WO2021227138A1

WO2021227138A1 - Monoclonal antibody for novel coronavirus, or antigen binding part thereof

Info

Publication number: WO2021227138A1
Application number: PCT/CN2020/092957
Authority: WO
Inventors: 朱凤才; 张黎; 潘红星; 郭喜玲
Original assignee: 江苏省疾病预防控制中心(江苏省公共卫生研究院)
Priority date: 2020-05-09
Filing date: 2020-05-28
Publication date: 2021-11-18

Abstract

A monoclonal antibody for a novel coronavirus, or an antigen binding part thereof. The monoclonal antibody specifically binds to an S protein of the novel coronavirus. The monoclonal antibody contains a heavy chain variable region comprising amino acid sequences as shown in SEQ ID NO.1-3, SEQ ID NO.9-11, and SEQ ID NO.17-19, and a light chain variable region comprising amino acid sequences as shown in SEQ ID NO.5-7, SEQ ID NO.13-15, and SEQ ID NO.21-23. The monoclonal antibody can be used for detecting the existence of the novel coronavirus. In addition, the monoclonal antibody has neutralizing activity, and therefore can be used for the development of drugs for preventing or treating novel coronavirus infection.

Description

Monoclonal antibody or its antigen binding part against new coronavirus

Technical field

The invention belongs to the fields of cellular immunology and molecular biology, and relates to a monoclonal antibody or an antigen binding part thereof against a novel coronavirus.

Background technique

The International Virus Classification Committee named the new coronavirus SARS-CoV-2, and the World Health Organization called the pneumonia caused by infection with this virus COVID-19. This virus is highly contagious and spreads widely. The virus can quickly adapt to the human environment, and has the ability to spread during the incubation period after infection. At the same time, some asymptomatic infections have been reported, and viral nucleic acids have even been detected in a variety of animals. These factors make the prevention and control of the virus very complicated, and there are currently no effective therapeutic drugs and vaccines on the market.

SARS-CoV-2 belongs to the genus Coronavirus. It is a single-stranded positive-stranded RNA virus with a size of about 30kb. The similarity to SARS-CoV is 79%, and the highest similarity to Coronavirus (CoV) isolated from bats is about 88%. . SARS-CoV-2 has typical coronavirus characteristics, with typical spinous processes on the virus envelope, resembling a corona. Spike protein is the most important surface membrane protein of coronavirus, which determines the host range and specificity of the virus. It is an important site for host neutralizing antibodies and a key target for vaccine design.

Since specific therapeutic drugs and effective vaccines have not been successfully developed, there have been attempts to treat severe patients with plasma from convalescent patients, and they have had obvious effects. Because the components of plasma and plasma products are complex, and may have potential risk factors. Virus neutralizing antibodies, especially fully human monoclonal antibodies, are particularly important in virus diagnosis and treatment. Monoclonal antibodies can recognize a single epitope of the virus, and some neutralizing monoclonal antibodies can bind to specific sites of the virus, such as receptor binding sites, protease cleavage sites, and attachments to membrane fusion sites, thereby infecting Adherent host cells in the virus life cycle use mechanisms such as membrane fusion and surface proteolysis to neutralize them. Among them, the fully human monoclonal antibodies obtained from patients in the convalescent stage have the potential to become drugs. First of all, because the immune system of the convalescent patients undergoes a sufficient immune response, the B cells undergo sufficient somatic high frequency mutations, so that the antibody affinity is matured to the greatest extent. Secondly, because the human immune system does not produce an immune response with fully human antibodies, the human antibody medicines are safer. Therefore, human antibodies with high affinity and high neutralizing activity have great application value for the control of the new coronavirus epidemic and the treatment of critically ill patients.

Summary of the invention

The present invention provides a monoclonal antibody or an antigen-binding portion thereof against a novel coronavirus, which comprises one or more CDRs in the variable region of the heavy chain, and/or one or more CDRs in the variable region of the light chain;

Among them, the CDR of the variable region of the heavy chain includes selected from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID The amino acid sequence shown in at least one of NO. 17, SEQ ID NO. 18, and SEQ ID NO. 19 or a conservatively modified form thereof;

The CDRs of the light chain variable region include SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 21. The amino acid sequence shown in at least one of SEQ ID NO. 22 and SEQ ID NO. 23 or a conservatively modified form thereof.

Further, the CDR1 of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 9, and SEQ ID NO. 17.

Further, the CDR2 of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 10, and SEQ ID NO. 18.

Further, the CDR3 of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 11, and SEQ ID NO. 19.

Further, the CDR1 of the light chain variable region comprises an amino acid sequence selected from SEQ ID NO. 5, SEQ ID NO. 13, and SEQ ID NO. 21.

Further, the CDR2 of the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 6, SEQ ID NO. 14, and SEQ ID NO. 22.

Further, the CDR3 of the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 7, SEQ ID NO. 15, and SEQ ID NO. 23.

In a specific embodiment of the present invention, the CDR of the heavy chain variable region of the monoclonal antibody or antigen binding portion of the monoclonal antibody against the novel coronavirus of the present invention includes the CDR1 shown in SEQ ID NO.1 and the CDR1 shown in SEQ ID NO.2. CDR2 shown in SEQ ID NO.3; CDR of the light chain variable region includes CDR1 shown in SEQ ID NO.5, CDR2 shown in SEQ ID NO.6, and CDR2 shown in SEQ ID NO.7 CDR3.

Preferably, the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO. 4; the light chain variable region comprises an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO. 8 The amino acid sequence of the source.

More preferably, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.4; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.8.

In the specific embodiment of the present invention, the CDR of the heavy chain variable region of the monoclonal antibody or antigen binding portion of the monoclonal antibody against the novel coronavirus of the present invention includes the CDR1 shown in SEQ ID NO.9 and the CDR1 shown in SEQ ID NO.10. The CDR2 shown in SEQ ID NO.11; the CDR of the light chain variable region includes the CDR1 shown in SEQ ID NO.13, the CDR2 shown in SEQ ID NO.14, and the CDR2 shown in SEQ ID NO.15. CDR3.

Preferably, the heavy chain variable region contains an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO. 12; the light chain variable region contains an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO. The amino acid sequence of the source.

More preferably, the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO. 12; the light chain variable region includes the amino acid sequence shown in SEQ ID NO. 16.

In the specific embodiment of the present invention, the CDR of the heavy chain variable region of the monoclonal antibody or antigen binding portion of the monoclonal antibody against the novel coronavirus of the present invention includes the CDR1 shown in SEQ ID NO. 17, and the CDR shown in SEQ ID NO. 18. CDR2 shown in SEQ ID NO.19; the CDR of the light chain variable region includes CDR1 shown in SEQ ID NO.21, CDR2 shown in SEQ ID NO.22, and CDR2 shown in SEQ ID NO.23 CDR3.

Preferably, the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO. 20; the light chain variable region comprises an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO. 24 The amino acid sequence of the source.

More preferably, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.20; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.24.

The present invention also provides a bispecific molecule comprising the aforementioned monoclonal antibody or its antigen-binding portion connected to a second functional module, and the second functional module has the ability to bind to the monoclonal antibody or its antigen. Partially different binding specificities.

The present invention also provides a composition containing the monoclonal antibody or antigen binding portion thereof, or bispecific molecule of the present invention.

The present invention also covers nucleic acid molecules encoding the monoclonal antibodies of the present invention or antigen-binding portions thereof, as well as expression vectors containing such nucleic acids and host cells containing such expression vectors.

The nucleic acid molecule of the present invention includes the sequence shown in SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, or SEQ ID NO. 30.

"Antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains connected to each other by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (herein abbreviated as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (herein abbreviated as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into hyperdenatured regions, called complementarity determining regions (CDR), which are interspersed in more conserved regions, called framework regions (FR). Each VH and VL consists of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulin to host tissues or factors, which includes various cells of the immune system (for example, effector cells) and the first component (Clq) of the classical complement system.

The term "antigen-binding portion" as used herein refers to one or more antibody fragments that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies. Examples of binding fragments covered by the term "antigen-binding portion" of the antibody include (i) Fab fragments, monovalent fragments composed of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, including A bivalent fragment of two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragment, composed of VH and CH1 domains; (iv) Fv fragment, composed of VL and VH domains of one antibody arm; (v) ) dAb fragment (Ward et al. (1989) Nature 341:544-546), consisting of VH domain; and (vi) isolated complementarity determining region (CDR). In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be connected through a synthetic linker using recombinant methods, so that they can be prepared into a single protein chain, in which the VL and VH regions are paired to form Monovalent molecules (referred to as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also intended to be encompassed by the "antigen-binding portion" of the term antibody. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and can be used to screen fragments in the same manner as intact antibodies.

"Isolated monoclonal antibody" as used herein is intended to refer to an antibody that is substantially free of other antibodies with different antigenic specificities. In addition, the isolated antibody may be substantially free of other cellular materials and/or chemicals.

"Monoclonal antibody" or "monoclonal antibody composition" when used herein refers to a preparation of antibody molecules of a single molecular composition. The monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope.

Homologous antibody

The amino acid sequence of the heavy chain and light chain variable regions contained in the antibody of the present invention is homologous to the amino acid sequence of the preferred antibody described herein, and wherein the antibody retains the desired functional characteristics of the anti-new coronavirus antibody of the present invention .

For example, the present invention provides an isolated monoclonal antibody or antigen binding portion thereof, which comprises a heavy chain variable region and a light chain variable region, wherein:

(a) The heavy chain variable region comprises an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO. 4;

(b) The light chain variable region comprises an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO.8.

In other embodiments, the VH and/or VL amino acid sequence may have 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with the aforementioned sequence. Antibodies whose VH and VL regions have high (i.e. 80% or higher) homology with the VH and VL regions of the above sequence can be nucleic acid molecules encoding amino acid sequences by mutagenesis (for example, site-directed mutagenesis or PCR-mediated mutagenesis) get.

As used herein, the percent homology between two amino acid sequences is equal to the percent identity between the two sequences. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences (ie% homology = number of identical positions/total number of positions x 100), in which the number of gaps and the number of gaps that need to be introduced to produce the optimal alignment of the two sequences should be considered. The length of each gap. As shown in the following non-limiting examples, a mathematical algorithm can be used to compare sequences and determine the percent identity between two sequences.

The algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) can be used to determine the percent identity between two amino acid sequences, which has been included in the ALIGN program (version 2.0) , It uses the PAM120 residue weight table, the gap length penalty is 12, and the gap penalty is 4. In addition, the algorithm of Needleman and Wunsch (J.Mol.Biol.48:444-453 (1970)) can be used to determine the percent identity between two amino acid sequences, which has been incorporated into the GCG software package (available at www.gcg In the GAP program in .com), it uses the Blossum 62 matrix or the PAM250 matrix, the gap weight is 16, 14, 12, 10, 8, 6, or 4, and the length weight is 1, 2, 3, 4, 5, or 6. .

Additionally/or, the protein sequence of the present invention can be further used as a "query sequence" to search public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. The XBLAST program can be used to perform BLAST protein search with score=50 and word length=3 to obtain amino acid sequences homologous to the antibody molecule of the present invention. To obtain gap-containing alignment results for comparison, Gapped BLAST was used as described in Altschul et al. (1997) Nucleic Acids Res. 25(17): 3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of each program (for example, XBLAST and NBLAST) can be used. (See www.ncbi.nlm.nih.gov).

Conservatively modified antibodies

In certain embodiments, the antibody of the present invention comprises a heavy chain variable region containing CDR1, CDR2, and CDR3 sequences and a light chain variable region containing CDR1, CDR2, and CDR3 sequences, wherein one or more of these CDR sequences It contains a specific amino acid sequence or conservative modification based on the preferred antibody described herein, and wherein the antibody retains the desired functional characteristics of the anti-new coronavirus antibody of the present invention.

As used herein, the term "conservative sequence modification" is intended to mean that the amino acid modification does not significantly affect or change the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated advantages. Conservative amino acid substitution refers to the replacement of amino acid residues with amino acid residues having similar side chains. In the art, the family of amino acid residues with similar side chains has been described in detail. These families include basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g. alanine, valine, leucine, isoleucine) , Proline, phenylalanine, methionine), β-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine) , Tryptophan, histidine) amino acids. Therefore, one or more amino acid residues in the CDR region of the antibody of the present invention can be replaced with other amino acid residues from the same side chain family.

Engineered and modified antibodies

The antibody of the present invention can further be prepared by using an antibody having one or more of the VH and/or VL sequences disclosed herein as a starting material to engineer a modified antibody, wherein the modified antibody may have the same starting material Different characteristics of antibodies. Antibodies can be engineered by modifying one or more of the two variable regions (ie VH and/or VL), for example, one or more residues in one or more CDR regions and/or one or more framework regions Transformation. Additionally/or alternatively, antibodies can be engineered by modifying residues in the constant region to, for example, alter the antibody's effector functions.

One type of variable region engineering that can be performed is CDR grafting. The interaction between the antibody and the target antigen is mainly through the amino acid residues located in the six complementarity determining regions (CDR) of the heavy and light chains. For this reason, the differences in the amino acid sequence of the CDR between antibody individuals are greater than those outside the CDR. Because CDR sequences are responsible for most of the antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally-occurring antibodies by constructing expression vectors that contain CDR sequences from specific naturally-occurring antibodies, which are grafted from different Characteristics of different antibodies on the framework sequence (see, for example, Riechmann, L. et al. (1998) Nature 332: 323-327; Jones, P. et al. (1986) Nature 321: 522-525; Queen, C. et al. ( 1989) Proc. Natl. Acad. See. USA 86: 10029-10033; U.S. Patent Nos. 5,225,539 to Winter and U.S. Patent Nos. 5,530,101 to Queen et al.; 5,585,089; 5,693,762 and 6,180,370).

Another type of variable region modification is to mutate amino acid residues in the VH and/or VK CDR1, CDR2 and/or CDR3 regions to improve one or more binding properties (such as affinity) of the antibody of interest. Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis. It is preferred to introduce conservative modifications (as described above). The mutation may be an amino acid substitution, addition, or deletion, but is preferably a substitution. In addition, the residue changes in the CDR regions usually do not exceed one, two, three, four, or five.

The engineered antibodies of the present invention include those in which the framework residues in VH and/or VK have been modified, for example, to improve antibody properties. Such framework modifications are usually made to reduce the immunogenicity of the antibody. For example, one method is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody undergoing somatic mutation may contain framework residues that are different from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody is derived.

In addition or alternatively to the modifications made in the framework or CDR regions, the antibodies of the invention can be engineered to include modifications in the Fc region, which is generally used to change one or more functional properties of the antibody, such as serum Half-life, complement fixation, Fc receptor binding, and/or antibody-dependent cellular cytotoxicity. In addition, the antibody of the present invention can be chemically modified (for example, by attaching one or more chemical modules to the antibody) or modified to change its glycosylation, and then used to change one or more functional properties of the antibody. The numbering of residues in the Fc region is that of Kabat's EU index.

In one embodiment, the hinge region of CH1 is modified to change the number of cysteine residues in the hinge region, for example, increase or decrease. This method is further described in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the CH1 hinge region is changed, for example, to facilitate the assembly of the light chain and the heavy chain or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of the antibody is mutated to shorten the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment, so that the antibody has Staphylococcal protein A ( SpA) combined. This method is described in further detail in US Patent No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to extend its biological half-life. There are multiple methods that are feasible. For example, as described in Ward, U.S. Patent No. 6,277,375, one or more of the following mutations are introduced: T252L, T254S, T256F. Alternatively, as described in US Patent Nos. 5,869,046 and 6,121,022 of Presta et al., in order to extend the biological half-life, the antibody can be altered in the CH1 or CL region to include a salvage receptor binding epitope. Taken from the two loops of the CH2 domain of the Fc region of IgG.

In yet another embodiment, the glycosylation of the antibody is modified. For example, an aglycoslated antibody can be prepared (ie, the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be achieved, for example, by changing one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions are made to remove one or more variable region framework glycosylation sites, thereby removing glycosylation at that site. Such aglycosylation can increase the affinity of the antibody to the antigen. This method is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

Another modification of the antibodies herein contemplated by the present invention is PEGylation. Antibodies can be PEGylated, for example, to extend the biological (e.g., serum) half-life of the antibody. For PEGylated antibodies, the antibody or fragment thereof is usually reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups are attached to the antibody or antibody fragment . Preferably, PEGylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to include any form of PEG that has been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or poly Ethylene glycol-maleimide. In certain embodiments, the antibody to be PEGylated is an aglycosylated antibody. Methods of PEGylating proteins are known in the art and can be used in the antibodies of the present invention. See, for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

Nucleic acid molecules encoding antibodies of the invention

Another aspect of the invention relates to nucleic acid molecules encoding the antibodies of the invention. The nucleic acid may be present in intact cells, in cell lysates, or in a partially purified or substantially pure form. When purified by standard techniques, including alkali/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and other techniques well known in the art to remove other cellular components or other contaminants, such as other cellular nucleic acids or In the case of proteins, the nucleic acid is "isolated" or "made substantially pure". See F. Ausubel et al. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acid of the present invention may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

Standard molecular biology techniques can be used to obtain the nucleic acid of the present invention. Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments are further manipulated by standard recombinant DNA techniques to, for example, convert variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, a DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" when used herein is intended to mean the connection of two DNA fragments so that the amino acid sequences encoded by the two DNA fragments remain in the same reading frame (in-frame).

The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the DNA encoding the VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3). The sequence of the human heavy chain constant region gene is known in the art.

The isolated DNA encoding the VL region can be converted into a full-length light chain gene (and Fab light chain gene) by operably linking the DNA encoding VL to another DNA molecule encoding the light chain constant region CL. The sequence of the human light chain constant region gene is known in the art.

Bispecific molecule

The present invention comprises a bispecific molecule of the monoclonal antibody of the present invention or an antigen-binding portion thereof.

The monoclonal antibody or antigen-binding portion thereof of the present invention can be derivatized or linked to another functional molecule, such as another peptide or protein (for example, another antibody or ligand of a receptor) to produce at least two A bispecific molecule that binds to different binding sites or target molecules. The antibodies of the present invention can in fact be derivatized or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to be Covered by the term "bispecific molecule" as used herein. To create the bispecific molecule of the present invention, the antibody of the present invention can be functionally linked (for example, by chemical coupling, gene fusion, non-covalent binding or other means) to one or more other binding molecules, such as another An antibody, antibody fragment, peptide, or binding mimetics to produce a bispecific molecule.

The present invention also provides an expression vector containing the aforementioned nucleic acid molecule.

The present invention also provides a host cell containing the aforementioned expression vector.

The present invention also provides a composition comprising the aforementioned monoclonal antibody or antigen-binding portion thereof.

As an example of the composition, the composition may be a conjugate, the conjugate is formed by coupling the aforementioned monoclonal antibody or its antigen-binding portion with other substances, and the other substances may be therapeutic Agent or diagnostic agent. Therapeutic agents can include cytotoxins, drugs, and radiotoxins.

Cytotoxins or cytotoxic agents include any agent that is harmful to cells (e.g., kills cells). Examples include paclitaxel, cerebellin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, and more Ruubicin, daunorubicin, dihydroxyanthracisin diketone, mitoxantrone, mitoxantrone, mitoxin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, butanol Caine, lidocaine, propranolol and puromycin, and their analogs or homologs.

Therapeutic agents also include, for example, antimetabolites (e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine (decarbazine)), alkylating agents (e.g. Dichloroethyl methylamine, thioepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan , Dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracycline antibiotics (such as daunorubicin (formerly known as Daunorubicin) And doxorubicin), antibiotics (e.g. actinomycin D (formerly known as actinomycin), bleomycin, mithramycin, and amoxicillin (AMC)), and Antimitotic agents (for example, vincristine and vinblastine). duocarmycin, calicheamicin, maytansine and auristatin, and their derivatives.

The cytotoxin can be coupled to the antibody of the present invention by using linker technology available in the art. Examples of the types of linkers that have been used to couple cytotoxins to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers.

The antibodies of the present invention can also be coupled with radioisotopes to generate cytotoxic radiopharmaceuticals. Examples of radioisotopes that can be conjugated to antibodies for diagnosis or therapy include, but are not limited to, iodine ¹³¹ , indium ¹¹¹ , yttrium ^90, and lutetium ¹⁷⁷ .

Diagnostic agent

The diagnostic agents that can be used in the present invention include: radionuclides, contrast agents, fluorescent agents, chemiluminescent agents, bioluminescent agents, paramagnetic ions, enzymes, and photosensitive diagnostic agents.

Radionuclides include ¹¹⁰ In, ¹¹¹ In, ¹⁷⁷ Lu, ¹⁸ F, ⁵² Fe, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁹⁰ Y, ⁸⁹ Zr, ^94m Tc, ⁹⁴ Tc, ^99m Tc, ¹²⁰ I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ^154-158 Gd, ³² F, ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸⁶ Re, ¹⁸⁸ Re, ⁵¹ Mn, ^52m Mn, ⁵⁵ Co, ⁷² As, ⁷⁵ Br, ⁷⁶ Br, ^82m Rb, ⁸³ Sr or other gamma emitters, beta emitters or positron emitters.

Paramagnetic ions include: chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), Ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III).

Fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine.

Chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts and oxalate esters.

Bioluminescent compounds include luciferin, luciferase, and aequorin.

As an example of the composition, the composition may be a pharmaceutical composition, which comprises the monoclonal antibody of the present invention or an antigen-binding portion thereof, and is formulated with a pharmaceutically acceptable carrier. The pharmaceutical composition may also contain the aforementioned conjugate or bispecific molecule.

The pharmaceutical composition of the present invention can also be administered in combination therapy, that is, in combination with other agents.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and other physiologically compatible carriers. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). According to the route of administration, the active ingredient (antibody or its antigen-binding portion, conjugate or bispecific molecule) can be coated in a substance to protect the active ingredient from acid and other inactivation of the active ingredient The role of natural conditions.

The pharmaceutical composition must generally be sterile and stable under the conditions of manufacture and storage. The pharmaceutical composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof.

For example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants, proper fluidity can be maintained. In many cases, it is preferable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of the injectable composition can be caused by including in the composition an agent that delays absorption, such as monostearate and gelatin.

The present invention provides a new type of coronavirus detection product, which contains the aforementioned monoclonal antibody or its antigen binding part.

Further, the products include products that use enzyme-linked immunosorbent assay, immunofluorescence detection, radioimmunoassay, luminescence immunoassay, colloidal gold immunochromatography, agglutination, and immunoturbidimetry to detect antigen-antibody binding.

The present invention also provides a method for producing an antibody, the method comprising culturing the aforementioned host cell in a culture medium under conditions suitable for the production of the antibody.

The present invention also provides antibodies produced according to the methods described above.

The present invention also provides a method for preventing or treating a novel coronavirus infection, the method comprising administering to an individual the aforementioned monoclonal antibody or antigen-binding portion thereof, or the aforementioned bispecific molecule, or the aforementioned combination Things.

The monoclonal antibody or antigen binding portion thereof, or the bispecific molecule, or the composition administered to the individual is used for passive immunization.

The present invention also provides a method for preventing or treating a disease caused by a novel coronavirus infection, the method comprising administering to an individual the aforementioned monoclonal antibody or antigen-binding portion thereof, or the aforementioned bispecific molecule, or the aforementioned Compositions.

The present invention also provides a method for detecting the novel coronavirus, which includes the following steps:

(1) Provide samples suspected of having a new type of coronavirus;

(2) Contact the sample with the aforementioned monoclonal antibody or antigen binding part;

(3) Detect the formation of a complex containing the monoclonal antibody or antigen-binding portion and the antigen, and the presence of the complex indicates that the sample contains a novel coronavirus.

The present invention also provides a method for diagnosing a novel coronavirus infection, the method includes the following steps:

(1) Provide samples from individuals suspected of being infected with the new coronavirus;

(3) Detect the formation of a complex containing the monoclonal antibody or antigen-binding portion and the antigen. The presence of the complex indicates that the individual is infected with the new coronavirus.

The present invention also provides the application of the aforementioned monoclonal antibody or its antigen binding part in the preparation of new coronavirus detection products.

The present invention also provides the application of the aforementioned monoclonal antibody or its antigen binding part in the preparation of a diagnostic product for novel coronavirus infection.

The present invention also provides the application of the aforementioned composition in the preparation of new coronavirus detection products or diagnostic products.

The present invention also provides the application of the aforementioned monoclonal antibody or its antigen binding part in the preparation of drugs for preventing or treating novel coronavirus infections.

The present invention also provides the application of the aforementioned monoclonal antibody or its antigen binding part in the preparation of drugs for preventing or treating diseases caused by novel coronavirus infection.

The present invention also provides the application of the aforementioned composition in the preparation of medicines for preventing or treating novel coronavirus infections.

The present invention also provides the application of the aforementioned composition in the preparation of medicines for preventing or treating diseases caused by novel coronavirus infection.

The aforementioned monoclonal antibodies or their antigen-binding parts, as well as the aforementioned preventive effects of the composition are achieved by the fact that they can trigger an immune response in the body to generate antibodies against the novel coronavirus.

The aforementioned monoclonal antibody or its antigen-binding portion, and the therapeutic effect of the aforementioned composition can be achieved by inhibiting the novel coronavirus by the neutralizing activity of the monoclonal antibody or its antigen-binding portion.

Description of the drawings

Figure 1 shows the result of using indirect ELISA to detect the specific binding of the antibody of the present invention to recombinant S-ECD;

Figure 2 shows the result of using indirect ELISA to detect the specific binding of the antibody of the present invention to recombinant S-RBD;

Figure 3 shows a protein electrophoresis diagram of the binding of the antibody of the present invention to S-RBD and S-ECD by immunoprecipitation;

Figure 4 shows the results of using the SPR experiment to detect the affinity of the antibody of the present invention to S-RBD and S-ECD, where A: FC05; B: FC08; C: FC11;

Figure 5 shows a graph showing the results of using an in vitro neutralization experiment to detect the neutralizing activity of the antibody of the present invention.

Detailed ways

The following examples further illustrate the present invention. It should be understood that the embodiments of the present invention are used to illustrate the present invention and not to limit the present invention. Simple improvements made to the present invention based on the essence of the present invention all fall within the scope of protection of the present invention.

Example 1 Antibody screening

1. Phage library construction

1. Collect the peripheral blood of COVID-19 patients during the recovery period, and separate mononuclear cells (PBMC) from the peripheral blood

On February 14, 2020, with informed consent, this project collected 20ml of peripheral blood from 5 COVID-19 confirmed patients from a city in Jiangsu Province before discharge from the hospital. The 5 patients belonged to the same transmission chain. One of them was a returnee from Wuhan. After bathing in the hotel bathroom, the other 3 patients were infected. The fifth patient and one of the 3 patients who shared the bath was a colleague. All 5 persons were not severely ill, and were separated from the hospital from February 15 to 22 after treatment. Using GE's Ficoll-Paque PLUS, mononuclear cells (PBMC) in 20ml heparin anticoagulated blood were separated by density gradient centrifugation.

2. RNA extraction and cDNA synthesis in PBMC

Use QIAGEN's RNeasy Mini Kit to extract RNA from PBMC cells, and then use Roche's First Strand cDNA Synthesis Kit (Roche, Cat No.: 04896866001) to reverse-transcribe the RNA into cDNA.

3. PCR amplification of VK, VL and VH (EX Taq, Takara, Cat No.: DRR001A)

(1) The amplified VK&VL system is shown in Table 1.

Table 1 Amplification of the VK&VL system

溶液或组分Solution or component	体积(μL)Volume (μL)
cDNA cDNA	11
EX Buffer(10x)EX Buffer(10x)	55
dNTPs(10mM each)dNTPs(10mM each)	44
P1(10μM)P1(10μM)	22
P2(10μM)P2(10μM)	22
EX Taq 1U/μlEX Taq 1U/μl	0.30.3
dH ₂O dH ₂ O	35.735.7

(2) The system of amplified heavy chain Fd segment is shown in Table 2.

Table 2 Amplified heavy chain Fd segment system

溶液或组分Solution or component	体积(μL)Volume (μL)
cDNA cDNA	22
EX Buffer(10x)EX Buffer(10x)	1010
dNTPs(10mM each)dNTPs(10mM each)	88
P1(10μM)P1(10μM)	22
P2(10μM)P2(10μM)	22
EX Taq 1U/μlEX Taq 1U/μl	0.60.6
dH ₂O dH ₂ O	75.475.4

(3) The reaction procedure is shown in Table 3.

Table 3 Response procedures

The PCR product was subjected to 2% agarose gel electrophoresis, and a fragment of about 750 bp was recovered.

4. Light chain cloning (cloning VK and VL into pComb3H vector)

VK and VL were digested with Xba Ⅰ and Sac Ⅰ and ligated with pComb3H vector that was also digested with Xba Ⅰ and Sac Ⅰ. The ligation product was recovered and then electrotransfected into XL1-Blue competent cells.

Coat a 15cm large plate with electric shock bacteria solution, scrape the bacteria the next day, and extract the plasmid to form the light chain library. At this time, the recombinant plasmids are pComb3H-VK and pComb3H-VL.

5. Heavy chain cloning (clone the VH gene into the pComb3H-VK and pComb3H-VL light chain libraries)

The pComb3-L and Fd fragments of the light chain library were digested with XhoI and SpeI, respectively, and connected with pComb3H-VK and pComb3H-VL which were also digested with XhoI and SpeI, and then electrotransformed to obtain the antibody library.

6. Packaging of antibody library

(1) Take out the antibody library from the refrigerator at -80°C, take 1ml after melting on ice, add 10ml A+(20μg/ml) 2YT medium, shake at 37°C 200rpm for 1 hour;

(2) Add 100ml A+(100μg/ml), T+(20μg/ml) 2YT medium, shake at 200rpm for 1 hour;

(3) Add 10 ¹² pfu of VCSM13 helper phage, stand at 37°C for 20 minutes, shake at 200 rpm for 2 hours;

(4) Add the final concentration of 70μg/ml kana at 30℃ and shake at 200rpm overnight;

(5) Centrifuge at 6000 rpm for 20 minutes the next day, pour out the supernatant, add 4% PEG8000 (4g) and 3% NaCl (3g), mix well, and place on ice for more than 30 minutes;

(6) Dispense into a 50ml centrifuge tube and centrifuge at 9000rpm for 25min, discard the supernatant, control it to dry, and resuspend the pellet in 1ml PBS to form the packaging library.

2. Phage library screening

1. Coat the recombinant SARS-CoV-2 spike protein extracellular domain (extra cellular domain of spike protein, S-ECD, purchased from Nanjing Baod Biotechnology Co., Ltd., catalog number NCP0030P) in an immune tube, press 50μg /The tube was coated with 3 tubes, placed overnight at 4°C, and the immune tube was sealed with 2% skimmed milk for 1 hour the next day.

2. Add 1.75ml of PBS containing 2% skim milk and 250μl of phage library to the immunotube, shake at 37°C for 1h, and then stand at 37°C for 1h.

3. Pour out the phage library and wash it with PBST 20 times, shaking for 5 minutes each time.

4. Use 1ml Gly-HCl with pH=2.2 to elute the immune tube, leave it at room temperature for 5 minutes, then shake at 37°C for 5 minutes, then pipet it into a 1.5ml EP tube, and add 57μl 2M Tris to neutralize to pH=7.

5. Transfer the eluate to a new 50ml centrifuge tube, immediately add 10ml of fresh XL1-Blue with OD=1, mix and incubate at 37°C for 30min, add 10ml of 2YT (Amp 100μg/ml, Tet 20μg/ml) .

6. Take 10μl of bacterial solution to measure the capacity of the elution library, and pour the remaining 20ml medium into a 500ml Erlenmeyer flask, shake at 230rpm for 1h.

7. Add 130ml 2YT (Amp 100ug/ml, Tet 20μg/ml), shake at 230rpm for 1h.

8. Add helper phage with MOI=20 and incubate at 37°C for 30 min.

9. Centrifuge at 3000g for 10min, resuspend the pellet to 150ml 2YT (Amp 100μg/ml, Tet 20μg/ml), shake at 37℃, 230rpm for 2h.

10. Add 110μl 70mg/ml kanamycin, 30℃ 230rpm overnight. The next day, add 1/5 volume of PEG-NaCl (40ml), mix well and ice bath for at least 1h, then centrifuge at 10000g 4℃ for 20min, resuspend the pellet in 2-3ml PBS, and then centrifuge to remove the bacteria, pass 0.45μm The filter is used for the next round of screening.

11. Repeat the above screening steps 3 times to achieve the purpose of enrichment and screening of the phage library.

12. After the third round of enrichment, select 2*96 clones. After induction by IPTG, ELISA was performed the next day.

3. ELISA to detect the binding specificity of 2*96 clones

1. Coat 2 pieces of anti-human Fab antibody (1:3000) and 2 pieces of S-ECD protein (2μg/ml) respectively, and coat overnight at 4°C.

2. The next day, it was blocked with 3% skimmed milk for 1 hour, then 50μl of induction supernatant and 50μl of skimmed milk were added, incubated at 37°C for 1h, and washed with PBST.

3, 4 plates were added with HRP-labeled anti-human Fab antibody (1:3000), incubated at 37°C for 1 h, washed with PBST, TMB developed color.

After screening, 159 strains of phage antibody fragments that can bind to the S-ECD protein were obtained. The antibody fragments were human-derived Fab segments, including the full-length light chain and the Fd segment of the heavy chain. 159 single colonies were amplified and then sent for sequencing to obtain qualified sequences with complete light and heavy chains.

Example 2 Indirect ELISA to detect the binding specificity of antibodies to S-RBD and S-ECD

Three human antibodies were selected from the 159 strains of antibodies obtained from the screening and constructed into IgG full-molecular antibodies (the three antibodies were named FC05, FC08, and FC11), expressed in 293F cells, and purified with Protein A Back up.

The sequence of the FC05 antibody is as follows:

The CDR1 sequence of the heavy chain variable region is shown in SEQ ID NO.1, the sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO.2, and the CDR3 sequence of the heavy chain variable region is shown in SEQ ID NO.3 The CDR1 sequence of the light chain variable region is shown in SEQ ID NO.5, the CDR2 sequence of the light chain variable region is shown in SEQ ID NO.6, and the CDR3 sequence of the light chain variable region is shown in SEQ ID NO.7. Show. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 4, and the nucleotide sequence is shown in SEQ ID NO. 25; the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8, and the nucleotide sequence is shown in SEQ ID NO. The sequence is shown in SEQ ID NO.26.

The sequence of the FC08 antibody is as follows:

The CDR1 sequence of the heavy chain variable region is shown in SEQ ID NO.9, the sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO.10, and the CDR3 sequence of the heavy chain variable region is shown in SEQ ID NO.11. The CDR1 sequence of the light chain variable region is shown in SEQ ID NO.13, the CDR2 sequence of the light chain variable region is shown in SEQ ID NO.14, and the CDR3 sequence of the light chain variable region is shown in SEQ ID NO.15. Show. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 12, and the nucleotide sequence is shown in SEQ ID NO. 27; the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 16, and the nucleotide sequence is shown in SEQ ID NO. The sequence is shown in SEQ ID NO.28.

The FC11 antibody sequence is as follows:

The CDR1 sequence of the heavy chain variable region is shown in SEQ ID NO.17, the sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO.18, and the CDR3 sequence of the heavy chain variable region is shown in SEQ ID NO.19. The CDR1 sequence of the light chain variable region is shown in SEQ ID NO.21, the CDR2 sequence of the light chain variable region is shown in SEQ ID NO.22, and the CDR3 sequence of the light chain variable region is shown in SEQ ID NO.23. Show. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 20, and the nucleotide sequence is shown in SEQ ID NO. 29; the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 24, and the nucleotide sequence is shown in SEQ ID NO. 24. The sequence is shown in SEQ ID NO.30.

Recombinant SARS-CoV-2 spike protein receptor binding domain (Recepor binding domain of spike protein, S-RBD, purchased from Nanjing Baod Biotechnology Co., Ltd., catalog number NCP0029P) and recombinant S-ECD with PBS at 1μg/ The concentration of ml is coated on the ELISA plate, and the concentration of all antibodies is diluted to 1mg/ml, and then dilute to 8 dilutions starting from 1:2500. Use patient serum as a positive control and healthy adult serum as a negative control, starting from 1:100 Start to dilute 8 gradients. After the specimen is diluted, incubate at 37°C for 30min, then wash 3 times with PBST, add HRP-labeled anti-human Fc (1:5000), incubate at 37°C for 30min, wash 3 times with PBST, develop color with TMB, and read the OD450 absorbance after termination value. Repeat 3 batches under the same conditions, take the average of the absorbance value of each well and analyze it with GraphPad software.

The results of indirect ELISA with recombinant S-ECD are shown in Figure 1. FC05, FC08 and FC11 can bind specifically to recombinant S-ECD. With 1mg/mL as the starting concentration, the cutoff value is defined as

The antibody titers can reach 1:320000, 1:320000 and 1:40000, respectively, indicating that the three strains of antibodies can specifically bind to S-ECD.

The results of indirect ELISA with recombinant S-RBD are shown in Figure 2. Only FC08 and FC11 have higher binding activity to S-RBD. Among them, FC08 antibody and S-RBD are significantly stronger than FC11, and FC05 does not bind to S-RBD protein.

This result shows that FC05, FC08 and FC11 can all recognize S-ECD, among which FC08 and FC11 recognize the RBD area in S-ECD, and FC05 is combined in the area outside the RBD.

Example 3 Immunoprecipitation experiment of antibody and S-RBD and S-ECD

In the early stage, Western Blot was used to detect the binding specificity of the three antibodies to S-RBD and S-ECD, and it was found that none of the three antibodies reacted with S-RBD and S-ECD after SDS-PAGE, indicating that the three antibodies are all Conformational epitope. Therefore, the immunoprecipitation (Immunoprecipitation, IP) method is used to detect the binding specificity of the antibody and the target protein, and the method is as follows:

Combine the FC05, FC08 and FC11 antibodies with 20 μL Protein A beads for 2 minutes at room temperature, and then wash off the unbound antibodies with 20 mM sodium phosphate. Then add 20 μg of target antigens (S-RBD and S-ECD) to the antibody and Protein A gel mixture, and bind for 2 minutes at room temperature. Wash off the unbound antigen with 20mM sodium phosphate, elute the antigen-antibody complex with 30μl Gly-HCl buffer (pH 3.0), add 1uL 1M Tris (pH 9.0) to neutralize the system, and add the eluate to SDS-PAGE SDS-PAGE analysis was performed after the Loading Buffer was boiled for 10 minutes.

The IP results are shown in Figure 3. FC05 can bind to ECD. ECD protein bands with a size of about 140kDa can be seen in Line 1, as well as the heavy chain (58kDa) and light chain (28kDa) of the antibody, while in Line 2 there is only antibody Two bands, no RBD protein band. FC08 and FC11 are considered to be able to bind to RBD and ECD based on the previous ELISA results.

Lines

3 and 5 show that these two antibodies can bind to ECD. Lines 4 and 6 are lanes that bind to RBD. Because the molecular weight of RBD is lighter than that of antibody The chain size is similar to about 28kDa, in Lin4, 6 lanes can see the scattered band after the antibody light chain overlaps with the RBD. Note: In the figure, M: protein marker; 1, 3, and 5 are the lanes where the 3 antibodies bind to the ECD protein; 2, 4, and 6 are the lanes where the 3 antibodies bind to the RBD protein.

Example 4 SPR determination of the affinity of antibody and S-ECD

The affinity determination is completed by the Biacore 8K workstation. First, the recombinant S-ECD protein labeled with streptomycin is immobilized on the CM5 chip using the NHS/EDC method and the response unit (RUs) reaches about 600. The serially diluted antibodies are injected sequentially from 125nM to 7.8nM; the injection concentration of ACE2 protein with HIS tag is sequentially from 500nM to 31.25nM. In the competition experiment, the first sample was first flowed through the chip at 20μl/min for 120s, and then the second sample was injected into the chip at the same speed and time, the response signal was collected, and the BIAevaluation (version 4.1) software was used for global fitting Curve to obtain binding affinity.

The SPR results are shown in Figure 4. The SPR results show that FC05, FC08 and FC11 antibodies can efficiently bind to S-ECD protein, with affinities of 0.1 nM, 0.8 nM and 0.5 nM, respectively. FC08 and FC11 can bind to the RBD region of the virus with high affinity, and can play a neutralizing effect by affecting the binding of the virus to the receptor. FC05 does not bind to RBD, but its affinity with ECD can reach 0.1nM.

Example 5 Identification of antibody neutralization activity

1. Source of the virus

The virus is derived from SARS-CoV-2 Jiangsu isolate, GISAID number: EPI_ISL_411953, strain name: BetaCoV/JS03/human/2020

2. Dilute the antibody

5 serums are diluted from 1:10 (100μl serum+900μlPBS);

3 antibodies are diluted from 1:80 (15μl antibody + 1185μl PBS)

3. Prepare the cells

Transfer Vero E6 cells to a 96-well plate at a rate of 1*10 ⁴ /well, and place them overnight at 37°C with 5% CO _{2. The} cells can be used after they grow to a single layer the next day.

4. Prepare the virus and antibody mixture

1) Take a 96-well plate, add 100μl of antibody (or serum) to wells A1-H1, add 50μl of PBS to the other wells, and then use a row gun to dilute from left to right, and make 4 replicate holes for each antibody.

2) Dilute the virus to a concentration of 100TCID/50μl, add 50μl of virus solution (that is, add 100TCID50 virus) to all wells, and incubate at 37°C for 1 hour.

3) Change the medium of Vero E6 cells in the 96-well plate, add 100 μl of virus antibody complex to each well, place in a 37°C 5% CO2 incubator, and continue to observe the cytopathic changes until 5 days (120h) later.

4) 100TCID 50, 10TCID50, 1TCID50, 0.1TCID50 virus control wells are required each time, and a positive serum control and a normal cell control are required at the same time.

5. Results

Table 4 shows the neutralization titer information of the antibodies of the present invention or patient serum.

Table 4 Antibody neutralization titer information

The statistical results are shown in Table 5 and Figure 5.

Table 5 IC50 value of antibody

抗体Antibody	IC50(ng/ml)IC50(ng/ml)
FC08FC08	325325
FC11FC11	818818
FC05FC05	142142
FC05+FC08FC05+FC08	44
FC05+FC11FC05+FC11	1919
FC08+FC11FC08+FC11	102102
FC05+FC08+FC11FC05+FC08+FC11	99

The IC50 values of the three monoclonal antibodies are between 142-818ng/mL, of which FC05 has the highest neutralizing activity, reaching 142ng/mL. After combining the three antibodies with each other to form a cocktail preparation, it showed a stronger synergistic effect. Among them, the IC50 of the two RBD region antibodies (FC08 and FC11) was 102 ng/mL after mixing, which was 5.6 times higher than the average value of the single monoclonal antibody, and had a certain synergistic effect. For example, after mixing the FC05 in the S-ECD region and the two antibodies in the S-RBD region, the neutralization effect has a synergistic effect. The IC50 value of the FC05 and FC11 combination is 19ng/mL, the IC50 value of the FC05 and FC08 combination is 4ng/mL, and the IC50 value of the combination of the three antibodies is 9ng/mL. From the results, it can be found that the neutralizing activity of the non-RBD region antibody FC05 is higher than that of the two RBD region antibodies. After the RBD region neutralizing antibody is combined with another non-RBD region antibody to form a cocktail, the neutralizing effect of the mixture can be increased by approximately 100 times. This reveals that the neutralization site of SARS-CoV-2 virus has other more important neutralization sites besides the RBD region.

Although only examples of specific implementations of the present invention are described above, those skilled in the art should understand that these are only examples, and the protection scope of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principle and essence of the present invention, but these changes or modifications all fall within the protection scope of the present invention.

Claims

A monoclonal antibody or antigen-binding portion thereof against a novel coronavirus, which contains one or more CDRs in the variable region of the heavy chain, and/or one or more CDRs in the variable region of the light chain;

Among them, the CDR of the variable region of the heavy chain includes selected from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID The amino acid sequence shown in at least one of NO. 17, SEQ ID NO. 18, and SEQ ID NO. 19 or a conservatively modified form thereof;

The CDRs of the light chain variable region include SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 21. The amino acid sequence shown in at least one of SEQ ID NO. 22 and SEQ ID NO. 23 or a conservatively modified form thereof.
The monoclonal antibody or antigen-binding portion thereof according to claim 1, wherein the CDR of the variable region of the heavy chain comprises CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, and SEQ ID CDR3 shown in NO.3; the CDR of the light chain variable region includes CDR1 shown in SEQ ID NO.5, CDR2 shown in SEQ ID NO.6, and CDR3 shown in SEQ ID NO.7.
The monoclonal antibody or antigen-binding portion thereof according to claim 2, wherein the variable region of the heavy chain comprises an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO. 4; The region contains an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO.8.
The monoclonal antibody or antigen-binding portion thereof according to claim 1, wherein the CDR of the variable region of the heavy chain comprises CDR1 shown in SEQ ID NO.9, CDR2 shown in SEQ ID NO.10, and SEQ ID CDR3 shown in NO.11; the CDR of the light chain variable region includes CDR1 shown in SEQ ID NO.13, CDR2 shown in SEQ ID NO.14, and CDR3 shown in SEQ ID NO.15.
The monoclonal antibody or antigen-binding portion thereof according to claim 4, wherein the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO. 12; the light chain variable region The region contains an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO.16.
The monoclonal antibody or antigen-binding portion thereof according to claim 1, wherein the CDR of the variable region of the heavy chain comprises CDR1 shown in SEQ ID NO.17, CDR2 shown in SEQ ID NO.18, and SEQ ID CDR3 shown in NO.19; the CDR of the light chain variable region includes CDR1 shown in SEQ ID NO.21, CDR2 shown in SEQ ID NO.22, and CDR3 shown in SEQ ID NO.23.
The monoclonal antibody or antigen-binding portion thereof according to claim 6, wherein the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO. 20; the light chain variable region The region contains an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO.24.
A bispecific molecule comprising the monoclonal antibody according to any one of claims 1-7 or an antigen-binding portion thereof connected to a second functional module, and the second functional module has a combination with the monoclonal antibody or The antigen binding part has different binding specificities.
An isolated nucleic acid molecule that encodes the monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-7.
The nucleic acid molecule of claim 9, which comprises SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, or SEQ ID NO. 30 the sequence of.
An expression vector comprising the nucleic acid molecule of claim 9 or 10.
A host cell comprising the expression vector of claim 11.
A composition comprising the monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-7, or the bispecific molecule according to claim 8.
The composition according to claim 13, wherein the composition is a conjugate; the conjugate is a monoclonal antibody or an antigen-binding portion thereof according to any one of claims 1-7 It is coupled with other substances, and the other substances include therapeutic agents or diagnostic agents.
The composition according to claim 14, wherein the therapeutic agent comprises a cytotoxin, a drug, and a radiotoxin.
The composition according to claim 14, wherein the diagnostic agent comprises a radionuclide, a contrast agent, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme, and a photosensitive diagnostic agent.
The composition according to claim 13, wherein the composition is a pharmaceutical composition, and the pharmaceutical composition includes a pharmaceutically acceptable carrier.
A new type of coronavirus detection product, which contains the monoclonal antibody or antigen binding portion thereof according to any one of claims 1-7.
The composition according to claim 18, wherein the product comprises the use of enzyme-linked immunosorbent assay, immunofluorescence detection, radioimmunoassay, luminescence immunoassay, colloidal gold immunochromatography, agglutination, immunoassay The turbidimetric method detects the product of antigen-antibody binding.
A method for producing an antibody, characterized in that the method comprises culturing the host cell according to claim 12 in a medium under conditions suitable for producing the antibody.
An antibody produced by the method of claim 20.
A method for preventing or treating a novel coronavirus infection, characterized in that the method comprises administering to an individual the monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-7, or the bispecific antibody according to claim 8. Sex molecules, or the composition of any one of claims 13-17.
The method of claim 27, wherein the monoclonal antibody or antigen binding portion thereof, or the bispecific molecule, or the composition administered to the individual is used for passive immunization.
A method for preventing or treating a disease caused by a novel coronavirus infection, characterized in that the method comprises administering to an individual the monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-7, or the method according to claim 8. A bispecific molecule, or the composition of any one of claims 13-17.
The method of claim 24, wherein the monoclonal antibody or antigen-binding portion thereof, or the bispecific molecule, or the composition administered to the individual is used for passive immunization.
The method for detecting a novel coronavirus is characterized in that the method includes the following steps:

(1) Provide samples suspected of having a new type of coronavirus;

(2) Contacting the sample with the monoclonal antibody or antigen-binding portion of any one of claims 1-7;

(3) Detect the formation of a complex containing the monoclonal antibody or antigen-binding portion and the antigen, and the presence of the complex indicates that the sample contains a novel coronavirus.
The method for diagnosing a novel coronavirus infection is characterized in that the method comprises the following steps:

(1) Provide samples from individuals suspected of being infected with the new coronavirus;

(2) Contacting the sample with the monoclonal antibody or antigen-binding portion of any one of claims 1-7;

(3) Detect the formation of a complex containing the monoclonal antibody or antigen-binding portion and the antigen. The presence of the complex indicates that the individual is infected with the new coronavirus.
An application, which includes any of the following applications:

(1) The use of the monoclonal antibody or antigen-binding portion of any one of claims 1-7 in the preparation of new coronavirus detection products or diagnostic products;

(2) The use of the monoclonal antibody or antigen-binding portion of any one of claims 1-7 in the preparation of drugs for the prevention or treatment of novel coronavirus infections;

(3) The use of the monoclonal antibody or antigen-binding portion of any one of claims 1-7 in the preparation of drugs for preventing or treating diseases caused by novel coronavirus infection;

(4) The application of the composition according to any one of claims 13-16 in the preparation of new coronavirus detection products or diagnostic products;

(5) The use of the composition according to any one of claims 13-17 in the preparation of a medicine for preventing or treating a novel coronavirus infection;

(6) The use of the composition according to any one of claims 13-17 in the preparation of a medicine for preventing or treating diseases caused by a novel coronavirus infection.