VL comprises the amino acid sequence shown in SEQ ID NO.2.

In certain embodiments, the isolated antigen binding protein comprises an antibody light chain constant region.

In certain embodiments, the isolated antigen-binding protein comprises at least one CDR in the VH of the light chain variable region, wherein the VH comprises the amino acid sequence shown in SEQ ID NO.1.

In some embodiments, the VH comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO.3.

In some embodiments, the VH comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO.4.

In some embodiments, the VH comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO.5.

In some embodiments, the VH comprises HCDR1 and HCDR2, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO.3, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO.4.

In some embodiments, the VH comprises HCDR1 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO.3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO.5.

In some embodiments, the VH comprises HCDR2 and HCDR3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO.4, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO.5.

In some embodiments, the VH comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO.3; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO.4; the HCDR3 Comprising the amino acid sequence shown in SEQ ID NO.5.

In certain embodiments, the VH includes framework regions H-FR1, H-FR2, H-FR3 and H-FR4, 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 the amino acid sequence shown in SEQ ID NO.13.

In some embodiments, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO.14.

In some embodiments, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO.15.

In some embodiments, the N-terminal of the H-FR4 is directly or indirectly connected to the C-terminal of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO.16.

In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO.1. In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region.

In certain embodiments, the isolated antigen binding protein has neutralizing activity against SARS-CoV-2.

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

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

In certain embodiments, the antibody is a fully human antibody.

In another aspect, the application provides isolated one or more nucleic acid molecules encoding said VL in the isolated antigen binding proteins described herein.

In another aspect, the application provides isolated one or more nucleic acid molecules encoding said VH in the isolated antigen binding proteins described herein.

In another aspect, the application provides isolated one or more nucleic acid molecules encoding the isolated antigen binding proteins described herein.

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

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

In certain embodiments, the cells express the isolated antigen binding proteins described herein.

On the other hand, the application provides a method for preparing the isolated antigen-binding protein described in the application, the method comprising culturing the antigen-binding protein described in the application under conditions that allow the expression of the isolated antigen-binding protein described in the application. the aforementioned cells.

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

In another aspect, the present application provides the isolated antigen-binding protein described herein, the nucleic acid molecule described herein, the carrier described herein, the cell described herein and/or the pharmaceutical combination described herein The purposes of the medicine in the preparation of medicine, described medicine is used for preventing, alleviating and/or treating the infection of coronavirus.

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

In another aspect, the present application provides a method for preventing, alleviating and/or treating coronavirus infection, which comprises administering the isolated antigen-binding protein described in the present application, the nucleic acid molecule described in the present application, the nucleic acid molecule described in the present application, The carrier of the application, the cell described in the application and/or the pharmaceutical composition described in the application.

In another aspect, the present application provides the isolated antigen-binding protein described herein, the nucleic acid molecule described herein, the carrier described herein, the cell described herein and/or the pharmaceutical combination described herein A substance, and its application in preventing, alleviating and/or treating coronavirus infection.

In another aspect, the present application provides a method for detecting SARS-CoV-2, comprising the steps of administering the isolated antigen-binding protein described in the present application, the nucleic acid molecule described in the present application, the carrier described in the present application, The cells described herein and/or the pharmaceutical compositions described herein.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make changes to the specific embodiments which are disclosed without departing from the spirit and scope of the invention to which this application relates. Correspondingly, the drawings and descriptions in the specification of the present application are only exemplary rather than restrictive.

Description of drawings

The particular features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates can be better understood with reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the accompanying drawings is as follows:

Figure 1 shows the neutralizing activity of the isolated antigen-binding protein described in the present application to the pseudovirus of SARS-CoV-2.

Figure 2 shows the neutralizing activity of the isolated antigen-binding protein described in the present application to the pseudovirus of the mutant strain of SARS-CoV-2.

Figure 3 shows the construction method of the mouse infection model, and the results show that the isolated antigen-binding protein described in this application makes the virus titer in the lung and brain of the virus undetectable, and realizes the complete neutralization of the virus.

Figure 4 shows the binding ability of the isolated antigen-binding protein described in this application to the S protein trimer of Omicron.

Figure 5 shows the therapeutic effect of the isolated antigen-binding protein described in this application on a mouse infection model.

Detailed ways

The implementation of the invention of the present application will be described in the following specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

Definition of Terms

In this application, the term "SARS-CoV-2" generally refers to Severe Acute Respiratory Syndrome Coronavirus Type 2, and its full English name is Severe Acute Respiratory Syndrome Coronavirus 2. SARS-CoV-2 belongs to the Betacoronavirus genus of the Coronaviridae family and the Sarbecovirus subgenus. SARS-CoV-2 is an enveloped, non-segmented, positive-sense, single-stranded RNA virus. SARS-CoV-2 can cause novel coronavirus pneumonia (COVID-19). In this application, the SARS-CoV-2 may include S protein (spike protein, spike protein).

In this application, the term "COVID-19" generally refers to Corona Virus Disease 2019, or Coronavirus Disease 2019, which is a respiratory disease caused by the SARS-CoV-2 virus. Common symptoms of COVID-19 can include fever, cough, fatigue, shortness of breath, and loss of smell and taste, with some symptoms progressing to viral pneumonia, multiple organ failure, or a cytokine storm. The disease spreads mainly through close contact between people, 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 "coronavirus S protein" generally refers to the spike protein (spike protein) of corona protein. The S protein can be combined into a trimer (ie S protein trimer), which contains about 1300 amino acids. The S protein may belong to the first class of membrane fusion protein (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 the receptor of the cell. S2 contains the basic elements required for the membrane fusion process, including an intrinsic membrane fusion peptide (fusion peptide), two heptad repeats (HR), and a membrane proximal external rich in aromatic amino acids. region, MPER), and the transmembrane region (transmembrane, TM). The S1 protein can be further divided into two domains, namely the N-terminal domain (N-terminal domain, NTD) and the C-terminal domain (C-terminal domain, CTD). The S protein can determine the host range and specificity of a virus (such as 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 refer to Daniel Wrapp et al., Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation, Science.

In this 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 can catalyze the conversion of angiotensin I into angiotensin-(1-9) or angiotensin II into angiotensin-(1-7). The ACE2 may include an N-terminal PD region (peptidase domain, peptidase domain) and a C-terminal CLD region (Collectrin-like domain). The angiotensin-converting enzyme II can be a receptor of SARS-CoV-2, for example, the extracellular domain of the ACE2 (for example, the PD region of the ACE2) can bind to the S protein of SARS-CoV-2 RBD. The accession number of human angiotensin-converting enzyme II in 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 Table 1 of as a captopril-insensitive carboxypeptidase. J.Biol.Chem.275:33238-33243,2000. In the present application, the ACE2 may include truncations or variants of the complete ACE2 protein, as long as the functional fragments still have the ability to function as receptors for coronaviruses (such as SARS-CoV and/or SARS-CoV-2) function.

In this application, the term "coronavirus infection" generally refers to diseases and/or symptoms caused by coronavirus infection. The coronavirus belongs to the genus Coronavirus of the order Nidovirales and the family Coronaviridae. The coronavirus can be a single-stranded RNA virus. The coronavirus infection may include respiratory tract infection, such as upper respiratory tract infection. The infection of the coronavirus may include symptoms such as fever, runny nose, chills, vomiting and/or fatigue.

In this application, the term "neutralization" generally refers to the neutralizing activity of the antigen-binding protein, that is, the antigen-binding protein can prevent and/or neutralize the biochemical activity of its corresponding antigen. In some cases, an antigen-binding protein with such neutralizing activity can counteract and inactivate an antigen that attacks the immune system (eg, a retrovirus, for example, the antigen can be SARS-CoV-2). In some cases, the antigen-binding protein having the neutralizing activity does not require the participation of leukocytes when neutralizing the biochemical activity of its corresponding antigen.

In the present application, the term "antigen binding protein" generally refers to a protein comprising a moiety that binds an antigen, and optionally a scaffold or backbone moiety that allows the moiety that binds the antigen 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 dAb), immunoconjugates substances, multispecific antibodies (such as bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.

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 contains 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 is different from Fab by adding a small number of residues (including one or more cysteines from the antibody hinge region) to the carboxyl terminus of the CH1 domain of the heavy chain; the term "F(ab ')2" generally refers to a dimer of Fab', an antibody fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region. The term "Fv" generally refers to the smallest antibody fragment that contains a complete antigen recognition and binding site. In some cases, the fragment may consist of a dimer of a heavy chain variable domain and a light chain variable domain in tight non-covalent association; the term "dsFv" generally refers to a disulfide bond stabilized Fv fragment, The linkage between its single light chain variable domain and its single heavy chain variable domain 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 molecule formed by pairing one heavy chain variable domain and one light chain variable domain of an antibody through a flexible peptide linker; such scFv molecules may have general Structure: NH ₂ -VL-Linker-VH-COOH or NH ₂ -VH-Linker-VL-COOH.

In this application, the term "antibody" generally refers to an immunoglobulin that can specifically bind to a corresponding antigen. The antibodies may be secreted by immune cells such as effector B cells. The antibody can be a monoclonal antibody (including a full-length monoclonal antibody comprising two light chains and two heavy chains), a polyclonal antibody, a multispecific antibody (such as a bispecific antibody), a humanized antibody, a fully Human antibodies, chimeric antibodies and/or camelized single domain antibodies. An "antibody" may generally comprise a protein of at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain comprises 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 consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), which alternate with more conserved regions called framework regions (FRs). Each VH and VL comprises 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) , most 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 in close proximity by the FR regions and, together with the CDRs from the other chain, form 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 certain parts of the sequence of the variable domains of antibodies vary 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 domains of antibodies. 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 portions of variable domains are called the frameworks (FRs). In the art, the CDRs of antibodies can be defined by various methods, such as the Kabat definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, 5th edition, National Institutes of Health, Besse Star, MD (1991)), Chothia definition rules based on the location of structural ring regions (see, A1-Lazikani et al., JMol Biol 273:927-48, 1997) and concepts based on the IMGT ontology (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 immunoglobulin (IG) or antibodies from humans and other vertebrates, T cell receptor (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, separated and/or recovered from a component of the environment in which it was produced (eg, natural or recombinant). Contaminating components of the environment in which it is produced are usually substances that interfere with its research, diagnostic or therapeutic use and can include enzymes, hormones and other proteinaceous or nonproteinaceous solutes. Isolated antigen binding protein or antibody will usually 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 usually highly specific against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by hybridoma cultures without contamination from other immunoglobulins. The modifier "monoclonal" denote the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies used herein can be produced in hybridoma cells, or can be produced by recombinant DNA methods.

In this application, the term "fully human antibody" generally refers to the antibody that is expressed by transferring the gene encoding the human antibody into a genetically engineered animal lacking the antibody gene. All portions of the antibody, including the variable and constant regions of the antibody, are encoded by genes of human origin. Fully human antibodies can greatly reduce the immune side effects caused by heterologous antibodies on the human body. Methods for obtaining fully human antibodies in this field include phage display technology, transgenic mouse technology, ribosome display technology and RNA-polypeptide technology.

In this application, the terms "bind", "specifically bind" 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 in the presence of a molecular The presence of a target in the context of a heterogeneous population (including biological molecules). For example, an antibody binds an epitope through 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 this target with greater affinity, avidity, easier and/or for a greater duration than it binds other targets. An antibody is said to "specifically bind" an antigen when it binds an epitope more readily through its antigen-binding domain than it would bind a random, unrelated epitope. "Epitope" refers to a specific atom on an antigen that binds to an antigen-binding protein (such as an antibody) Click or click here to enter text. Click or tap here to enter text. Click or tap here to enter text. groups (eg, sugar side chains, phosphoryl, sulfonyl) or amino acids.

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

In this application, the term "between" usually means that the C-terminal of a certain amino acid fragment is directly or indirectly connected to the N-terminal of the first amino acid fragment, and its N-terminal is directly or indirectly connected to the C-terminal of the second amino acid fragment. indirect connection. In the light chain, for example, the N-terminal of the L-FR2 is directly or indirectly connected to the C-terminal of the LCDR1, and the C-terminal of the L-FR2 is directly or indirectly connected to the N-terminal of the LCDR2. For another example, the N-terminal of the L-FR3 is directly or indirectly connected to the C-terminal of the LCDR2, and the C-terminal of the L-FR3 is directly or indirectly connected to the N-terminal of the LCDR3. In the heavy chain, for example, the N-terminal of the H-FR2 is directly or indirectly connected to the C-terminal of the HCDR1, and the C-terminal of the H-FR2 is directly or indirectly connected to the N-terminal of the HCDR2. For another example, the N-terminal of the H-FR3 is directly or indirectly connected to the C-terminal of the HCDR2, and the C-terminal of the H-FR3 is directly or indirectly connected to the N-terminal of the HCDR3. In the present application, "first amino acid fragment" and "second amino acid fragment" may be any amino acid fragment that is the same or different.

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. The isolated nucleic acid molecule may not be associated with all or a portion of a polynucleotide found in nature, or linked to a polynucleotide to which it is not associated 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 an inserted nucleic acid molecule into and/or between host cells. The vectors may include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and vectors mainly used for expression of transcription and/or translation of DNA or RNA. The carrier also includes a carrier having various functions as described above. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce the desired expression product by culturing an appropriate host cell containing the vector.

In the present application, the term "cell" generally refers to individual cells, cell lines or cell culture. The cells may include progeny of a single host cell. Due to natural, accidental or deliberate mutations, the progeny cells may not necessarily be completely identical in shape or genome to the original parent cells, but it is sufficient to be able to express the antibodies or antigen-binding fragments thereof described in this application. The cells can be obtained by transfecting cells in vitro with the vectors described in this application. The cells can be prokaryotic cells (such as Escherichia coli) or eukaryotic cells (such as yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells or myeloma cells). In the present application, the cell may include a cell into which the vector is introduced. The cells include not only specific cells but also descendants of these cells.

In this application, the term "pharmaceutically acceptable adjuvant" generally includes a pharmaceutically acceptable carrier, excipient or stabilizer which is incompatible with the cells or mammals to which it is exposed at the dosage and concentration employed. poisonous. Typically, the physiologically acceptable carrier is a pH buffered aqueous solution.

As used herein, the term "administering" generally refers 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" can refer to therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of cells can include contacting a reagent (eg, a reagent comprising the isolated antigen binding protein) with the cell, as well as contacting the reagent with a fluid, and contacting the fluid with the cell. "Administering" also means by an agent, a diagnostic, a binding composition, or by another in vitro and ex vivo treatment of a cell. "Treatment" when applied to a human, animal or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnosis; for example may include the association of the isolated antigen binding protein with a human or animal, subject, Contact of cells, tissues, physiological compartments or physiological fluids.

As used herein, the term "treatment" refers to giving a patient an internal or external therapeutic agent, such as comprising any one of the isolated antigen-binding proteins of the present application, and/or a pharmaceutical composition comprising the isolated antigen-binding proteins, The patient has one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. Generally, an amount of the therapeutic agent effective to alleviate one or more symptoms of the disease (therapeutically effective amount) is administered to a patient. Desirable effects of treatment include decreased rate of disease progression, amelioration or palliation of the 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 symptoms resulting from the disease, improving the quality of life of those individuals with the disease , reducing the dose of other drugs needed to treat the disease, delaying the progression of the disease, and/or prolonging the survival of the individual, the individual is successfully "treated".

In this application, the term "comprises" generally refers to the meanings comprising, encompassing, comprising or encompassing. In some cases, it also means "for" and "consisting of".

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%, above or below the specified value. 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 that specifically binds to SARS-CoV-2, comprising at least one CDR in the VL of the light chain variable region, wherein the VL comprises the protein shown in SEQ ID NO.2 amino acid sequence.

In the present application, the VL may comprise LCDR1, and the LCDR1 may comprise SEQ ID NO.6. For example, the sequence may be a sequence determined according to KABAT definition rules.

In the present application, the VL may comprise LCDR2, and the LCDR2 may comprise SEQ ID NO.7. For example, the sequence may be a sequence determined according to KABAT definition rules.

In the present application, the VL may comprise LCDR3, and the LCDR3 may comprise SEQ ID NO.8. For example, the sequence may be a sequence determined according to KABAT definition rules.

For example, the VL may comprise LCDR1 and LCDR2, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO.6, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO.7.

For example, the VL may comprise LCDR1 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO.6, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO.8.

For example, the VL may comprise LCDR2 and LCDR3, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO.7, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO.8.

For example, the VL may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO.6, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO.7; the LCDR3 may comprise The amino acid sequence shown in SEQ ID NO.8.

For example, the VL may comprise the amino acid sequence shown in SEQ ID NO.2.

For example, the isolated antigen-binding protein may comprise a heavy chain variable region VH, the VH may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO.3. For example, the sequence may be a sequence determined according to KABAT definition rules.

For example, the VH may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO.4. For example, the sequence may be a sequence determined according to KABAT definition rules.

For example, the VH may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO.5. For example, the sequence may be a sequence determined according to KABAT definition rules.

For example, the VH may comprise HCDR1 and HCDR2, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO.3, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO.4.

For example, the VH may comprise HCDR1 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO.3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO.5.

For example, the VH may comprise HCDR2 and HCDR3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO.4, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO.5.

For example, the isolated antigen-binding protein may comprise a heavy chain variable region VH, the VH may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO.SEQ ID NO.3; The HCDR2 may comprise the amino acid sequence shown in SEQ ID NO.4; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO.5.

For example, the VH may comprise the amino acid sequence shown in SEQ ID NO.1.

The isolated antigen-binding protein described in 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, so The heavy chain variable region of the reference antibody may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO.3, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO.4, And the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO.5, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO.6, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO.7, and The LCDR3 may comprise the amino acid sequence shown in SEQ ID NO.8.

In the present application, the antigen binding protein of described separation can comprise antibody light chain variable region CDR---LCDR1, LCDR2 and LCDR3, and described LCDR1 can comprise the aminoacid sequence shown in SEQ ID NO.6, and described LCDR2 can comprise The amino acid sequence shown in SEQ ID NO.7, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO.8.

In the present application, the antigen binding protein of described separation can comprise antibody heavy chain variable region CDR---HCDR1, HCDR2 and HCDR3, and described HCDR1 can comprise the aminoacid sequence shown in SEQ ID NO.3, and described HCDR2 can comprise The amino acid sequence shown in SEQ ID NO.4, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO.5.

In the present application, the antigen binding protein of described separation can comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, described HCDR1 can comprise the amino acid sequence shown in SEQ ID NO.3, and described HCDR2 can comprise SEQ ID NO The amino acid sequence shown in .4, and the HCDR3 can include the amino acid sequence shown in SEQ ID NO.5, the LCDR1 can include the amino acid sequence shown in SEQ ID NO.6, and the LCDR2 can include the amino acid sequence shown in SEQ ID NO. The amino acid sequence shown in 7, and the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO.8.

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

For example, the C-terminal of the L-FR1 may be directly or indirectly connected to the N-terminal of the LCDR1, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO.9.

For example, the L-FR2 may be located between the LCDR1 and the LCDR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO.10.

For example, the L-FR3 may be located between the LCDR2 and the LCDR3, and the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO.11.

For example, the N-terminal of the L-FR4 may be directly or indirectly connected to the C-terminal of the LCDR3, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO.12.

In the present application, the VL may comprise the amino acid sequence shown in SEQ ID NO.2.

For example, the isolated antigen binding protein can comprise an antibody light chain constant region, and the antibody light chain constant region comprises a human Ig kappa constant region or a human Ig lambda constant region.

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

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

For example, the C-terminal of the H-FR1 may be directly or indirectly connected to the N-terminal of the HCDR1, and the H-FR1 may comprise the amino acid sequence of SEQ ID NO.9.

For example, the H-FR2 may be located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10.

For example, the H-FR3 may be located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO.11.

For example, the N-terminal of the H-FR4 may be directly or indirectly connected to the C-terminal of the HCDR3, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO.12.

For example, the VH may comprise the amino acid sequence shown in SEQ ID NO.1.

In the present application, the antigen binding protein of described isolation can comprise light chain variable region VL and heavy chain variable region VH, and described VL can comprise the aminoacid sequence shown in SEQ ID NO.2, and described VH can comprise SEQ ID NO.2 Amino acid sequence shown in ID NO:1.

The protein, polypeptide and/or amino acid sequence involved in the present application should also be understood to include at least the following scope: variants or homologues having the same or similar functions as the protein or polypeptide.

In the present application, the variant may be, in the amino acid sequence of the protein and/or the polypeptide (for example, the antigen-binding protein described in the application) substituted, deleted or added one or more amino acids protein or peptide. For example, the functional variant may comprise at least 1, such as 1-30, 1-20 or 1-10, further such as 1, 2, 3, 4 or 5 amino acid substitutions , proteins or polypeptides with amino acid changes by deletion and/or insertion. Said functional variant may substantially retain the biological properties of said protein or said polypeptide prior to 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 ability) of the protein or polypeptide prior to the alteration. For example, the substitutions may be conservative substitutions.

In the present application, a part of the amino acid sequence of the antigen-binding protein may be homologous to the corresponding amino acid sequence in an antibody from a specific species, or belong to a specific class. For example, both the variable and constant portions of the antigen binding protein can be derived from the variable and constant regions of an antibody of one animal species, such as a human. In the present application, the homolog may be at least about 85% (for example, having at least about 85% amino acid sequence) with the protein and/or the polypeptide (for example, the antigen binding protein described in the application). %, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology of proteins or peptides.

In this application, the homology generally refers to the similarity, similarity or association between two or more sequences. The "percentage of sequence homology" can be calculated by comparing the two sequences to be aligned in the comparison window, and determining the presence of the same nucleic acid base (for example, A, T, C, G) or Positions of identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) Number To obtain the number of matching positions, the number of matching positions was divided by the total number of positions in the comparison window (ie, window size), and the result was multiplied by 100 to yield the percent sequence identity. Alignment for purposes of determining percent sequence homology can be accomplished in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared or over the sequence region 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 "An Improved Tool for Biological Sequence Comparison" by W.R.Pearson and D.J. Lipman, Proc. Natl. Acad. Sci., 85:2444-2448, 1988; and D.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.

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

For example, the isolated antigen binding protein can comprise an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a human IgG1 constant region.

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

In the present application, the isolated antigen-binding protein may comprise an antibody or an antigen-binding fragment thereof. For example, isolated antigen binding proteins described herein may include, but are not limited to, recombinant antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single chain antibodies, diabodies, triabodies , tetrabody, Fv fragment, scFv fragment, Fab fragment, Fab' fragment, F(ab')2 fragment and camelized single domain antibody.

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, either 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.

In the present application, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and/or dAb.

The antigen binding protein (eg, SARS-CoV-2 antibody) described in the present application can specifically bind the RBD of the S protein of SARS-CoV-2. Antigen-binding proteins (e.g., antibodies) that "specifically bind" to a SARS-CoV-2 antigen (e.g., the RBD of the S protein of SARS-CoV-2) can typically bind with an EC50 value of about 10 or more with an affinity (e.g., about RBD of the S protein of SARS-CoV-2, but not other proteins lacking SARS-CoV-2 sequences. 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. For example, detected by flow cytometry and ELISA.

The antigen-binding protein (eg, SARS-CoV-2 antibody) described in the present application can block the binding of RBD of the S protein of SARS-CoV-2 or its functional fragments to human ACE2. Blocking assays can be tested using a competition assay, for example, combining the antigen-binding protein (e.g., SARS-CoV-2 antibody) with an antigen (or, a cell expressing the antigen) and a ligand for the antigen (or, expressing the ligand). body cells), the ability of the antigen-binding protein to compete with the ligand of the antigen for binding to the antigen is reflected based on the intensity (eg, fluorescence intensity or concentration) of the detectable label.

The protein and/or amino acid sequence involved in this application should also be understood to include at least the following range: variants or homologues having the same or similar functions as the protein.

In the present application, the variant may be a protein or polypeptide with substitution, deletion or addition of one or more amino acids in the amino acid sequence of the protein (eg, the antigen-binding protein described in the present application). For example, the functional variant may comprise at least 1, such as 1-30, 1-20 or 1-10, further such as 1, 2, 3, 4 or 5 amino acid substitutions , proteins or polypeptides with amino acid changes by deletion and/or insertion. Said functional variant may substantially retain the biological properties of said protein or said polypeptide prior to 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 ability) of the protein or polypeptide prior to the alteration. For example, the substitutions may be conservative substitutions.

In the present application, a part of the amino acid sequence of the antigen-binding protein may be homologous to the corresponding amino acid sequence in an antibody from a specific species, or belong to a specific class. For example, both the variable and constant portions of an antibody can be derived from the variable and constant regions of an antibody from one animal species, such as a human. In the present application, the homolog may be at least about 85% (for example, having at least about 85% amino acid sequence) with the protein and/or the polypeptide (for example, the antigen binding protein described in the application). %, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology of proteins or peptides.

In this application, the homology generally refers to the similarity, similarity or association between two or more sequences. The "percentage of sequence homology" can be calculated by comparing the two sequences to be aligned in the comparison window, and determining the presence of the same nucleic acid base (for example, A, T, C, G) or Positions of identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) Number To obtain the number of matching positions, the number of matching positions was divided by the total number of positions in the comparison window (ie, window size), and the result was multiplied by 100 to yield the percent sequence identity. Alignment for purposes of determining percent sequence homology can be accomplished in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared or over a region of 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 "An Improved Tool for Biological Sequence Comparison" by W.R.Pearson and D.J. Lipman, Proc. Natl. Acad. Sci., 85:2444-2448, 1988; and D.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.

pharmaceutical composition

On the other hand, the present application provides a pharmaceutical composition, which may comprise the isolated antigen-binding protein described in the present application, the nucleic acid molecule described in the present application, the carrier described in the present application and/or the nucleic acid molecule described in the present application. cells, and optionally a pharmaceutically acceptable adjuvant.

The pharmaceutical composition described in this application can be directly used to bind 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 (such as 0.001-99wt%) of the antigen-binding protein described in the present application and a pharmaceutically acceptable adjuvant (may include a carrier or excipient). The pharmaceutical formulation should match the mode of administration. The pharmaceutical composition described in this application can be prepared in the form of injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably produced under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. In addition, the antigen binding proteins described herein can also be used with other therapeutic agents.

The antigen binding proteins or pharmaceutical compositions described herein can be formulated, dosed and administered in a manner consistent with good medical practice. Considerations in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the etiology of the condition, the site of delivery of the agent, the method of administration and other factors known to the medical practitioner. Therapeutic agents (e.g., the antigen binding proteins described herein and/or the pharmaceutical compositions described herein) need not, but are optionally formulated with one or more agents currently used to prevent or treat the disorder under consideration and/or or at the same time. The effective amount of such other agents depends on the amount of therapeutic agent present in the formulation (e.g., the antigen binding proteins described herein and/or the pharmaceutical compositions described herein), the type of disorder or treatment, and other factors discussed above . These agents can generally be administered in any dosage and by any route empirically/clinically determined to be appropriate. The dose of antibodies administered in combination therapy can be reduced compared to the individual treatments. The progress of this therapy is easily monitored by conventional techniques.

use

In another aspect, the present application provides an isolated antigen-binding protein described herein, a nucleic acid molecule described herein, a carrier described herein, a cell described herein and/or a drug described herein Use of the composition in the preparation of medicaments for preventing, alleviating and/or treating coronavirus infection.

The present application provides a method for preventing, alleviating and/or treating coronavirus infection, which comprises administering the isolated antigen-binding protein described in the present application, the nucleic acid molecule described in the present application, the nucleic acid molecule described in the present application to a subject in need. The carrier, the cell described in the application and/or the pharmaceutical composition described in the application.

The application provides an isolated antigen binding protein, a nucleic acid molecule described herein, a carrier described herein, a cell described herein and/or a pharmaceutical composition described herein, which can prevent, relieve and/or Or treat coronavirus infection.

In this application, the coronavirus infection may include COVID-19.

In the present application, administration of the isolated antigen binding protein described herein, the nucleic acid molecule described herein, the carrier described herein, the cell described herein and/or the pharmaceutical composition described herein can be It has effective neutralizing ability against pseudoviruses of COVID-19 (such as pseudoviruses prepared from WA1/2020, Alpha, Beta, Gamma, Kappa, Delta and Omicron's Spike trimer).

In the present application, administration of the isolated antigen binding protein described herein, the nucleic acid molecule described herein, the carrier described herein, the cell described herein and/or the pharmaceutical composition described herein can be Different strains of COVID-19 (eg SARS-CoV-2 WA1/2020 (US_WA-1/2020 isolate), Alpha (B.1.1.7/UK, strain: SARS-CoV-2/human/USA/CA_CDC_5574 /2020), Beta (B.1.351/SA, Strain: hCoV-19/USA/MD-HP01542/2021), Gamma (P.1/Brazil, Strain: SARS-CoV-2/human/USA/MD-MDH -0841/2021), Delta variant (B.1.617.2/Indian, strain: GNL-751) and Omicron variant (B.1.1.529)) have potent neutralizing capacity.

In the present application, administration of the isolated antigen binding protein described herein, the nucleic acid molecule described herein, the carrier described herein, the cell described herein and/or the pharmaceutical composition described herein can be Different strains that have been infected with COVID-19 (such as SARS-CoV-2 WA1/2020 (US_WA-1/2020 isolate), Alpha (B.1.1.7/UK, strain: SARS-CoV-2/human/USA /CA_CDC_5574/2020), Beta (B.1.351/SA, Strain: hCoV-19/USA/MD-HP01542/2021), Gamma (P.1/Brazil, Strain: SARS-CoV-2/human/USA/MD - MDH-0841/2021), Delta variant (B.1.617.2/Indian, strain: GNL-751) and Omicron variant (B.1.1.529)) animal models (e.g. mouse model, rhesus monkey model) has a good therapeutic effect.

In another aspect, the present application provides a method for detecting SARS-CoV-2, which includes the following steps of administering the isolated antigen-binding protein described in the present application, the nucleic acid molecule described in the present application, and the carrier described in the present application , the cells described herein and/or the pharmaceutical compositions described herein. In the present application, the isolated antigen-binding protein, the nucleic acid molecule described in the present application, the carrier described in the present application, the cell described in the present application and/or the pharmaceutical composition described in the present application can specifically And/or bind SARS-CoV-2 with high affinity, such as binding to the Spike trimer of strains WA1/2020, Alpha, Beta, Gamma, Kappa, Delta and Omicron.

The antigen binding proteins of the present application can be used in detection applications, for example for detection of samples, thereby providing diagnostic information. For example, the antibodies and/or methods described in this application can be used to treat samples (for example, throat swabs) of subjects (for example, patients suspected of being infected by SARS-CoV-2, or patients who have been infected by SARS-CoV-2). Sub-test samples, such as serum, whole blood, sputum, oral/nasopharyngeal secretions or washings, urine, feces, pleural and peritoneal effusions, cerebrospinal fluid, and tissue samples) are used as indicators for curative effect observation and whether they are infectious Indicators of sex and need for isolation. For example, the antibodies and/or methods described herein can provide a monitoring scheme for therapeutic intervention.

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

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

Without intending to be bound by any theory, the following examples are only for explaining the isolated antigen-binding protein of the present application, the preparation method and application, etc., and are not intended to limit the scope of the invention of the present application.

Example

Preparation of Example 1 Candidate Antibody

From the peripheral blood of recovered patients with COVID-19, memory B cells that can recognize SARS-CoV-2 RBD were isolated by flow cytometry, and single B cell sorting was performed on these isolated memory B cells. By single-cell PCR method, the V region gene of the antibody is cloned, and the IgG type antibody is reconstituted.

The resulting candidate antibody contains the amino acid sequence shown in Table 1:

Table 1

序号serial number	克隆号clone number	VHVH		VLVL
11	8G38G3	SEQ ID NO.1SEQ ID NO.1	SEQ ID NO.2SEQ ID NO.2

Example 2 Affinity Determination of Candidate Antibody Binding to SARS-CoV-2S Trimeric Protein

The day before the experiment, add the antigen SARS-CoV-2 Spike trimer protein (ie, S protein trimer) to the coating buffer (pH 9.6, 0.05M carbonate buffer) to a final concentration of 2 μg/mL. Add 100 μL to each well and shake slightly until the liquid in each well is evenly spread on the bottom. Put the ELISA plate containing the antigen into a sealed bag, seal it and put it in a refrigerator at 4°C for overnight antigen adsorption;

The next day, discard the supernatant, pat dry on clean absorbent paper, add 250 μL/well washing solution (pH 7.4 PBST), keep for 5 min each time, discard the supernatant and pat dry on clean absorbent paper, repeat 3 times. Add 250 μL/well blocking solution (PBST+3% skimmed milk powder), put into a new sealed bag, and block at 37° C. for 1 hour. Discard the supernatant, pat dry on clean absorbent paper, wash with 250 μL/well washing solution, keep for 5 min each time, discard supernatant and pat dry on clean absorbent paper, repeat 3 times.

The candidate antibody 8G3 prepared in Example 1 was serially diluted using antibody diluent (pH7.4PBS).

Pipette the diluted candidate antibody sample at 100 μL/well and add it to the treated microtiter plate, put it into a new sealed bag, and incubate at 37°C for 1 hour. Discard the supernatant, pat dry on clean absorbent paper, wash with 250 μL/well washing solution, keep for 5 min each time, discard supernatant and pat dry on clean absorbent paper, repeat 3 times. According to 100 μL/well, add anti-human IgG HRP secondary antibody diluted 1:5000, put into a new sealed bag, and incubate at 37°C for 1h. Discard the supernatant, pat dry on clean absorbent paper, wash with 250 μL/well washing solution, keep for 5 min each time, discard supernatant and pat dry on clean absorbent paper, repeat 3 times. Add TMB chromogenic solution at 100 μL/well, wrap in tin foil and develop color at room temperature for 15 minutes in the dark, observe the blue reaction, add stop solution (2M H ₂ SO ₄ ) at 50 μL/well, mix well and immediately read at 450 nm on a microplate reader.

The results showed that 8G3 had a higher affinity for S trimer protein.

The neutralizing activity assay of embodiment 3 candidate antibody pseudo-SARS-CoV-2 virus

The day before the experiment, the HEK293T-ACE2 cells to be infected were inoculated in a 96-well cell culture plate with an inoculation amount of about 1 × ¹⁰⁴ cells/well, and cultured overnight at 37°C in a 5% CO ₂ incubator. On the second day, virus infection was carried out when the cell density was about 30%, and the frozen pseudovirus was taken out and thawed on ice or completely thawed at 4°C. The amount of virus used was 0.25 μL/well, respectively Diluted concentrations of the candidate antibodies prepared in Example 1 were mixed and incubated at 37° C. for 30 min, and the mixture was added to the cell culture system to infect the target cells. After 6 hours of virus infection, the supernatant was sucked off, and 100 μL of complete medium was added to continue culturing for 48 hours. Forty-eight hours after the cells were infected with the pseudovirus, the expression of green fluorescent protein and the activity of luciferase were observed by fluorescence microscope to determine the infection efficiency. Add 100 μL of One-Glo luciferase to each well, shake and mix well, and read with a microplate reader after 3 minutes.

The results are shown in Figure 1 and Table 2. The results show that the candidate antibodies all have good neutralizing activity against the pseudo-SARS-CoV-2 virus, and can effectively inhibit the continued amplification of the SARS-CoV-2 virus. Wherein the control is human CPmAp.7 antibody (WA1 strain neutralizing antibody, the amino acid sequences of its VH and VL are respectively shown in SEQ ID NO.17, SEQ ID NO.18).

Table 2

序号serial number	克隆号clone number	EC50(μg/ml)EC50(μg/ml)
11	8G38G3	0.0130.013
22	对照control	603.7603.7

The neutralizing activity assay of embodiment 4 candidate antibody true SARS-CoV-2 virus

The day before the experiment, Vero-E6 cells to be infected were inoculated on cell culture plates and cultured overnight. On the second day, virus infection was carried out. After the frozen virus was taken out and thawed, they were mixed and incubated with different dilution concentrations of candidate antibodies, and the mixture was added to the cell culture system to infect the target cells. After virus infection, aspirate the supernatant and add complete medium to continue culturing. Observe the cytopathic changes in 3 to 5 days, and judge the neutralizing activity.

The specific steps are as follows. The following experimental operations are all completed in the BSL-3 laboratory:

(1) The sample is prepared into a 200μg/ml solution with MEM medium (containing 1% double antibody), and then serially diluted 10 times, 200μg/ml, 20μg/ml, 2μg/ml, 0.2μg/ml, 0.02μg/ml , a total of 6 dilutions of 0.002 μg/ml, 2 replicate wells for each concentration, 50 μl per well, and then add an equal volume of 100 TCID ₅₀ virus to each well, and incubator at 37 ° C, 5% CO ₂ for 1.5 h;

(2) After 1.5 hours, add cell culture medium to the 96-well culture plate, and add 100 μl of Vero cell suspension with a concentration of 1×10 ⁵ cells/mL to each well;

(3) Set up cell control and virus back titration control at the same time;

Cell control: Add 100 μL of Vero cell suspension to 100 μL of MEM medium (containing 1% double antibody) in each well into a 96-well culture plate, with 4 duplicate wells in total;

Virus back titration control: 100TCID ₅₀ virus was serially diluted 10-fold three times with MEM medium (containing 1% double antibody) to obtain 10TCID ₅₀ , 1TCID ₅₀ , and 0.1TCID ₅₀ . Add 50 μL of MEM medium (containing 1% double antibody) to each well of the 96-well culture plate, and then add an equal volume of 100 TCID _{50 ,} 10 TCID ₅₀ , 1 TCID ₅₀ , 0.1 TCID ₅₀ virus to each well, and make 4 duplicate wells for each dilution. 37°C, 5% CO ₂ incubator for 1.5h, after 1.5h, add 100 μL of Vero cell suspension with a concentration of 1×10 ⁵ cells/mL to each well. The results of the virus back titration control were in the range of 32-320TCID50/50μl, and the experiment was valid.

(4) Cells were incubated at 37°C in a 5% CO ₂ incubator for 3-5 days;

(5) Cytopathic changes (CPE) were observed under an optical microscope. Cells with CPE changes were recorded as "+", and cells without CPE changes or normal cell morphology were recorded as "-".

Inhibition effect calculation: Inhibition virus half effective concentration (EC ₅₀ )

Wherein A: percentage greater than 50% inhibition rate, B: percentage less than 50% inhibition rate, C: log (dilution factor), D: log (sample concentration corresponding to less than 50% inhibition rate). IC ₅₀ cannot be measured if the sample has no virus-inhibiting effect. The results are shown in Table 3.

table 3

克隆号clone number	IC ₅₀(μg/ml) _IC50 (μg/ml)
8G38G3	0.011730.01173

The results in Table 3 show that the 8G3 antibody can effectively neutralize the true virus of SARS-CoV-2, with an IC50 of 0.01173 μg/ml.

It can be seen that the 8G3 antibody has good neutralizing activity against the true SARS-CoV-2 virus, and can effectively inhibit the continued amplification of the SARS-CoV-2 virus.

The neutralizing activity assay of the SARS-CoV-2 virus of embodiment 5 candidate antibody in animal body

The candidate antibody prepared in Example 1 was administered to an animal model infected with SARS-CoV-2 virus. The neutralizing activity of the candidate antibody against SARS-CoV-2 virus after administration is determined by quantitative PCR detection of virus content. As a result, it was found that all the candidate antibodies had good neutralizing activity against the candidate antibodies in animals. As shown in Figure 3, on the 4th day after 8G3 treatment, the virus titers in the lungs and brain were undetectable, and the virus was completely neutralized.

Example 6 Candidate Antibody Detection of Binding Kinetics to SARS-CoV-2

The CM5 chip (Cytiva 29149603) was used to detect the binding kinetics of the monoclonal antibody using WA-1S1-His or Spike trimer as the antigen.

1. Antigen coupling

Buffer: PBS (Cytiva BR100672)

Flow rate: 10μL/min

Antigen diluent: Acetate pH 5.0 (Cytiva BR100351)

Antigen concentration: 1μg/mL

Amino coupling kit (Cytiva BR100050): activator EDC+NHS 1:1 mixture, blocking agent ethanolamine

Activate the chip for 700s, couple the diluted antigen to a level of about 70RU, and block redundant unreacted sites.

2. Antibody binding

Buffer: HBS-EP (Cytiva BR100669)

Flow rate: 30μL/min

Antibody concentration: 0.2μg/mL 2-fold dilution to 0.0125μg/mL

Regeneration buffer: Glycine pH 1.5 (Cytiva BR100354)

According to the set concentration arrangement, add diluted antibodies of each concentration into the corresponding 96-well plate wells, and bind for 120s, dissociate for 120s, and regenerate and elute for 30s as a cycle, from low concentration to high concentration sample.

It was found that all IgG monoclonal antibodies can efficiently bind to the S protein trimer, with an affinity of -10 to -15M.

Example 7 Affinity Research of Candidate Antibodies to Spike Protein

In order to avoid the influence of "dancing effect", monoclonal antibody 8G3 was digested with papain to obtain Fab fragments, and the monovalent Fab and S protein trimers of WA1/2020, Alpha, Beta, Gamma, Kappa, Delta and Omicron (Spike trimer) binding kinetics.

The CM5 chip (Cytiva 29149603) was used to detect the binding kinetic properties of monoclonal antibodies using WA1/2020, Alpha, Beta, Gamma, Kappa, Delta and Omicron S protein trimers as antigens.

1. Antigen coupling

Buffer: PBS (Cytiva BR100672)

Flow rate: 10μL/min

Antigen diluent: Acetate pH 5.0 (Cytiva BR100351)

Antigen concentration: 1μg/mL

2. Antibody binding

Buffer: HBS-EP (Cytiva BR100669)

Flow rate: 30μL/min

Antibody concentration: 0.2μg/mL 2-fold dilution to 0.0125μg/mL

Regeneration buffer: Glycine pH 1.5 (Cytiva BR100354)

It was found that 8G3 had an association constant Ka of 2.66×10 ⁵ 1/M, a dissociation constant Kd of 2.78×10 ^-4 1/s, and an affinity KD of 1.05×10 ^-9 M for the S protein trimer of Omicron. In addition, the affinity for B.1.1.529RBD is 3.86×10 ^-9 M, the affinity for BA.1.1RBD is <10 ^-12 M, the affinity for BA.2RBD is 2.18×10 ^-10 M, and the affinity for BA.2.12 The affinity for .1RBD was 4.95×10 ^-12 M, the affinity for BA.4RBD was 2.3×10 ^-10 M, and the affinity for BA.5RBD was 3.25×10 ^-10 M.

The neutralization ability detection of embodiment 8 candidate antibody to pseudovirus

The pseudovirus contains Spike protein on the surface of SARS-CoV-2, which can specifically infect ACE2-positive cells. Select WA1/2020, D614G, Cluster 5, Alpha, Beta, Gamma, Delta, and Omicron’s Spike protein prepared pseudovirus, According to the following steps, the pseudovirus neutralization assay was performed.

1. Plate ACE2-293T cells in the logarithmic growth phase at 1×10 ⁴ /well into a white transparent-bottom 96-well plate (Corning, 3903);

2. The next day, dilute different concentrations of neutralizing antibodies (20, 2, 0.2, 0.02, 0.002, 0.0002, 0.00002 μg/mL) with DMEM medium (Gibco, C11995500BT) + 10% FBS (Gibco, 10270-106) ); and dilute an appropriate volume of 0.2 μL/100 μL of the COV2-S protein pseudovirus in the P2 laboratory.

3. Pipette 55 μL of different concentrations of antibodies and 55 μL of diluted pseudovirus and mix well, set up negative control and positive control wells, and incubate at 37°C for 30 minutes;

4. Aspirate the medium in the 96-well plate, add 100 μL of the medium containing the corresponding antibody-virus mixture, and continue to cultivate in the CO ₂ incubator;

After 5.6 hours, inhale the culture medium containing the virus into the waste liquid tank containing 84 disinfectant, and the proportion of 84 disinfectant should not be less than 30%. Then add 100 μL of fresh medium, and continue to cultivate in the CO ₂ incubator for 48 hours;

6. Seal the 96-well plate with parafilm in a biological safety cabinet, and disinfect the outer surface of the 96-well plate with 75% alcohol, then take it out, add 90 μL of luciferase substrate (Promega, E6120) to each well, and incubate for 3- After 5 min, the fluorescence value of each well was read with a microplate reader.

7. Calculate the inhibition rate at each concentration according to the fluorescence value.

The results are shown in Figure 2, which shows that the monoclonal antibody 8G3 can effectively neutralize various pseudoviruses. The neutral IC50 is WA1/2020 0.01173 μg/ml, Alpha 0.08249 μg/ml, beta 0.02105 μg/ml, GAMMA 0.004397 μg/ml, Delta 0.0147 μg/ml, KAPPA 0.02191ug/ml, omicron 0. 01173 μg/ml.

Example 9 Candidate Antibody Detection of Neutralizing Ability to True Viruses

The neutralization ability of 8G3 to mutant strain true virus.

Using SARS-CoV-2WA1/2020 (US_WA-1/2020 isolate), Alpha (B.1.1.7/UK, strain: SARS-CoV-2/human/USA/CA_CDC_5574/2020), Beta (B.1.351 /SA,Strain:hCoV-19/USA/MD-HP01542/2021), Gamma (P.1/Brazil,Strain:SARS-CoV-2/human/USA/MD-MDH-0841/2021), Delta variant (B.1.617.2/Indian, strain: GNL-751) and Omicron variant (B.1.1.529) mutant true virus, for virus neutralization experiments. The brief method involves serial 3-fold dilution of antibodies at a concentration of 20 μg/mL in MEM medium (Gibco) to prepare working solutions. Add the dilution to an equal volume of 100 TCID50 virus and incubate for 1 hr at room temperature. The mixture was added to a 96-well plate with confluent Vero cells. At the same time set the cell blank control and virus infection control. After culturing at 37°C and 5% CO ₂ for 3 days, the cytopathic effect (CPE) was observed under a microscope, and the plaques were counted to evaluate the curative effect. Wells with a change in CPE were recorded as "+", otherwise as "-".

IC50 values were calculated according to the following equation: IC50 = Antilog(DC x (50-B)/(AB)). Wherein A represents the inhibition rate greater than 50%, B represents the inhibition rate less than 50%, C is 1g (dilution factor), and D is 1g (sample concentration when the inhibition rate is less than 50%). All experiments were performed in a biosafety level 3 laboratory. It was found that 8G3 has high virus neutralization ability against WA1/2020, Alpha, Beta, Gamma, Delta, and Omicron. Table 4 shows that in the true virus system species, the IC ₅₀ of 8G3 for WA1/2020 is 0.37ug/ml, the IC ₅₀ for Alpha is 1.111ug/ml, the IC ₅₀ for Beta is 1.111ug/ml, and the IC ₅₀ for Gamma The IC _{50 of Delta Plus is 0.641ug/ml, the IC 50} of Delta Plus is 0.370ug/ml, the IC ₅₀ of Delta Plus is 0.123ug/ml, and the IC ₅₀ of Omicron is 0.213ug/ml.

Table 4

In vivo biological activity research of the candidate antibody of embodiment 10

10.1 Mouse Model

AC70 is a human ACE2 transgenic mouse (Taconic Biosciences, Cat#18222). AC70 mice were divided into three groups, control group (PBS), low dose (2.2mg/mL monoclonal antibody 8G3) medium dose group (6.7mg/mL mL mAb 8G3) and high dose (20 mg/kg mAb 8G3), 14 mice per group. All mice were mutated with 100LD50 of SARS-CoV-2 (US_WA-1/2020 isolate), Beta-(B.1.351/SA, strain: hCoV-19/USA/MD-HP01542/2021), Delta and Omicron. body to infect. The first dose of mAb 8G3 and PBS was administered 4 hours post-infection; the second and third doses were administered on day 2 and day 4 post-infection, respectively. Mice were clinically observed at least once per day and as described in clinical health. On a scale of 1 to 4, on a standardized 1 to 4 scale, 1 is healthy; 2 is piloerection and lethargy; and 3 is additional clinical symptoms such as hunched posture , orbital tightening, increased respiratory rate, and/or weight loss >15%; a score of 4 indicates dyspnea and/or cyanosis, reluctance to move when stimulated, or weight loss ≥20% requiring immediate euthanasia. Four mice in each group were euthanized on day 4 post-infection to assess viral load and lung and brain histopathology. Continue to monitor the remaining mice for morbidity and motility for up to 14 days post-infection.

See Figure 3 for the construction method of the mouse infection model. The results in Fig. 5 show that the monoclonal antibody 8G3 has good therapeutic effects on the above-mentioned mouse infection models. Both 6.7mg/kg and 20mg/kg can neutralize the virus, prevent the mice from losing weight, and can effectively relieve the clinical symptoms. The clinical symptoms disappeared on the 6th to 7th day after administration, and the mice returned to a normal state. By detecting the virus in the brain and lung, it was found that the virus in the brain and lung was cleared on the 4th day after 8G3 injection.

The embodiments of the present application have been described in detail above, but 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. These simple modifications are all Belong to the protection scope of this application. In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way if there is no contradiction. The combination method will not be described separately. In addition, any combination of various implementations of the present application can also be made, 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.

Claims

An isolated antigen-binding protein specifically binding to SARS-CoV-2, which comprises at least one CDR in the VL of the light chain variable region, wherein the VL comprises the amino acid sequence shown in SEQ ID NO.2.
The isolated antigen-binding protein according to claim 1, wherein the VL comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.6.
The isolated antigen-binding protein according to any one of claims 1-2, wherein the VL comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO.7.
The isolated antigen-binding protein according to any one of claims 1-3, wherein the VL comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.8.
The isolated antigen-binding protein according to any one of claims 1-4, wherein said VL comprises LCDR1 and LCDR2, said LCDR1 comprises the amino acid sequence shown in SEQ ID NO.6, and said LCDR2 comprises SEQ ID NO . The amino acid sequence shown in 7.
The isolated antigen-binding protein according to any one of claims 1-5, wherein said VL comprises LCDR1 and LCDR3, said LCDR1 comprises the amino acid sequence shown in SEQ ID NO.6, and said LCDR3 comprises SEQ ID NO . The amino acid sequence shown in 8.
The isolated antigen-binding protein according to any one of claims 1-6, wherein said VL comprises LCDR2 and LCDR3, said LCDR2 comprises the amino acid sequence shown in SEQ ID NO.7, and said LCDR3 comprises SEQ ID NO . The amino acid sequence shown in 8.
The isolated antigen-binding protein according to any one of claims 1-7, wherein said VL comprises LCDR1, LCDR2 and LCDR3, said LCDR1 comprises the amino acid sequence shown in SEQ ID NO.6, and said LCDR2 comprises SEQ ID NO.6 The amino acid sequence shown in ID NO.7; The LCDR3 comprises the amino acid sequence shown in SEQ ID NO.8.
The isolated antigen binding protein according to any one of claims 1-8, wherein said VL comprises framework regions L-FR1, L-FR2, L-FR3 and L-FR4, wherein C of said L-FR1 The terminal is directly or indirectly connected to the N-terminal of the LCDR1, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO.9.
The isolated antigen-binding protein according to claim 9, wherein said L-FR2 is located between said LCDR1 and said LCDR2, and said L-FR2 comprises the amino acid sequence shown in SEQ ID NO.10.
The isolated antigen-binding protein according to any one of claims 9-10, wherein said L-FR3 is located between said LCDR2 and said LCDR3, and said L-FR3 comprises SEQ ID NO.11 amino acid sequence.
The isolated antigen-binding protein according to any one of claims 9-11, wherein the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3, and the L-FR4 comprises SEQ ID NO . The amino acid sequence shown in 12.
The isolated antigen-binding protein according to any one of claims 1-12, wherein the VL comprises the amino acid sequence shown in SEQ ID NO.2.
The isolated antigen binding protein according to any one of claims 1-13, which comprises an antibody light chain constant region.
The isolated antigen-binding protein according to any one of claims 1-14, which comprises at least one CDR in the light chain variable region VH, wherein said VH comprises the amino acid sequence shown in SEQ ID NO.1.
The isolated antigen-binding protein according to any one of claims 1-15, which comprises a heavy chain variable region VH, said VH comprising HCDR1, said HCDR1 comprising the amino acid sequence shown in SEQ ID NO.3.
The isolated antigen-binding protein according to any one of claims 1-16, which comprises a heavy chain variable region VH, said VH comprising HCDR2, said HCDR2 comprising the amino acid sequence shown in SEQ ID NO.4.
The isolated antigen-binding protein according to any one of claims 1-17, which comprises a heavy chain variable region VH, said VH comprising HCDR3, said HCDR3 comprising the amino acid sequence shown in SEQ ID NO.5.
The isolated antigen-binding protein according to any one of claims 1-18, wherein said VH comprises HCDR1 and HCDR2, said HCDR1 comprises the amino acid sequence shown in SEQ ID NO.3, and said HCDR2 comprises SEQ ID NO . The amino acid sequence shown in 4.
The isolated antigen-binding protein according to any one of claims 1-19, wherein the VH comprises HCDR1 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO.3, and the HCDR3 comprises SEQ ID NO . The amino acid sequence shown in 5.
The isolated antigen-binding protein according to any one of claims 1-20, wherein the VH comprises HCDR2 and HCDR3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO.4, and the HCDR3 comprises SEQ ID NO . The amino acid sequence shown in 5.
The isolated antigen-binding protein according to any one of claims 1-21, which comprises a heavy chain variable region VH, said VH comprising HCDR1, HCDR2 and HCDR3, said HCDR1 comprising SEQ ID NO.3 Amino acid sequence; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO.4; the HCDR3 comprises the amino acid sequence shown in SEQ ID NO.5.
The isolated antigen binding protein according to any one of claims 1-22, wherein said VH comprises framework regions H-FR1, H-FR2, H-FR3 and H-FR4, wherein C of said H-FR1 The terminal is directly or indirectly connected to the N-terminal of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO.13.
The isolated antigen-binding protein according to claim 23, wherein said H-FR2 is located between said HCDR1 and said HCDR2, and said H-FR2 comprises the amino acid sequence shown in SEQ ID NO.14.
The isolated antigen-binding protein according to any one of claims 23-24, wherein said H-FR3 is located between said HCDR2 and said HCDR3, and said H-FR3 comprises SEQ ID NO.15 amino acid sequence.
The isolated antigen-binding protein according to any one of claims 23-25, wherein the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO . The amino acid sequence shown in 16.
The isolated antigen-binding protein according to any one of claims 15-26, wherein the VH comprises the amino acid sequence shown in SEQ ID NO.1.
The isolated antigen binding protein of any one of claims 1-27, which comprises an antibody heavy chain constant region.
The isolated antigen binding protein according to any one of claims 1-28, which has the activity of neutralizing SARS-CoV-2.
The isolated antigen-binding protein of any one of claims 1-29, which comprises an antibody or antigen-binding fragment thereof.
The isolated antigen binding protein according to claim 30, wherein said 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 according to any one of claims 30-31, wherein said antibody is a fully human antibody.
Isolated one or more nucleic acid molecules encoding said VL in the isolated antigen binding protein of any one of claims 1-32.
Isolated one or more nucleic acid molecules encoding said VH in the isolated antigen binding protein of any one of claims 1-32.
Isolated one or more nucleic acid molecules encoding the isolated antigen binding protein of any one of claims 1-32.
A vector comprising a nucleic acid molecule according to any one of claims 33-35.
A cell comprising a nucleic acid molecule according to any one of claims 33-35 or a vector according to claim 36.
The cell of claim 37 expressing the isolated antigen binding protein of any one of claims 1-32.
A method for preparing the isolated antigen-binding protein of any one of claims 1-32, said method comprising culturing the antigen-binding protein of any one of claims 1-32 under conditions that express A cell according to claim 37.
A pharmaceutical composition comprising the isolated antigen-binding protein of any one of claims 1-32, the nucleic acid molecule of any one of claims 33-35, the carrier of claim 36 and/or the The cell of any one of claims 37-38, and optionally a pharmaceutically acceptable adjuvant.
The isolated antigen binding protein of any one of claims 1-32, the nucleic acid molecule of any one of claims 33-35, the vector of claim 36, any one of claims 37-38 Use of the cells and/or the pharmaceutical composition according to claim 40 in the preparation of medicines for preventing, alleviating and/or treating coronavirus infections.
The use according to claim 41, wherein the infection of the coronavirus comprises COVID-19.
A method for preventing, alleviating and/or treating coronavirus infection, comprising administering the isolated antigen-binding protein according to any one of claims 1-32, the nucleic acid according to any one of claims 33-35 A molecule, a carrier according to claim 36, a cell according to any one of claims 37-38 and/or a pharmaceutical composition according to claim 40.
The isolated antigen binding protein of any one of claims 1-32, the nucleic acid molecule of any one of claims 33-35, the vector of claim 36, any one of claims 37-38 The application of the cell and/or the pharmaceutical composition according to claim 40 in preventing, alleviating and/or treating coronavirus infection.
A method for detecting SARS-CoV-2 comprising the steps of administering the isolated antigen binding protein of any one of claims 1-32, the nucleic acid molecule of any one of claims 33-35, the right The carrier according to claim 36, the cell according to any one of claims 37-38 and/or the pharmaceutical composition according to claim 40.