CN114751986A - Multispecific antibodies that neutralize 2019-nCoV - Google Patents

Abstract

The present invention relates to a multispecific antibody or an antigen-binding molecule thereof that neutralizes a novel coronavirus, and a homodimer thereof, a nucleic acid molecule encoding the multispecific antibody or an antigen-binding molecule thereof, a carrier comprising the nucleic acid molecule, Host cells comprising the vector, recombinant proteins or immunoconjugates comprising the multispecific antibody or antigen-binding molecule thereof, and their applications in the preparation of medicines for the treatment or prevention of diseases caused by the novel coronavirus, and in detection products Aspects of application; the multispecific antibody or its antigen-binding molecule for neutralizing the new coronavirus of the present invention is effective against SARS-CoV-2 virus, and various SARS-CoV-2 including Alpha, Beta, Gamma, Delta and Omicron The mutant strains all have significant neutralizing ability, showing excellent broad-spectrum in neutralizing the new coronavirus and its mutant strains, and have good clinical application prospects in the future.

Description

Multispecific antibodies that neutralize 2019-nCoV

technical field

The present invention relates to a multispecific antibody or an antigen-binding molecule thereof that neutralizes a novel coronavirus, and a homodimer thereof, a nucleic acid molecule encoding the multispecific antibody or an antigen-binding molecule thereof, a carrier comprising the nucleic acid molecule, Host cells comprising the vector, recombinant proteins or immunoconjugates comprising the multispecific antibody or antigen-binding molecule thereof, and their applications in the preparation of medicines for the treatment or prevention of diseases caused by the novel coronavirus, and in detection products The application belongs to the field of biomedicine.

Background technique

The novel coronavirus pneumonia (COVID-19) outbreak caused by the new coronavirus (SARS-CoV-2) has brought severe challenges to human health and global public health security.

SARS-CoV-2 belongs to the family Coronaviridae, and belongs to the β-coronavirus genus as the SARS coronavirus that broke out in 2003, with amino acid homology as high as 77.2%. The main envelope protein of the SARS-CoV-2 virus is the spike protein (also known as the Spike protein, or S protein for short). The spike protein is hydrolyzed into two parts, S1 and S2, by intracellular proteases during virus infection. S2 is a transmembrane protein, and S1 has the ability to recognize and bind to the cellular receptor angiotensin-converting enzyme-2 (ACE-2). Receptor binding domain (Receptor Binding Domain, referred to as RBD). The spike protein composed of S1 and S2 is a viral protein that SARS-CoV-2 virus specifically recognizes, binds to target cell receptors, and mediates virus infection, so it is also the recognition target of neutralizing antibodies.

So far, the clinical treatment of COVID-19 is mostly symptomatic and supportive treatment. The complexity of the clinical symptoms of the new coronary pneumonia has brought great challenges to doctors, and the development of specific drugs for the treatment of new coronary pneumonia has also become the current drug Research hotspots and frontiers.

Many studies at home and abroad have reported neutralizing antibodies isolated from patients with new crowns. These neutralizing antibodies mainly target the RBD region or NTD (N-terminal domain) region on the SARS-CoV-2 spike protein, preventing the virus from entering cells. achieve protective effect. In animal experiments and clinical experiments, many antibodies have also been confirmed to have a certain role in the prevention and treatment of new coronavirus infection, and some SARS-CoV-2 neutralizing antibodies have been approved by the FDA for the clinical treatment of COVID-19. For example, Regeneron's REGN-COV (casirivimab and imdevimab) combination antibody treatment for COVID-19 can reduce hospitalizations and deaths by 100%.

However, SARS-CoV-2 is an RNA virus, and the genome sequence of the virus is prone to mutation during the epidemic; for example, the Alpha British mutant B.1.1.7, Beta mutant B.1.351, Gamma Brazilian mutants P.1, etc.; in particular, more contagious circulating strains have recently emerged, the Delta mutant B.1.617.2 and the Omicron mutant B.1.1.529. According to reports, there are as many as 30 mutations in the spike protein (S protein) of the Omicron mutant strain, and many of these mutations lead to stronger resistance to the new crown vaccine currently used in various countries. Clinical treatment Antibodies against the new crown are basically ineffective.

Therefore, the development of antibodies that can broadly neutralize a variety of new coronavirus mutant strains, especially antibodies that can neutralize emerging new coronavirus mutant strains, is a very urgent research topic for researchers in the field.

In order to improve the neutralization breadth and ability of neutralizing antibodies to SARS-CoV-2, researchers genetically engineer existing SARS-CoV-2 neutralizing antibodies, hoping to construct multiple epitopes that can target the virus (effectively inhibiting escape phenomenon) , antibodies with better broad-spectrum and neutralizing activities. Multi/bispecific antibodies are a promising research direction.

Multispecific antibodies, such as bispecific antibodies (BsAbs), contain two or more specific antigen-binding sites and are capable of simultaneously binding two or more antigens, or two or more antigens present on one antigen. artificial antibodies with different epitopes. BsAb can not only target two epitopes of the same molecule to play the role of multi-site specific binding, but also can target two epitopes of different target molecules to play a bridge role between different target molecules, so it can be used as a therapeutic agent great potential for medicines.

Bispecific antibodies were originally prepared using hybridoma technology by fusing Fab fragments of two different antibodies to form bispecific F(ab')2 molecules. Since each hybridoma is capable of producing different immunoglobulins, the resulting hybridomas or tetraploids can theoretically produce antibodies with the antigen specificity of both the first parental hybridoma and the second parental hybridoma, However, the antibody light and heavy chain pairing combinations produced by this method are complex, and the correct pairing ratio is low, which cannot achieve the desired effect of drug production. Scientists have also developed methods such as "knob-into-hole" and the construction of single-gene-encoded bispecific antibodies.

Therefore, those skilled in the art hope to develop new multi-/bispecific antibodies capable of neutralizing 2019-nCoV, especially multiple mutant strains of 2019-nCoV through the above-mentioned methods.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned technical problems, a first aspect of the present invention provides a multispecific antibody or an antigen-binding molecule thereof, wherein,

The multispecific antibody or antigen-binding molecule thereof comprises a first antigen-binding moiety and a second antigen-binding moiety;

The first antigen binding moiety comprises a light chain variable region VL-1 and a heavy chain variable region VH-1;

The second antigen binding moiety comprises a light chain variable region VL-2 and a heavy chain variable region VH-2;

The light chain variable region VL-1 comprises the LCDR1-1 sequence, the LCDR2-1 sequence and the LCDR3-1 sequence of the light chain variable region; the heavy chain variable region VH-1 comprises the HCDR1 of the heavy chain variable region -1 sequence, HCDR2-1 sequence and HCDR3-1 sequence; wherein,

The LCDR1-1 sequence is shown in SEQ ID NO.1, the LCDR2-1 sequence is shown in SEQ ID NO.2, and the LCDR3-1 sequence is shown in SEQ ID NO.3; the HCDR1-1 The sequence is shown in SEQ ID NO.4, the HCDR2-1 sequence is shown in SEQ ID NO.5, and the HCDR3-1 sequence is shown in SEQ ID NO.6; and,

The light chain variable region VL-2 comprises the LCDR1-2 sequence, the LCDR2-2 sequence and the LCDR3-2 sequence of the light chain variable region; the heavy chain variable region VH-2 of the scFv-2 comprises the heavy chain variable region. HCDR1-2 sequence, HCDR2-2 sequence and HCDR3-2 sequence of the variable region; wherein,

The LCDR1-2 sequence is shown in SEQ ID NO.9, the LCDR2-2 sequence is shown in SEQ ID NO.10, and the LCDR3-2 sequence is shown in SEQ ID NO.11; the HCDR1-2 The sequence is shown in SEQ ID NO.12, the HCDR2-2 sequence is shown in SEQ ID NO.13, and the HCDR3-2 sequence is shown in SEQ ID NO.14; or,

The LCDR1-2 sequence is shown in SEQ ID NO.17, the LCDR2-2 sequence is shown in SEQ ID NO.18, and the LCDR3-2 sequence is shown in SEQ ID NO.19; the HCDR1-2 The sequence is shown in SEQ ID NO.20, the HCDR2-2 sequence is shown in SEQ ID NO.21, and the HCDR3-2 sequence is shown in SEQ ID NO.22.

In a specific embodiment of the present invention, the sequence of the light chain variable region VL-1 is as shown in SEQ ID NO.7, or it is more than 80% identical to the sequence shown in SEQ ID NO.7 The sequence of the heavy chain variable region VH-1 is shown in SEQ ID NO.8, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.8.

In a preferred embodiment of the present invention, the sequence of the light chain variable region VL-2 is shown in SEQ ID NO. 15, or it is more than 80% identical to the sequence shown in SEQ ID NO. 15 homology; the sequence of the heavy chain variable region VH-2 is shown in SEQ ID NO.16, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.16; or,

The sequence of the light chain variable region VL-2 is shown in SEQ ID NO.23, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.23; the heavy chain variable region The sequence of VH-2 is shown in SEQ ID NO.24, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.24.

In a specific embodiment of the present invention, the above-mentioned light chain variable region VL-1 can be subjected to a small amount of amino acid deletion, insertion or amino acid mutation on the basis of the above-mentioned sequence to obtain an amino acid sequence with a homology of more than 80%. Substitution of a small number of amino acids (deletion or insertion, or amino acid mutation, or substitution of similar amino acids), especially variants obtained by conservative amino acid substitutions in the framework region, which have higher homology to the above sequences (80 % homology), and retains the original properties and functions of the variable region of the light chain, that is, the properties and functions of antibodies that specifically bind to the coronavirus, then these variants also fall within the protection scope of the present invention. Inside. Similarly, the above-mentioned heavy chain variable region VH-1 can also be obtained by a small amount of amino acid deletion, insertion or amino acid mutation on the basis of the above-mentioned sequence, especially the variant obtained by conservative amino acid substitution in the framework region. The variants of α-β retain the original properties and functions of the variable region of the heavy chain, that is, the properties and functions of antibodies that specifically bind to coronaviruses, and these variants also fall within the protection scope of the present invention. Similarly, the above situation also applies to the light chain variable region VL-2 and the heavy chain variable region VH-2, and details are not repeated here.

In a specific embodiment of the present invention, the first antigen binding moiety is selected from any one of Fv, Fab, Fab', dsFv or scFv; the second antigen binding moiety is selected from Fv, Fab, Fab', Either dsFv or scFv.

Preferably, the first antigen-binding moiety and the second antigen-binding moiety select the structure of a single-chain antibody fragment (scFv); specifically, the first antigen-binding moiety is referred to as a single-chain antibody fragment scFv-1; The two-antigen binding moiety is called the single-chain antibody fragment scFv-2; the C-terminus of the scFv-1 is linked to the N-terminus of the scFv-2 through a first linker peptide, or the C-terminus of scFv-2 is linked through a first linker peptide Linked to the N-terminus of the scFv-1.

Preferably, the scFv-1 comprises the light chain variable region VL-1, the second linker peptide and the heavy chain variable region VH-1 in sequence from the N-terminus to the C-terminus; or, the scFv-1 The heavy chain variable region VH-1, the second linker peptide and the light chain variable region VL-1 are sequentially included from the N-terminus to the C-terminus;

The scFv-2 comprises the light chain variable region VL-2, the third linker peptide and the heavy chain variable region VH-2 in sequence from the N-terminus to the C-terminus; or, the scFv-2 from the N-terminus The heavy chain variable region VH-2, the third linker peptide and the light chain variable region VL-2 are included in sequence to the C-terminus.

Preferably, the sequence of the first linker peptide is GlySer(Gly ₄ Ser) ₄ pattern, and the sequences of the second linker peptide and the third linker peptide are (Gly ₄ Ser) ₃ pattern.

In another alternative embodiment of the invention, the first and second antigen binding moieties are Fab or Fab' fragments. In yet another alternative embodiment of the present invention, one of the first and second antigen binding moieties is a Fab or Fab' fragment and the other is a Fv, dsFv or scFv.

In an alternative embodiment of the present invention, the multispecific antibody or antigen-binding molecule thereof of the present invention may further comprise more antigen-binding moieties, which may be the same as the first/second antigen-binding moiety, or may be different, such as Can be an antigen binding moiety that binds other antigens.

In an alternative embodiment of the invention, the first/second antigen binding moiety is selected from murine antibodies, humanized antibodies or chimeric antibodies.

In a specific embodiment of the present invention, the multispecific antibody or antigen-binding molecule thereof comprises a heavy chain constant region; the heavy chain constant region is preferably a heavy chain constant region of human IgG1, 2, 3, and 4.

Preferably, the heavy chain constant region is the Fc domain of human IgG1; the C-terminus of the scFv-1 is connected to the N-terminus of the scFv-2 through a first linker peptide, and the C-terminus of the scFv-2 is connected by a first linker peptide. The hinge peptide is connected to the Fc domain of human IgG1, or, the C-terminus of the scFv-2 is connected to the N-terminus of the scFv-1 through a first linker peptide, and the C-terminus of the scFv-1 is connected to human IgG1 through a hinge peptide Fc domain.

Preferably, the Fc domain of the human IgG1 sequentially comprises a heavy chain constant region CH2 and a heavy chain constant region CH3 from the N-terminus to the C-terminus;

The sequence of the heavy chain constant region CH2 is shown in SEQ ID NO.25;

The sequence of the heavy chain constant region CH3 is shown in SEQ ID NO.26;

The sequence of the hinge peptide is shown in SEQ ID NO.27.

The second aspect of the present invention provides a multispecific antibody or a homodimer of an antigen-binding molecule thereof, wherein,

The homodimer of the multispecific antibody or its antigen-binding molecule is: when the above-mentioned multispecific antibody or its antigen-binding molecule is expressed in a host cell, the domain of the constant region of the heavy chain undergoes homology. homodimers formed by the dimerization of the source.

A third aspect of the present invention provides a nucleic acid molecule encoding a nucleic acid molecule of a multispecific antibody or an antigen-binding molecule thereof as described above.

The fourth aspect of the present invention provides a vector comprising the above-mentioned nucleic acid molecule, that is, a vector comprising the nucleic acid molecule encoding the above-mentioned multispecific antibody or its antigen-binding molecule, especially a vector expressing the above-mentioned multispecific antibody or its antigen-binding molecule Expression vector.

The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. A vector can be transformed, transduced or transfected into a host cell so that the elements of genetic material it carries are expressed in the host cell. Vectors may contain various elements to control expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, reporter genes, and the like. Additionally, the vector may also contain an origin of replication site. The carrier may also include components to assist its entry into the cell, such as viral particles, liposomes or protein coats, but not only these substances. In embodiments of the present invention, the vector may be selected from, but is not limited to, plasmids, phagemids, cosmids, artificial chromosomes (such as yeast artificial chromosomes YAC, bacterial artificial chromosomes BAC or P1-derived artificial chromosomes PAC), phage (such as lambda phage or M13 phage) and animal viruses used as vectors, for example, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (eg, herpes simplex virus), poxviruses, baculoviruses, Papillomavirus, papillomavirus (eg SV40).

A fifth aspect of the present invention provides a host cell comprising the above-mentioned vector.

Regarding the "host cell", one can choose, but is not limited to: prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, S2 fruit fly cells or insect cells such as Sf9, or fibroblasts, CHO cells, COS Cells, NSO cells, HeLa cells, BHK cells, HEK293 cells and other animal cell models. Preferably, the host cells are HEK293 cells.

The sixth aspect of the present invention provides a method for producing the above-mentioned multispecific antibody or an antigen-binding molecule thereof, wherein by culturing a host cell containing a nucleic acid molecule encoding the above-mentioned multispecific antibody or its antigen-binding molecule , to produce the above-mentioned multispecific antibodies or antigen-binding molecules thereof.

The above-mentioned multispecific antibody or its antigen-binding molecule of the present invention can be produced by the above-mentioned recombinant method, and can also be produced by a hybridoma method.

A seventh aspect of the present invention provides a method for producing the above-mentioned multispecific antibody or a homodimer of an antigen-binding molecule thereof, culturing the above-mentioned host cell, and when the above-mentioned multispecific antibody or its antigen-binding molecule is in the When expressed in the host cell, the domain of the heavy chain constant region homodimerizes to produce the multispecific antibody or a homodimer of the antigen-binding molecule thereof.

Other aspects of the present invention also provide glycosylation variants, cysteine-engineered antibody variants, antibody derivatives, and immunoconjugates of the above-mentioned multispecific antibodies or antigen-binding molecules thereof.

The eighth aspect of the present invention provides a recombinant protein comprising the above-mentioned multispecific antibody or its antigen-binding molecule, or the above-mentioned homodimer.

The ninth aspect of the present invention provides an immunoconjugate comprising the above-mentioned multispecific antibody or its antigen-binding molecule, or the above-mentioned homodimer.

Preferably, the conjugated part of the immunoconjugate uses one or more heterologous molecules, such as heterologous molecules with cytotoxicity that can be applied to immunoconjugates.

A tenth aspect of the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the above-mentioned multispecific antibody or its antigen-binding molecule, or the above-mentioned homodimer, or the above-mentioned nucleic acid molecule, Or comprise the above-mentioned carrier, or comprise the above-mentioned host cell, or comprise the above-mentioned recombinant protein, or comprise the above-mentioned immunoconjugate, and a pharmaceutically acceptable carrier.

The eleventh aspect of the present invention provides the above-mentioned multispecific antibody or its antigen-binding molecule, or the above-mentioned homodimer, or the above-mentioned nucleic acid molecule, or the above-mentioned vector, or the above-mentioned host cell, or the above-mentioned recombinant protein, Or the use of the above-mentioned immunoconjugate in the preparation of a medicament for the treatment or prevention of diseases caused by coronavirus.

In a preferred embodiment of the present invention, the use refers to the use in the preparation of medicines for the treatment or prevention of diseases caused by SARS-CoV-2 and its mutants, SARS-CoV or SARS-like coronaviruses.

In a more preferred embodiment of the present invention, the SARS-CoV-2 mutant is an Alpha, Beta, Gamma, Delta or Omicron mutant.

A twelfth aspect of the present invention provides a detection product, wherein the detection product comprises the above-mentioned multispecific antibody or its antigen-binding molecule, or the above-mentioned homodimer, or the above-mentioned nucleic acid molecule, or It contains the above-mentioned vector, or the above-mentioned host cell, or the above-mentioned recombinant protein, or the above-mentioned immunoconjugate.

The detection product is used to detect the presence or level of coronavirus in a sample.

In a specific embodiment of the present invention, the detection products include, but are not limited to, detection reagents, detection kits, detection chips or test strips, and the like.

The above-mentioned multispecific antibody or its antigen-binding molecule of the present invention can be labeled by chemical methods or genetic engineering methods, and the labeled antibody or its antigen-binding molecule can be used for detection; the labeled antibody or its antigen-binding molecule can be into the protection scope of the present invention.

The specific detection method can adopt the following steps: 1) providing a sample; 2) contacting the sample with the multispecific antibody or its antigen-binding molecule of the present invention; 3) detecting the relationship between the sample and the antibody or its antigen-binding molecule immune response between.

Another aspect of the present invention also provides a method for treating or preventing diseases caused by novel coronavirus, by administering to a patient a therapeutically effective amount of the above-mentioned multispecific antibody or antigen-binding molecule thereof, or a homodimer thereof; or administering to the patient A pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned multispecific antibody or antigen-binding molecule thereof, or a homodimer thereof is administered. Preferably, the disease caused by the new coronavirus is the disease caused by the SARS-CoV-2 virus and its mutants. More preferably, the SARS-CoV-2 mutant is Alpha, Beta, Gamma, Delta or Omicron mutant.

The present invention relates to a multispecific antibody or an antigen-binding molecule thereof that neutralizes a novel coronavirus, and a homodimer thereof, a nucleic acid molecule encoding the multispecific antibody or an antigen-binding molecule thereof, a carrier comprising the nucleic acid molecule, Host cells comprising the vector, recombinant proteins or immunoconjugates comprising the multispecific antibody or antigen-binding molecule thereof, and their applications in the preparation of medicines for the treatment or prevention of diseases caused by the novel coronavirus, and in detection products Aspects of the application; the multispecific antibody or its antigen-binding molecule for neutralizing the new coronavirus of the present invention is effective against SARS-CoV-2 virus, and a variety of SARS-CoV-2 including Alpha, Beta, Gamma, Delta and Omicron The mutant strains all have significant neutralizing ability, showing excellent broad-spectrum in neutralizing 2019-nCoV and its mutant strains, and have good clinical application prospects in the future.

Detailed ways

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Furthermore, the materials, methods and examples described herein are illustrative only and not intended to be limiting. Other features, objects and advantages of the present invention will be apparent from the description and from the appended claims.

For the purpose of interpreting this specification, the following definitions will be used and where appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

The term "about" when used in conjunction with a numerical value is intended to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

As used herein, the term "comprising" or "comprising" means the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps.

The term "antibody" is used herein in the broadest sense to encompass natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), Single-chain antibodies, intact antibodies, and antigen-binding molecules, antigen-binding fragments, antigen-binding proteins, fusion proteins, recombinant proteins, and the like exhibiting the desired antigen-binding activity.

The terms "antigen-binding molecule" and "antibody-binding fragment" are used interchangeably herein to refer to a molecule that is not an intact antibody, which is the specific portion of an intact antibody that binds to an antigen. Antigen-binding molecules can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.

The term "multispecific" antibody refers to an antibody having at least two antigen-binding sites/antigen-binding moieties, each of which is associated with a different epitope of the same antigen or with a different Binding to different epitopes of an antigen.

The terms "antigen-binding moiety", "antigen-binding site" refer to the region of an antibody molecule that actually binds to an antigen, including, for example, consisting of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH) the VH/VL pair. In some embodiments of the invention, bispecific antibodies are provided having two antigen binding sites/antigen binding moieties capable of binding to two different epitopes of the coronavirus.

In some embodiments of the invention, the antigen binding moiety is selected from any one of Fv, Fab, Fab', dsFv or scFv.

The Fab fragment is a monovalent fragment consisting of VL, VH, CL and CH1 domains, eg, Fab fragments can be obtained by papain digestion of complete antibodies. The Fab' monomer is essentially a Fab fragment with a hinge region (for a more detailed description of other antibody fragments see: Fundamental Immunology, edited by W.E. Paul, Raven Press, N.Y. (1993)). F(ab')2 is obtained by pepsin digestion of the entire IgG antibody, removing most of the Fc region while retaining some hinge region, and has two antigen-binding F(ab) moieties linked together by disulfide bonds; F The (ab')2 fragment is a dimer of Fab' and is a bivalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bond in the hinge region, thereby converting the F(ab')2 dimer to a Fab' monomer. The Fv fragment consists of the VL and VH domains of the antibody one-arm. Additionally, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, using recombinant methods, they can be linked by a synthetic linker that enables the production of the two domains as a single protein chain, described in The VL and VH domains of a single protein chain are paired to form a single-chain Fv (scFv, also known as a single-chain antibody). The dsFv refers to disulfide-stabilized Fv (dsFv), which is a new type of small molecule antibody developed on the basis of scFv. It mutates one amino acid residue of VH and VL into Cysteine, an antibody that links the VH and VL variable domains through interchain disulfide bonds, enhances Fv stability. The above-mentioned antigen binding moieties can be obtained by chemical methods, recombinant DNA methods or protease digestion methods.

Regarding the combination of the two antigen-binding modules of the bispecific antibody, according to the different structures of the antigen-binding modules, it mainly includes Fab-Fab combination, Fab-Fv combination and Fv-Fv combination.

Among them, the bispecific antibodies of the Fab-Fab combination mainly include: Triomab produced by the re-fusion technology of rat and mouse hybridoma cells, hybrid hybridomas using various Fc heterodimer technologies (such as Knob-in-Hole , charge pairing, SEED, BEAT, LUZ-Y and Duobody, etc.) and avoid Fab mismatch technology (such as CrossMab, shared light chain, single chain Fab, κλ-body, Orthogonal Fab, Duetmab and TCR-CαCβ, etc.) Class bsIgG, class IgG molecules produced by a variety of Fab tandem methods (such as Tandem orthogonal Fab-IgG, FIT-IgG and BiXAb, etc.), IgG-IgG produced by chemical cross-linking technology and Fab connections produced by various Fab cross-linking technologies Molecules (such as F(ab')2, Dock and Lock, etc.). Fab-Fab double antibody combines two antigen recognition and binding domains at the same time, using Fab can completely retain the high affinity of the original antibody, the structure is closer to the natural IgG, and has higher stability. However, in order to avoid the mismatch of heavy and light chains between two different Fabs, it is necessary to use a common light chain or introduce mutations to form a favorable pairing.

In the bispecific antibody of the Fab-Fv combination, one of the binding domains that recognize the antigen or epitope is Fab, and the other is Fv. The generalized Fv can include single-chain variable region antibody (scFv), engineered polypeptide or protein domain with specific recognition function (such as Anticalins, Bicyclic peptides, DARPins, Fynomers, etc.), ligand or receptor molecules and Engineered ligand or receptor molecules, etc. Representative structures are: scFv-Fab, scFv-IgG, DVD-IgG, Fab-scFv-Fc and so on.

In the bispecific antibody of the Fv-Fv combination, the binding domains that recognize the antigen or antigenic epitope are all Fv. Representative structures include: BiTE, Diabody, DART, TandAb, scFv-scFv-Fc, etc. This combinatorial approach is very flexible and facilitates the construction of multivalent and multispecific binding molecules.

The above-mentioned bispecific antibodies in different combinations may have different structural stability, but the affinity for antigen/antigen epitopes and the neutralization ability to viruses mainly depend on the "antigen binding module/site" of the bispecific antibody. "; in other words, a bispecific antibody with a defined "antigen binding moiety/site" may exist in any of the known combinations above.

In a specific embodiment of the present invention, a bispecific antibody solution of scFv-scFv-Fc is provided; those skilled in the art can use existing The diabody technology transforms it into any one of the above known combinations, such as Fab-Fab combination, Fab-Fv combination or other Fv-Fv combination bispecific antibodies.

In a specific embodiment of the invention, the antigen binding moiety is an scFv comprising a heavy chain variable region (VH region) and a light chain variable region (VL region).

The variable region of the heavy chain (VH region) and the variable region of the light chain (VL region) can be further subdivided into complementarity determining regions (CDR) and framework regions (FR); CDRs are hypervariable regions with more conserved intervening regions. FR region. Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In a given VH or VL amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known protocols including, for example, Chothia (Chothia et al. (1989) Nature 342:877-883), Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath) and Contact ( University College London), International ImMunoGeneTics database (IMGT) (https://www.imgt.org/). The CDRs of the antibodies of the invention can be bounded according to any protocol in the art or a combination thereof and human evaluation.

The terms "Fc domain" or "Fc region" are used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains, a CH2 domain, a CH3 domain and an optional CH4 domain. For example, in native antibodies, the immunoglobulin Fc domain comprises the second and third constant domains (CH2 and CH3 domains) derived from the two heavy chains of antibodies of the IgG, IgA and IgD classes; or The second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) of both heavy chains of IgM and IgE class antibodies. Unless otherwise stated herein, the numbering of amino acid residues in the Fc region or heavy chain constant region is according to, for example, Kabat et al., Sequences of Proteins of Immunological Interes, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991 The EU numbering system (also known as the EU index) is used for numbering.

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein to generate Fc region variants in order to enhance effectiveness. Modifications of the Fc region include amino acid changes (substitutions, deletions, and insertions), glycosylation or deglycosylation, and addition of multiple Fcs. Modifications to the Fc can also alter the half-life of the antibody in a therapeutic antibody, enabling less frequent dosing and thus increased convenience and reduced material usage.

The term "linker peptide" refers to a linker peptide consisting of amino acids, such as glycine and/or serine residues, used alone or in combination, to link the various variable domains in an antibody. In certain embodiments, the linker peptide can be about 1 to about 100 amino acids long, eg, about 1 to 50 amino acids long. In one embodiment, the linker peptide is a G/S linker peptide comprising the amino acid sequences (GGGGS) _n , GS(GGGGS) _n , wherein n is a positive integer equal to or greater than 1, eg, n is a positive number from 1-7 Integer. Non-limiting examples of linkers are disclosed in the literature (Shen et al., Anal. Chem. 80(6): 1910-1917 (2008)) and patents (WO 2014/087010), the contents of which are incorporated herein by reference in their entirety.

As used herein, the term "binding" or "specific binding" means that binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding moiety/site to bind to a particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art.

"Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. One specific method used to measure affinity is biofilm layer interferometry.

The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune cells, or both. The skilled artisan will understand that any macromolecule, including substantially any protein or peptide, can be used as an antigen. In addition, antigens can be derived from recombinant or genomic DNA.

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

The percentage of "sequence homology" with respect to amino acid sequences is generated by determining the number of amino acid residues present in the two sequences to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and dividing the result Multiply by 100 to yield the percent homology of the sequence. Optimal alignment to determine percent sequence homology can be achieved in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software . Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length sequences being compared or within the region of the sequence of interest.

In the present invention, with respect to antibody sequences, the percent amino acid sequence homology is determined by optimally aligning the candidate antibody sequence with the reference antibody sequence, and in a preferred embodiment, performing optimal alignment according to the Kabat numbering rule. . In some embodiments, for antibodies, sequence homology may be distributed over the entire heavy chain variable region and/or the entire light chain variable region, or the percent sequence homology may be limited to the framework regions only, while the corresponding The sequences of the CDR regions remained 100% identical.

Similarly, for antibody sequences, based on the alignment, candidate antibodies can be identified that have amino acid changes in the region of the antibody of interest relative to the reference antibody.

In the present invention, "conservative substitution" refers to an amino acid change that results in the substitution of a certain amino acid with a chemically similar amino acid. Amino acid modifications, such as substitutions, can be introduced into the antibodies of the invention by standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

Conservative substitution tables providing functionally similar amino acids are well known in the art. In a preferred aspect, the conservatively substituted residues are from the following conservative substitution table, preferably the preferred conservatively substituted residues shown in the table below.

conservative substitution table

The term "N-terminal" refers to the last amino acid of the N-terminal, and the term "C-terminal" refers to the last amino acid of the C-terminal.

Antibody CDR regions, "complementarity determining regions" or "CDR regions" or "CDRs" (which are used interchangeably with "hypervariable regions" and "HVRs" herein), are the variable regions of antibodies that are primarily responsible for interacting with antigenic epitopes. bound amino acid region. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus. The CDRs located within the variable domains of antibody heavy chains are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the variable domains of antibody light chains are referred to as LCDR1, LCDR2 and LCDR3.

Various protocols are known in the art for determining the CDR sequences of a given VH or VL amino acid sequence. For example, Kabat complementarity determining regions (CDRs) are determined based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). )). Whereas Chothia refers to the position of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM HVR is a compromise between Kabat HVR and Chothia structural loops, and is used by Oxford Molecular's AbM antibody modeling software. The Contact HVR is based on the analysis of the complex crystal structures available.

Unless otherwise specified, in the present invention, when referring to a residue position in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering system according to the Kabat numbering system. numbered position.

Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM and Contact methods, the region of minimum overlap can be determined, thereby providing the "minimum binding unit" for antigen binding. The minimal binding unit can be a sub-portion of a CDR. The residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody, as will be apparent to those skilled in the art. Accordingly, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be replaced by conservative amino acid residues.

"Hinge peptide" or "hinge region" generally refers to amino acids Glu216 to Pro230 of human IgGl (see Burton, Molec. Immunol. 22:161-206 (1986)). In certain embodiments, the hinge regions of other IgG isotypes can be aligned with the IgGl sequence by placing the first and last cysteine residues that form the S-S bond between heavy chains in the same position.

The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. A host cell is any type of cellular system that can be used to produce the antibody molecules of the invention, including eukaryotic cells, eg, mammalian cells, insect cells, yeast cells; and prokaryotic cells, eg, E. coli cells. Host cells include cultured cells and also include transgenic animals, transgenic plants, or cells within cultured plant or animal tissue.

The terms "individual" or "subject" are used interchangeably and refer to a mammal. Mammals include, but are not limited to, domesticated animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, mice and large mouse). In particular, the individual is a human being.

Example 1

The bispecific antibody of Example 1 comprises a first antigen-binding moiety and a second antigen-binding moiety, which are single-chain antibody fragments scFv-1 and scFv-2, respectively.

In a specific embodiment of the present invention, the light chain variable region VL-1 of the scFv-1 comprises the LCDR1-1, LCDR2-1 and LCDR3-1 sequences of the light chain variable region; the scFv- The heavy chain variable region VH-1 of 1 comprises the HCDR1-1 sequence, HCDR2-1 sequence and HCDR3-1 sequence of the heavy chain variable region; wherein, the LCDR1-1 sequence is shown in SEQ ID NO. The LCDR2-1 sequence is shown in SEQ ID NO.2, the LCDR3-1 sequence is shown in SEQ ID NO.3; the HCDR1-1 sequence is shown in SEQ ID NO.4, and the HCDR2-1 sequence is shown in SEQ ID NO.4 As shown in SEQ ID NO.5, the HCDR3-1 sequence is shown in SEQ ID NO.6.

In a preferred embodiment of the present invention, the sequence of the light chain variable region VL-1 of the scFv-1 is as shown in SEQ ID NO.7, or it is more than 80% of the sequence shown in SEQ ID NO.7 The sequence homology of the heavy chain variable region VH-1 of the scFv-1 is shown in SEQ ID NO.8, or it has more than 80% sequence identity with the sequence shown in SEQ ID NO.8 origin.

Specifically in this example, the sequence of the light chain variable region VL-1 of the scFv-1 is shown in SEQ ID NO. 7, and the sequence of the heavy chain variable region VH-1 of the scFv-1 is shown in SEQ ID NO. ID NO.8.

Specifically, the light chain variable region VL-1 of the single-chain antibody fragment scFv-1 in Example 1 of the present application adopts the sequence of the light chain variable region of the monoclonal antibody 4L12, and the heavy chain variable region VH-1 adopts the sequence of the light chain variable region of the monoclonal antibody 4L12. Sequence of the heavy chain variable region of mAb 4L12. For the technical content of monoclonal antibody 4L12, please refer to the patent application publication document with publication number CN112159469A (that is, the patent application with application number 202011065506.2 submitted by the inventor of this application on September 30, 2020); according to the published information, Monoclonal antibody 4L12 is a fully human antibody that can specifically bind to the receptor binding region RBD of the S1 protein of SARS-CoV-2.

In a specific embodiment of the present invention, the light chain variable region VL-2 of the scFv-2 comprises the LCDR1-2, LCDR2-2 and LCDR3-2 sequences of the light chain variable region; the scFv- The heavy chain variable region VH-2 of 2 comprises the HCDR1-2 sequence, HCDR2-2 sequence and HCDR3-2 sequence of the heavy chain variable region; wherein, the LCDR1-2 sequence is shown in SEQ ID NO. The LCDR2-2 sequence is shown in SEQ ID NO.10, the LCDR3-2 sequence is shown in SEQ ID NO.11; the HCDR1-2 sequence is shown in SEQ ID NO.12, and the HCDR2-2 sequence is shown in SEQ ID NO.12 As shown in SEQ ID NO.13, the HCDR3-2 sequence is shown in SEQ ID NO.14.

In a preferred embodiment of the present invention, the sequence of the light chain variable region VL-2 of the scFv-2 is as shown in SEQ ID NO.15, or it is more than 80% of the sequence shown in SEQ ID NO.15 The sequence homology of the heavy chain variable region VH-2 of the scFv-2 is shown in SEQ ID NO.16, or it has more than 80% sequence identity with the sequence shown in SEQ ID NO.16 origin.

Specifically in this example, the sequence of the light chain variable region VL-2 of the scFv-2 is shown in SEQ ID NO. 15; the sequence of the heavy chain variable region VH-2 of the scFv-2 is shown in SEQ ID NO. ID NO.16.

Specifically, the light chain variable region VL-2 of the single-chain antibody fragment scFv-2 in Example 1 of the present application adopts the sequence of the light chain variable region of the monoclonal antibody REGN10987, and the heavy chain variable region VH-2 adopts the sequence of the light chain variable region of the monoclonal antibody REGN10987. Sequence of the heavy chain variable region of mAb REGN10987. For the technical content of monoclonal antibody REGN10987, see the published document "Studies in Humanized Mice and Convalescent Humans Yield a SARS-CoV-2 Antibody Cocktail, DOI: 10.1126/science.abd0827"; according to published information, monoclonal antibody REGN10987 is fully human An antibody that can specifically bind to the receptor binding region RBD of the S1 protein of SARS-CoV-2.

In a specific embodiment of the present application, the C-terminus of scFv-1 is linked to the N-terminus of said scFv-2 through a first linker peptide. In another specific embodiment of the present application, the C-terminus of scFv-2 may also be linked to the N-terminus of the scFv-1 through a first linker peptide.

In this example, the C-terminus of scFv-1 is connected to the N-terminus of scFv-2 through a first linker peptide (Linker-a), and scFv-1 sequentially includes the light chain variable region VL- 1. The second linker peptide (Linker-b) and the heavy chain variable region VH-1, scFv-2 from the N-terminus to the C-terminus sequentially contains the light chain variable region VL-2, the third linker peptide (Linker-c) and heavy chain variable region VH-2.

That is, the bispecific antibody of Example 1, from N terminus to C terminus, is: scFv-1—Linker-a—scFv-2; more specifically, the bispecific antibody of Example 1, from N terminus to C terminus The terminals are as follows: VL-1—Linker-b—VH-1—Linker-a—VL-2—Linker-c—VH-2.

Of course, in other embodiments of the present application, the structure of the antibody can also be VH-1—Linker-b—VL-1—Linker-a—VL-2—Linker-c—VH-2, VL-1—Linker -b—VH-1—Linker-a—VH-2—Linker-c—VL-2 or VH-1—Linker-b—VL-1—Linker-a—VH-2—Linker-c—VL-2 .

In other embodiments of the present application, the structure of the antibody can also be VL-2—Linker-c—VH-2—Linker-a—VL-1—Linker-b—VH-1, VH-2—Linker-c —VL-2—Linker-a—VL-1—Linker-b—VH-1, VL-2—Linker-c—VH-2—Linker-a—VH-1—Linker-b—VL-1 or VH -2—Linker-c—VL-2—Linker-a—VH-1—Linker-b—VL-1.

In this example, the sequence of the first linker peptide (Linker-a) between scFv-1 and scFv-2 adopts the pattern of GlySer (Gly ₄ Ser) ₄ .

In this example, the sequences of the second linker peptide (Linker-b) and the third linker peptide (Linker-c) both adopt (Gly ₄ Ser) ₃ mode.

In a specific embodiment of the present application, the bispecific antibody further comprises the Fc domain of human IgGl.

In a specific embodiment of the present application, the C-terminus of scFv-1 is connected to the N-terminus of scFv-2 through a first linker peptide, and the C-terminus of scFv-2 is connected to the Fc domain of human IgG1 through a hinge peptide (Hinge). In another specific embodiment of the present application, the C-terminus of scFv-2 can also be connected to the N-terminus of the scFv-1 through a first linker peptide, and the C-terminus of scFv-1 can be connected to the human through a hinge peptide (Hinge). Fc domain of IgG1.

In this example, the sequence of the bispecific antibody, from the N-terminus to the C-terminus, is: scFv-1-Linker-a-scFv-2-Hinge-Fc.

In a specific embodiment of the present application, the Fc domain of human IgG1 comprises a heavy chain constant region CH2 and a heavy chain constant region CH3 in sequence from the N-terminus to the C-terminus;

That is, the sequence of the bispecific antibody of Example 1, from the N-terminus to the C-terminus, is:

VL-1—Linker-b—VH-1—Linker-a—VL-2—Linker-c—VH-2—Hinge—CH2—CH3.

The sequence of the heavy chain constant region CH2 is shown in SEQ ID NO.25; the sequence of the heavy chain constant region CH3 is shown in SEQ ID NO.26; the sequence of the hinge peptide Hinge is shown in SEQ ID NO.27.

Preparation of Bispecific Antibody Neutralizing Novel Coronavirus of Example 1

Step 1) Construct the antibody expression vector pcDNA3.4-Fc containing the Fc gene fragment

The gene fragments of fully human IgG1 signal peptide gene SP, hinge peptide Hinge, heavy chain constant region CH2 and heavy chain constant region CH3, namely SP-Fc gene, were synthesized by GenScript. Insert AgeI and BamHI enzyme cleavage sites between the signal peptide and hinge peptide genes, and the middle is separated by GTACGC sequence, that is, the SP-AgeI-BamHI-Fc sequence is synthesized and the SP-AgeI-BamHI-Fc gene is cloned by TA. Connected to the pcDNA3.4 vector, and finally obtained the pcDNA3.4-Fc expression vector, wherein the pcDNA3.4 vector was purchased from Thermo Fisher Scientific Co., Ltd. For the specific plasmid map, please refer to the invention patent application publication document with publication number CN113461811A .

Step 2) Synthesize antibody gene sequence

As mentioned above, the light chain variable region VL-1 of the single-chain antibody fragment scFv-1 of Example 1 adopts the sequence of the light chain variable region of the monoclonal antibody 4L12 (as shown in SEQ ID NO. 7), and the heavy chain The variable region VH-1 adopts the sequence of the heavy chain variable region of monoclonal antibody 4L12 (as shown in SEQ ID NO. 8); the amino acid sequence of the second linker peptide (Linker-b) between the two is (Gly ₄ Ser) ₃ .

Correspondingly, for the nucleic acid sequence encoding scFv-1, please refer to the publication of the invention patent application publication number CN112159469A.

The light chain variable region VL-2 of the single-chain antibody fragment scFv-2 of Example 1 adopts the sequence of the light chain variable region of the monoclonal antibody REGN10987 (as shown in SEQ ID NO. 15), and the heavy chain variable region VH -2 The sequence of the heavy chain variable region of the monoclonal antibody REGN10987 was adopted (as shown in SEQ ID NO. 16); the amino acid sequence of the third linker peptide (Linker-c) between the two was (Gly ₄ Ser) ₃ .

Correspondingly, for the nucleic acid sequence encoding scFv-2, please refer to the document of the above-mentioned monoclonal antibody REGN10987.

The nucleotide sequence corresponding to the single-chain antibody scFv-1-Linker-a-scFv-2 was synthesized by Nanjing GenScript according to conventional methods.

Step 3) construct the expression vector of bispecific antibody gene

The N-terminus and C of the nucleotide sequence of the single-chain antibody scFv-1-Linker-a-scFv-2 synthesized in the above step 2) were digested by AgeI and BamHI, respectively, and the target fragment recovered by ligation gel purification was obtained. The pcDNA3.4-Fc expression vector constructed in the above step 1) was transformed into DH5α competent cells to construct the final bispecific antibody expression plasmid, which was named pcDNA3.4-4L12-REGN10987.

Step 4) Expression of bispecific antibodies in mammalian cells 293F

The expression plasmid of the bispecific antibody was purified by a plasmid purification kit (Megi Bio), and co-transfected into HEK293F cells using EZ Trans cell transfection reagent (Li Ji Bio) for expression.

The specific transfection steps are: one day before transfection, 50ml of 293F cells were plated in a 250ml cell culture shake flask at a density of 1.2×10 ⁶ cells/ml, and the transfection reagent EZ-Trans was used on the day of transfection and constructed in step 3). The expression plasmids were thoroughly mixed (the mass-to-volume ratio was DNA:EZ-Trans=1:3) and dissolved in serum-free OPM medium to obtain the EN-Trans mixture (i.e. 60 μg DNA and 180 μL EZ-Trans were dissolved in 4 mL medium), After standing for 15 minutes, the EZ-Trans-DNA mixture was evenly added to HEK293F cells in the form of raindrops. Six days after transfection, the cell culture supernatant was obtained by centrifugation for subsequent extraction and purification of bispecific antibodies. (Note: When the antibody is expressed in a host cell, the Fc domain of human IgG1 undergoes homodimerization to form a homodimer; it is identified after subsequent extraction and purification.)

Step 5) Extraction and purification of antibodies

The cell supernatant collected in the above step 4) was filtered with a 0.45 μM filter, diluted with binding buffer 1 × PBS, and purified by protein-G column (Tian Di Ren Bio Technology Co., Ltd., Changzhou). For the bispecific antibody of IgG1 Fc, please refer to the instruction of protein-G column for the purification method. The obtained bispecific antibody was purified and named as 4L12-REGN10987.

Absorbance at 280 nm was measured using Nanodrop2000 (ThermoFisher) and antibody concentration was calculated. After affinity purification, the purity of the antibody was analyzed and identified by SDS-PAGE. 5 μl of the purified sample was mixed with 20 μl of 5× loading buffer, placed in a metal water bath at 100 °C and heated for 10 minutes, and the heated sample was mixed. 10 μl of the solution was loaded on PAGE gels (Nanjing GenScript Biotechnology Co., Ltd.), and the samples were separated by molecular weight by electrophoresis. GelDoc Go Gel Imaging System (BIO-RAD) photographed to obtain the SDS-PAGE detection results of the expressed and purified bispecific antibodies.

Example 2

The sequence structure of the antibody of Example 2, from N terminus to C terminus, is: scFv-2—Linker-a—scFv-1; more specifically, VL-2—Linker-c—VH-2—Linker-a— VL-1—Linker-b—VH-1—Hinge—CH2—CH3.

Wherein, the VL-1 and VH-1 sequences of the single-chain antibody fragment scFv-1 are the same as the corresponding sequences in Example 1, that is, the light chain variable region sequence and the heavy chain variable region sequence of the monoclonal antibody 4L12.

The VL-2 and VH-2 sequences of the single-chain antibody fragment scFv-2 are the same as the corresponding sequences in Example 1, that is, the light chain variable region sequence and the heavy chain variable region sequence of the monoclonal antibody REGN10987.

Other technical contents of the antibody in Example 2 are the same as those in Example 1, and details are not repeated here.

The antibody preparation process of Example 2 is basically the same as that of Example 1, except that step 2) is synthesized according to the antibody sequence of Example 2, and details are not repeated here.

The antibody of Example 2 was named double anti-REGN10987-4L12.

Example 3

The bispecific antibody for neutralizing the novel coronavirus of Example 3 comprises two single-chain antibody fragments, scFv-1 and scFv-2.

The single-chain antibody fragment scFv-1 of Example 3 is the same as that of Example 1, that is, the single-chain antibody fragment of 4L12;

In a specific embodiment of the present invention, the light chain variable region VL-2 of the single chain antibody fragment scFv-2 comprises the LCDR1-2 sequence, LCDR2-2 sequence and LCDR3-2 sequence of the light chain variable region; said The heavy chain variable region VH-2 of scFv-2 comprises the HCDR1-2 sequence, HCDR2-2 sequence and HCDR3-2 sequence of the heavy chain variable region; wherein, the LCDR1-2 sequence is shown in SEQ ID NO.17 , the LCDR2-2 sequence is shown in SEQ ID NO.18, the LCDR3-2 sequence is shown in SEQ ID NO.19; the HCDR1-2 sequence is shown in SEQ ID NO.20, the HCDR2-2 The sequence is shown in SEQ ID NO.21, and the HCDR3-2 sequence is shown in SEQ ID NO.22.

In a preferred embodiment of the present invention, the sequence of the light chain variable region VL-2 of the scFv-2 is as shown in SEQ ID NO.23, or it has more than 80% of the sequence shown in SEQ ID NO.23 The sequence homology of the heavy chain variable region VH-2 of the scFv-2 is shown in SEQ ID NO.24, or it has more than 80% sequence identity with the sequence shown in SEQ ID NO.24 origin.

Specifically in this Example 3, the sequence of the light chain variable region VL-2 of the scFv-2 is shown in SEQ ID NO. 23, and the sequence of the heavy chain variable region VH-2 of the scFv-2 is shown in Shown in SEQ ID NO.24.

Specifically, the light chain variable region VL-2 of the single-chain antibody fragment scFv-2 in Example 3 of the present application adopts the sequence of the light chain variable region of the monoclonal antibody 16L9, and the heavy chain variable region VH-2 adopts the sequence of the light chain variable region of the monoclonal antibody 16L9. Sequence of the heavy chain variable region of mAb 16L9. For the technical content of monoclonal antibody 16L9, please refer to the patent application publication document with publication number CN112159469A (that is, the patent application with application number 202011065506.2 submitted by the inventor of this application on September 30, 2020); according to the published information, Monoclonal antibody 16L9 is a fully human antibody that can specifically bind to the receptor binding region RBD of the S1 protein of SARS-CoV-2.

The sequence of the bispecific antibody of Example 3, from N terminus to C terminus, is: scFv-1—Linker-a—scFv-2; specifically, VL-1—Linker-b—VH-1—Linker-a -VL-2-Linker-c-VH-2-Hinge-CH2-CH3.

The sequence of the heavy chain constant region CH2 is shown in SEQ ID NO.25; the sequence of the heavy chain constant region CH3 is shown in SEQ ID NO.26; the sequence of the hinge peptide Hinge is shown in SEQ ID NO.27.

The antibody preparation process of Example 3 is basically the same as that of Example 1, except that step 2) is synthesized according to the antibody sequence of Example 3, and details are not repeated here.

The antibody of Example 3 was named double antibody 4L12-16L9.

performance data

1. Production of SARS-CoV-2 and its mutants Alpha, Beta, Gamma, Delta, Omicron pseudoviruses

SARS-CoV-2 and its mutants Alpha, Beta, Gamma, Delta, and Omicron pseudoviruses are non-replication-deficient reverse transcriptases with corresponding spike membrane proteins (Spike, S) on the surface and carrying a luciferase reporter gene. Viral particles can simulate the infection process of SARS-CoV-2 and its mutant viruses on host cells (such as human liver cancer cell line Huh-7, 293T cell line 293T-ACE2 stably expressing human ACE2 receptor), and in infected cells Internally expressed luciferase reporter gene. Since pseudovirus infection does not produce infectious virus particles, it can be safely performed in a biosafety secondary laboratory.

The pseudoviruses of SARS-CoV-2 and its mutants were obtained by co-transfecting 293T cells with their respective S protein expression plasmids and HIV Env-deficient backbone plasmids (pNL4-3.Luc.R-E-) with a luciferase reporter gene, respectively. .

The S gene sequence of SARS-CoV-2 was designed according to the NCBI GenBank sequence NC_045512. After codon optimization, the gene sequence was synthesized by Nanjing GenScript and connected to the pcDNA3.1 eukaryotic expression vector to construct the SARS-CoV-2 protein. expression plasmid. Among them, the pseudoviruses Alpha, Beta, Gamma, Delta, and Omicron of SARS-CoV-2 mutant strains require corresponding point mutation and deletion mutation of the S protein expression plasmid. The pNL4-3.Luc.R-E-backbone plasmid was obtained from the NIHAIDS Reagent Program in the United States. All plasmids were amplified by transforming DH5α competent cells, and purified by using the plasmid purification kit produced by MegiBio. The purification operation process refers to the kit instructions.

293T cells were cultured in DMEM medium containing 10% fetal bovine serum (Gibco) and seeded into 10 cm cell dishes before transfection. After culturing for 24 hours, the backbone plasmid (pNL4-3.Luc.R-E-) was co-transfected with the expression plasmid expressing SARS-CoV-2 and its mutant strain at a ratio of 3:1 using EZTrans cell transfection reagent (Liji Biotechnology). 293T cells were transfected. For the detailed transfection method, please refer to the instruction manual of EZ Trans cell transfection reagent. 48 hours after transfection, the supernatant containing pseudovirus was collected, centrifuged at 2500 rpm for 10 minutes to remove cell debris, and then aliquoted and stored in a -80°C refrigerator for the detection of neutralizing antibodies.

2. Detection of the neutralizing activity of the bispecific antibodies of Examples 1-3 of the present application to the pseudoviruses of SARS-CoV-2 and its mutants (Alpha, Beta, Gamma, Delta and Omicron)

Different concentrations of bispecific antibodies were tested on 96-well cell plates to inhibit pseudovirus infection of Huh-7 cells to detect their neutralizing ability against SARS-CoV-2 and its mutants.

The detection method is roughly as follows: 1) Huh-7 cells were seeded in a 96-well cell plate, 1×10 ⁴ cells per well, and cultured in a 37°C, 5% CO ₂ cell incubator for 24 hours; 2) The antibodies of the examples and comparative examples were incubated Dilute with cell culture medium to different concentrations, mix with an equal volume of pseudovirus dilution containing 100 TCID50, and incubate at 37 °C for 1 hour; 3) Discard the cell culture medium, add 50 μl of virus-antibody complex to each well, set up duplicate wells, and at the same time Set up an antibody-free group, a virus-free group and a positive antibody control group; 4) After culturing for 12 hours, add 150 μl maintenance solution to each well, and continue to culture at 37°C for 48 hours; 5) Use a luciferase detection kit (Luciferase Assay System, Promega) Cat.#E1500) lyse cells and detect the luciferase activity of each well, the specific detection method refers to the kit instructions; use a multi-function microplate reader (Perkin Elmer) to detect the chemiluminescence RLU value of each well; 6) According to the antibody and virus control The ratio of RLU values was used to calculate the neutralization inhibition percentage of different concentrations of antibodies against pseudovirus, and the half inhibitory dose IC50 (unit μg/ml) of the antibody to inhibit virus was calculated using PRISM7 software (GraphPad).

Example 1-3: bispecific antibodies 4L12-REGN10987, REGN10987-4L12 and 4L12-16L9 of Example 1-3 of the present application;

Comparative Example 1: mAb 4L12; Comparative Example 2: mAb REGN10987; Comparative Example 3: mAb 16L9; the test results are shown in Table 1 below:

Table 1

Table 1 shows the anti-SARS- Neutralization IC50 results of CoV-2 virus and its mutants.

It can be seen from Table 1 that:

1) The bispecific antibodies of Example 1-3 (4L12-REGN10987, REGN10987-4L12 and 4L12-16L9) have potent neutralizing ability against SARS-CoV-2 virus and its mutants Alpha, Beta, Gamma, Delta , it is proved that the bispecific antibodies of Examples 1-3 can neutralize SARS-CoV-2 virus and its various mutant strains, and have a good broad-spectrum in neutralizing the new coronavirus;

2) On the whole, the bispecific antibodies (4L12-REGN10987 and REGN10987-4L12) of Examples 1 and 2 are slightly better than Comparative Examples 1 and 2 in neutralizing SARS-CoV-2 virus and its various mutants. 2 (ie their corresponding mAb 4L12 and mAb REGN10987);

3) On the whole, the effect of the bispecific antibody (4L12-16L9) of Example 3 in neutralizing SARS-CoV-2 virus and its various mutants is slightly better than that of Comparative Examples 1 and 3 (that is, their corresponding mAb 4L12 and mAb 16L9);

4) The surprising finding is that for the recently emerged Omicron mutants, the IC50 values of Comparative Examples 1 and 2 (mAb 4L12 and mAb REGN10987) are both greater than 10 μg/ml, while the bispecific antibodies after their combination (4L12-REGN10987) has an IC50 value of 0.0152 μg/ml. This shows that mAb 4L12 and mAb REGN10987 each have poor neutralizing effect on Omicron mutants, but the bispecific antibody (4L12-REGN10987) formed by their combination unexpectedly shows significant neutralizing ability;

5) The surprising finding is that for the recently emerged Omicron mutants, the IC50 values of Comparative Examples 1 and 3 (mAb 4L12 and mAb 16L9) are both greater than 10 μg/ml, but the bispecific antibodies after their combination (4L12-16L9) has an IC50 value of 0.158 μg/ml. This shows that mAb 4L12 and mAb 16L9 each have poor neutralizing effect on Omicron mutants, but the bispecific antibody (4L12-16L9) formed by their combination unexpectedly shows significant neutralizing ability.

This discovery made the inventor feel very unexpected and pleasantly surprised, and hoped that it could be disclosed in a timely manner and clinically promoted, so as to contribute to the prevention and control of a new round of epidemics caused by Omicron mutant strains.

In conclusion, the bispecific antibodies (4L12-REGN10987, REGN10987-4L12 and 4L12-16L9) of Examples 1-3 of the present application have potent neutralizing ability against SARS-CoV-2 virus and its various mutants, and the geometric mean The semi-neutralizing concentration (GM IC50) is at the level of 0.0014 to 0.0068 μg/ml, showing an excellent broad spectrum in neutralizing the new coronavirus and its mutants, which is significantly better than the current FDA-approved clinical treatment for COVID-19. Antibodies such as casirivimab and imdevimab; in particular, the bispecific antibodies of Examples 1 and 3 of the present application (4L12-REGN10987 and 4L12-16L9) also unexpectedly showed significant neutralizing ability against the recently emerged Omicron mutant strains.

On the basis that those skilled in the art obtain the disclosure content and spirit of the present application, some simple adjustments can be made on the basis of the double antibody sequences in the examples of the present application. On the basis of changing the sequence of light chain and heavy chain, obtain such as VH-1—Linker-b—VL-1—Linker-a—VL-2—Linker-c—VH-2, VL-1—Linker-b — Antibodies for VH-1—Linker-a—VH-2—Linker-c—VL-2 or VH-1—Linker-b—VL-1—Linker-a—VH-2—Linker-c—VL-2 . It is also possible to exchange the sequence of the light chain and the heavy chain on the basis of the double antibody sequence of Example 2 to obtain, for example, VH-2—Linker-c—VL-2—Linker-a—VL-1—Linker-b—VH -1, VL-2—Linker-c—VH-2—Linker-a—VH-1—Linker-b—VL-1 or VH-2—Linker-c—VL-2—Linker-a—VH-1 - Antibodies of Linker-b-VL-1; it can be reasonably inferred that they all have similar effects to the double antibodies in Examples 1-3; these equivalent alternatives all fall within the protection scope of the present application.

Those skilled in the art can also make routine equivalent substitutions for the linker peptide sequence, Fc domain sequence, hinge peptide sequence, etc. on the basis of the double antibody sequences in Examples 1-3 of the present application; On the basis of the double antibody sequence of the invention, amino acid insertion, substitution or deletion processing that does not affect the overall effect of the antibody is performed; these equivalent substitution schemes all fall within the protection scope of the present application.

On the basis of obtaining the bispecific antibody scheme of scFv-scFv and the sequence of its antigen-binding module of the present application by those skilled in the art, it can be transformed into Fab-Fab combination, Fab-Fv Bi/multispecific antibodies in combination or other Fv-Fv combinations.

As can be seen from the above content, the multispecific antibody for neutralizing SARS-CoV-2 virus or its antigen-binding molecule of the present invention has excellent broad-spectrum and potent neutralizing ability for the new coronavirus. Based on the knowledge of this technical content, corresponding recombinant proteins, fusion proteins and immunoconjugates, as well as drugs for the treatment or prevention of diseases caused by the new coronavirus, and detection products for the detection of the new coronavirus can be further developed.

Application example

This application example describes the method for treating diseases caused by the SARS-CoV-2 virus and its mutants by the bispecific antibodies of Examples 1-3 of the present application.

Although specific methods, dosages and modes of administration are provided, those skilled in the art will understand that changes may be made without materially affecting treatment. Based on the guidelines disclosed herein, coronavirus infection can be reduced or eliminated by administering a therapeutically effective amount of a bispecific antibody described herein to treat or prevent coronavirus infection.

The specific application method is as follows:

1) Pre-treatment of the subject: In particular embodiments, the subject is treated prior to administration of a therapeutic agent comprising one or more antiviral drug therapies known to those of skill in the art. However, such pre-treatment is not always required and can be determined by the skilled clinician.

2) Administration of the therapeutic composition: after screening the object, the above-mentioned therapeutically effective dose of the bispecific antibody of Examples 1-3 of the present application is administered to the object (such as being at risk of being infected with SARS-CoV-2 virus or known to be infected with SARS-CoV-2). -CoV-2 virus in adults or newborn babies). Additional drugs, such as antiviral agents, can be administered to the subject at the same time as, before, or after administration of the disclosed agents. Administration is accomplished by any method known in the art such as oral administration, inhalation, intravenous, intramuscular, intraperitoneal or subcutaneous. To prevent, reduce, inhibit and/or treat the condition of a subject, the amount of the composition administered will depend on the subject being treated, the severity of the disorder and the mode of administration to the subject being treated. Ideally, a therapeutically effective amount of an agent is an amount sufficient to prevent, reduce, and/or inhibit, and/or treat a condition in a subject without causing substantial cytotoxic effects in the subject. An effective amount can be readily determined by one of skill in the art, eg, using routine experiments to establish dose-response curves. Likewise, these compositions can be formulated with inert diluents or pharmaceutically acceptable carriers. In a specific example, the antibody is administered at 5 mg/kg every two weeks or 10 mg/kg every two weeks, depending on the particular stage of SARS-CoV-2 viral infection. In one example, the antibody is administered continuously. In another example, the antibody is administered at 50 μg per kg twice a week for 2-3 weeks. Therapeutic compositions can be administered chronically (eg, over a period of months or years).

3) Evaluation: Following administration of one or more therapies, subjects infected with SARS-CoV-2 are monitored for a reduction in the level of SARS-CoV-2 virus, or a reduction in one or more clinical symptoms associated with COVID-19 disease. In certain instances, starting after 2 days of treatment, subjects are subjected to one or more analyses. Subjects are monitored using any method known in the art. For example, biological samples including throat swabs from subjects can be obtained and assessed for changes in SARS-CoV-2 virus levels.

4) Additional treatment: In specific embodiments, if the subject is stable or has a minimal, mixed or partial response to treatment, additional treatment may be performed after re-evaluation with the same regimen and formulation of substances that they had previously received for the desired period of time. Treatment.

It should be understood that although this specification is described in terms of embodiments, not every embodiment only includes an independent technical solution, and this description in the specification is only for the sake of clarity, and those skilled in the art should take the specification as a whole, and each The technical solutions in the embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

The series of detailed descriptions listed above are only specific descriptions for the feasible embodiments of the present invention, and they are not used to limit the protection scope of the present invention. Changes should all be included within the protection scope of the present invention.

Claims (15)

1. a multispecific antibody or its antigen-binding molecule, characterized in that: The multispecific antibody or antigen-binding molecule thereof comprises a first antigen-binding moiety and a second antigen-binding moiety; The first antigen binding moiety comprises a light chain variable region VL-1 and a heavy chain variable region VH-1; The second antigen binding moiety comprises a light chain variable region VL-2 and a heavy chain variable region VH-2; The light chain variable region VL-1 comprises the LCDR1-1 sequence, the LCDR2-1 sequence and the LCDR3-1 sequence of the light chain variable region; the heavy chain variable region VH-1 comprises the HCDR1 of the heavy chain variable region -1 sequence, HCDR2-1 sequence and HCDR3-1 sequence; wherein, The LCDR1-1 sequence is shown in SEQ ID NO.1, the LCDR2-1 sequence is shown in SEQ ID NO.2, and the LCDR3-1 sequence is shown in SEQ ID NO.3; the HCDR1-1 The sequence is shown in SEQ ID NO.4, the HCDR2-1 sequence is shown in SEQ ID NO.5, and the HCDR3-1 sequence is shown in SEQ ID NO.6; and, The light chain variable region VL-2 comprises the LCDR1-2 sequence, the LCDR2-2 sequence and the LCDR3-2 sequence of the light chain variable region; the heavy chain variable region VH-2 comprises the HCDR1 of the heavy chain variable region -2 sequence, HCDR2-2 sequence and HCDR3-2 sequence; wherein, The LCDR1-2 sequence is shown in SEQ ID NO.9, the LCDR2-2 sequence is shown in SEQ ID NO.10, and the LCDR3-2 sequence is shown in SEQ ID NO.11; the HCDR1-2 The sequence is shown in SEQ ID NO.12, the HCDR2-2 sequence is shown in SEQ ID NO.13, and the HCDR3-2 sequence is shown in SEQ ID NO.14; or, The LCDR1-2 sequence is shown in SEQ ID NO.17, the LCDR2-2 sequence is shown in SEQ ID NO.18, and the LCDR3-2 sequence is shown in SEQ ID NO.19; the HCDR1-2 The sequence is shown in SEQ ID NO.20, the HCDR2-2 sequence is shown in SEQ ID NO.21, and the HCDR3-2 sequence is shown in SEQ ID NO.22. 2. multispecific antibody or its antigen-binding molecule as claimed in claim 1, is characterized in that: The sequence of the light chain variable region VL-1 is shown in SEQ ID NO.7, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.7; the heavy chain variable region The sequence of VH-1 is shown in SEQ ID NO. 8, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO. 8; and, The sequence of the light chain variable region VL-2 is shown in SEQ ID NO.15, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.15; the heavy chain variable region The sequence of VH-2 is shown in SEQ ID NO.16, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.16; or, The sequence of the light chain variable region VL-2 is shown in SEQ ID NO.23, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.23; the heavy chain variable region The sequence of VH-2 is shown in SEQ ID NO.24, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.24. 3. multispecific antibody or its antigen-binding molecule as claimed in claim 1 or 2, is characterized in that: The first antigen binding moiety is selected from any one of Fv, Fab, Fab', dsFv or scFv; The second antigen binding moiety is selected from any one of Fv, Fab, Fab', dsFv or scFv. 4. multispecific antibody or its antigen-binding molecule as claimed in claim 3, is characterized in that: The first antigen-binding moiety is a single-chain antibody fragment scFv-1; the second antigen-binding moiety is a single-chain antibody fragment scFv-2; The C-terminus of the scFv-1 is linked to the N-terminus of the scFv-2 through a first linker peptide, or the C-terminus of the scFv-2 is linked to the N-terminus of the scFv-1 through a first linker peptide. 5. multispecific antibody or its antigen-binding molecule as claimed in claim 4, is characterized in that: The scFv-1 comprises the light chain variable region VL-1, the second linker peptide and the heavy chain variable region VH-1 in sequence from the N-terminus to the C-terminus; or, the scFv-1 from the N-terminus To the C-terminus, the heavy chain variable region VH-1, the second linker peptide and the light chain variable region VL-1 are included in sequence; The scFv-2 comprises the light chain variable region VL-2, the third linker peptide and the heavy chain variable region VH-2 in sequence from the N-terminus to the C-terminus; or, the scFv-2 from the N-terminus The heavy chain variable region VH-2, the third linker peptide and the light chain variable region VL-2 are included in sequence to the C-terminus. 6. The multispecific antibody or its antigen-binding molecule of claim 5, wherein: The multispecific antibody or its antigen-binding molecule comprises a heavy chain constant region; the heavy chain constant region is preferably the heavy chain constant region of human IgG1, 2, 3, and 4; Preferably, the heavy chain constant region is the Fc domain of human IgG1; the C-terminus of the scFv-1 is connected to the N-terminus of the scFv-2 through a first linker peptide, and the C-terminus of the scFv-2 is connected by a first linker peptide. The hinge peptide is connected to the Fc domain of human IgG1, or, the C-terminus of the scFv-2 is connected to the N-terminus of the scFv-1 through a first linker peptide, and the C-terminus of the scFv-1 is connected to human IgG1 through a hinge peptide Fc domain; Preferably, the Fc domain of the human IgG1 sequentially comprises a heavy chain constant region CH2 and a heavy chain constant region CH3 from the N-terminus to the C-terminus; The sequence of the heavy chain constant region CH2 is shown in SEQ ID NO.25; The sequence of the heavy chain constant region CH3 is shown in SEQ ID NO.26; The sequence of the hinge peptide is shown in SEQ ID NO.27. 7. A homodimer of a multispecific antibody or an antigen-binding molecule thereof, characterized in that: The homodimer of the multispecific antibody or its antigen-binding molecule is: when the multispecific antibody or its antigen-binding molecule described in claim 6 is expressed in a host cell, the heavy chain constant region homodimers formed by homodimerization of the domains. 8. A nucleic acid molecule, characterized in that: the nucleic acid molecule encodes the multispecific antibody or its antigen-binding molecule according to any one of claims 1 to 6. 9. A vector comprising the nucleic acid molecule as claimed in claim 8; preferably, the vector is an expression vector. 10. A host cell comprising the vector of claim 9. 11. A recombinant protein, characterized in that: the recombinant protein comprises a multispecific antibody or an antigen-binding molecule thereof as described in any one of claims 1 to 6, or a homology as claimed in claim 7 dimer. 12. An immunoconjugate comprising the multispecific antibody or antigen-binding molecule thereof of any one of claims 1 to 6, or a homodimer of claim 7. 13. A pharmaceutical composition, characterized in that: the pharmaceutical composition comprises the multispecific antibody or the antigen-binding molecule thereof according to any one of claims 1 to 6, or comprises the Homodimer, or comprises the nucleic acid molecule as claimed in claim 8, or comprises the vector as claimed in claim 9, or comprises the host cell as claimed in claim 10, or comprises the recombinant protein as claimed in claim 11 , or comprising the immunoconjugate of claim 12, and a pharmaceutically acceptable carrier. 14. A detection product, characterized in that: the detection product comprises the multispecific antibody or the antigen-binding molecule thereof according to any one of claims 1 to 6, or the homologous antibody according to claim 7 A dimer, or a nucleic acid molecule as claimed in claim 8, or a vector as claimed in claim 9, or a host cell as claimed in claim 10, or a recombinant protein as claimed in claim 11, or The immunoconjugate of claim 12 is included. 15. The multispecific antibody or antigen-binding molecule thereof of any one of claims 1 to 6, or the homodimer of claim 7, or the nucleic acid molecule of claim 8, Or the vector as claimed in claim 9, or the host cell as claimed in claim 10, or the recombinant protein as claimed in claim 11, or the immunoconjugate as claimed in claim 12, in the preparation of treatment or prevention by the novel coronavirus Medicinal uses for diseases caused by viruses.