CN115087667A

CN115087667A - Antigen binding proteins that specifically bind to SARS-CoV-2

Info

Publication number: CN115087667A
Application number: CN202180013680.9A
Authority: CN
Inventors: 朱建伟; 韩雷; 肖晓东; 江华; 谢跃庆; 张亮
Original assignee: Jecho Laboratories Inc; Jieku Shanghai Biomedical Research Co ltd; Jecho Biopharmaceuticals Co ltd
Current assignee: Jecho Laboratories Inc; Jieku Shanghai Biomedical Research Co ltd; Jecho Biopharmaceuticals Co ltd
Priority date: 2020-12-14
Filing date: 2021-12-13
Publication date: 2022-09-20
Anticipated expiration: 2041-12-13
Also published as: CN115087667B; TW202229330A; WO2022127739A1

Abstract

An isolated antigen binding protein that specifically binds SARS-CoV-2, comprising at least one CDR in the light chain variable region VL, wherein the CDR comprises the amino acid sequence set forth in SEQ ID NO:95, methods of making the antigen binding protein, and pharmaceutical uses thereof are provided.

Description

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

Technical Field

The application relates to the field of biological medicine, in particular to an antigen binding protein specifically binding SARS-CoV-2.

Background

The outbreak of COVID-19 caused by SARS-Cov-2 has become a major public health event worldwide. Strategies for the prevention and treatment of COVID-19 are being developed in preclinical and clinical studies, and there are currently no very powerful drugs for treating COVID-19.

Antibody-based therapy is one viable therapeutic option. Neutralizing antibodies are an important component of the host 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. The existing monoclonal antibody preparation technologies include a hybridoma technology, an EBV (Epstein-Barr virus) B lymphocyte transformation technology, a phage display technology, a transgenic mouse technology, a single B cell antibody preparation technology and the like.

Disclosure of Invention

The present application provides an isolated antigen binding protein that specifically binds to SARS-CoV-2. The isolated antigen binding proteins described herein have at least the following beneficial effects: 1) specifically binds to SARS-CoV-2; 2) has the activity of neutralizing SARS-CoV-2; 3) has good prevention, treatment and/or alleviation effects on SARS-CoV-2 infection. The 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 present application provides an isolated antigen binding protein that specifically binds SARS-CoV-2 comprising at least one CDR in the light chain variable region VL, wherein the CDR comprises the amino acid sequence set forth in SEQ ID NO. 95.

In certain embodiments, the VL comprises LCDR1 and the LCDR1 comprises the amino acid sequence set forth in SEQ ID No. 95.

In certain embodiments, the VL comprises LCDR1 and the LCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, and SEQ ID NO 50.

In certain embodiments, the VL comprises LCDR2 and the LCDR2 comprises the amino acid sequence set forth in any one of SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, and SEQ ID No. 54.

In certain embodiments, the VL comprises LCDR3 and the LCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO: 61.

In certain embodiments, the VL comprises LCDR1 and LCDR2, the LCDR1 comprises the amino acid sequence set forth in SEQ ID No. 95, and the LCDR2 comprises the amino acid sequence set forth in any one of SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, and SEQ ID No. 54.

In certain embodiments, the VL comprises LCDR1 and LCDR3, the LCDR1 comprises the amino acid sequence set forth in SEQ ID No. 95, and the LCDR3 comprises the amino acid sequence set forth in any one of SEQ ID No. 55, SEQ ID No. 56, SEQ ID No. 57, SEQ ID No. 58, SEQ ID No. 59, SEQ ID No. 60, and SEQ ID No. 61.

In certain embodiments, the VL comprises LCDR1, LCDR2, and LCDR3, the LCDR1 comprises the amino acid sequence set forth in SEQ ID No. 95, the LCDR2 comprises the amino acid sequence set forth in any one of SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, and SEQ ID No. 54; the LCDR3 comprises an amino acid sequence shown in any one of SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60 and SEQ ID NO. 61.

In certain embodiments, the VL comprises the framework regions L-FR1, L-FR2, L-FR3 and L-FR4, wherein the C-terminus of L-FR1 is linked directly or indirectly to the N-terminus of LCDR1 and the L-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO: 64.

In certain embodiments, the L-FR2 is located between the LCDR1 and the LCDR2 and the L-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, and SEQ ID NO 71.

In certain embodiments, the L-FR3 is located between the LCDR2 and the LCDR3 and the L-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NO 72, SEQ ID NO 73, SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, and SEQ ID NO 78.

In certain embodiments, the N-terminus of L-FR4 is linked directly or indirectly to the C-terminus of LCDR3 and the L-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NO:79 and SEQ ID NO: 80.

In certain embodiments, the VL comprises an amino acid sequence 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.

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

In certain embodiments, the isolated antigen binding protein comprises a heavy chain variable region VH comprising HCDR1, wherein HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID No.1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, and SEQ ID No. 7.

In certain embodiments, the VH comprises HCDR2 and the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, and SEQ ID NO 14.

In certain embodiments, the VH comprises HCDR3 and the HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, and SEQ ID NO 21.

In certain embodiments, the isolated antigen binding protein comprises a heavy chain variable region VH comprising HCDR1, HCDR2, and HCDR3, the HCDR1 comprising the amino acid sequence set forth in any one of SEQ ID No.1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, and SEQ ID No. 7; the HCDR2 comprises an amino acid sequence shown in any one of SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 14; the HCDR3 comprises an amino acid sequence shown in any one of SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21.

In certain embodiments, the VH comprises the framework regions H-FR1, H-FR2, H-FR3 and H-FR4, wherein the C-terminus of H-FR1 is linked directly or indirectly to the N-terminus of the HCDR1 and the H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27 and SEQ ID NO 28.

In certain embodiments, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, and SEQ ID NO 34.

In certain embodiments, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises an amino acid sequence set forth in any one of SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, and SEQ ID NO 41.

In certain embodiments, the N-terminus of H-FR4 is linked directly or indirectly to the C-terminus of the HCDR3 and the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NO 42, SEQ ID NO 43, and SEQ ID NO 44.

In certain embodiments, the VH comprises the amino acid sequence set forth in any one of SEQ ID NO 81, SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86, and SEQ ID NO 87.

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

In certain embodiments, the isolated antigen binding protein has an activity of neutralizing 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 a 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 present application provides an isolated nucleic acid molecule or molecules encoding the VL of an isolated antigen binding protein described herein.

In another aspect, the present application provides an isolated nucleic acid molecule or molecules encoding the VH in an isolated antigen binding protein described herein.

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

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

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

In certain embodiments, the cell expresses an isolated antigen binding protein described herein.

In another aspect, the present application provides a method of making an isolated antigen binding protein described herein, the method comprising culturing a cell according to the present application under conditions such that the isolated antigen binding protein described herein is expressed.

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

In another aspect, the present application provides the use of an isolated antigen binding protein as described herein, a nucleic acid molecule as described herein, a vector as described herein, a cell as described herein and/or a pharmaceutical composition as described herein in the manufacture of a medicament for the prevention, amelioration and/or treatment of an infection by a coronavirus.

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

In another aspect, the present application provides a method of preventing, ameliorating, and/or treating an infection by a coronavirus comprising administering an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein, and/or a pharmaceutical composition described herein.

In another aspect, the present application provides an isolated antigen binding protein as described herein, a nucleic acid molecule as described herein, a vector as described herein, a cell as described herein and/or a pharmaceutical composition as described herein, for use in preventing, ameliorating and/or treating an infection by a coronavirus.

In another aspect, the present application provides a method of detecting SARS-CoV-2 comprising the step of administering an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein, and/or a pharmaceutical composition described herein.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

The specific 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 will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The brief description of the drawings is as follows:

FIG. 1 shows the results of specific binding of an isolated antigen binding protein described herein to a trimer of SARS-CoV-2S protein.

FIG. 2 shows the neutralizing activity of the isolated antigen binding proteins described herein against SARS-CoV-2 pseudovirus.

FIG. 3 shows the neutralizing activity of the isolated antigen binding proteins described herein against SARS-CoV-2 pseudovirus.

FIG. 4 shows a method for constructing a mouse infection model.

FIG. 5 shows the effect of isolated antigen binding proteins described herein on body weight in a mouse infection model.

FIG. 6 shows the results of clinical scoring of mouse infection models by the isolated antigen binding proteins described herein.

FIG. 7 shows the effect of the isolated antigen binding proteins described herein on the survival curves of mouse infection models.

FIG. 8 shows a method for constructing a rhesus monkey infection model.

FIG. 9 shows the effect of the isolated antigen binding proteins described herein on viral RNA content in a rhesus monkey infection model (throat swab assay).

FIG. 10 shows the effect of isolated antigen binding proteins described herein on viral RNA content in a rhesus monkey infection model (anal swab assay)

FIG. 11 shows the effect of isolated antigen binding proteins described herein on viral RNA content in various tissues and organs in a rhesus monkey infection model.

FIGS. 12a-12g show cryo-electron microscopy analysis of isolated antigen binding protein and S protein complexes described herein.

FIGS. 13a-13b show a cryo-electron microscopy procedure for isolated antigen-binding protein and S-protein complexes described herein.

FIG. 14 shows data collection, 3D model reconstruction, and model statistical parameters for the isolated antigen binding protein and S protein complexes described herein.

FIGS. 15a-15e show cryo-electron microscopic structures of complexes of the isolated antigen binding proteins and S protein described herein.

FIGS. 16a-16c are graphical representations of binding patterns of isolated antigen binding protein and S protein complexes described herein.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.

Definition of terms

In the present application, the term "SARS-CoV-2" generally refers to Severe Acute Respiratory Syndrome Coronavirus type 2, which is collectively referred to in English as Severe acid Respiratory Syndrome Virus Syndrome Coronavir 2. SARS-CoV-2 belongs to the family Coronaviridae (Coronaviridae) genus B coronavirus (Betaconoviridus) subfamily Sarbecoviridus (Sarbecovirus). SARS-CoV-2 is a non-segmented positive-stranded RNA virus with an envelope. SARS-CoV-2 can cause novel coronavirus pneumonia (COVID-19). In the present application, the SARS-CoV-2 may include an S protein (spike protein).

In the present application, the term "COVID-19" generally refers to a novel coronavirus pneumonia (Corona Virus Disease 2019), or 2019 coronavirus Disease, which is a respiratory Disease caused by SARS-CoV-2 Virus. Common symptoms of COVID-19 may include fever, cough, fatigue, shortness of breath, and loss of smell and taste, some of which may progress to viral pneumonia, multiple organ failure, or cytokine storm. The disease is mainly transmitted when there is close contact between people, for example, by coughing, sneezing and the production of small droplets by speech. The world health organization announced that the outbreak of COVID-19 was pandemic (pandemics) on day 11/3/2020. There is currently no vaccine or specific therapeutic approach available against COVID-19.

In the present application, the term "S protein of a coronavirus" generally refers to the spike protein (spike protein) of a coronavirus. The S protein can be combined into a trimer (i.e., S protein trimer) which contains about 1300 amino acids. The S protein may belong to the first membrane fusion protein (Class I viral fusion protein). The S protein may typically contain two subunits (subbunit), S1 and S2. S1 mainly contains a receptor binding domain RBD (receptor binding domain RBD), which can be responsible for recognizing cellular receptors. S2 contains essential elements required for the membrane fusion process, including an intrinsic membrane fusion peptide (fusion peptide), two 7-peptide repeats (HR), an aromatic amino acid-rich membrane proximal region (MPER), and a transmembrane region (TM). The S1 protein can be further divided into two regions (domains), an N-terminal region (NTD) and a C-terminal region (CTD). The S protein may determine the host range and specificity of the virus (e.g., coronavirus SARS-CoV-2), may also be an important site of action for host neutralizing antibodies, and/or may be a key target for vaccine design. The S protein may be that of SARS-CoV-2, for example, the structure of which may be found in Daniel Wrapp et al, Cryo-EM structure of the 2019-nCoV spike in the fusion formation, Science.

In the present application, the term "ACE 2" refers generally to Angiotensin-converting enzyme II (Angiotensin-converting enzyme 2) or a functional fragment thereof. The angiotensin-converting enzyme II can catalyze the conversion of angiotensin I to angiotensin- (1-9) or the conversion of angiotensin II to exopeptidase of angiotensin- (1-7). The ACE2 may include a PD region (peptidase domain) at the N-terminus and a CLD region (Collectrin-like domain) at the C-terminus. The angiotensin converting enzyme II may be a receptor of SARS-CoV-2, for example, the extracellular domain of ACE2 (e.g., the PD region of ACE 2) may bind to the RBD of the S protein of SARS-CoV-2. Human angiotensin-converting enzyme II has accession number Q9BYF1 in the UniProt database. The human ACE2 gene may contain 18 exons as shown in Table 1 of 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-sensitive carbon typeptidase.J.biol.chem.275: 33238-33243, 2000. In the present application, the ACE2 may include a truncation or variant of the intact ACE2 protein, as long as the functional fragment still functions as a coronavirus (e.g., SARS-CoV and/or SARS-CoV-2) receptor.

In the present application, the term "infection by a coronavirus" generally refers to a disease and/or condition caused by infection by a coronavirus. The Coronavirus belongs to the genus of the order of the nested viruses (Nidovirales) Coronaviridae (Coronaviridae) genus of the family of Coronavirus (Coronavir). The coronavirus may be a single-stranded RNA virus. The infection by the coronavirus may comprise a respiratory infection, such as an upper respiratory infection. Infection by the coronavirus may include symptoms of fever, runny nose, chills, vomiting, and/or fatigue.

In the present application, the term "neutralizing" generally refers to the neutralizing activity of an antigen binding protein, i.e., the biochemical activity of an antigen binding protein that can block and/or neutralize its corresponding antigen. In some cases, an antigen binding protein having such neutralizing activity can be resistant to and inactivate an antigen that attacks the immune system (e.g., a retrovirus, for example, the antigen can be SARS-CoV-2). In some cases, an antigen binding protein having such neutralizing activity does not require the participation of leukocytes in neutralizing the biochemical activity of its corresponding antigen.

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

In the present application, the term "Fab" generally refers to a fragment containing a heavy chain variable domain and a light chain variable domain, and also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1); the term "Fab'" generally refers to fragments other than Fab that have a small number of residues (including one or more cysteines from the antibody hinge region) added at 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 connected by a disulfide bridge at the hinge region. The term "Fv" generally refers to the smallest antibody fragment that contains an intact antigen recognition and binding site. In some cases, the fragment may consist of a dimer of one heavy chain variable region and one light chain variable region in tight, non-covalent association; the term "dsFv" generally refers to disulfide-stabilized Fv fragments whose bond between the single light chain variable region and the 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 the present application, the term "scFv" generally refers to a monovalent molecule formed by the covalent linkage pairing of one heavy chain variable domain and one light chain variable domain of an antibody via a flexible peptide linker; such scFv molecules can have the general structure: NH (NH) ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH.

In the present application, the term "antibody" generally refers to an immunoglobulin that can undergo a specific binding reaction with a corresponding antigen. The antibody may be secreted by immune cells (e.g., effector B cells). The antibody may be a monoclonal antibody (including a full length monoclonal antibody comprising two light chains and two heavy chains), a polyclonal antibody, a multispecific antibody (e.g., bispecific antibody), a humanized antibody, a fully human antibody, a chimeric antibody, and/or a camelized single domain antibody. An "antibody" may generally comprise proteins of at least two Heavy Chains (HC) and two Light Chains (LC) that are linked to each other by disulfide bonds, or antigen-binding fragments 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 CH 3. 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 comprises one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), which alternate with more conserved regions termed 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 FR 4. 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), largely adopting a β -sheet configuration, linked by three CDRs, forming a loop junction, and in some cases forming part of a β -sheet structure. The CDRs in each chain are held in close proximity by the FR region and form, together with the CDRs from the other chain, the antigen-binding site of the antibody. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

In the present application, the term "variable" generally refers to the fact that certain portions of the sequences of the variable domains of antibodies vary strongly, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable region of the 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 portions of the variable domains are called the Framework (FR). In the art, the CDRs of an antibody can be defined by a variety of methods, such as Kabat definition rules based on sequence variability (see Kabat et al, immunological protein sequences, fifth edition, national institutes of health, Besserda, Md. (1991)), Chothia definition rules based on the position of the structural loop region (see A1-Lazikani et al, Jmol Biol 273: 927. sup. 48,1997) and KABAT definition rules based on the concept of the IMGT ONTOLOGY (IMGT-ONTOGIY) and the rules of the IMGT Scientific chart. IMGT refers to the International ImmunoGeneTiCs information System, a global reference database for ImMunoGeneTics and immunoinformatics (http:// www.imgt.org). IMGT is specialized for the study of Immunoglobulins (IG) or antibodies, T cell receptors (TR), Major Histocompatibility (MH) from humans and other vertebrates, as well as immunoglobulin superfamily (IgSF), MH superfamily (MhSF) and immune system Related Proteins (RPI) from vertebrates and non-vertebrates.

In the present application, the term "isolated" antigen binding protein generally refers to an antigen binding protein that has been identified, isolated and/or recovered from a component of its production environment (e.g., native or recombinant). The contaminating components of the environment that they produce are often substances that interfere with their research, diagnostic or therapeutic uses and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. An isolated antigen binding protein or antibody will typically be prepared by at least one purification step.

In the present application, the term "monoclonal antibody" generally refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies in the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are typically highly specific for a single antigenic site. Moreover, 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, monoclonal antibodies have the advantage that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use herein can be prepared in hybridoma cells, or can be prepared by recombinant DNA methods.

In this application, the term "fully human antibody" generally refers to an antibody that is expressed by an animal by transferring a gene encoding the antibody from the human to a genetically engineered antibody gene-deleted animal. All parts of the antibody (including the variable and constant regions of the antibody) are encoded by genes of human origin. The fully human antibody can greatly reduce the immune side reaction of the heterologous antibody to the human body. The method for obtaining the fully human antibody in the field can be a phage display technology, a transgenic mouse technology, a ribosome display technology, an RNA-polypeptide technology and the like.

In the present application, the terms "binding", "specific binding" or "specific for …" generally refer to a measurable and reproducible interaction, such as binding between an antigen and an antibody, that can determine 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 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, more readily, and/or for a greater duration than it binds other targets. An antibody is said to "specifically bind" to a random, unrelated epitope when it is more likely to bind to the epitope through its antigen binding domain than it would. "epitope" refers to the single click or click of a particular atom on an antigen that binds to an antigen binding protein (e.g., an antibody) where the text is entered. Click or click on the text entered here. Click or click on the text entered here. A group (e.g., sugar side chain, phosphoryl, sulfonyl) or an amino acid.

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

In this application, the term "between … …" generally means that the C-terminus of an amino acid fragment is directly or indirectly linked to the N-terminus of a first amino acid fragment and that the N-terminus is directly or indirectly linked to the C-terminus of a second amino acid fragment. In the light chain, for example, the N-terminus of the L-FR2 is linked directly or indirectly to the C-terminus of the LCDR1, and the C-terminus of the L-FR2 is linked directly or indirectly to the N-terminus of the LCDR 2. For another example, the N-terminus of the L-FR3 is directly or indirectly linked to the C-terminus of the LCDR2, and the C-terminus of the L-FR3 is directly or indirectly linked to the N-terminus of the LCDR 3. In the heavy chain, for example, the N-terminus of the H-FR2 is linked directly or indirectly to the C-terminus of the HCDR1 and the C-terminus of the H-FR2 is linked directly or indirectly to the N-terminus of the HCDR 2. For another example, the N-terminus of the H-FR3 is directly or indirectly linked to the C-terminus of the HCDR2, and the C-terminus of the H-FR3 is directly or indirectly linked to the N-terminus of the HCDR 3. In the present application, the "first amino acid fragment" and the "second amino acid fragment" may be any one of the same or different amino acid fragments.

In the present 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 linked in nature.

In the present 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. The vector may include a vector mainly for inserting a DNA or RNA into a cell, a vector mainly for replicating a DNA or RNA, and a vector mainly for expression of transcription and/or translation of a DNA or RNA. The vector also includes vectors having a plurality of the above-described 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 will produce the desired expression product by culturing an appropriate host cell containing the vector.

In the present application, the term "cell" generally refers to an individual cell, cell line or cell culture that may or may already contain a plasmid or vector comprising a nucleic acid molecule described herein, or that is capable of expressing an antibody or antigen-binding fragment thereof described herein. The cell may comprise progeny of a single host cell. Due to natural, accidental, or deliberate mutation, the progeny cells may not necessarily be identical in morphology or in genome to the original parent cell, but may be capable of expressing an antibody or antigen-binding fragment thereof as described herein. The cells can be obtained by in vitro transfection of cells using the vectors described herein. The cell may be a prokaryotic cell (e.g., E.coli) or a eukaryotic cell (e.g., a yeast cell, such as a COS cell, a Chinese Hamster Ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, an NS0 cell, or a myeloma cell). In the present application, the cell may include a cell into which the vector is introduced. The cell includes not only a specific cell but also a progeny of the cell.

In the present application, the term "pharmaceutically acceptable adjuvant" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers which are non-toxic to the cells or mammals to which they are exposed at the dosages and concentrations employed. Typically, the physiologically acceptable carrier is an aqueous pH buffered 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. "administration" may refer to therapeutic, pharmacokinetic, diagnostic, research and experimental methods. The treatment of the cell may include contacting the cell with an agent (e.g., an agent comprising the isolated antigen binding protein), and contacting the cell with a fluid, and a reagent. "administering" also means treating in vitro and ex vivo by a reagent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, animal or study subject refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnosis; for example, contacting the isolated antigen binding protein with a human or animal, a subject, a cell, a tissue, a physiological compartment, or a physiological fluid can be included.

As used herein, the term "treatment" refers to the administration of a therapeutic agent, e.g., a pharmaceutical composition comprising any one of the isolated antigen binding proteins of the present application, and/or comprising the isolated antigen binding protein, either internally or externally to a patient having one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the patient in an amount effective to alleviate one or more symptoms of the disease (therapeutically effective amount). Desirable effects of treatment include reducing the rate of disease progression, ameliorating or alleviating the disease state, and regression or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are reduced or eliminated, including, but not limited to, reducing (or destroying) cancer cell proliferation, reducing symptoms resulting from the disease, increasing the quality of life of those individuals with the disease, reducing the dosage of other drugs required to treat the disease, delaying the progression of the disease, and/or prolonging survival of the individual.

In this application, the term "comprising" is used in a generic sense to mean including, summarizing, containing or encompassing. In some cases, the meaning of "is", "consisting of … …" is also indicated.

In this application, the term "about" generally means varying by 0.5% -10% above or below the stated value, for example, varying by 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% above or below the stated value.

Detailed Description

Antigen binding proteins

In one aspect, the present application provides an isolated antigen binding protein that specifically binds SARS-CoV-2 comprising at least one CDR in the light chain variable region VL, wherein the CDR comprises the amino acid sequence set forth in SEQ ID NO. 95. For example, the CDR may comprise the amino acid sequence shown in SEQ ID NO 96, 97, 98 or 99.

In the present application, the VL may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence set forth in SEQ ID No. 95: TG X ₃ SS X ₆ X ₇ G X ₉ X ₁₀ X ₁₁ X ₁₂ V X ₁₄ Wherein X is ₃ Ser or Thr; x ₆ Asp or Asn, X ₇ Is Ile or Val, X ₉ Is Ala, Gly or Ser; x ₁₀ Gly, Ser or Tyr; x ₁₁ Asp, Phe, Asn or Tyr; x ₁₂ Asp, Leu or Tyr; x ₁₄ Is His or Ser. For example, the sequence may be a sequence determined according to the KABAT definition rule.

In the present application, the VL may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 96: TG X ₃ SSDVGX ₉ X ₁₀ X ₁₁ X ₁₂ VS, wherein X ₃ Ser or Thr; x ₉ Is Gly or Ser; x ₁₀ Is Ser or Tyr; x ₁₁ Asp or Asn; x ₁₂ Is Leu or Tyr. For example, the sequence may be a sequence determined according to the KABAT definition rule.

In the present application, the VL may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 97: TGTSSDVGX ₉ X ₁₀ NX ₁₂ VS, wherein X ₉ Is Gly or Ser; x ₁₀ Is Ser or Tyr; x ₁₂ Is Leu or Tyr. For example, the sequence may be a sequence determined according to the KABAT definition rule.

In the present application, the VL may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 98: TGTSSDVGGX ₁₀ NYVS wherein X ₁₀ Is Ser or Tyr. For example, the sequence may be a sequence determined according to the KABAT definition rule.

In the present application, the VL may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 99: TGSSSNIGAG X ₁₁ DVH, wherein X ₁₁ Is Phe or Tyr. For example, the sequence may be a sequence determined according to the KABAT definition rule.

For example, the VL may comprise LCDR1 and the LCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, and SEQ ID NO 50.

In the present application, the VL may comprise LCDR2, and the LCDR2 may comprise an amino acid sequence set forth in any one of SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 53, and SEQ ID NO. 54.

In the present application, the VL may comprise LCDR3 and the LCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO: 61.

For example, the VL may comprise LCDR1 and LCDR2, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID No. 95, and the LCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, and SEQ ID No. 54.

For example, the VL may comprise LCDR1 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 95, and the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, and SEQ ID NO. 61.

For example, the VL may comprise LCDR1, LCDR2, and LCDR3, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID No. 95, the LCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, and SEQ ID No. 54; the LCDR3 may comprise an amino acid sequence set forth in any one of SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, and SEQ ID NO 61.

For example, the VL may comprise an amino acid sequence set forth in any one of SEQ ID NO 88, 89, 90, 91, 92, 93 and 94.

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 set forth in any one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, and SEQ ID NO 7.

For example, the VH may comprise HCDR2 and the HCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 14.

For example, the VH may comprise HCDR3 and the HCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, and SEQ ID NO 21.

For example, the isolated antigen binding protein may comprise a heavy chain variable region VH, which may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID No.1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7; the HCDR2 may comprise an amino acid sequence as set forth in any one of SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, and SEQ ID NO 14; the HCDR3 may comprise an amino acid sequence as set forth in any one of SEQ ID NO 15, 16, 17, 18, 19, 20 and 21.

For example, the VH may comprise an amino acid sequence shown in any one of SEQ ID NO 81, SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86 and SEQ ID NO 87.

The isolated antigen binding protein described herein, is capable of competing with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein the reference antibody can comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody can comprise HCDR1, HCDR2, and HCDR3, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 2, the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 8, and the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 15, the LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 45, the LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 51, and the LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 55.

The isolated antigen binding protein described herein is capable of competing for binding to the RBD of the S protein of SARS-CoV-2 with a reference antibody, wherein the reference antibody can comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody can comprise HCDR1, HCDR2, and HCDR3, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID No.1, the HCDR2 can comprise the amino acid sequence set forth in SEQ ID No. 9, and the HCDR3 can comprise the amino acid sequence set forth in SEQ ID No. 16, the LCDR1 can comprise the amino acid sequence set forth in SEQ ID No. 46, the LCDR2 can comprise the amino acid sequence set forth in SEQ ID No. 51, and the LCDR3 can comprise the amino acid sequence set forth in SEQ ID No. 56.

The isolated antigen binding protein described herein, is capable of competing with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein the reference antibody can comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody can comprise HCDR1, HCDR2, and HCDR3, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 3, the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO.10, and the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 17, the LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 47, the LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 52, and the LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 57.

An isolated antigen binding protein described herein, capable of competing with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein the reference antibody can comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody can comprise HCDR1, HCDR2, and HCDR3, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 4, the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 11, and the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 18, the LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 48, the LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 53, and the LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 58.

The isolated antigen binding protein described herein, is capable of competing with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein the reference antibody can comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody can comprise HCDR1, HCDR2, and HCDR3, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 5, the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 12, and the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 19, the LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 64, the LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 53, and the LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 59.

The isolated antigen binding protein described herein is capable of competing for binding to the RBD of the S protein of SARS-CoV-2 with a reference antibody, wherein the reference antibody can comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody can comprise HCDR1, HCDR2, and HCDR3, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID No. 7, the HCDR2 can comprise the amino acid sequence set forth in SEQ ID No. 14, and the HCDR3 can comprise the amino acid sequence set forth in SEQ ID No. 21, the LCDR1 can comprise the amino acid sequence set forth in SEQ ID No. 49, the LCDR2 can comprise the amino acid sequence set forth in SEQ ID No. 54, and the LCDR3 can comprise the amino acid sequence set forth in SEQ ID No. 60.

The isolated antigen binding protein described herein, is capable of competing with a reference antibody for binding to the RBD of the S protein of SARS-CoV-2, wherein the reference antibody can comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody can comprise HCDR1, HCDR2, and HCDR3, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 6, the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 13, and the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 20, the LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 50, the LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 51, and the LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 61.

In the present application, the isolated antigen binding protein may comprise the antibody light chain variable region CDRs LCDR1, LCDR2, and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:45, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:51, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 55.

In the present application, the isolated antigen binding protein may comprise the antibody light chain variable region CDRs LCDR1, LCDR2, and LCDR3, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:46, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:51, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 56.

In the present application, the isolated antigen binding protein may comprise the antibody light chain variable region CDRs LCDR1, LCDR2, and LCDR3, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:47, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:52, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 57.

In the present application, the isolated antigen binding protein may comprise the antibody light chain variable region CDRs LCDR1, LCDR2, and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:48, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:53, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 58.

In the present application, the isolated antigen binding protein may comprise the antibody light chain variable region CDRs LCDR1, LCDR2, and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:64, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:53, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 59.

In the present application, the isolated antigen binding protein may comprise the antibody light chain variable region CDRs LCDR1, LCDR2, and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID No. 49, the LCDR2 may comprise the amino acid sequence shown in SEQ ID No. 54, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID No. 60.

In the present application, the isolated antigen binding protein may comprise the antibody light chain variable region CDRs LCDR1, LCDR2, and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:50, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:51, 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 the antibody heavy chain variable region CDRs HCDR1, HCDR2, and HCDR3, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID No. 2, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID No. 8, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID No. 15.

In the present application, the isolated antigen binding protein may comprise the antibody heavy chain variable region CDRs HCDR1, HCDR2, and HCDR3, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID No.1, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID No. 9, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID No. 16.

In the present application, the isolated antigen binding protein may comprise the antibody heavy chain variable region CDRs HCDR1, HCDR2, and HCDR3, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID No. 3, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID No.10, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID No. 17.

In the present application, the isolated antigen binding protein may comprise the antibody heavy chain variable region CDRs HCDR1, HCDR2, and HCDR3, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID No. 4, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID No. 11, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID No. 18.

In the present application, the isolated antigen binding protein may comprise the antibody heavy chain variable region CDRs HCDR1, HCDR2, and HCDR3, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID No. 5, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID No. 12, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID No. 19.

In the present application, the isolated antigen binding protein may comprise the antibody heavy chain variable region CDRs HCDR1, HCDR2, and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID No. 7, the HCDR2 may comprise the amino acid sequence shown in SEQ ID No. 14, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID No. 21.

In the present application, the isolated antigen binding protein may comprise the antibody heavy chain variable region CDRs-HCDR 1, HCDR2, and HCDR3, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID No. 6, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID No. 13, and the HCDR3 may comprise the amino acid sequence set forth 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, the HCDR2 may comprise the amino acid sequence shown, and the HCDR3 may comprise the amino acid sequence shown, the LCDR1 may comprise the amino acid sequence shown, the LCDR2 may comprise the amino acid sequence shown, and the LCDR3 may comprise the amino acid sequence shown.

For example, the VL may comprise the framework regions L-FR1, L-FR2, L-FR3, and L-FR 4.

For example, the C-terminus of the L-FR1 may be linked directly or indirectly to the N-terminus of the LCDR1, and the L-FR1 may comprise an amino acid sequence set forth in any one of SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO: 64.

For example, the L-FR2 may be located between the LCDR1 and the LCDR2, and the L-FR2 may comprise an amino acid sequence shown in any one of SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, and SEQ ID NO 71.

For example, the L-FR3 may be located between the LCDR2 and the LCDR3, and the L-FR3 may comprise an amino acid sequence shown in any one of SEQ ID NO 72, SEQ ID NO 73, SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, and SEQ ID NO 78.

For example, the N-terminus of the L-FR4 may be linked directly or indirectly to the C-terminus of the LCDR3, and the L-FR4 may comprise the amino acid sequence shown in any one of SEQ ID NO:79 and SEQ ID NO: 80.

As used herein, the VL may comprise the amino acid sequences shown in 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.

For example, the isolated antigen binding protein may 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 Ig κ constant region may be as shown in GenBank accession No. 50802 of the NCBI database; the gene encoding the human Ig λ constant region can be as shown in GenBank accession No. 3535 of the NCBI database.

For example, the VH may comprise the framework regions H-FR1, H-FR2, H-FR3, and H-FR 4.

For example, the C-terminus of the H-FR1 may be linked directly or indirectly to the N-terminus of the HCDR1, and the H-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27 and SEQ ID NO 28.

For example, the H-FR2 may be located between the HCDR1 and the HCDR2, and the H-FR2 may comprise an amino acid sequence set forth in any one of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO: 34.

For example, the H-FR3 may be located between the HCDR2 and the HCDR3, and the H-FR3 may comprise an amino acid sequence shown in any one of SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, and SEQ ID NO 41.

For example, the N-terminus of the H-FR4 may be linked directly or indirectly to the C-terminus of the HCDR3, and the H-FR4 may comprise the amino acid sequence shown in any one of SEQ ID NO 42, SEQ ID NO 43 and SEQ ID NO 44.

In the present application, the isolated antigen binding protein may comprise a light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO:88 and a heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID NO: 81.

In the present application, the isolated antigen binding protein may comprise a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO. 89 and a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO. 82.

In the present application, the isolated antigen binding protein may comprise a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO. 90 and a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO. 83.

In the present application, the isolated antigen binding protein may comprise a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO. 91 and a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO. 84.

In the present application, the isolated antigen binding protein may comprise a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO. 92 and a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO. 85.

In the present application, the isolated antigen binding protein may comprise a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO:93 and a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 87.

In the present application, the isolated antigen binding protein may comprise a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO. 94 and a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO. 86.

Reference in the present application to protein, polypeptide and/or amino acid sequences is also to be understood as including at least the following ranges: variants or homologues having the same or similar function as said protein or polypeptide.

In the present application, the variant may be a protein or polypeptide having a substitution, deletion or addition of one or more amino acids in the amino acid sequence of the protein and/or the polypeptide (e.g., the antigen binding protein described herein). For example, the functional variant may comprise a protein or polypeptide that has been altered by at least 1, such as 1-30, 1-20 or 1-10, and further such as 1, 2, 3,4 or 5 amino acid substitutions, deletions and/or insertions. The functional variant may substantially retain the biological properties of the protein or the polypeptide prior to the alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding capacity) of the protein or the polypeptide prior to the alteration. For example, the substitution may be a conservative substitution.

In the present application, a portion of the amino acid sequence of the antigen binding protein may be homologous to a corresponding amino acid sequence in an antibody from a particular species, or belong to a particular class. For example, the variable and constant portions of the antigen binding protein can both be from the variable and constant regions of an antibody from one animal species (e.g., human). In the present application, the homolog may be a protein or polypeptide having at least about 85% (e.g., 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 more) sequence homology to the amino acid sequence of the protein and/or the polypeptide (e.g., the antigen binding protein described herein).

In the present application, homology generally refers to similarity, similarity or relatedness between two or more sequences. The "percentage of sequence homology" can be calculated by: the two sequences to be aligned are compared in a comparison window, the number of positions in the two sequences at which the same nucleobase (e.g., A, T, C, G) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, gin, Cys, and Met) is determined to yield the number of matched positions, the number of matched positions is divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to yield the percentage of sequence homology. Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared or over a region of the target sequence. The homology can also be determined by the following method: FASTA and BLAST. The FASTA algorithm is described in "improved tools for biological sequence comparison" by w.r.pearson and d.j.lipman, proceedings of the national academy of sciences of the united states (proc.natl.acad.sci.), 85: 2444 2448, 1988; and "rapid and sensitive protein similarity search" by d.j.lipman and w.r.pearson, Science, 227: 1435-1441, 1989. BLAST algorithms are described in "a basic local contrast (alignment) search tool" by s.altschul, w.gish, w.miller, e.w.myers and d.lipman, journal of molecular biology, 215: 403-410, 1990.

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 may 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 the NCBI database.

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

Humanized antibodies may be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE. In the present application, the antibody is an IgG antibody, using the IgG1 subtype. 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 the present application.

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

The antigen binding proteins described herein (e.g., SARS-CoV-2 antibodies) are capable of specifically binding to the RBD of the S protein of SARS-CoV-2. An antigen binding protein (e.g., an antibody) that "specifically binds" to a SARS-CoV-2 antigen (e.g., the RBD of the S protein of SARS-CoV-2) can generally bind to the RBD of the S protein of SARS-CoV-2 with an EC50 value of about or greater affinity (e.g., about), but not to other proteins lacking the SARS-CoV-2 sequence. Whether an antigen binding protein (e.g., an antibody) binds to a SARS-CoV-2 antigen (e.g., the RBD of the S protein of SARS-CoV-2) can be determined using any assay known in the art. For example, by flow assay techniques and enzyme-linked immunoassays.

The antigen binding proteins described herein (e.g., SARS-CoV-2 antibodies; e.g., monoclonal antibody 2G1) are capable of specifically binding to the S protein trimer of WA1/2020, Alpha, Beta, Gamma, Kappa, and Delta. The antigen binding proteins described herein (e.g., SARS-CoV-2 antibodies; e.g., monoclonal antibody 2G1) are capable of neutralizing WA1/2020, Alpha, Beta, Gamma, Kappa, and Delta pseudoviruses. The antigen binding proteins described in the application (e.g., SARS-CoV-2 antibodies; e.g., monoclonal antibody 2G1) are capable of neutralizing ARS-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) and Delta variants (B.1.617.2/Indianan, Strain: GNL-751) mutants of the true virus.

The antigen binding proteins described herein (e.g., SARS-CoV-2 antibodies; e.g., monoclonal antibody 2G1) are capable of treating animal models (e.g., mouse animal models; and/or rhesus monkey models) infected with SARS-CoV-2(US _ WA-1/2020 isolate), Beta- (B.1.351/SA, strain: hCoV-19/USA/MD-HP01542/2021), or Delta variants.

The antigen binding proteins described herein (e.g., SARS-CoV-2 antibodies) are capable of blocking the binding of the RBD of the S protein of SARS-CoV-2 or a functional fragment thereof to human ACE 2. Blocking assays can be performed using competition methods, e.g., by mixing the antigen-binding protein (e.g., SARS-CoV-2 antibody) with the antigen (or, cells that can express the antigen) and the ligand for the antigen (or, cells that express the ligand), and reacting the antigen-binding protein's ability to competitively bind the antigen with the ligand for the antigen based on the intensity (e.g., fluorescence intensity or concentration) of the detectable label.

Reference to protein and/or amino acid sequences in the present application should also be understood to include at least the following ranges: variants or homologues having the same or similar function as said protein.

In the present application, the variant may be a protein or polypeptide having one or more amino acids substituted, deleted, or added in the amino acid sequence of the protein (e.g., the antigen binding protein described herein). For example, the functional variant may comprise a protein or polypeptide that has been altered by at least 1, such as 1-30, 1-20 or 1-10, and further such as 1, 2, 3,4 or 5 amino acid substitutions, deletions and/or insertions. The functional variant may substantially retain the biological properties of the protein or the polypeptide prior to the alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding capacity) of the protein or the polypeptide prior to alteration. For example, the substitution may be a conservative substitution.

In the present application, a portion of the amino acid sequence of the antigen binding protein may be homologous to a corresponding amino acid sequence in an antibody from a particular species, or belong to a particular class. For example, both the variable and constant regions of an antibody can be from the variable and constant regions of an antibody from one animal species (e.g., human). In the present application, the homolog may be a protein or polypeptide having at least about 85% (e.g., 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 more) sequence homology to the amino acid sequence of the protein and/or the polypeptide (e.g., the antigen binding protein described herein).

In the present application, homology generally refers to similarity, similarity or relatedness between two or more sequences. The "percentage of sequence homology" can be calculated by: the two sequences to be aligned are compared in a comparison window, the number of positions in the two sequences at which the same nucleobase (e.g., A, T, C, G) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, gin, Cys, and Met) is determined to yield the number of matched positions, the number of matched positions is divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to yield the percentage of sequence homology. Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared or over a region of the target sequence. The homology can also be determined by the following method: FASTA and BLAST. The FASTA algorithm is described in "improved tools for biological sequence comparison" by w.r.pearson and d.j.lipman, proceedings of the national academy of sciences of the united states (proc.natl.acad.sci.), 85: 2444 2448, 1988; and "rapid and sensitive protein similarity search" by d.j.lipman and w.r.pearson, Science, 227: 1435-1441, 1989. See "an essential local contrast (alignment) search tool" by s.altschul, w.gish, w.miller, e.w.myers, and d.lipman, journal of molecular biology, 215: 403-410, 1990.

Pharmaceutical composition

In another aspect, the present application provides a pharmaceutical composition that may comprise an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, and/or a cell described herein, and optionally a pharmaceutically acceptable adjuvant.

The pharmaceutical compositions described herein can be used directly to bind to the S protein of SARS-CoV-2 and thus can be used to prevent and treat diseases associated with coronavirus infection (e.g., COVID-19). In addition, other therapeutic agents may also be used simultaneously.

The pharmaceutical compositions of the present application may contain a safe and effective amount (e.g., 0.001-99 wt%) of an antigen binding protein as described herein, and a pharmaceutically acceptable adjuvant (which may include a carrier or excipient). The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical compositions described herein may be prepared in the form of injections, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured 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, 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 agent delivery, the method of administration, and other factors known to medical practitioners. Therapeutic agents (e.g., the antigen binding proteins described herein and/or the pharmaceutical compositions described herein) need not be formulated and/or administered concurrently with one or more agents currently used to prevent or treat the condition of interest. The effective amount of such other agents depends on the amount of therapeutic agent (e.g., the antigen binding protein described herein and/or the pharmaceutical composition described) present in the formulation, the type of disorder or treatment, and other factors discussed above. These agents can generally be used at any dosage and by any route empirically/clinically determined to be appropriate. The dose of antibody administered in the combination therapy can be reduced compared to the individual treatments. The progress of this therapy is readily monitored by conventional techniques.

Use of

In another aspect, the present application provides a use of an isolated antigen binding protein as described herein, a nucleic acid molecule as described herein, a vector as described herein, a cell as described herein and/or a pharmaceutical composition as described herein in the manufacture of a medicament for preventing, ameliorating and/or treating an infection by a coronavirus.

The present application provides a method of preventing, ameliorating, and/or treating an infection by a coronavirus comprising administering to a subject in need thereof an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein, and/or a pharmaceutical composition described herein.

The present application provides isolated antigen binding proteins, nucleic acid molecules described herein, vectors described herein, cells described herein, and/or pharmaceutical compositions described herein that can prevent, ameliorate, and/or treat infection by a coronavirus.

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

In the present application, administration of an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein, and/or a pharmaceutical composition described herein can have an effective neutralizing ability against CoVID-19 pseudoviruses (e.g., pseudoviruses prepared against WA1/2020, Alpha, Beta, Gamma, Kappa, and Delta S protein trimers (Spike trimers)).

In the present application, administering an isolated antigen binding protein described herein, a nucleic acid molecule described herein, the vectors described herein, cells described herein and/or pharmaceutical compositions described herein may have effective neutralizing ability against different strains of COVID-19 (e.g., 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) and Delta variant (B.1.617.2/Indian, Strain: GNL-751)).

In the present application, the isolated antigen binding proteins described herein, the nucleic acid molecules described herein, the vectors described herein, the cells described herein and/or the pharmaceutical compositions described herein may be administered to animal models (e.g., mouse models) of different strains that have been infected with COVID-19 (e.g., 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/MDH-0841/2021) and Delta variants (B.1.617.2/Indain, Strain: GNL-751)) Rhesus monkey model) has good therapeutic effect.

In another aspect, the present application provides a method of detecting SARS-CoV-2 comprising the step of administering an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein, and/or a pharmaceutical composition described herein. In the present application, the isolated antigen binding protein, the nucleic acid molecule described herein, the vector described herein, the cell described herein and/or the pharmaceutical composition described herein are capable of specifically and/or high affinity binding to SARS-CoV-2, e.g., S protein trimer (Spike trimer) of strains WA1/2020, Alpha, Beta, Gamma, Kappa, and Delta.

The antigen binding proteins of the present application can be used in detection applications, for example, to detect a sample, thereby providing diagnostic information. For example, the antibodies and/or methods described herein can be used to detect a sample (e.g., a throat swab test sample, such as serum, whole blood, sputum, oral/nasopharyngeal secretions or washes, urine, feces, pleural effusion, cerebrospinal fluid, and tissue samples) from a subject (e.g., a patient suspected of being infected with SARS-CoV-2, or having been infected with SARS-CoV-2) as an indicator of efficacy and whether infectivity is present and isolation is required. For example, the antibodies and/or methods described herein can provide a monitoring regimen for therapeutic intervention.

In the present application, the samples (specimens) used include cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to the person skilled in the art. A biopsy as used in the present application may thus comprise a tissue sample prepared, for example, by endoscopic methods or by a needle or needle biopsy of an organ. For example, the sample may comprise 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 a container, instructions for use, buffers, and the like. For example, a protobinding protein of the present application may be immobilized to a detection plate.

Without wishing to be bound by any theory, the following examples are intended only to illustrate the isolated antigen binding proteins, methods of preparation, uses, etc. of the present application, and are not intended to limit the scope of the invention of the present application.

Examples

EXAMPLE 1 preparation of candidate antibodies

Memory B cells capable of recognizing SARS-CoV-2 RBD are obtained from peripheral blood of a convalescent patient with COVID-19 by flow cytometry, and the separated memory B cells are subjected to single B cell sorting. Cloning to obtain the V region gene of the antibody by a single cell PCR method, and reconstructing the IgG type antibody.

The resulting candidate antibodies comprise the amino acid sequences shown in table 1:

TABLE 1

Serial number	Clone number	VH	VL
1	13H8	SEQ ID NO：81	SEQ ID NO：88
2	2G1	SEQ ID NO：82	SEQ ID NO：89
3	5H10	SEQ ID NO：83	SEQ ID NO：90
4	7G10	SEQ ID NO：84	SEQ ID NO：91
5	8G4	SEQ ID NO：85	SEQ ID NO：92
6	8G9	SEQ ID NO：87	SEQ ID NO：93

7

6F12

SEQ ID NO：86

SEQ ID NO：94

EXAMPLE 2 affinity assay for binding of candidate antibodies to SARS-CoV-2S trimer protein

The day before the experiment, the antigen SARS-CoV-2 Spike trimer protein (i.e., the S protein trimer) was added to the coating buffer (pH 9.6, 0.05M carbonate buffer) to a final concentration of 2. mu.g/mL. Add 100. mu.L per well and shake gently until the liquid spreads evenly on the bottom of each well. Placing the ELISA plate containing the antigen into a sealing bag, sealing and placing the sealing bag into a refrigerator at 4 ℃ for antigen adsorption overnight;

the next day, the supernatant was discarded, patted dry on clean absorbent paper, 250. mu.L/well of washing solution (pH7.4 PBST) was added, each time for 5min, and patted dry on clean absorbent paper, and repeated 3 times. Add 250. mu.L/well blocking solution (PBST + 3% skim milk powder), put into a new sealed bag, and block for 1h at 37 ℃. Discarding the supernatant, patting dry on clean absorbent paper, washing with 250 μ L/well washing solution, keeping for 5min each time, discarding the supernatant, patting dry on clean absorbent paper, and repeating for 3 times.

The candidate antibody prepared in example 1 was subjected to gradient dilution using an antibody diluent (pH7.4 PBS).

And sucking the diluted candidate antibody sample by 100 mu L/hole, adding the candidate antibody sample into the processed enzyme label plate, putting the candidate antibody sample into a new sealed bag, and incubating for 1h at 37 ℃. Discarding the supernatant, patting dry on clean absorbent paper, washing with 250 μ L/well washing solution, keeping for 5min each time, discarding the supernatant, patting dry on clean absorbent paper, and repeating for 3 times. Add 1:5000 dilution of anti-human IgG HRP secondary antibody per 100. mu.L/well, put in a new sealed bag, and incubate for 1h at 37 ℃. Discarding the supernatant, patting dry on clean absorbent paper, washing with 250 μ L/hole washing solution, keeping for 5min each time, discarding the supernatant, patting dry on clean absorbent paper, and repeating for 3 times. Adding TMB color development solution at 100 μ L/hole, wrapping with tinfoil, dark, developing at room temperature for 15min, observing blue reaction, and adding stop solution (2M H) at 50 μ L/hole ₂ SO ₄ ) And reading by a microplate reader at 450nm immediately after mixing. Wherein the control is human ACE2-Fc fusion protein (which comprises the amino acid sequence shown as SEQ ID NO: 100).

The results are shown in FIG. 1 and Table 2. As a result, it was found that all of the candidate antibodies had a high affinity for the S trimer protein.

TABLE 2

Serial number	Clone number	EC50(μg/ml)	Serial number	Clone number	EC50(μg/ml)
1	13H8	0.013	5	8G4	0.014
2	2G1	0.135	6	8G9	0.088
3	5H10	0.020	7	6F12	0.012
4	7G10	0.011	8	Control	1.065

EXAMPLE 3 determination of the neutralizing Activity of the candidate antibody pseudoSARS-CoV-2 Virus

One day prior to the experiment, HEK293T-ACE2 cells to be infected were seeded in 96-well cell culture plates at approximately 1X 10 ⁴ Individual cells/well, 5% CO ₂ The incubator was incubated overnight at 37 ℃. On the second day, virus infection is carried out when the cell density is about 30%, the frozen pseudovirus is taken out and placed on ice to be melted or is completely melted at 4 ℃, the using amount of the virus is 0.25 mu L/hole, the candidate antibodies prepared in the embodiment 1 with different dilution concentrations are mixed and incubated at 37 ℃ for 30min, and the mixture is added into a cell culture system to infect target cells. After 6h after viral infection, the supernatant was aspirated and 100. mu.L of complete medium was added and incubation continued for 48 hours. And (3) observing the expression of green fluorescent protein and detecting the activity of luciferase by a fluorescence microscope 48 hours after the cells are infected with pseudovirus and the liquid is changed, and judging the infection efficiency. Add 100. mu.L of One-Glo luciferase to each well, mix well with shaking, read by microplate reader after 3 min.

The results are shown in FIG. 2 and Table 3. The results show that the candidate antibodies all have good neutralizing activity on the pseudoSARS-CoV-2 virus and can effectively inhibit the continuous amplification of the SARS-CoV-2 virus. Wherein the control is human ACE2-Fc fusion protein (which comprises the amino acid sequence shown as SEQ ID NO: 100).

TABLE 3

Serial number	Clone number	EC50(μg/ml)	Serial number	Clone number	IC50(μg/ml)
1	13H8	0.013	5	8G4	0.061
2	2G1	0.011	6	8G9	0.042
3	5H10	0.058	7	6F12	0.100
4	7G10	0.322	8	Control of	0.404

EXAMPLE 4 determination of neutralizing Activity of candidate antibody EuSARS-CoV-2 Virus

The day before the experiment, Vero-E6 cells to be infected were seeded on a cell culture plate and cultured overnight. And on the second day, performing virus infection, taking out the frozen virus, thawing, mixing and incubating with candidate antibodies with different dilution concentrations, and adding the mixture into a cell culture system to infect target cells. After viral infection, the supernatant was aspirated, and complete medium was added to continue the culture. Cytopathic effects were observed for 3 to 5 days and the neutralizing activity was judged.

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

(1) samples were prepared in MEM medium (containing 1% diabody) as a 200. mu.g/ml solution, then diluted 10-fold serially, 200. mu.g/ml, 20. mu.g/ml, 2. mu.g/ml, 0.2. mu.g/ml, 0.02. mu.g/ml, 0.002. mu.g/ml for 6 dilutions, each at 2 replicates, 50. mu.l per well, and then an equal volume of 100TCID was added to each well ₅₀ Virus, 37 ℃, 5% CO ₂ The incubator is used for 1.5 h;

(2) after 1.5h, the 96-well plate was filled with cell culture medium at a concentration of 1X 10 per 100. mu.l per well ⁵ Individual cells/mL Vero cell suspension;

(3) simultaneously setting cell control and virus back drop control;

cell control: add 100. mu.L of Vero cell suspension to 96-well culture plates in 100. mu.L of MEM medium (containing 1% diabody) per well for 4 replicates;

virus back-drop control: will 100TCID ₅₀ The virus was diluted 10-fold in MEM medium (containing 1% double antibody) for 3 times to obtain 10TCID ₅₀ ，1TCID ₅₀ ，0.1TCID ₅₀ . In 96-well plates, 50. mu.L of MEM medium (containing 1% diabody) was added to each well, and then an equal volume of 100TCID was added to each well ₅₀ ，10TCID ₅₀ ，1TCID ₅₀ ，0.1TCID ₅₀ Virus, 4 replicate wells per dilution, 37 ℃, 5% CO ₂ Incubating for 1.5h, and adding 100 μ L of the culture solution into each well after 1.5h ⁵ Individual cells/mL Vero cell suspension. The result of the virus back drop control is in the range of 32-320TCID50/50 μ l, and the experiment is effective.

(4) Cells at 37 ℃ and 5% CO ₂ Incubating in an incubator for 3-5 days;

(5) cytopathic effect (CPE) was observed under an optical microscope, and changes in CPE in the cells were marked as "+", changes in CPE in the cells were not observed, or normal cell morphology was marked as "-".

And (3) calculating an inhibition effect: inhibitory virus half Effective Concentration (EC) ₅₀ )

Wherein A: percent of inhibition greater than 50%, B: percent of inhibition less than 50%, C: log (dilution factor), D: log (sample concentration corresponding to less than 50% inhibition). If the sample does not inhibit the virus, EC50 cannot be detected. The results are shown in Table 4.

TABLE 4

Clone number	IC ₅₀ (μg/ml)
9E12	0.03
9D11	0.3
5B2	0.3
13A12	0.03
2G1	0.003
3A4	0.03
10D4	0.03
9A6	3.16
8G9	31.6

The results in Table 4 demonstrate that the above antibodies achieve effective neutralization of both SARS-CoV-2 true virus. Neutralization IC of SARS-CoV-2 true virus ₅₀ As a result, 9E12 was 0.03. mu.g/mL, 9D11 was 0.3. mu.g/mL, 5B2 was 0.3. mu.g/mL, 13A12 was 0.03. mu.g/mL, 2G1 was 0.003. mu.g/mL, 3A4 was 0.03. mu.g/mL, 10D4 was 0.03. mu.g/mL, 9A6 was 3.16. mu.g/mL, and 8G9 was 31.6. mu.g/mL. The antibodies can neutralize SARS-CoV-2 virus.

Therefore, the candidate antibodies have good neutralizing activity on the true SARS-CoV-2 virus and can effectively inhibit the continuous amplification of the SARS-CoV-2 virus.

EXAMPLE 5 measurement of SARS-CoV-2 Virus neutralizing Activity of candidate antibody in animals

The candidate antibody prepared in example 1 was administered to an animal model infected with SARS-CoV-2 virus. And (3) determining the neutralizing activity of the candidate antibody on the SARS-CoV-2 virus after the administration by a method for detecting the virus content by quantitative PCR. As a result, it was found that the candidate antibodies all had good neutralizing activity against the candidate antibodies in the animal body.

EXAMPLE 6 detection of binding kinetics of candidate antibodies to SARS-CoV-2

The binding kinetics of the monoclonal antibodies were tested using a CM5 chip (Cytiva 29149603) using WA-1S1-His or a S protein trimer (Spike trimer) as antigen.

Antigen coupling

Buffer solution: PBS (Cytiva BR100672)

Flow rate: 10 mu L/min

Antigen dilution: acetate pH 5.0(Cytiva BR100351)

Antigen concentration: 1. mu.g/mL

Amino coupling kit (Cytiva BR 100050): mixing activator EDC + NHS 1: 1, and blocking agent ethanolamine

The chip was activated for 700s and the diluted antigen was coupled to a level of about 70RU, blocking excess unreacted sites.

Antibody binding

Buffer solution: HBS-EP (Cytiva BR100669)

Flow rate: 30 mu L/min

Antibody concentration: 2-fold dilution of 0.2. mu.g/mL to 0.0125. mu.g/mL

Regeneration of buffer solution: glycine pH 1.5(Cytiva BR100354)

According to the set concentration arrangement, the diluted antibodies with each concentration are respectively added into the corresponding 96-well plate holes so as to combine for 120s, dissociate for 120s, regenerate and elute for 30s, and sequentially load from low concentration to high concentration.

The affinity results are shown in table 5, and the IgG monoclonal antibodies can be combined with the S protein trimer with high efficiency, and the affinity reaches-10 to-15M.

TABLE 5

Example 7 affinity study of candidate antibodies to Spike protein

To avoid the effect of "dance effect", monoclonal antibody 2G1 WAs subjected to papain digestion to obtain Fab fragments, and binding kinetics of monovalent Fab fragments to WA1/2020, Alpha, Beta, Gamma, Kappa, and Delta S protein trimer (Spike trimer) were examined.

The binding kinetics of monoclonal antibodies were tested using CM5 chip (Cytiva 29149603) using the S protein trimer of WA1/2020, Alpha, Beta, Gamma, Kappa, and Delta as antigen.

One, antigen coupling

Buffer solution: PBS (Cytiva BR100672)

Flow rate: 10 mu L/min

Antigen dilution: acetate pH 5.0(Cytiva BR100351)

Antigen concentration: 1. mu.g/mL

Amino coupling kit (Cytiva BR 100050): activator EDC + NHS 1: 1 mixing, blocking agent ethanolamine to activate the chip for 700s, coupling the diluted antigen to about 70RU level, and blocking excess unreacted sites.

II, antibody binding

Buffer solution: HBS-EP (Cytiva BR100669)

Flow rate: 30 mu L/min

Antibody concentration: 2-fold dilution of 0.2. mu.g/mL to 0.0125. mu.g/mL

Regeneration of buffer solution: glycine pH 1.5(Cytiva BR100354)

According to the set concentration arrangement, the diluted antibodies with each concentration are respectively added into the corresponding 96-well plate holes to combine 120s, dissociate 120s and regenerate and elute 30s, so that the samples are sequentially loaded from low concentration to high concentration.

The results are shown in Table 6.

TABLE 6

SARS-CoV-2S protein trimer

K _a (Ms ^-1 )

K _d (s ^-1 )

K _D (nM)

WA1/2020	1.03×10 ⁶	1.05×10 ^-3	1.02
Alpha	8.72×10 ⁵	7.55×10 ^-4	0.86
Beta	7.96×10 ⁵	2.20×10 ^-3	2.77
Gamma	8.73×10 ⁵	2.01×10 ^-3	2.30
Kappa	8.22×10 ⁵	8.53×10 ^-4	1.04
Delta	2.80×10 ⁶	4.27×10 ^-2	15.30

Example 8 detection of neutralizing Capacity of candidate antibodies to pseudoviruses

The pseudovirus contains SARS-CoV-2 surface Spike protein, can specifically infect ACE2 positive cells, selects pseudoviruses prepared from WA1/2020, D614G, Cluster 5, Alpha, Beta, Gamma and Delta Spike protein, and performs a pseudovirus neutralization activity test according to the following steps.

1. ACE2-293T cells in logarithmic growth phase at 1 × 10 ⁴ Wells were plated into white primed 96-well plates (Corning, 3903);

2. the next day, different concentrations of neutralizing antibody (20, 2, 0.2, 0.02, 0.002, 0.0002, 0.00002. mu.g/mL) were diluted with DMEM medium (Gibco, C11995500BT) + 10% FBS (Gibco, 10270-106); and an appropriate volume of 0.2. mu.L/100. mu.L of COV2-S protein pseudovirus (Yeasen, 11903ES50) was diluted in P2 laboratory.

3. Respectively sucking 55 μ L of antibody with different concentrations and 55 μ L of diluted pseudovirus, mixing, setting negative control and positive control holes, and incubating at 37 deg.C for 30 min;

4. the medium in the 96-well plate was aspirated off, 100. mu.L of the medium containing the corresponding antibody-virus mixture was added, and the mixture was incubated in CO ₂ Continuously culturing in an incubator;

after 5.6 h, the virus-containing medium was aspirated into a waste tank containing 84 sterilizing fluid, the proportion of 84 sterilizing fluid being not less than 30%. Then 100. mu.L of fresh medium was added and the reaction was continued in CO ₂ Culturing for 48h in an incubator;

6. sealing the 96-well plate in a biological safety cabinet by using a sealing film, sterilizing the outer surface of the 96-well plate by using 75% alcohol, taking out the plate, adding 90 mu L of luciferase substrate (Promega, E6120) into each well, incubating for 3-5min, and reading the fluorescence value of each well by using a microplate reader.

7. The inhibition rate at each concentration was calculated from the fluorescence value.

The results are shown in FIG. 3, and the results in FIG. 3 demonstrate that monoclonal antibody 2G1 is effective in neutralizing various pseudoviruses. The neutralization IC50 is WA 1/20200.0032 mu g/mL, D614G 0.0038.0038 mu g/mL, Cluster 50.0002 mu g/mL, Alpha 0.0013 mu g/mL, Beta 0.0028 mu g/mL, Gamma 0.0005 mu g/mL and Delta 0.0082 mu g/mL.

Example 9 detection of neutralizing Capacity of candidate antibodies to Euviruses

2G1 study on neutralizing ability of mutant euviruses.

Virus neutralization experiments were performed using the 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) and Delta variant (B.1.617.2/Indian, Strain: GNL-751) mutant true viruses. The brief method comprises the following steps: the antibody was serially diluted 3-fold in MEM medium (Gibco) at a concentration of 20. mu.g/mL to prepare a working solution. The dilutions were added to an equal volume of 100TCID50 virus and incubated for 1 hour at room temperature. The mixture was added to 96-well plates with confluent Vero cells. A cell blank control and a virus infection control were set simultaneously. 37 ℃ and 5% CO ₂ After 3 days of culture, cytopathic effect (CPE) was observed under a microscope, and plaque was counted for efficacy evaluation. Holes with CPE changes were recorded as "+", otherwise as "-".

The IC50 value was calculated according to the following equation: IC50 ═ Antilog (D-C × (50-B)/(a-B)). Wherein A represents an inhibition rate of more than 50%, B represents an inhibition rate of less than 50%, C is lg (dilution factor), and D is lg (concentration of the sample at which the inhibition rate is less than 50%). All experiments were performed in a biosafety class 3 laboratory. As shown in Table 7, 2G1 has high virus neutralizing ability to WA1/2020, Alpha, Beta, Gamma, Delta.

TABLE 7

Neutralizing power of true virus	IC ₅₀ (μg/ml)	IC ₁₀₀ (μg/ml)
WA1/2020	0.0240	0.0411
Alpha	0.0138	0.0411
Beta	0.0046	0.0137
Gamma	0.0079	0.0137
Delta	0.0079	0.0411

Example 10 in vivo biological Activity Studies of candidate antibodies

10.1 mouse model

AC70 was human ACE2 transgenic mice (Taconic Biosciences, Cat #18222), AC70 mice were divided into three groups, a control group (PBS), a low dose (2.2mg/mL monoclonal antibody 2G1) medium dose group (6.7mg/mL monoclonal antibody 2G1) and a high dose (20mg/kg monoclonal antibody 2G1), with 14 mice per group. All mice were infected with 100LD 50. The first dose of monoclonal antibody 2G1 and PBS was given 4 hours after infection; the second and third were administered on

days

2 and 4 post-infection, respectively. Mice were subjected to at least one clinical observation per day and according to the clinical health profile. Scoring on a scale of 1 to 4, with score 1 being healthy in a standardized scale of 1 to 4 scoring system; 2 points are upright fur and drowsiness; a score of 3 is an additional clinical symptom, such as hunched posture, tightening of the orbit, increased breathing rate and/or > 15% weight loss; score 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. The remaining mice were continuously monitored for morbidity and motility for up to 14 days post infection.

The mouse infection model was constructed as shown in FIG. 4.

The body weight of the mice after infection was measured and the results are shown in FIG. 5. The results in FIG. 5 show that there WAs no significant weight loss in the upper, middle, or lower three doses for WA1/2020 and Beta infected mice, indicating that even a low dose of 2.2mg/mL of monoclonal antibody 2G1 WAs sufficient to neutralize the virus. Delta infection group, the animal weight of the 20mg/kg high dose group did not significantly reduce, and the weight reduction phenomenon occurred at the 6.7mg/kg dose and the 2.2mg/kg dose.

The WA1/2020, Beta and Delta mouse infection models were observed and clinically scored and the results are shown in FIG. 6. The results in FIG. 6 demonstrate that even at a low dose of 2.2mg/kg of monoclonal antibody 2G1, the WA1/2020, Beta model has no significant clinical symptoms. In the Delta model, no clinical symptoms appear at the high dose of 20mg/kg, and clinical response appears at the medium and low dose.

According to the principle of euthanasia of the mice, the mice are difficult to breathe and/or cyanosis, do not want to move when stimulated, or need to euthanasia immediately when the weight loss is more than or equal to 20 percent, and the mice are considered to die, and a survival curve is drawn. Survival curves for the mouse infection model of WA1/2020, Beta and Delta were observed and plotted, and the results are shown in FIG. 7. The results in FIG. 7 show that in both the WA1/2020 and Beta infection models, mice treated with high, medium and low doses did not die and the survival rate reached 100% and were able to recover to a healthy state. In the Delta infection model, the survival rate of the low-dose mice is 10 percent, the survival rate of the middle-dose mice is 55.6 percent and the survival rate of the high-dose mice is 100 percent.

10.2 rhesus monkey model

Rhesus monkeys of 6-7 years old were randomized into control, low (10mg/kg) and high (50mg/kg) dose groups, one male and one female per group. 4mL of 1X 10 per animal ⁵ TCID50 virus infects animals through tracheal intubation. Antibody 2G1 antibody and PBS were administered intravenously 24 hours post infection. Disease-related changes in rhesus monkeys were monitored continuously, body weight and body temperature were measured daily, and pharyngeal and anal swab samples were collected for virus titration. See figure 8 for a method of constructing a rhesus infection model.

On day 7 post-infection, animals were euthanized and tissue samples were collected. Viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen). According to the supplier's instructions (

II One Step qRT-PCR

Green Kit, Vazyme Biotech co., Ltd) quantitates viral RNA using one-step real-time quantitative PCR and primers for the RBD gene, using quantitative primers: RBD-qF 1: 5'-CAATGGTTAAGGCAGG-3' (SEQ ID NO. 101); RBD-qR 1: 5'-CTCAAGGTCTGGATCACG-3' (SEQ ID NO. 102).

The content of viral RNA in the rhesus monkey infection model was measured by throat swabs, and the results are shown in fig. 9. The results in FIG. 9 demonstrate that viral RNA loads were detected only at

days

3,4 and 5 after challenge in control animals, ranging from 10e3 to 10e7 copies/mL, and that the peak period of viral replication fluctuated slightly at day 2 after challenge. In general, the law of viral load changes indicates the propagation process of the virus in vivo. The viral RNA load of the animals tested in the high dose group 2 was always in a decreasing trend, no virus could be detected by decreasing from 10e6 copies/mL to 10e3 copies/mL below the detection threshold on

days

3 and 4 after challenge, respectively, and the viral RNA load of the animals tested in the low dose group 2 was always in a decreasing trend, from 10e6 copies/mL to 10e3 copies/mL, and below the detection threshold on day 4 after challenge, no virus could be detected.

The content of viral RNA in the rhesus monkey model was measured by anal swabs, and the results are shown in fig. 10. The results in FIG. 10 demonstrate that the control animals detected viral RNA at between 10e3 and 10e5 copies/mL on

days

4, 5, and 7, while no virus was detected at both the high and low doses.

To further understand the distribution of the virus in different tissues of the upper respiratory tract and lung, tissues of different parts of trachea, bronchi and lung of control group and low and high dose group animals were collected at day 7 after challenge, and the viral load in different organs and tissues was determined. The results are shown in FIG. 11, and the results of FIG. 11 show that: on day 7 post-infection, approximately 1 × 10e 5-1 × 10e7 copies/g of viral RNA were detected in both the trachea and the left and right bronchi of control animals. Virus was detected in the right middle lung, left lower lung and left bronchus in the high dose group, and virus was found only in the trachea in the low dose group.

Example 11 in vivo biological Activity Studies of candidate antibodies

In order to analyze the mode of interaction between the Spike (S) protein and the antigen and antibody of the 2G1 antibody and the binding site, the three-dimensional structure of the complex of the extracellular domain of the trimeric S protein of the novel coronavirus (SARS-CoV-2) WA-1 strain and the 2G1 antibody is analyzed by using a single particle reconstruction technology of a cryoelectron microscope, and the binding site of the antibody on the S protein is confirmed.

Procedure of the test

1. Expression and purification of trimeric S proteins

1.1 construction of trimeric S protein recombinant expression plasmid

The modified S protein is used for improving the stability of the protein, and the specific scheme is as follows: proline mutations are introduced at positions 817, 892, 899, 942, 986 and 987 of the extracellular region of the S protein (amino acids 1-1208, Genbank ID: QHD 43416.1); meanwhile, the furin restriction enzyme cutting site RRAR from 682 to 685 is mutated into GSAS; fusing T4 fibritin foldon at the C end of the S protein extracellular region to assist the S protein extracellular region to form a trimer; finally, the vector is cloned into a pCAG vector with a 1xFlag tag at the C terminal end.

1.2 expression purification of S protein

HEK 293F cells were used to transiently transfect recombinant expression plasmids for secretory expression of the S protein. When the density of HEK 293F cells cultured in suspension reaches 2.0 multiplied by 10 ⁶ Transfection was performed at/mL. In 1L HEK 293F cells, 1mg S plasmid and 3mg PEI 4000 mixed incubation 15min after adding to the cells, continued to culture for 60h after collecting cell supernatant for purification.

The supernatant obtained from the transfection was filtered, and the cell culture medium was removed by concentration and replacement with a buffer (25mM Tris-HCl, 150mM NaCl, pH 8.0). Purification was performed using Anti-Fag M2 resin, and 60mL of buffer (25mM Tris-HCl, 150mM NaCl, pH8.0) was used to wash away the contaminating proteins, followed by elution with 1Xflag peptide. The eluate was concentrated to 2mL and further purified by using a molecular sieve column (Superose 6 Increate 10/300GL, GE Co.) to obtain a trimeric S protein.

And (3) incubating the obtained S protein and 2G1 antibody for 1h at a molar ratio of 1:5, concentrating, further purifying by using molecular sieve column chromatography, and removing excessive 2G1 antibody to obtain an S-2G1 compound for preparing a cryoelectron microscope sample.

2. Preparation of frozen samples, data collection and three-dimensional reconstruction by single particle method

The S-2G1 complex is concentrated to 2.5mg/mL, 3.3 mu L is dripped on a hydrophilized grid (Quantifoil Au R1.2/1.3), and a Vitrobot (Mark IV, Thermo Scientific) sampling robot is used to prepare a frozen electron microscope sample through three steps of sample adsorption, excess sample suction and quick freezing in sample liquid ethane.

Data collection was performed using Titan Krios (FEI)300kV electron microscopy equipped with a Gatan K3 camera. And transferring the prepared frozen electron microscope sample into an electron microscope lens barrel, debugging the electron microscope to an optimal state, and automatically acquiring Movie stacks data by using Autoanimation software. The defocusing range is set to be 1.2-2.2 mu m, the magnification of a K3 camera is set to be 81000 times, and the corresponding pixel size is

Each picture collected had 32 frames, each frame was exposed for 0.08s, and the total exposure time was 2.56 s. The total electron dose for taking a picture is about

And (3) performing drift correction on the collected original picture by using MotionCor2, then manually screening the corrected picture, and manually selecting a uniform and clear electron microscope picture to remove the picture with poor quality or serious pollution. Particles of the S protein and 2G1 complex were automatically sorted using Relion 3.0.6. After two-dimensional classification is carried out on all the particles, the matched particles are selected for carrying out three-dimensional model reconstruction. The method comprises the steps of firstly carrying out two rounds of three-dimensional classification through cryoSPARC, then selecting proper particles for 3D remodeling, and then correcting by means of Relion to obtain a three-dimensional model of the S protein and 2G1 compound. To further improve the resolution of the interaction interface of the S protein and 2G1, model correction and optimization were performed for this local region, resulting in a three-dimensional model of the RBD and 2G1 fractions. The resolution was determined using the fourier shell correlation function curve of the gold standard, with the threshold determined set to 0.143. The detailed processes and parameters of complex purification, data collection and three-dimensional reconstruction by single particle method are shown in fig. 12-13 and fig. 14. Wherein FIG. 12a shows that molecular sieve chromatography purifies 2G1 and S protein complex; FIG. 12b shows the E μ Ler angle distribution of the final 3D model of S-2G1 complex; FIGS. 12c-12d show the local resolution of the S-2G1 complex overall structure (c) and the local RBD-2G1 structure (d); FIG. 12e shows an FSC plot of the resolution of the S-2G1 (blue) and RBD-2G1 (orange) complexes; FIGS. 12f and 12G show the optimized FSC curve of the S-2G1 complex model. FIG. 13a shows a representative cryoelectron microscopy of S-2G1 complex and a 2D classification with a 10nm scale in the 2D classification; fig. 13b shows the data processing step.

The FSC curve of the refined model of the RBD-2G1 complex is the same as f.

In order to build an atomic model of the S protein of SARS-CoV-2 and 2G1 complex, 4A8(PDB ID: 7C2L) is used as a template, and the corresponding atomic model is superimposed into the cryo-electron microscope density files of the whole S-2G1 and the local RBD-2G1 obtained by the previous single-particle reconstruction method by molecular dynamics flexible matching. Firstly, an antibody 4A8 is taken as a template to obtain a 2G1 Chainaw model; taking a locally optimized RBD-2G1 density file as a standard, further manually adjusting an atomic model by using Coot software, and compounding the chemical characteristics of each amino acid residue with the surrounding density cloud; and finally, optimizing the whole atomic model by using Phenix software, and correcting by using a secondary structure and geometric constraint to prevent overfitting. Detailed data for data collection, 3D model reconstruction and atomic model building is shown in fig. 14.

Test results

To investigate the binding pattern of 2G1 antibody to S protein, revealing the epitope of 2G1 antibody, S-2G1 complex was analyzed using cryoelectron microscopy

Resolution structure (fig. 15a, fig. 15-16). FIG. 15a is a cryo-electron density plot of S-2G1 composites shown in orthogonal orientation. The heavy and light chains of 2G1 were blue and cyan, respectively. Each monomeric structure of the trimeric S protein is gray, orange and pink, respectively. FIGS. 15b-e show the interaction between 2G1 and an RBD and an adjacent RBD'. RBD and 2G1 primarily through hydrophobic interactions (fig. 15c and 15 d). The 2G1 heavy chains (CDRH3 and CDRH1) were located above the adjacent RBD' (fig. 15 e).

FIG. 16a shows the epitope boundaries of three similar antibodies (S2E12, B1-182.1 and REGN10933) in different colors, respectively, and the epitopes of S2E12, B1-182.1 and REGN10933 are red, orange and green, respectively. FIG. 16B shows a comparison of the binding angles of 2G1, S2E12, B1-182.1 and REGN 10933. The 2G1 epitope boundary is blue. The epitope boundaries of the ACE2 binding site, 2G1, S2E12, B1-182.1, and REGN10933 are superimposed on the RBD, shown in black, blue, red, orange, and green, respectively. The central axis is the line between the center point of 2G1 antibody Fab and the center of RBD, the angle between S2E12, B1-182.1 and the central axis is about 6 degrees, and the angle between REGN10933 and the main axis is about 13 degrees. FIG. 16c shows statistics of amino acid positions of epitopes on RBDs for 2G1, ACE2, S2E12, B1-182.1, and REGN 10933.

However, in the overall structure, the structure density at the interaction interface of the RBD and 2G1 is unclear, so that the partial complex of the 2G1 antibody and the RBD binding domain is resolved by adopting a local optimization calculation method

Resolution structure, providing a structural basis for accurate analysis of 2G1 interaction with RBD (fig. 15 b). In the S-2G1 complex structure, three soluble 2G1 antibody Fab regions bound to the three RBD domains of the trimeric S spike protein, respectively. Also in this structure, all RBDs are in the "down" conformation and the trimeric S protein is in a locked conformation as a whole (fig. 15 a). In addition, it was found that an additional density of fatty acid Linoleic Acid (LA) is present in the S protein, consistent with the position of the LA binding pocket in the locked conformation S trimer structure reported in the literature.

Through detailed analysis of the 2G1 and RBD binding interface, the antibody 2G1 was found to bind to the loop region at the tip of the RBD, which partially overlaps with the ACE2 receptor binding site and does not belong to the mutational hotspot region of VOCs. The heavy chain of 2G1 is primarily involved in RBD interactions via three Complementarity Determining Regions (CDRs) CDRH1 (amino acid residues 30-35), CDRH2 (amino acid residues 50-65) and CDRH3 (amino acid residues 98-111); the light chain is involved in the interaction mainly by the two CDR regions CDRL1 (amino acid residues 23 to 36) and CDRL3 (amino acid residues 91 to 100) (fig. 15 b-e). The binding interface between RBD and 2G1 is mainly stabilized by a network of extensive hydrophobic interactions, among which the more important ones are: phe486 of the top loop region of RBD binds to Tyr33, Tyr52 on the heavy chain and Tyr34, Tyr93, Trp99 on the light chain by hydrophobic and/or-interactions (fig. 15 c). CDRH1 and CDRH3 of the 2G1 heavy chain were located directly above the LA binding pocket in the adjacent RBD' (fig. 15b and 15 e). 2G1 was compared to three antibodies with similar epitopes (S2E12, B1-182.1 and REGN10933) (FIGS. 16 a-c). Structural comparison analysis showed that the epitope of 2G1 partially overlapped the three antibodies (S2E12, B1-182.1, and REGN10933), but they had different binding directions (fig. 16B). Furthermore, 2G1 has a relatively narrow binding epitope (F456, a475, G476, S477, T478, E484, G485, F486, N487, Y489), which may have the advantage of being less susceptible to viral mutations, thus achieving a broad spectrum of virus neutralization capacity (fig. 16 c).

By resolving the complex cryo-electron microscopy structure of the S protein with 2G1, it was revealed that 2G1 is capable of binding the S protein in a locked conformation in which the RBD domain is in a "down" conformation in each monomer located in the S trimer. No other antibodies in the structural database have been found to bind this locked conformation. Although the epitopes of 2G1, S2E12 and B1-182.1 are relatively similar and partially overlap, it is reported that S2E12 and B1-182.1 bind RBDs in the "up" conformation in structure, while 2G1 is able to bind the S protein in the locked conformation, probably due to the particular angle of binding of the 2G1 antibody. In the current research stage, it is speculated that the reason for the neutralizing activity of 2G1 is not only to block the binding of ACE2 to RBD, but also to prevent the conformational change of S in the fused state by binding to the S protein in the locked conformation. In addition, the amino acid of the epitope of 2G1 antibody at a specific position on the RBD tip deviates from the mutational hot spot of VOCs, and may increase the broad-spectrum neutralizing activity of the antibody. Therefore, the complex structure of S-2G1 may provide a good reference for the development of vaccines and the optimization of optimal combination therapies.

The embodiments of the present application have been described in detail, but the present application is not limited to the details of the above embodiments, and various simple modifications can be made to the technical solution of the present application within the technical idea of the present application, and the simple modifications belong to the protection scope of the present application. It should be noted that, in the above embodiments, the various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described separately in the present application. In addition, any combination of the various embodiments of the present application is also possible, and the same should be considered as disclosed in the present application as long as it does not depart from the idea of the present application.

Claims

An isolated antigen binding protein that specifically binds SARS-CoV-2 comprising at least one CDR in the light chain variable region VL, wherein the CDR comprises the amino acid sequence set forth in SEQ ID NO: 95.
The isolated antigen binding protein of claim 1, wherein the VL comprises LCDR1 and the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO 95.
The isolated antigen binding protein of any of claims 1-2, wherein the VL comprises LCDR1, the LCDR1 comprising the amino acid sequence set forth in any one of SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 49, and SEQ ID No. 50.
The isolated antigen binding protein of any of claims 1-3, wherein the VL comprises LCDR2 and the LCDR2 comprises the amino acid sequence set forth in any of SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, and SEQ ID NO 54.
The isolated antigen binding protein of any of claims 1-4, wherein the VL comprises LCDR3, the LCDR3 comprises an amino acid sequence set forth in any of SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, and SEQ ID NO 61.
The isolated antigen binding protein of any of claims 1-5, wherein the VL comprises LCDR1 and LCDR2, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO 95, and the LCDR2 comprises the amino acid sequence set forth in any of SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, and SEQ ID NO 54.
The isolated antigen binding protein of any of claims 1-6, wherein the VL comprises LCDR1 and LCDR3, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO 95, and the LCDR3 comprises the amino acid sequence set forth in any of SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, and SEQ ID NO 61.
The isolated antigen binding protein of any of claims 1-7, wherein the VL comprises LCDR1, LCDR2, and LCDR3, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO 95, the LCDR2 comprises the amino acid sequence set forth in any of SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, and SEQ ID NO 54; the LCDR3 comprises an amino acid sequence shown in any one of SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60 and SEQ ID NO. 61.
The isolated antigen binding protein of any of claims 1-8, wherein said VL comprises framework regions L-FR1, L-FR2, L-FR3, and L-FR4, wherein the C-terminus of said L-FR1 is linked directly or indirectly to the N-terminus of said LCDR1, and said L-FR1 comprises the amino acid sequence set forth in any one of SEQ ID No. 62, SEQ ID No. 63, and SEQ ID No. 64.
The isolated antigen binding protein of claim 9, wherein said L-FR2 is located between said LCDR1 and said LCDR2, and said L-FR2 comprises the amino acid sequence set forth in any one of SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, SEQ ID No. 68, SEQ ID No. 69, SEQ ID No. 70, and SEQ ID No. 71.
The isolated antigen binding protein of any of claims 9-10, wherein said L-FR3 is located between said LCDR2 and said LCDR3, and said L-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NO 72, SEQ ID NO 73, SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, and SEQ ID NO 78.
The isolated antigen binding protein of any of claims 9-11, wherein the N-terminus of L-FR4 is linked directly or indirectly to the C-terminus of LCDR3 and L-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs 79 and 80.
The isolated antigen binding protein of any of claims 1-12, wherein the VL comprises an amino acid sequence 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.
The isolated antigen binding protein of any one of claims 1-13, comprising an antibody light chain constant region.
The isolated antigen binding protein of any of claims 1-14, comprising a heavy chain variable region VH comprising HCDR1, the HCDR1 comprising the amino acid sequence set forth in any one of SEQ ID No.1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, and SEQ ID No. 7.
The isolated antigen binding protein of any of claims 1-15, comprising a heavy chain variable region VH comprising HCDR2, the HCDR2 comprising the amino acid sequence set forth in any one of SEQ ID No. 8, SEQ ID No. 9, SEQ ID No.10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14.
The isolated antigen binding protein of any of claims 1-16, comprising a heavy chain variable region VH comprising HCDR3, the HCDR3 comprising the amino acid sequence set forth in any one of SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, and SEQ ID NO 21.
The isolated antigen binding protein of any one of claims 1-17, comprising a heavy chain variable region VH, said VH comprising HCDR1, HCDR2, and HCDR3, said HCDR1 comprising the amino acid sequence set forth in any one of SEQ ID No.1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, and SEQ ID No. 7; the HCDR2 comprises an amino acid sequence shown by any one of SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 14; the HCDR3 comprises an amino acid sequence shown in any one of SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21.
The isolated antigen binding protein of any of claims 1-18, wherein said VH comprises the framework regions H-FR1, H-FR2, H-FR3, and H-FR4, wherein the C-terminus of said H-FR1 is directly or indirectly linked to the N-terminus of said HCDR1, and said H-FR1 comprises the amino acid sequence set forth in any of SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, and SEQ ID No. 28.
The isolated antigen binding protein of claim 19, wherein said H-FR2 is located between said HCDR1 and said HCDR2, and said H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, and SEQ ID No. 34.
The isolated antigen binding protein of any one of claims 19-20, wherein said H-FR3 is located between said HCDR2 and said HCDR3, and said H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID No. 35, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, and SEQ ID No. 41.
The isolated antigen binding protein of any of claims 19-21, wherein the N-terminus of H-FR4 is linked directly or indirectly to the C-terminus of HCDR3 and the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs 42, 43, and 44.
The isolated antigen binding protein of any of claims 19-22, wherein said VH comprises the amino acid sequence set forth in any one of SEQ ID NO 81, SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86, and SEQ ID NO 87.
The isolated antigen binding protein of any one of claims 1-23, comprising an antibody heavy chain constant region.
The isolated antigen binding protein of any of claims 1-24, having an activity of neutralizing SARS-CoV-2.
The isolated antigen binding protein of any one of claims 1-25, comprising an antibody or antigen binding fragment thereof.
The isolated antigen binding protein of claim 26, wherein the antigen binding fragment comprises a Fab, Fab ', F (ab)2, Fv fragment, F (ab')2, scFv, di-scFv and/or dAb.
The isolated antigen binding protein of any of claims 26-27, wherein said antibody is a fully human antibody.
An isolated one or more nucleic acid molecules encoding the VL in the isolated antigen binding protein of any one of claims 1-28.
An isolated nucleic acid molecule or molecules encoding the VH in the isolated antigen binding protein of any one of claims 1-28.
An isolated one or more nucleic acid molecules encoding the isolated antigen binding protein of any one of claims 1-28.
A vector comprising the nucleic acid molecule of any one of claims 29-31.
A cell comprising the nucleic acid molecule of any one of claims 29-31 or the vector of claim 32.
The cell of claim 33, which expresses the isolated antigen binding protein of any one of claims 1-28.
A method of making the isolated antigen binding protein of any one of claims 1-28, the method comprising culturing the cell of claim 33 under conditions such that the isolated antigen binding protein of any one of claims 1-28 is expressed.
A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-28, the nucleic acid molecule of any one of claims 29-31, the vector of claim 32, and/or the cell of any one of claims 33-34, and optionally a pharmaceutically acceptable adjuvant.
Use of the isolated antigen binding protein of any one of claims 1-28, the nucleic acid molecule of any one of claims 29-31, the vector of claim 32, the cell of any one of claims 33-34, and/or the pharmaceutical composition of claim 36 in the manufacture of a medicament for preventing, ameliorating, and/or treating an infection by a coronavirus.
The use of claim 37, wherein the infection by a coronavirus comprises COVID-19.
A method of preventing, ameliorating, and/or treating an infection by a coronavirus comprising administering the isolated antigen binding protein of any one of claims 1-28, the nucleic acid molecule of any one of claims 29-31, the vector of claim 32, the cell of any one of claims 33-34, and/or the pharmaceutical composition of claim 36.
The isolated antigen binding protein of any one of claims 1-28, the nucleic acid molecule of any one of claims 29-31, the vector of claim 32, the cell of any one of claims 33-34, and/or the pharmaceutical composition of claim 36 for use in preventing, ameliorating, and/or treating an infection by a coronavirus.
A method of detecting SARS-CoV-2 comprising the step of administering the isolated antigen binding protein of any one of claims 1-28, the nucleic acid molecule of any one of claims 29-31, the vector of claim 32, the cell of any one of claims 33-34, and/or the pharmaceutical composition of claim 36.