Monoclonal antibodies against coxsackievirus b1 and uses thereof

Abstract

The present invention relates to the field of immunology and virology, in particular the field of diagnosis, prevention and treatment of Coxsackievirus B1. In particular, the present invention relates to monoclonal antibodies or antigen-binding fragments thereof directed against Coxsackievirus B1, as well as compositions (e.g. diagnostic and therapeutic agents) comprising said antibodies or antigen-binding fragments thereof. Furthermore, the present invention also relates to the use of said antibodies or antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof of the present invention can be used for the diagnosis, prevention and / or treatment of an infection with Coxsackievirus B1 and / or a disease caused by said infection.

Description

[0001] This application is a divisional application of application number 202010339682.4, filed on April 26, 2020, entitled "Monoclonal Antibody Against Coxsackievirus B1 and Its Use Thereof". Technical Field

[0002] This invention relates to the fields of immunology and virology, particularly to the diagnosis, prevention, and treatment of Coxsackievirus B1. Specifically, this invention relates to monoclonal antibodies against Coxsackievirus B1 or antigen-binding fragments thereof, and compositions comprising said antibodies or antigen-binding fragments thereof (e.g., diagnostic and therapeutic agents). Furthermore, this invention relates to the use of said antibodies or antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof of this invention can be used for the diagnosis, prevention, and / or treatment of Coxsackievirus B1 infection and / or diseases caused by said infection. Background Technology

[0003] Coxsackievirus B1 (CVB1) belongs to the Enterovirus genus of the Picornaviridae family. Studies have shown that CVB1 infection can lead to aseptic meningitis, myocarditis, meningoencephalitis, hand-foot-mouth disease, chest pain, and neonatal fulminant hepatitis with coagulation disorders (US National CDC, 2010, Morbidity and Mortality Weekly Report 59(48):1577-80). There is also evidence that CVB1 infection is associated with some chronic diseases, such as type 1 diabetes (Oikarinen et al., 2014, Diabetes 63(2):655-62). CVB1 is a single-stranded positive-sense RNA virus with a genome length of approximately 7500 bp. Its viral particles have an icosahedral structure, no envelope or protrusions, and a diameter of about 30 nm. Its protein coat is composed of structural proteins VP1, VP2, VP3 and VP4. Neutralizing epitopes are mainly on VP1, VP2 and VP3. Among them, VP1 protein is its main antigenic epitope determining region, while VP4 is enclosed inside the capsid, its sequence is highly conserved and there are few reported neutralizing epitopes.

[0004] Since the 1990s, the overall circulating level of Coxsackievirus B1 has gradually increased. The virus identification results of 52,812 enterovirus patients reported by the National Enterovirus Detection System in the United States from 1970 to 2005 showed that the infection rate of CVB1 was about 2.3%. At the same time, during the 36-year study period, CVB1 was among the top 15 circulating enterovirus types in 17 years, and even became the second most prevalent strain in 1977 (Khetsuriani et al., 2006, Morbidity and Mortality Weekly Report (MMWR) 59(48):1577-80). In 2010, data from the US National CDC showed that CVB1 was the leading enterovirus pathogen between 2006 and 2008. In 2006, CVB1 ranked ninth among the annual circulating strains (3.7%), while in 2007 and 2008 it accounted for 23.6% and 18.6% of non-polio enterovirus positive samples, respectively, ranking first. Simultaneously, CVB1 accounted for the highest proportion of all enterovirus positive samples over the three years (16.5%) (US National CDC, 2010, Morbidity and Mortality Weekly Report (MMWR) 59(48); 1577-1580). CVB1 prevalence was found in cases of viral encephalitis in Zhejiang Province, China, between 2002 and 2012, and in patients with aseptic meningitis in Shandong Province, China, between 2006 and 2012. Furthermore, CVB1 prevalence was also found in South Korea between 2008 and 2009.

[0005] Currently, vaccination is considered the most effective and convenient method to prevent enterovirus infection. Studies have found that many enteroviruses exist in two types of viral particles: hollow particles (no RNA, non-infectious) and solid particles (containing RNA, infectious), and both types of viral particles are immunogenic. The polio vaccine is currently the most mature and successfully applied vaccine. The poliovirus, also belonging to the enterovirus genus, also exists in both solid and hollow particle types. Furthermore, the solid particle antigen can stimulate the body to produce neutralizing antibodies, which are the main protective antigens (Mayer et al., 1957, Journal of Immunology 78, 435-455). Therefore, the effective component of the polio vaccine is measured by the content of solid particle antigen. Studies have shown that CVB1 virus particles, after inactivation, possess good immunogenicity. Mice inoculated with inactivated virus particles can produce strong neutralizing antibodies in vivo, protecting themselves from lethal attacks by CVB1 virus (Hankaniemi et al., 2017, Vaccine, 35(30):3718-3725). Related studies on other enteroviruses such as EV71 (Wu et al., 2019, PLoS one, 14(1):e0210553) and CVA16 (Chong et al., 2012, PLoS one, 7(11):e49973) have also shown that solid particle antigens may play an important role in the immunogenicity of viral vaccines. Therefore, it is essential to conduct research on the detection of CVB1 virus solid particle antigens. Currently, there are no reports on detection methods for CVB1 solid particle antigens. Therefore, obtaining monoclonal antibodies that can specifically bind to solid particles of CVB1 virus and establishing a detection method for CVB1 solid particle antigens is of great significance for the research and development and quality control of CVB1 vaccines. Summary of the Invention

[0006] The inventors of this application, through in-depth research and creative labor, have obtained monoclonal antibodies capable of specifically binding to solid and / or hollow particles of CVB1 virus, and based on this, have established a detection method for specifically detecting solid particles of CVB1 virus. Furthermore, the obtained monoclonal antibody can also efficiently neutralize CVB1 virus, blocking or inhibiting viral infection of cells; therefore, this monoclonal antibody has the potential to prevent and treat CVB1 infection and diseases related to CVB1 infection. The following invention is thus provided.

[0007] The antibody of the present invention

[0008] In one aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof that specifically binds to Coxsackievirus B1 (CVB1), comprising the following complementarity-determining regions (CDRs) as defined according to the IMGT numbering system:

[0009] (1) The amino acid sequences are VH CDR1-3 as shown in SEQ ID NO:5-7, and / or the amino acid sequences are VL CDR1-3 as shown in SEQ ID NO:8-10;

[0010] (2) The amino acid sequences are VH CDR1-3 as shown in SEQ ID NO:15-17, and / or the amino acid sequences are VL CDR1-3 as shown in SEQ ID NO:18-20; or

[0011] (3) The amino acid sequences are VH CDR1-3 as shown in SEQ ID NO:25-27, and / or the amino acid sequences are VL CDR1-3 as shown in SEQ ID NO:28-30.

[0012] In some embodiments, the monoclonal antibody or its antigen-binding fragment comprises: VH CDR1-3 with amino acid sequences as shown in SEQ ID NO:5-7, and / or VLCDR1-3 with amino acid sequences as shown in SEQ ID NO:8-10.

[0013] In some embodiments, the antibody or its antigen-binding fragment comprises: (a) three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:1; and / or, (b) three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:2. In some embodiments, the three CDRs contained in the heavy chain variable region (VH) and / or the three CDRs contained in the light chain variable region (VL) are defined by the Kabat, Chothia, or IMGT numbering system. In some exemplary embodiments, the three CDRs contained in the heavy chain variable region (VH) and / or the three CDRs contained in the light chain variable region (VL) are defined by the IMGT numbering system.

[0014] In some embodiments, the monoclonal antibody or its antigen-binding fragment comprises:

[0015] (i) Heavy chain variable region comprising an amino acid sequence selected from the following: the sequence shown in SEQ ID NO: 1, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) with SEQ ID NO: 1;

[0016] And / or,

[0017] (ii) A light chain variable region comprising an amino acid sequence selected from the following: the sequence shown in SEQ ID NO: 2, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) with SEQ ID NO: 2.

[0018] In some embodiments, the monoclonal antibody or its antigen-binding fragment comprises: VH as shown in SEQ ID NO:1 and VL as shown in SEQ ID NO:2.

[0019] In some embodiments, the monoclonal antibody or its antigen-binding fragment is 5F5 or its antigen-binding fragment, its chimeric antibody, its humanized antibody, or variants thereof, which substantially retain the biological function of the monoclonal antibody or its antigen-binding fragment from which they are derived.

[0020] In some embodiments, the monoclonal antibody or its antigen-binding fragment has one or more of the following biological functions:

[0021] (a) Specific binding to CVB1 solid particles (infectious particles), for example, by ELISA assay;

[0022] (b) Non-infectious or unbound CVB1 hollow particles, for example, as determined by ELISA;

[0023] (c) Detect the presence or level of CVB1 virus (e.g., CVB1 solid particles) in a sample;

[0024] (d) Diagnose whether the subject is infected with CVB1 virus;

[0025] (e) Neutralize CVB1 in vitro or in a subject (e.g., a human); for example, neutralize CVB1 in vitro with a neutralizing potency of not less than 6,000 (e.g., 6,000-10,000, 7,000-10,000, 7,000-9,000, or 8,000-9,000, e.g., about 8,100-8,200), as determined by the neutralization experiment described in Example 7;

[0026] (f) Inhibit or block CVB1 infection of cells;

[0027] (g) Prevention and / or treatment of CVB1 infection or diseases associated with CVB1 virus infection.

[0028] In some embodiments, the monoclonal antibody or its antigen-binding fragment comprises: VH CDR1-3 as shown in SEQ ID NO:15-17, and / or VLCDR1-3 as shown in SEQ ID NO:18-20.

[0029] In some embodiments, the monoclonal antibody or its antigen-binding fragment comprises: (a) three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:11; and / or, (b) three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:12. In some embodiments, the three CDRs contained in the heavy chain variable region (VH) and / or the three CDRs contained in the light chain variable region (VL) are defined by the Kabat, Chothia, or IMGT numbering system. In some exemplary embodiments, the three CDRs contained in the heavy chain variable region (VH) and / or the three CDRs contained in the light chain variable region (VL) are defined by the IMGT numbering system.

[0030] In some embodiments, the monoclonal antibody or its antigen-binding fragment comprises:

[0031] (i) Heavy chain variable region comprising an amino acid sequence selected from the following: the sequence shown in SEQ ID NO: 11, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 11, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to SEQ ID NO: 11;

[0032] And / or,

[0033] (ii) A light chain variable region comprising an amino acid sequence selected from the following: the sequence shown in SEQ ID NO: 12, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 12, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) with SEQ ID NO: 12.

[0034] In some embodiments, the monoclonal antibody or its antigen-binding fragment comprises: VH as shown in SEQ ID NO:11 and VL as shown in SEQ ID NO:12.

[0035] In some embodiments, the monoclonal antibody or its antigen-binding fragment is 8A10 or its antigen-binding fragment, its chimeric antibody, its humanized antibody, or variants thereof, which substantially retain the biological function of the monoclonal antibody or its antigen-binding fragment from which they are derived.

[0036] In some embodiments, the monoclonal antibody or its antigen-binding fragment has one or more of the following biological functions:

[0037] (a) Specific binding to CVB1 solid particles (infectious particles), for example, by ELISA assay;

[0038] (b) Specific binding to CVB1 hollow particles (non-infectious particles), for example, by ELISA assay;

[0039] (c) Detect the presence or level of CVB1 virus (e.g., solid and hollow CVB1 particles) in the sample;

[0040] (d) Diagnose whether the subject is infected with CVB1 virus;

[0041] (e) Neutralize CVB1 in vitro or in a subject (e.g., a human); for example, neutralize CVB1 in vitro with a neutralizing potency of not less than 10,000 (e.g., 10,000-15,000, 11,000-15,000, 12,000-15,000, 12,000-14,000, or 13,000-14,000, e.g., about 131,000-131,100), as determined by the neutralization experiment described in Example 7;

[0042] (f) Inhibit or block CVB1 infection of cells;

[0043] (g) Prevention and / or treatment of CVB1 infection or diseases associated with CVB1 virus infection.

[0044] In some embodiments, the monoclonal antibody or its antigen-binding fragment comprises: VH CDR1-3 as shown in SEQ ID NO:25-27, and / or VLCDR1-3 as shown in SEQ ID NO:28-30.

[0045] In some embodiments, the antibody or its antigen-binding fragment comprises: (a) three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:21; and / or, (b) three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:22. In some embodiments, the three CDRs contained in the heavy chain variable region (VH) and / or the three CDRs contained in the light chain variable region (VL) are defined by the Kabat, Chothia, or IMGT numbering system. In some exemplary embodiments, the three CDRs contained in the heavy chain variable region (VH) and / or the three CDRs contained in the light chain variable region (VL) are defined by the IMGT numbering system.

[0046] In some embodiments, the monoclonal antibody or its antigen-binding fragment comprises:

[0047] (i) Heavy chain variable region comprising an amino acid sequence selected from the following: the sequence shown in SEQ ID NO: 21, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 21, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to SEQ ID NO: 21;

[0048] And / or,

[0049] (ii) A light chain variable region comprising an amino acid sequence selected from the following: the sequence shown in SEQ ID NO: 22, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 22, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) with SEQ ID NO: 22.

[0050] In some embodiments, the monoclonal antibody or its antigen-binding fragment comprises: VH as shown in SEQ ID NO:21 and VL as shown in SEQ ID NO:22.

[0051] In some embodiments, the monoclonal antibody or its antigen-binding fragment is 9A3 or its antigen-binding fragment, its chimeric antibody, its humanized antibody, or variants thereof, which substantially retain the biological function of the monoclonal antibody or its antigen-binding fragment from which they are derived.

[0052] In some embodiments, the monoclonal antibody or its antigen-binding fragment has one or more of the following biological functions:

[0053] (a) Specific binding to CVB1 solid particles (infectious particles), for example, by ELISA assay;

[0054] (b) Specific binding to CVB1 hollow particles (non-infectious particles), for example, by ELISA assay;

[0055] (c) Detect the presence or level of CVB1 virus (e.g., solid and hollow CVB1 particles) in the sample;

[0056] (d) Diagnose whether the subject is infected with CVB1 virus;

[0057] (e) Neutralize CVB1 in vitro or in a subject (e.g., a human); for example, neutralize CVB1 in vitro with a neutralizing potency of not less than 1000 (e.g., 1000-5000, 1000-4000, 1000-3000, or 2000-3000, e.g., about 2000-2100), as determined by the neutralization experiment described in Example 7;

[0058] (f) Inhibit or block CVB1 infection of cells;

[0059] (g) Prevention and / or treatment of CVB1 infection or diseases associated with CVB1 virus infection.

[0060] In some embodiments, any of the monoclonal antibodies or their antigen-binding fragments further comprise a constant region sequence or a variant thereof derived from a mammalian (e.g., mouse or human) immunoglobulin, the variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence. In some embodiments, the variant has one or more conserved amino acid substitutions compared to its derived wild-type sequence.

[0061] In some embodiments, the heavy chain of the monoclonal antibody or its antigen-binding fragment comprises the heavy chain constant region (CH) of a mouse immunoglobulin or a variant thereof, the variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and / or,

[0062] The light chain of the monoclonal antibody or its antigen-binding fragment comprises the light chain constant region (CL) of mouse immunoglobulin or a variant thereof, the variant having one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids).

[0063] In some embodiments, the heavy chain of the monoclonal antibody or its antigen-binding fragment comprises the heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, the variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and / or,

[0064] The light chain of the monoclonal antibody or its antigen-binding fragment comprises the light chain constant region (CL) of human immunoglobulin or a variant thereof, the variant having one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids).

[0065] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region is a mouse IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region is a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

[0066] In some embodiments, the light chain constant region is the κ light chain constant region. In some embodiments, the light chain constant region is the mouse κ light chain constant region. In some embodiments, the light chain constant region is the human κ light chain constant region.

[0067] In some embodiments, the antigen-binding fragment of any of the above monoclonal antibodies is selected from scFv, di-scFv, (scFv)2, Fab, Fab', (Fab')2, Fv, or disulfide-stable Fv (dsFv).

[0068] In some implementations, any of the above monoclonal antibodies are murine antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, or multispecific antibodies.

[0069] In this application, the monoclonal antibody or antigen-binding fragment thereof of the present invention may include variants that differ from the antibody or antigen-binding fragment from which they are derived only in the conserved substitution of one or more (e.g., up to 20, 15, 10, or 5 amino acid substitutions) amino acid residues, or have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the antibody or antigen-binding fragment from which they are derived, and substantially retain the biological function of the antibody or antigen-binding fragment from which they are derived.

[0070] Derived antibodies

[0071] The antibodies or antigen-binding fragments of the present invention can be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Generally, derivatization (e.g., labeling) of monoclonal antibodies or antigen-binding fragments of the present invention does not adversely affect their binding to the CVB1 virus. Therefore, the antibodies or antigen-binding fragments of the present invention are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (e.g., forming a bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide capable of mediating the binding of the antibody or antigen-binding fragment to another molecule (e.g., avidin or a multihistidine tag). Furthermore, the antibodies or antigen-binding fragments of the present invention can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl groups. These groups can be used to improve the biological properties of the antibody, such as increasing serum half-life.

[0072] In some embodiments, the monoclonal antibody or its antigen-binding fragment of the present invention carries a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters), a fluorescent dye, a radionuclide, or biotin.

[0073] In this document, the detectable markers described in this invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.) and radionuclides (e.g., 3 H, 125 I, 35 S, 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent materials (e.g., chemiluminescent materials, such as acridine esters), magnetic beads (e.g., ), thermal markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers.

[0074] In some embodiments, the detectable marker can be used for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.).

[0075] In some embodiments, the detectable marker described above can be linked to the antibody or its antigen-binding fragment of the present invention via linkers of different lengths to reduce potential steric hindrance.

[0076] Antibody preparation

[0077] The antibodies of the present invention can be prepared by various methods known in the art, such as through genetic engineering recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into an expression vector and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0078] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can also be directly produced from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, the Fab' fragment can be obtained directly from host cells; the Fab' fragment can be chemically coupled to form the F(ab')2 fragment (Carter et al., Bio / Technology, 10:163-167 (1992)). Furthermore, the Fv, Fab, or F(ab')2 fragments can also be directly isolated from the recombinant host cell culture medium. Other techniques for preparing these antigen-binding fragments are fully known to those skilled in the art.

[0079] Therefore, in another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody of the present invention or an antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof. In some embodiments, the isolated nucleic acid molecule encodes an antibody of the present invention or an antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof.

[0080] In another aspect, the present invention provides a vector (e.g., a cloning vector or an expression vector) containing the isolated nucleic acid molecules of the present invention. In some embodiments, the vector of the present invention is, for example, a plasmid, a granule, a bacteriophage, etc.

[0081] In another aspect, the present invention provides a host cell comprising the isolated nucleic acid molecules of the present invention or the vectors of the present invention. Such host cells include, but are not limited to, prokaryotic cells such as *Escherichia coli* cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). In some embodiments, the host cell of the present invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHOG44).

[0082] In another aspect, a method for preparing the antibody or antigen-binding fragment thereof of the present invention is provided, comprising culturing the host cell of the present invention under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0083] Detection methods and kits

[0084] The monoclonal antibody or its antigen-binding fragment of the present invention can specifically bind to the CVB1 virus, thereby being used to detect the presence or level of the CVB1 virus in a sample, and optionally to diagnose whether a subject is infected with the CVB1 virus based on the presence or level of the CVB1 virus in a sample.

[0085] Therefore, in another aspect, the present invention provides a kit comprising the monoclonal antibody of the present invention or an antigen-binding fragment thereof.

[0086] In some embodiments, the antibody or antigen-binding fragment of the present invention carries a detectable label. In some embodiments, the kit further includes a secondary antibody that specifically recognizes the monoclonal antibody or antigen-binding fragment of the present invention. In some embodiments, the secondary antibody further includes a detectable label.

[0087] In some exemplary embodiments, the kit comprises: an antibody of the present invention or an antigen-binding fragment thereof with a detectable label. In some exemplary embodiments, the kit comprises: an antibody of the present invention or an antigen-binding fragment thereof (e.g., without a detectable label), and a secondary antibody with a detectable label.

[0088] In some embodiments, the kit comprises a first antibody and a second antibody capable of specifically binding to CVB1, wherein the first antibody and the second antibody are different from each other; wherein,

[0089] The first antibody is selected from monoclonal antibodies or antigen-binding fragments thereof containing the following CDRs: VH CDR1-3 as shown in SEQ ID NO:5-7, and / or VLCDR1-3 as shown in SEQ ID NO:8-10, respectively.

[0090] The second antibody is selected from other monoclonal antibodies or their antigen-binding fragments that can specifically bind to CVB1, or polyclonal antibodies.

[0091] In some embodiments, the polyclonal antibody is an antiserum containing a polyclonal antibody. The antiserum refers to the serum of an immunized animal (e.g., a non-human mammal, such as a mouse, rabbit, sheep, etc.) immunized against CVB1 virus, which contains a polyclonal antibody against CVB1.

[0092] In some embodiments, the second antibody is selected from monoclonal antibodies comprising the following CDRs or antigen-binding fragments thereof:

[0093] (i) amino acid sequences as shown in SEQ ID NO:15-17 (VH CDR1-3), and / or amino acid sequences as shown in SEQ ID NO:18-20 (VL CDR1-3); or

[0094] (ii) The amino acid sequences are VH CDR1-3 as shown in SEQ ID NO:25-27, and / or the amino acid sequences are VL CDR1-3 as shown in SEQ ID NO:28-30.

[0095] In some embodiments, the second antibody carries a detectable label. In some embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters), fluorescent dyes, radionuclides, or biotin.

[0096] In some embodiments, the kit further comprises a solid-phase support. In some embodiments, the solid-phase support comprises a well plate, test tube, beads (e.g., latex particles), or film (e.g., nitrocellulose membrane) made of or coated with a polymeric material (e.g., polyvinyl chloride, polystyrene, polyacrylamide, or cellulose), or magnetic beads pre-coated with functional groups (e.g., amino, carboxyl, biotin, or avidin). In some embodiments, the solid-phase support is selected from magnetic beads or microtiter plates (e.g., microplates or ELISA plates).

[0097] In some embodiments, the first antibody is coated on the surface of a solid-phase support.

[0098] In some exemplary embodiments, the kit comprises: a first antibody and a second antibody with a detectable label. In some exemplary embodiments, the kit comprises: a first antibody coated on the surface of a solid support and a second antibody with a detectable label.

[0099] In some embodiments, the kit may further comprise reagents for detecting the corresponding detectable label. For example, when the detectable label is an enzyme, the kit may also comprise a chromogenic substrate for the corresponding enzyme, such as o-phenylenediamine (OPD), tetramethylbenzidine (TMB), ABTS, or luminol compounds for horseradish peroxidase, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase. For example, when the detectable label is a chemiluminescent reagent (e.g., acrid ester compounds), the kit may also comprise a pre-excitation solution and / or an excitation solution for chemiluminescence.

[0100] In another aspect, the present invention provides a method for detecting the presence or level of CVB1 virus in a sample, comprising using the monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0101] In some embodiments, the method is used to detect solid particles of CVB1 virus. In some embodiments, the monoclonal antibody or its antigen-binding fragment comprises: VH CDR1-3 with amino acid sequences as shown in SEQ ID NO:5-7, and / or VL CDR1-3 with amino acid sequences as shown in SEQ ID NO:8-10.

[0102] In some embodiments, the method is used to detect solid and hollow CVB1 virus particles. In some embodiments, the monoclonal antibody or its antigen-binding fragment comprises: (i) amino acid sequences VH CDR1-3 as shown in SEQ ID NO:15-17, and / or, amino acid sequences VL CDR1-3 as shown in SEQ ID NO:18-20; or (ii) amino acid sequences VH CDR1-3 as shown in SEQ ID NO:25-27, and / or, amino acid sequences VL CDR1-3 as shown in SEQ ID NO:28-30.

[0103] In some implementations, the detection is an immunological detection, such as an enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0104] In some embodiments, the monoclonal antibody or its antigen-binding fragment includes a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridine esters), a fluorescent dye, a radionuclide, or biotin. In some embodiments, the method further includes detecting the monoclonal antibody or its antigen-binding fragment using a secondary antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridine esters), a fluorescent dye, a radionuclide, or biotin). In some embodiments, the method includes: (1) contacting the sample with the monoclonal antibody or its antigen-binding fragment of the present invention; and (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex. The formation of the immune complex indicates the presence of CVB1 virus or CVB1 virus particles.

[0105] In some embodiments, the method is a double-antibody sandwich method, which includes the following steps:

[0106] (1) The sample is contacted with a first antibody to form an antibody-antigen complex, wherein the first antibody is selected from monoclonal antibodies containing the following CDRs or antigen-binding fragments thereof: VHCDR1-3 with amino acid sequences as shown in SEQ ID NO:5-7, and / or VL CDR1-3 with amino acid sequences as shown in SEQ ID NO:8-10.

[0107] (2) The antibody-antigen complex is contacted with a second antibody capable of specifically binding to CVB1 to form an antibody-antigen-antibody complex, wherein the second antibody is selected from other monoclonal antibodies or their antigen-binding fragments capable of specifically binding to CVB1, or polyclonal antibodies; and

[0108] (3) Determine the amount of the antibody-antigen-antibody complex.

[0109] In some embodiments, the polyclonal antibody is an antiserum containing a polyclonal antibody. The antiserum refers to the serum of an immunized animal (e.g., a non-human mammal, such as a mouse, rabbit, sheep, etc.) immunized against CVB1 virus, which contains a polyclonal antibody against CVB1.

[0110] In some embodiments, the second antibody is selected from monoclonal antibodies comprising the following CDRs or antigen-binding fragments thereof:

[0111] (i) amino acid sequences as shown in SEQ ID NO:15-17 (VH CDR1-3), and / or amino acid sequences as shown in SEQ ID NO:18-20 (VL CDR1-3); or

[0112] (ii) The amino acid sequences are VH CDR1-3 as shown in SEQ ID NO:25-27, and / or the amino acid sequences are VL CDR1-3 as shown in SEQ ID NO:28-30.

[0113] In some embodiments, the first antibody is coated on the surface of a solid-phase support.

[0114] In some embodiments, the method can be used for diagnostic purposes, such as diagnosing whether a subject is infected with CVB1 virus based on the presence or level of CVB1 virus in a sample. In such embodiments, the sample can be a blood sample (e.g., whole blood, plasma, or serum), excrement, oral or nasal secretions, or bronchoalveolar lavage fluid from a subject (e.g., a mammal, preferably a human).

[0115] In some implementations, the method can be used for non-diagnostic purposes, such as when the sample is not from the subject, for example, an oncolytic virus sample or a vaccine sample.

[0116] In some implementations, the subject is a mammal, such as a human.

[0117] In another aspect, the use of the monoclonal antibody or antigen-binding fragment thereof of the present invention in the preparation of a kit for detecting the presence or level of CVB1 virus in a sample, and / or for diagnosing whether a subject is infected with CVB1 virus is provided.

[0118] In some embodiments, the detection is an immunological assay, such as an enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0119] In some embodiments, the kit detects the presence or level of CVB1 virus in a sample using the detection method described above, and optionally diagnoses whether a subject is infected with CVB1 virus based on the detection results.

[0120] In some embodiments, the sample is a blood sample (e.g., whole blood, plasma, or serum), excrement, oral or nasal secretions, or bronchoalveolar lavage fluid from a subject (e.g., a mammal, preferably a human).

[0121] Treatment methods and pharmaceutical compositions

[0122] The monoclonal antibody or its antigen-binding fragment of the present invention can be used in vitro or in vivo to neutralize CVB1 virus, block or inhibit CVB1 virus infection of cells, and for the prevention and / or treatment of CVB1 virus infection or CVB1 virus-related diseases in subjects.

[0123] Therefore, in another aspect, the present invention provides a pharmaceutical composition comprising the monoclonal antibody of the present invention or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier and / or excipient.

[0124] In some embodiments, the pharmaceutical composition further comprises additional pharmaceutically active agents, such as additional antiviral agents.

[0125] In some embodiments, the monoclonal antibody or antigen-binding fragment of the present invention, along with the additional pharmaceutically active agent, is provided as a separate component or as a component of a single composition in the pharmaceutical composition. Therefore, the antibody or antigen-binding fragment of the present invention, along with the additional pharmaceutically active agent, can be administered simultaneously, separately, or sequentially.

[0126] In some exemplary embodiments, the pharmaceutically acceptable carrier and / or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such a sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0127] In another aspect, the present invention provides a method for neutralizing Coxsackievirus B1 (CVB1), comprising using a monoclonal antibody or antigen-binding fragment thereof or a pharmaceutical composition of the present invention. This method can be used to neutralize CVB1 virus in vitro or in a subject (e.g., a human).

[0128] In some embodiments, the method is used to neutralize the virulence of Coxsackievirus B1 (CVB1) in a sample. In some embodiments, the method includes contacting a sample containing CVB1 virus with a monoclonal antibody or antigen-binding fragment thereof of the present invention or a pharmaceutical composition.

[0129] In some embodiments, the monoclonal antibody or its antigen-binding fragment is used alone or in combination with other pharmaceutically active agents (e.g., other antiviral agents).

[0130] In another aspect, the present invention provides a method for preventing or treating CVB1 infection or disease associated with CVB1 virus infection in a subject, comprising: administering an effective amount of the monoclonal antibody of the present invention or an antigen-binding fragment thereof or a pharmaceutical composition thereof to the subject in need.

[0131] In some embodiments, the monoclonal antibody or its antigen-binding fragment is used alone or in combination with another pharmaceutically active agent (e.g., another antiviral agent). The antibody or its antigen-binding fragment of the present invention can be administered simultaneously, separately, or sequentially with the other pharmaceutically active agent.

[0132] In some implementations, the diseases associated with CVB1 virus infection include aseptic meningitis, myocarditis, meningoencephalitis, hand-foot-mouth disease, chest pain, or neonatal fulminant hepatitis with coagulation disorders.

[0133] In some implementations, the subject is a mammal, such as a human.

[0134] In another aspect, the present invention relates to the use of the monoclonal antibody or antigen-binding fragment thereof of the present invention in the preparation of a medicament used for:

[0135] (1) Neutralize CVB1 virus in vitro or in a subject (e.g., human); and / or

[0136] (2) For the prevention or treatment of CVB1 virus infection or disease associated with CVB1 virus infection in subjects.

[0137] In some implementations, the diseases associated with CVB1 virus infection include aseptic meningitis, myocarditis, meningoencephalitis, hand-foot-mouth disease, chest pain, or neonatal fulminant hepatitis with coagulation disorders.

[0138] In some embodiments, the monoclonal antibody or its antigen-binding fragment is used alone or in combination with other pharmaceutically active agents (e.g., other antiviral agents).

[0139] In some implementations, the subject is a mammal, such as a human.

[0140] The monoclonal antibodies or antigen-binding fragments thereof of the present invention, or the pharmaceutical compositions of the present invention, can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The pharmaceutical compositions of the present invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the required dose of the recombinant protein of the present invention into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Alternatively, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0141] The monoclonal antibodies or antigen-binding fragments thereof, or pharmaceutical compositions of the present invention, may be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In a preferred embodiment, the monoclonal antibodies or antigen-binding fragments thereof, or pharmaceutical compositions of the present invention, are administered by intravenous injection or bolus.

[0142] The pharmaceutical compositions of the present invention may include a "therapeutic effective amount" or a "preventive effective amount" of the monoclonal antibody or its antigen-binding fragment of the present invention. A "preventive effective amount" refers to an amount sufficient to prevent, stop, or delay the onset of a disease. A "therapeutic effective amount" refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the monoclonal antibody or its antigen-binding fragment of the present invention may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the method of administration of the drug, and other concurrent treatments, etc.

[0143] In this invention, the dosing regimen can be adjusted to obtain the optimal target response (e.g., treatment or prevention). For example, it can be administered as a single dose, multiple times over a period of time, or the dose can be reduced or increased proportionally according to the urgency of the treatment situation.

[0144] In this invention, the subject can be a mammal, such as a human.

[0145] Terminology Definition

[0146] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used herein are all conventional procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0147] As used herein, the term "Coxsackivirus B1 (CVB1)" refers to a single-stranded positive-sense RNA virus belonging to the family Picomaviridae, genus Enterovirus, and group B of Coxsackivirus. The genome or cDNA sequence of CVB1 is well known in the art and is available in various public databases (e.g., GenBank accession number: MG780414). CVB1 exists in two types of viral particles: hollow particles (without RNA, non-infectious) and solid particles (containing RNA, infectious), both of which are immunogenic.

[0148] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding, but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). H and V LIt consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. The allocation of amino acids in each region or domain can follow the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0149] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residues in the variable region of an antibody responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0150] In this invention, the CDR contained in the antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined by the Kabat, Chothia, or IMGT numbering system. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined by the IMGT numbering system.

[0151] As used herein, the term “framework region” or “FR” residues refer to those amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0152] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0153] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety”. See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabody, linear antibody, nanobody (technology from Domantis), domain antibody (technology from Ablynx), and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0154] As used herein, the term "full-length antibody" refers to an antibody composed of two "full-length heavy chains" and two "full-length light chains." A "full-length heavy chain" is a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-terminal to C-terminal direction; and, optionally, a heavy chain constant region CH4 domain is also included when the full-length antibody is an IgE isotype. Preferably, the "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibody of this invention can be derived from a single species, such as humans; it can also be a chimeric antibody or a humanized antibody. The full-length antibody of this invention comprises two antigen-binding sites formed by VH and VL pairs, respectively, which specifically recognize / bind to the same antigen.

[0155] As used herein, the term “Fd fragment” refers to an antibody fragment consisting of VH and CH1 domains; the term “dAb fragment” refers to an antibody fragment consisting of VH domains (Ward et al., Nature 341:544 546 (1989)); the term “Fab fragment” refers to an antibody fragment consisting of VL, VH, CL and CH1 domains; the term “F(ab')2 fragment” refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; the term “Fab' fragment” refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and heavy chain Fd fragment (consisting of VH and CH1 domains).

[0156] As used herein, the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0157] As used herein, the term "Fc fragment" refers to an antibody fragment formed by the disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0158] As used herein, the term “scFv” refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between VH and VL of scFv. As used in this article, the term "di-scFv" refers to an antibody fragment formed by the linking of two scFvs.

[0159] As used herein, the term “biantibody” means that its VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0160] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0161] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0162] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0163] As used herein, the terms “monoclonal antibody,” “monoclonal antibody,” and “mAb” have the same meaning and are used interchangeably. They refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules; that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Furthermore, the modifier “monoclonal” only indicates that the antibody is derived from a highly homologous group of antibodies and should not be construed as requiring preparation by any particular method.

[0164] As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain portion is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and whose light chain and / or heavy chain portion is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains its binding activity to the target antigen in any case (USP4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:68516855 (1984)). For example, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the variable regions of the heavy and light chains of the antibody are derived from a first antibody (e.g., a mouse antibody), while the constant regions of the heavy and light chains of the antibody are derived from a second antibody (e.g., a human antibody).

[0165] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with human antibodies. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and ability to enhance immune responses. Donor antibodies can be mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibodies with the intended properties (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and / or ability to enhance immune responses).

[0166] The chimeric or humanized antibodies of the present invention can be prepared based on the sequence of the mouse monoclonal antibody prepared above. The DNA encoding the heavy and light chains can be obtained from the target mouse hybridoma and engineered using standard molecular biology techniques to contain non-mouse (e.g., human) immunoglobulin sequences.

[0167] To prepare chimeric antibodies, methods known in the art can be used to ligate the variable region of mouse immunoglobulins to the constant region of human immunoglobulins (see, for example, U.S. Patent No. 4,816,567, Cabilly et al.). For example, DNA encoding VH can be operatively ligated to another DNA molecule encoding the heavy chain constant region to obtain a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is generally preferred to be an IgG1 or IgG4 constant region. For example, DNA encoding VL can be operatively ligated to another DNA molecule encoding the light chain constant region CL to obtain a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant regions are known in the art (see, for example, Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. Light chain constant regions can be κ or λ constant regions, but κ constant regions are generally preferred.

[0168] To prepare humanized antibodies, mouse CDR regions can be inserted into human frame sequences using methods known in the art (see Winter’s U.S. Patent No. 5,225,539; Queen et al.’s U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004).

[0169] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K0) of that interaction. D () indicates. In this invention, the term "K" is used. D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. The specific binding properties between the two molecules can be determined using methods known in the art, such as surface plasmon resonance (SPR) in a BIACORE instrument.

[0170] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0171] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0172] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoIBiol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0173] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0174] The twenty common amino acids mentioned in this article are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. And in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0175] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintainers, absorption delayers, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintainers include, but are not limited to, sugars, NaCl, and their analogues. Absorption delayers include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art as being capable of stabilizing the desired activity of the active ingredient in the pharmaceutical product, including, but not limited to, monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In some exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such sterile injectable fluids are selected from water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0176] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to the expected survival (if no treatment was received).

[0177] As used in this article, the term "subject" refers to a mammal, such as a primate mammal, such as a human.

[0178] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for disease prevention is an amount sufficient to prevent, stop, or delay the onset of disease; an effective amount for disease treatment is an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0179] As used herein, the term "neutralizing activity" refers to the functional activity of an antibody or antibody fragment to bind to antigenic proteins on a virus, thereby preventing the virus from infecting cells and / or maturing and / or releasing viral progeny. Antibodies or antibody fragments with neutralizing activity can prevent viral amplification, thereby inhibiting or eliminating viral infection.

[0180] Beneficial effects of the invention

[0181] The monoclonal antibody or antigen-binding fragment of the present invention exhibits good affinity for CVB1 solid and / or hollow particles, enabling specific detection of CVB1 virus or virus particles. Furthermore, the monoclonal antibody or antigen-binding fragment of the present invention can neutralize the virulence of CVB1, thereby facilitating the prevention and treatment of CVB1 infection and related diseases. Therefore, the monoclonal antibody or antigen-binding fragment of the present invention has significant clinical value.

[0182] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0183] Figure 1 The results of transmission electron microscopy analysis of different types of CVB1 virus particles separated by sucrose gradient density centrifugation are shown.

[0184] Figure 2 The binding activity of different monoclonal antibodies to solid and hollow CVB1 virus particles was demonstrated.

[0185] Figure 3 The results of the detection of CVB1 virus particles are shown by a double-antibody sandwich ELISA method based on monoclonal antibody 5F5.

[0186] Figure 4 The results of immunofluorescence assays based on monoclonal antibodies 5F5, 8A10, and 9A3 on the detection of cells infected with CVB1 virus are shown.

[0187] Figure 5 The prophylactic activity of monoclonal antibodies 5F5, 8A10, and 9A3 against CVB1 infection in a mouse model was evaluated.

[0188] Figure 6 The therapeutic activity of monoclonal antibodies 5F5, 8A10, and 9A3 against CVB1 infection in a mouse model was evaluated.

[0189] Sequence information

[0190] Information on some of the sequences involved in this invention is provided in Table 1 below.

[0191] Table 1: Sequence Description

[0192]

[0193]

[0194] Detailed Implementation

[0195] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0196] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially in accordance with the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Susubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the manufacturer's recommendations. Those skilled in the art will appreciate that the examples illustrate the invention by way of illustration and are not intended to limit the scope of the invention as claimed.

[0197] Example 1: Preparation of CVB1 virus particles

[0198] 1.1 Virus culture:

[0199] The CVB1 strain used in this embodiment is 301 (GenBank No. MT129657), and the cells used to culture the virus are human rhabdomyosarcoma cells (RD). CCL-136 TM First, RD cells were cultured in 10cm culture dishes (NEST) in MEM medium (GIBCO) containing 10% fetal bovine serum (PAA). When the cell confluence reached 80%, the medium was replaced with serum-free MEM medium, and the virus was inoculated at an MOI of 0.1. The cells were cultured at 37°C, and the virus was harvested after 3 days when the cells showed complete cytopathic activity. The virus harvesting method was as follows: the cells were scraped off, frozen and thawed three times, centrifuged to remove cell debris, and the cell lysis supernatant was filtered through a 0.22μm filter to obtain the virus stock solution, which was then stored at -80°C for later use.

[0200] 1.2 Sucrose density gradient centrifugation was used to separate virus particles with different properties:

[0201] The viral stock solution obtained above was concentrated and precipitated using PEG 6000. The precipitation conditions were 6% PEG 6000, 0.3 mol / L NaCl, and precipitation overnight at 4°C. The precipitate was then collected by centrifugation at 10000g and resuspended in PBS. Subsequent sucrose gradient centrifugation (15%-50%) was performed at 120000g for 2.5 h using an SW41 Ti rotor. Samples were then taken for negative staining electron microscopy observation. The results are as follows: Figure 1 As shown, sucrose gradient centrifugation can effectively separate solid and hollow CVB1 virus particles.

[0202] Example 2: Preparation of Monoclonal Antibodies

[0203] The CVB1 virus stock solution prepared in Example 1 was emulsified with Freund's complete adjuvant and injected into 6-8 week old BALB / c female mice at multiple sites, including subcutaneous injection in the back, groin, footpads, and limbs, with a dose of 500 μL per mouse. Booster immunizations were performed every two weeks using the same method, with the immunogen being a mixture of the CVB1 virus stock solution prepared in Example 1 and Freund's incomplete adjuvant. Before each immunization, 20 μL of tail vein blood or 200 μL of ocular venous blood was collected for titer determination. Serum titers were measured using indirect ELISA. Once the mouse serum titer reached a plateau, immunization was stopped, and the mice were allowed to rest for two months before fusion. 72 hours before fusion, 100 μL of CVB1 virus stock solution (without adjuvant) was directly injected into the spleen of the mice for booster immunization. 72 hours later, mice were sacrificed and mouse antiserum (mouse polyantiserum) was collected. Simultaneously, mouse spleens were harvested and cell suspensions were prepared (suspended in RPMI 1640 medium) for cell counting using a cell counting chamber. One-sixth of the spleen cells were mixed with mouse myeloma cells Sp2 / 0, followed by cell fusion using 50% polyethylene glycol (PEG). The cell suspension was then mixed with an equal volume of feeder cells (BALB / c mouse macrophages and thymocytes) and placed in 96-well cell culture plates (200 μL / well) at 37°C with 5% CO2. After 3 days, the medium was partially replaced with RPMI 1640 medium containing hypoxanthine, aminopterin, and thymidine. After 7 days, ELISA plates were coated with CVB1 virus stock solution, and the hybridoma cell culture supernatant was detected using an indirect ELISA method. For ELISA-positive wells, cloning was performed using a limiting dilution method.

[0204] Purification of monoclonal antibodies: Healthy BALB / c mice aged 10 weeks were intraperitoneally injected with paraffin oil, 0.5 mL per mouse. Two to seven days later, cloned hybridoma cells were collected, centrifuged to remove the supernatant, and then added to serum-free 1640HT medium to adjust the cell density to 2 × 10⁻⁶ cells / mL. 5 -2×10 60.5 mL was injected per mouse. After 7-10 days, the mice's abdomens enlarged, and ascites fluid collection began. The fluid was centrifuged at 3000 rpm for 15 min, and the clear intermediate liquid was collected and stored at -20℃. Antibody purification was performed after ascites fluid collection. The ascites fluid was diluted twofold with 0.02 mol / L, pH 7.4 PBS, and an equal volume of saturated ammonium sulfate was slowly added with stirring. The solution was incubated overnight at 4℃. After centrifugation at 12000 rpm for 15 min at 4℃, the supernatant was discarded. The precipitate was dissolved in an appropriate amount of PBS, placed in a dialysis bag, and then placed in 50-100 volumes of 0.02 mol / L PB (pH 7.4). The solution was stirred and desalted at 4℃ for approximately 12 h, with the dialysis buffer changed 3 times during this period. The dialyzed antibody was purified using a Protein A column (GE) in an AKTA purification system. The column was affinity-coated with 0.02 mol / L PB (pH 7.4), and eluted with 0.1 M citric acid. The eluent was the purified antibody. Dialyze the eluent to 0.02 mol / L PB (pH 7.4) and store at -20°C.

[0205] Example 3: Screening for monoclonal antibodies that specifically bind to CVB1 virus particles

[0206] ELISA Assay (Indirect Method): The solid and hollow particle antigens of CVB1 virus prepared in Example 1 were diluted 100-fold with 20mM PB7.4 and coated onto 96-well microplates at 100 μL / well. The plates were incubated at 37°C for 2 h, washed once with PBST, and blocked at 200 μL / well with the relevant blocking buffer (20mM PB7.4 containing 150mM NaCl, 0.5% casein, and 0.002% gelatin) to block non-specific binding sites overnight at 4°C. 100 μL of the monoclonal antibody sample (0.1 mg / mL) prepared in Example 2 was added to the microplate and incubated at 37°C for 1 h. The plates were washed 5 times with PBST, and GAM-HRP (horseradish peroxidase-labeled goat anti-mouse antibody, purchased from Bio-Rad, USA; dilution formulation is the same as the blocking buffer) was added. The plates were incubated at 37°C for 30 min. Wash the plate 5 times with PBST, add TMB chromogenic buffer and develop for 15 min, then stop the reaction with stop solution. Read the values ​​using a TECAN sunrise microplate reader. Finally, from over 30 monoclonal antibodies, three monoclonal antibodies, 5F5, 8A10, and 9A3, were screened to specifically bind to CVB1 virus particles. Some antibody screening results are shown below. Figure 2 As shown, 5F5 can specifically bind to solid CVB1 particles, but has no obvious reaction with hollow particles. 8A10 and 9A3 can specifically bind to both solid and hollow CVB1 virus particles, and their binding activity is significantly better than other monoclonal antibodies.

[0207] Further variable region sequence analysis was performed on monoclonal antibodies 5F5, 8A10, and 9A3, and their VH and VL sequences are shown in the table below. Furthermore, the CDR sequences of the three monoclonal antibodies were determined based on the IMGT database (http: / / www.imgt.org / IMGT_vquest / analysis).

[0208] Table 2: Monoclonal Antibody Variable Region Sequence

[0209]

[0210] Example 4: Establishment of a method for detecting CVB1 solid particle antigen

[0211] In this embodiment, 5F5 was used as the coating monoclonal antibody to establish a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) method to detect CVB1 solid particle antigen.

[0212] 4.1 Preparation of horseradish peroxidase-labeled antibody:

[0213] Dissolve 1 mg each of HRP and NaIO4 in ultrapure water. Then, add NaIO4 solution dropwise to the HRP solution while shaking to mix. Place the mixture at 4°C in the dark for 30 minutes. Dissolve 1 μL of ethylene glycol in ultrapure water and add it dropwise while shaking to mix. Let it stand at room temperature in the dark for 30 minutes. This completes the enzyme oxidation process. During the HRP oxidation process, dialyze the purified antibody against 50 mM CB buffer (pH 9.6). After HRP oxidation, mix the dialyzed antibody with HRP in the required ratio and dialyze against 50 mM CB buffer for at least 6 hours. Then, terminate the process with freshly prepared NaBH4 solution (0.2 mg NaBH4), shake well, and let stand at 4°C for 2 hours. Finally, dialyze against 10 mM PBS (pH 7.2) overnight.

[0214] Three HRP-labeled antibodies were prepared using the above method: polyclonal antibody-HRP, a mouse polyclonal antibody that binds to CVB1 virus particles (the mouse polyclonal antibody serum prepared in Example 2); 8A10-HRP, a monoclonal antibody that binds to CVB1 virus particles (8A10 screened in Example 3); and 9A3-HRP, a monoclonal antibody that binds to CVB1 virus particles (9A3 screened in Example 3).

[0215] 4.2 ELISA assay (double antibody sandwich ELISA)

[0216] The 5F5 monoclonal antibody was diluted to a concentration of 0.1 μg / mL with 20 mM PB7.4 and coated onto a 96-well ELISA plate at 100 μL / well, incubating at 37°C for 2 h. The plate was washed once with PBST, and non-specific binding sites were blocked with the relevant blocking buffer (20 mM PB7.4 containing 150 mM NaCl, 0.5% casein, and 0.002% gelatin) at 200 μL / well, incubating overnight at 4°C. Different CVB1 virus samples were diluted 100-fold with blocking buffer and added to the ELISA plate at 100 μL / well, incubating at 37°C for 1 h. The plate was washed 5 times with PBST, and the HRP-labeled antibody (polyclonal antibody-HRP, 8A10-HRP, or 9A3-HRP) prepared in section 4.1 was added, incubating at 37°C for 30 min. The plate was washed 5 times with PBST, and TMB chromogenic buffer was added for 15 min of development. The incubation was stopped with stop buffer, and the readings were recorded using a TECAN sunrise microplate reader. Results are as follows: Figure 3 As shown, 5F5 can detect CVB1 solid particulate antigen but not hollow particulate antigen, regardless of whether it is paired with a polyclonal antibody or a monoclonal antibody. These results indicate that the double-antibody sandwich ELISA method using 5F5 as the coating antibody can be used for the detection of CVB1 solid particulate antigen.

[0217] Example 5: Identification of Antibody Subtypes

[0218] The CVB1 virus solid particle antigen prepared in Example 1 was diluted 100-fold with 20mM PB7.4 and coated onto a 96-well ELISA plate at 100 μL / well. Coating was carried out at 37°C for 2 h. The plate was washed once with PBST, and non-specific binding sites were blocked with a blocking buffer (20mM PB7.4 containing 150mM NaCl, 0.5% casein, and 0.002% gelatin) at 200 μL / well, and incubated overnight at 4°C. 100 μL of a 500-fold diluted monoclonal antibody was added to the ELISA plate and incubated at 37°C for 1 h. The plate was washed 5 times with PBST, and HRP-labeled anti-IgG1, IgG2a, IgG2b, IgG3, and IgM goat anti-mouse secondary antibodies (Thermo) were added. Incubation was carried out at 37°C for 30 min. The plate was washed 5 times with PBST, and TMB chromogenic buffer was added for 15 min of development. The reaction was stopped with stop buffer, and the readings were recorded using a TECAN sunrise ELISA reader. The results showed that monoclonal antibodies 5F5, 8A10 and 9A3 were all IgG2a subtypes.

[0219] Example 6: Monoclonal antibodies 5F5, 8A10, and 9A3 were used to detect CVB1-infected cells.

[0220] RD cells were seeded in 24-well cell culture plates (500 μL, 5 × 10⁶ cells / well). 4 / well), with a round coverslip pre-placed in each well. After the cells adhere, inoculate with CVB1 virus at 5000 TCID50. 50 / well, and set up a cell blank control. After 12 hours, aspirate the supernatant with a pipette tip, wash once with PBS, 1 mL / well. Aspirate the PBS, add 4% paraformaldehyde, 1 mL / well, and fix at room temperature in the dark for 15 min. Add 0.5% Triton X-100 (prepared with PBS) for permeabilization, 1 mL / well, and incubate at room temperature for 10 min. Wash three times with PBS, 3 min each time. Flatten a sealing film and fix it on the cover of the 24-well cell culture plate. Add 50 μL of goat serum to each well on the sealing film. After adding, use a curved needle to lift the coverslip containing cells, hold the edge with forceps, cover the film with the cell side down, and place it in a humidified chamber at 37°C for 1 hour. Put the coverslip back into the 24-well plate with the cell side up, and wash three times with PBS, 3 min each time. Dilute the antibody (1 mg / mL) 200-fold with 2% BSA and add it to the cells (using the same procedure as blocking). Block at 37°C for 1 hour. Place the coverslip back into the 24-well plate with the cells facing up. Wash three times with PBS, 3 min each time. Dilute the secondary antibody GAM-FITC (Sigma) 1:500 with 2% BSA and incubate in a humidified chamber at 37°C for 30 min in the dark (using the same procedure as blocking). Place the coverslip back into the 24-well plate with the cells facing up. Wash three times with PBS, 3 min each time. Dilute DAPI (Invitrogen) 1:2000 with PBS and add it to the cells (using the same procedure as blocking). Incubate at room temperature in the dark for 5 min. Place the coverslip back into the 24-well plate with the cells facing up. Wash three times with PBS, 3 min each time. Incubate in the dark. Mark a clean glass slide, drop approximately 30 μL of mounting medium (70% glycerol, 2.5% cell-inhibiting agent) onto the cleaned slide, cover with a coverslip containing cells, seal the edges with nail polish, and allow to air dry in the dark. Observe and photograph under a fluorescence microscope. Results are as follows: Figure 4 As shown, cells infected with CVB1 virus exhibited green fluorescence, while the blank control showed no green fluorescence. These results indicate that monoclonal antibodies 5F5, 8A10, and 9A3 can all be used to detect CVB1-infected cells.

[0221] Example 7: Neutralizing titers of monoclonal antibodies 5F5, 8A10, and 9A3

[0222] Neutralization assay: The neutralizing activity of hybridoma cell culture supernatant or crude purified antibody was identified using conventional neutralization assay methods. Human rhabdomyosarcoma cells (RD) were seeded in 96-well cell culture plates (5 × 10⁻⁶ cells / well). 3 / well). After 10 hours, serially dilute the test samples with serum-free MEM medium (starting with an 8-fold dilution, perform 2-fold serial dilutions, and repeat 10 times), with at least 4 replicates per sample. Dilute CVB1 virus to 100 TCID using serum-free MEM. 50At a concentration of 50 μL, 50 μL was added to each of the serially diluted monoclonal antibody sample wells. After incubation at 37°C for 1 h, 100 μL of the monoclonal antibody-virus mixture was added to a 96-well cell culture plate pre-coated with RD cells. The plate was incubated at 37°C with 5% CO2, and cytopathic effects were observed and recorded for 7 consecutive days. The highest dilution that inhibited cytopathic effects in wells exceeding 50% was used as the neutralizing titer of the sample. Results showed that monoclonal antibodies 5F5, 8A10, and 9A3 all exhibited high in vitro neutralizing activity, with neutralizing titers of approximately 8192, 131072, and 2048, respectively.

[0223] Example 8: Experiment on the prevention of CVB1 infection in mice by monoclonal antibodies 5F5, 8A10 and 9A3

[0224] Newborn mice were grouped as follows: One-day-old BALB / c mice were selected, with 15 mice in each group, forming an antibody prevention group and a PBS control group. After 4 hours of fasting, the newborn mice were intraperitoneally injected with monoclonal antibodies (5F5, 8A10, and 9A3) at a dose of 30 μg / mouse; the PBS control group received the same volume of PBS intraperitoneally. Four hours later, the newborn mice in both the antibody prevention and PBS control groups were intraperitoneally infected with CVB1 virus at a challenge dose of 100 TCID50. 50 All the animals were observed for 20 consecutive days.

[0225] Experimental results are as follows Figure 5 As shown, most mice in the PBS control group died 7 days after challenge; while the mice in the 5F5, 8A10, and 9A3 prevention groups showed normal physical signs and no abnormalities. This indicates that monoclonal antibodies 5F5, 8A10, and 9A3 all have a preventive effect against CVB1 infection.

[0226] Example 9: In vivo therapeutic experiments in mice with monoclonal antibodies 5F5, 8A10, and 9A3

[0227] Since monoclonal antibodies 5F5, 8A10 and 9A3 all have high neutralizing activity against CVB1 virus, their use in animal model experiments of monoclonal antibody treatment for CVB1 infection may protect CVB1-infected mice from developing the disease.

[0228] Newborn mice were grouped as follows: One-day-old BALB / c mice were selected, with an antibody protection group and a PBS control group, each group consisting of 15 mice. After being starved for 4 hours, the newborn mice in the antibody protection group and the PBS control group were intraperitoneally injected with CVB1 virus at a challenge dose of 100 TCID50. 50 The antibody protection group received intraperitoneal injections of monoclonal antibodies (5F5, 8A10, and 9A3) at a dose of 30 μg per animal 24 hours after challenge; the PBS control group received the same volume of PBS intraperitoneally. All animals were observed for 20 consecutive days.

[0229] The experimental results are shown in Figure 6In the PBS control group, mice exhibited emaciation, hind limb paralysis four days after challenge, and most died seven days after challenge. In contrast, the protective groups treated with monoclonal antibodies 5F5, 8A10, and 9A3 showed normal physical signs and no abnormalities. This indicates that monoclonal antibodies 5F5, 8A10, and 9A3 are all effective in treating mice infected with CVB1.

[0230] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof capable of specifically binding to Coxsackievirus B1 (CVB1), comprising: VH CDR1-3 with amino acid sequences as shown in SEQ ID NO: 25-27, and VL CDR1-3 with amino acid sequences as shown in SEQ ID NO: 28-30.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, wherein, The monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) as shown in SEQ ID NO: 21 and a light chain variable region (VL) as shown in SEQ ID NO:

22.

3. The monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The monoclonal antibody is selected from murine antibodies, chimeric antibodies, humanized antibodies; and / or the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv or single-chain antibodies.

4. The monoclonal antibody or its antigen-binding fragment according to claim 3, wherein, The single-chain antibody is scFv.

5. The monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The antibody is an IgG antibody.

6. The monoclonal antibody or its antigen-binding fragment according to claim 5, wherein, The IgG antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

7. The monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The monoclonal antibody or its antigen-binding fragment carries a detectable label selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides, or biotin.

8. The monoclonal antibody or its antigen-binding fragment according to claim 7, wherein, The enzyme is horseradish peroxidase or alkaline phosphatase.

9. The monoclonal antibody or its antigen-binding fragment according to claim 7, wherein, The chemiluminescent reagent is an acridine ester compound.

10. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described in any one of claims 1-6, or a variable region of the heavy chain and a variable region of the light chain thereof.

11. A vector comprising the nucleic acid molecule of claim 10.

12. The carrier of claim 11, wherein, The vector is a cloning vector or an expression vector.

13. A host cell comprising the nucleic acid molecule of claim 10 or the vector of claim 11 or 12.

14. A method for preparing a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1-6, comprising culturing a host cell according to claim 13 under conditions allowing expression of the monoclonal antibody or the antigen-binding fragment thereof, and recovering the monoclonal antibody or the antigen-binding fragment thereof from the cultured host cell culture.

15. A kit comprising the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-9.

16. The kit of claim 15, wherein, The monoclonal antibody or its antigen-binding fragment includes a detectable marker selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides, or biotin.

17. The kit of claim 15, wherein, The kit also includes a secondary antibody that specifically recognizes the monoclonal antibody or its antigen-binding fragment.

18. The kit of claim 17, wherein, The secondary antibody also includes a detectable marker selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides, or biotin.

19. A method for detecting the presence or level of Coxsackievirus B1 (CVB1) in a sample for non-diagnostic purposes, comprising using a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1-9.

20. The method of claim 19, wherein, The detection is an immunological detection, which is selected from enzyme immunoassay, chemiluminescent immunoassay, fluorescence immunoassay or radioimmunoassay.

21. The method of claim 20, wherein, The enzyme immunoassay method is ELISA.

22. The method of claim 19, wherein, The method is used to detect solid and hollow particles of CVB1 virus.

23. The method of claim 19, wherein, The monoclonal antibody or its antigen-binding fragment further includes a detectable label selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides, or biotin; or, the method further includes using a secondary antibody with a detectable label to detect the monoclonal antibody or its antigen-binding fragment, the detectable label selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides, or biotin.

24. The kit of claim 16 or 18 or the method of claim 23, wherein, The enzyme is horseradish peroxidase or alkaline phosphatase.

25. The kit of claim 16 or 18 or the method of claim 23, wherein, The chemiluminescent reagent is an acridine ester compound.

26. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-9 in the preparation of a kit for detecting the presence or level of CVB1 virus in a sample, and / or for diagnosing whether a subject is infected with CVB1 virus.

27. The use as described in claim 26, wherein, The samples are blood samples, excrement, oral or nasal secretions, or bronchoalveolar lavage fluid from the subject.

28. The use as described in claim 27, wherein, The blood sample is selected from whole blood, plasma, or serum.

29. The use as described in claim 27, wherein, The subjects were mammals.

30. The use as described in claim 29, wherein, The mammal in question is selected from humans.

31. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-6, and a pharmaceutically acceptable carrier and / or excipient.

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