CN116751288A

CN116751288A - Monoclonal antibody against coxsackievirus B1 and application thereof

Info

Publication number: CN116751288A
Application number: CN202310799919.0A
Authority: CN
Inventors: 程通; 徐龙发; 朱瑞; 尹志超; 王玮; 付文锟; 叶祥忠; 夏宁邵
Original assignee: Xiamen University; Beijing WanTai Biological Pharmacy Enterprise Co Ltd
Current assignee: Xiamen University; Beijing WanTai Biological Pharmacy Enterprise Co Ltd
Priority date: 2020-04-26
Filing date: 2020-04-26
Publication date: 2023-09-15
Also published as: CN116731164A; CN113549146B; CN113549146A

Abstract

The present application relates to the fields of immunology and virology, in particular to the fields of diagnosis, prevention and treatment of coxsackievirus B1. In particular, the application relates to monoclonal antibodies or antigen-binding fragments thereof directed against coxsackievirus B1, and compositions (e.g., diagnostic and therapeutic agents) comprising the antibodies or antigen-binding fragments thereof. Furthermore, the application relates to the use of said antibodies or antigen binding fragments thereof. The antibodies or antigen binding fragments thereof of the application are useful in the diagnosis, prevention and/or treatment of infection of coxsackievirus B1 and/or diseases caused by such infection.

Description

Monoclonal antibody against coxsackievirus B1 and application thereof

The present application is a divisional application of the application application with the application number of 202010339682.4, the application date of 2020, 4 months and 26 days, and the application name of "monoclonal antibody against coxsackievirus B1 and application thereof".

Technical Field

Background

Coxsackievirus type B1 (hereinafter referred to as CVB 1) belongs to the genus enterovirus of the family picornaviridae. Studies have shown that CVB1 infection can lead to aseptic meningitis, myocarditis, meningoepithymitis, hand-foot-and-mouth disease, chest pain, neonatal fulminant hepatitis with clotting disorders, etc. (us national CDC,2010, morbidity and mortality weekly report (Morbidity and Mortality Weekly Report) 59 (48): 1577-80), there is evidence that CVB1 infection is also associated with some chronic diseases, such as type I Diabetes (oikarien et al, 2014, diabetes (Diabetes) 63 (2): 655-62). CVB1 is a single-stranded positive strand RNA virus with a genome of about 7500bp in length. The virus particles are of a regular icosahedron structure, have no envelope and protrusion and have a diameter of about 30nm, the protein shell of the virus particles is composed of structural proteins VP1, VP2, VP3 and VP4, and the neutralizing epitope is mainly formed on VP1, VP2 and VP3, wherein VP1 protein is a main epitope determining region, and VP4 is highly conserved in sequence and reported by less neutralizing epitope due to being wrapped inside the shell.

The general circulating level of coxsackievirus B1 has increased gradually since the 90 s of the 20 th century. The results of virus identification of 52812 enterovirus patients reported by national enterovirus detection systems in 1970-2005 in the united states show that the infection rate of CVB1 is about 2.3%, and that CVB1 was 15 years old in 17 years during the 36-year study period, and was more a second-most annual epidemic strain in 1977 (Khetdurian et al, 2006, report on Morbidity and Mortality (MMWR) 59 (48): 1577-80). In 2010, the national CDC data showed that CVB1 was the major enterovirus pathogen in 2006-2008, with CVB1 in the ninth (3.7%) of annual epidemic strains, 23.6% and 18.6% of non-polio enterovirus positive samples in 2007 and 2008, respectively, were first, while CVB1 was the first (16.5%) of the total enterovirus positive sample proportion in 3 (national CDC,2010, morbidity and Mortality Weekly Report (MMWR) 59 (48); 1577-1580). The prevalence of CVB1 was found in both cases of viral encephalitis in the province of Zhejiang in 2002-2012 and in patients with aseptic meningitis in the province of Shandong in 2006-2012. In addition, CVB1 epidemic infection was also found in korea between 2008-2009.

Currently, vaccine immunization is considered to be the most effective and convenient method of preventing enterovirus infection. Two types of viral particles exist for various enteroviruses found: hollow particles (no RNA, no infectivity), solid particles (RNA, infectivity), and both viral particles are immunogenic. Polio vaccine is a mature and successfully applied vaccine at present, solid and hollow particles exist in the same polio virus belonging to enterovirus, and solid particle antigen can excite human body to generate neutralizing antibodies and is a main protective antigen (Mayer et al 1957, J.Immunol. (Journal of Immunology) 78, 435-455.), so that the effective components of polio vaccine are measured by the content of solid particle antigen. The study shows that CVB1 virus particles have better immunogenicity after inactivation, mice can generate stronger neutralizing antibodies in vivo after being inoculated with the inactivated virus particles, and the mice are protected from the lethal attack of CVB1 virus (Hankaniemi et al, 2017, vaccine,35 (30): 3718-3725). Other enteroviruses such as EV71 (Wu et al 2019, plosone, 14 (1): e 0210553) and CVA16 (Chong et al 2012, plosone, 7 (11): e 49973) have also shown that solid particle antigens may play an important role in the immunogenicity of viral vaccines, and thus it is necessary to develop studies on the detection of solid particle antigens of CVB1 viruses. At present, no report on a CVB1 solid particle antigen detection method exists. Therefore, the monoclonal antibody capable of specifically combining CVB1 virus solid particles is obtained, and a CVB1 solid particle antigen related detection method is established, so that the method has very important significance for developing and quality control of CVB1 vaccines.

Disclosure of Invention

The inventor of the present application has conducted intensive studies and creative efforts to obtain monoclonal antibodies capable of specifically binding to solid particles and/or hollow particles of CVB1 virus, and based on this, established a detection method capable of specifically detecting solid particles of CVB1 virus. In addition, the obtained monoclonal antibody can also efficiently neutralize CVB1 virus and block or inhibit the infection of the virus to cells, so that the monoclonal antibody has the potential of preventing and treating CVB1 infection and diseases related to CVB1 infection. The following application is thereby provided.

Antibodies of the application

In one aspect, the application provides a monoclonal antibody or antigen binding fragment thereof that specifically binds coxsackievirus B1 (CVB 1) comprising the following Complementarity Determining Regions (CDRs) defined according to the IMGT numbering system:

(1) VH CDR1-3 with amino acid sequence shown as SEQ ID NO 5-7 and/or VL CDR1-3 with amino acid sequence shown as SEQ ID NO 8-10;

(2) VH CDR1-3 with amino acid sequence shown as SEQ ID NO 15-17, and/or VL CDR1-3 with amino acid sequence shown as SEQ ID NO 18-20; or alternatively

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

In certain embodiments, the monoclonal antibody or antigen binding fragment thereof comprises: the amino acid sequences are shown as VH CDR1-3 shown in SEQ ID NO 5-7 and/or VL CDR1-3 shown in SEQ ID NO 8-10.

In certain embodiments, the antibody or antigen binding fragment thereof comprises: (a) 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO. 1; and/or (b) 3 CDRs contained in the light chain variable region (VL) as set forth in SEQ ID NO. 2. In certain embodiments, the 3 CDRs contained in the heavy chain variable region (VH) and/or the 3 CDRs contained in the light chain variable region (VL) are defined by Kabat, chothia or IMGT numbering system. In certain exemplary embodiments, the 3 CDRs contained in the heavy chain variable region (VH) and/or the 3 CDRs contained in the light chain variable region (VL) are defined by the IMGT numbering system.

In certain embodiments, the monoclonal antibody or antigen binding fragment thereof comprises:

(i) A heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:1, or a sequence as set forth 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 to SEQ ID NO:1 (e.g., substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids) as compared to a sequence having a substitution, deletion, or addition of one or more amino acids;

And/or the number of the groups of groups,

(ii) A light chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:2, or a sequence as set forth 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 to SEQ ID NO:2 (e.g., substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids) as compared to a sequence having a substitution, deletion, or addition of one or more amino acids.

In certain embodiments, the monoclonal antibody or antigen binding fragment thereof comprises: VH shown as SEQ ID NO. 1 and VL shown as SEQ ID NO. 2.

In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof is 5F5 or antigen-binding fragment thereof, a chimeric antibody thereof, or a humanized antibody thereof, or a variant thereof that substantially retains the biological function of the monoclonal antibody or antigen-binding fragment thereof from which it was derived.

In certain embodiments, the monoclonal antibody or antigen binding fragment thereof has one or more of the following biological functions:

(a) Specific binding to solid particles of CVB1 (infectious particles), e.g. as determined by ELISA;

(b) Substantially no binding or no binding to CVB1 hollow particles (non-infectious particles), e.g. as determined by ELISA;

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

(d) Diagnosing whether the subject is infected with CVB1 virus;

(e) Neutralizing CVB1 in vitro or in vivo in a subject (e.g., human); for example, CVB1 can be neutralized in vitro at a neutralization potency of no less than 6000 (e.g., 6000-10000, 7000-10000, 7000-9000, or 8000-9000, e.g., about 8100-8200), as determined, for example, by the neutralization assay described in example 7;

(f) Inhibit or block infection of cells by CVB1;

(g) Preventing and/or treating CVB1 infection or diseases related to CVB1 virus infection.

In certain embodiments, the monoclonal antibody or antigen binding fragment thereof comprises: the amino acid sequences are shown as VH CDR1-3 shown in SEQ ID NO 15-17 and/or VL CDR1-3 shown in SEQ ID NO 18-20.

In certain embodiments, the monoclonal antibody or antigen binding fragment thereof comprises: (a) 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO. 11; and/or (b) 3 CDRs contained in the light chain variable region (VL) as set forth in SEQ ID NO. 12. In certain embodiments, the 3 CDRs contained in the heavy chain variable region (VH) and/or the 3 CDRs contained in the light chain variable region (VL) are defined by Kabat, chothia or IMGT numbering system. In certain exemplary embodiments, the 3 CDRs contained in the heavy chain variable region (VH) and/or the 3 CDRs contained in the light chain variable region (VL) are defined by the IMGT numbering system.

(i) A heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:11, or a sequence as set forth 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 to SEQ ID NO:11 to sequences having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions);

and/or the number of the groups of groups,

(ii) A light chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:12, or a sequence as set forth 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 to SEQ ID NO:12 to sequences having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions).

In certain embodiments, the monoclonal antibody or antigen binding fragment thereof comprises: a VH shown as SEQ ID NO. 11 and a VL shown as SEQ ID NO. 12.

In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof is 8a10 or antigen-binding fragment thereof, a chimeric antibody thereof, or a humanized antibody thereof, or a variant thereof that substantially retains the biological function of the monoclonal antibody or antigen-binding fragment thereof from which it was derived.

(b) Specific binding to CVB1 hollow particles (non-infectious particles), e.g. as determined by ELISA;

(c) Detecting the presence or level of CVB1 virus (e.g., CVB1 solid particles and hollow particles) in a sample;

(d) Diagnosing whether the subject is infected with CVB1 virus;

(e) Neutralizing CVB1 in vitro or in vivo in a subject (e.g., human); for example, CVB1 can be neutralized in vitro at a neutralization potency of not less than 10000 (e.g., 10000-15000, 11000-15000, 12000-15000, 12000-14000, or 13000-14000, e.g., about 131000-131100), as determined by the neutralization assay described in example 7;

(f) Inhibit or block infection of cells by CVB 1;

In certain embodiments, the monoclonal antibody or antigen binding fragment thereof comprises: the amino acid sequences are shown as VH CDR1-3 shown in SEQ ID NO 25-27 and/or VL CDR1-3 shown in SEQ ID NO 28-30.

In certain embodiments, the antibody or antigen binding fragment thereof comprises: (a) 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO. 21; and/or (b) 3 CDRs contained in the light chain variable region (VL) as set forth in SEQ ID NO. 22. In certain embodiments, the 3 CDRs contained in the heavy chain variable region (VH) and/or the 3 CDRs contained in the light chain variable region (VL) are defined by Kabat, chothia or IMGT numbering system. In certain exemplary embodiments, the 3 CDRs contained in the heavy chain variable region (VH) and/or the 3 CDRs contained in the light chain variable region (VL) are defined by the IMGT numbering system.

(i) A heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:21, or a sequence as set forth 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 to SEQ ID NO:21 to sequences having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions);

And/or the number of the groups of groups,

(ii) A light chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:22, or a sequence as set forth 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 to SEQ ID NO:22 (e.g., substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids).

In certain embodiments, the monoclonal antibody or antigen binding fragment thereof comprises: VH shown as SEQ ID NO. 21 and VL shown as SEQ ID NO. 22.

In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof is 9A3 or antigen-binding fragment thereof, a chimeric antibody thereof, or a humanized antibody thereof, or a variant thereof that substantially retains the biological function of the monoclonal antibody or antigen-binding fragment thereof from which it was derived.

(d) Diagnosing whether the subject is infected with CVB1 virus;

(e) Neutralizing CVB1 in vitro or in vivo in a subject (e.g., human); for example, the CVB1 can be neutralized in vitro at a neutralization potency of not less than 1000 (e.g., 1000-5000, 1000-4000, 1000-3000, or 2000-3000, e.g., about 2000-2100), as determined, for example, by the neutralization assay described in example 7;

(f) Inhibit or block infection of cells by CVB1;

In certain embodiments, any of the monoclonal antibodies or antigen-binding fragments thereof described above further comprises a constant region sequence derived from a mammalian (e.g., murine or human) immunoglobulin or variant thereof having a substitution, deletion, or addition of one or more amino acids as compared to the wild-type sequence from which it was derived. In certain embodiments, the variant has a conservative substitution of one or more amino acids compared to the wild-type sequence from which it is derived.

In certain embodiments, the heavy chain of the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) of a murine immunoglobulin or variant thereof having one or more amino acid substitutions, deletions, or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) as compared to the wild-type sequence from which it is derived; and/or the number of the groups of groups,

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

In certain embodiments, the heavy chain of the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) of a human immunoglobulin or variant thereof having one or more amino acid substitutions, deletions, or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) as compared to the wild-type sequence from which it is derived; and/or the number of the groups of groups,

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

In certain embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, igG2, igG3, or IgG4 heavy chain constant region. In certain embodiments, the heavy chain constant region is a murine IgG1, igG2, igG3, or IgG4 heavy chain constant region. In certain embodiments, the heavy chain constant region is a human IgG1, igG2, igG3, or IgG4 heavy chain constant region.

In certain embodiments, the light chain constant region is a kappa light chain constant region. In certain embodiments, the light chain constant region is a murine kappa light chain constant region. In certain embodiments, the light chain constant region is a human kappa light chain constant region.

In certain embodiments, the antigen-binding fragment of any of the monoclonal antibodies described above is selected from the group consisting of scFv, di-scFv, (scFv) ₂ 、Fab、Fab’、(Fab’) ₂ Fv, or disulfide stabilized Fv (dsFv).

In certain embodiments, any of the monoclonal antibodies described above is a murine antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.

In the present application, a monoclonal antibody or antigen-binding fragment thereof of the application may comprise a variant that differs from the antibody or antigen-binding fragment thereof from which it is derived only by conservative substitutions of one or more (e.g., conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids), or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the antibody or antigen-binding fragment thereof from which it is derived, and substantially retains the biological function of the antibody or antigen-binding fragment thereof from which it is derived.

Derived antibodies

The antibodies or antigen binding fragments thereof of the application may be derivatized, e.g., linked to another molecule (e.g., another polypeptide or protein). In general, derivatization (e.g., labeling) of monoclonal antibodies or antigen-binding fragments thereof does not adversely affect their binding to CVB1 virus. Thus, the antibodies or antigen binding fragments thereof of the application are also intended to include such derivatized forms. For example, an antibody or antigen-binding fragment thereof of the application may be functionally linked (by chemical coupling, gene fusion, non-covalent linkage, or otherwise) 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 (e.g., avidin or polyhistidine tag) capable of mediating binding of the antibody or antigen-binding fragment to another molecule. Furthermore, the antibodies of the application or antigen binding fragments thereof may 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 antibodies, such as increasing serum half-life.

In certain embodiments, the monoclonal antibodies of the invention, or antigen binding fragments thereof, carry a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound), a fluorescent dye, a radionuclide, or biotin.

In this context, a detectable label according to the invention may be any substance that is detectable by fluorescence, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, examples of which include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), 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 substances (e.g., chemiluminescent substances, such as acridine esters), magnetic beads (e.g.,) A calorimetric label such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to the label-modified avidin (e.g., streptavidin) described above.

In certain embodiments, the detectable label can be suitable for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.).

In certain embodiments, a detectable label as described above may be attached to an antibody or antigen binding fragment thereof of the invention by linkers of different lengths to reduce potential steric hindrance.

Preparation of antibodies

The antibodies of the invention may be prepared by various methods known in the art, for example, by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then the host cell is transfected. The transfected host cells are then cultured under specific conditions and express the antibodies of the invention.

Antigen binding fragments of the invention may be obtained by hydrolysis of 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 be produced directly 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, fab' fragments can be obtained directly from the host cell; fab 'fragments can be chemically coupled to form F (ab') ₂ Fragments (Carter et al, bio/Technology,10:163-167 (1992)). In addition, fv, fab or F (ab') ₂ Fragments may also be isolated directly from recombinant host cell culture broth. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.

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

In another aspect, the invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the invention. In certain embodiments, the vectors of the invention are, for example, plasmids, cosmids, phages and the like.

In another aspect, the invention provides a host cell comprising an isolated nucleic acid molecule of the invention or a vector of the invention. Such host cells include, but are not limited to, prokaryotic cells, such as E.coli cells, and eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.). In certain embodiments, the host cell of the invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DG 44).

In another aspect, there is provided a method of producing an antibody or antigen-binding fragment thereof of the invention comprising culturing a host cell of the 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.

Detection method and kit

The monoclonal antibodies or antigen binding fragments thereof of the invention are capable of specifically binding to CVB1 virus and thus can be used to detect the presence or level of CVB1 virus in a sample and optionally to diagnose whether a subject is infected with CVB1 virus based on the presence or level of CVB1 virus in the sample.

Thus, in another aspect, the invention provides a kit comprising a monoclonal antibody of the invention or an antigen-binding fragment thereof.

In certain embodiments, the antibodies or antigen binding fragments thereof of the invention bear a detectable label. In certain embodiments, the kit further comprises a secondary antibody that specifically recognizes the monoclonal antibody of the invention or antigen-binding fragment thereof. In certain embodiments, the secondary antibody further comprises a detectable label.

In certain exemplary embodiments, the kit comprises: an antibody of the invention or antigen binding fragment thereof with a detectable label. In certain exemplary embodiments, the kit comprises: the antibodies of the invention or antigen-binding fragments thereof (e.g., without a detectable label), and secondary antibodies with a detectable label.

In certain embodiments, the kit comprises a first antibody and a second antibody capable of specifically binding to CVB1, the first and second antibodies being different from each other; wherein,,

the first antibody is selected from monoclonal antibodies or antigen binding fragments thereof comprising the following CDRs: VH CDR1-3 with amino acid sequence shown as SEQ ID NO 5-7 and/or VL CDR1-3 with amino acid sequence shown as SEQ ID NO 8-10;

the second antibody is selected from an additional monoclonal antibody or antigen-binding fragment thereof, or a polyclonal antibody, capable of specifically binding to CVB 1.

In certain embodiments, the polyclonal antibody is an antiserum comprising polyclonal antibodies. The antiserum refers to serum of an immunized animal (such as a non-human mammal, e.g., a mouse, a rabbit, a sheep, etc.) immunized with the CVB1 virus, and the serum contains polyclonal antibodies against CVB 1.

In certain embodiments, the second antibody is selected from a monoclonal antibody or antigen-binding fragment thereof comprising the following CDRs:

(i) VH CDR1-3 with amino acid sequence shown as SEQ ID NO 15-17, and/or VL CDR1-3 with amino acid sequence shown as SEQ ID NO 18-20; or alternatively

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

In certain embodiments, the second antibody is detectably labeled. In certain embodiments, the detectable label is selected from the group consisting of an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound), a fluorescent dye, a radionuclide, or biotin.

In certain embodiments, the kit further comprises a solid support. In certain embodiments, the solid support comprises a well plate, tube, bead (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 bead pre-coated with a functional group (e.g., amino, carboxyl, biotin, or avidin). In certain embodiments, the solid support is selected from magnetic beads or microtiter plates (e.g., microwell plates or elisa plates).

In certain embodiments, the first antibody is coated on the surface of a solid support.

In certain exemplary embodiments, the kit comprises: a primary antibody, and a secondary antibody with a detectable label. In certain exemplary embodiments, the kit comprises: a primary antibody coated on the surface of the solid support, and a secondary antibody with a detectable label.

In certain embodiments, the kit may further comprise reagents for causing the detection of the corresponding detectable label. For example, when the detectable label is an enzyme, the kit may further comprise a chromogenic substrate for the corresponding enzyme, such as o-phenylenediamine (OPD), tetramethyl benzidine (TMB), ABTS, or luminol for horseradish peroxidase, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase. The kit may further comprise a pre-excitation and/or excitation liquid for chemiluminescence, for example when the detectable label is a chemiluminescent reagent, such as an acridine ester compound.

In another aspect, the invention provides a method of detecting the presence or level of a CVB1 virus in a sample comprising using a monoclonal antibody or antigen binding fragment thereof according to the invention.

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

In certain embodiments, the methods are used to detect solid particles and hollow particles of CVB1 virus. In certain embodiments, the monoclonal antibody or antigen binding fragment thereof comprises: (i) VH CDR1-3 with amino acid sequence shown as SEQ ID NO 15-17, and/or VL CDR1-3 with amino acid sequence shown as SEQ ID NO 18-20; or (ii) VH CDR1-3 with the amino acid sequences shown in SEQ ID NO 25-27 and/or VL CDR1-3 with the amino acid sequences shown in SEQ ID NO 28-30 respectively.

In certain embodiments, the assay is an immunological assay, such as an enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.

In certain embodiments, the monoclonal antibody or antigen binding fragment thereof comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound), a fluorescent dye, a radionuclide, or biotin. In certain embodiments, the method further comprises detecting the monoclonal antibody or antigen binding fragment thereof using a secondary antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound), a fluorescent dye, a radionuclide, or biotin). In certain embodiments, the method comprises: (1) Contacting the sample with a monoclonal antibody or antigen binding fragment thereof of the invention; (2) Detecting the formation of an antigen-antibody immune complex or detecting the amount of said immune complex. The formation of the immune complex indicates the presence of CVB1 virus or CVB1 viral particles.

In certain embodiments, the method is a double antibody sandwich method comprising the steps of:

(1) Contacting the sample with a first antibody selected from a monoclonal antibody or antigen binding fragment thereof comprising the CDRs: VH CDR1-3 with amino acid sequence shown as SEQ ID NO 5-7 and/or VL CDR1-3 with amino acid sequence shown as SEQ ID NO 8-10;

(2) Contacting the antibody-antigen complex with a second antibody capable of specifically binding to CVB1 selected from the group consisting of an additional monoclonal antibody or antigen binding fragment thereof, or a polyclonal antibody, capable of specifically binding to CVB1, to form an antibody-antigen-antibody complex; and

(3) Determining the amount of the antibody-antigen-antibody complex.

In certain embodiments, the polyclonal antibody is an antiserum comprising polyclonal antibodies. The antiserum refers to serum of an immunized animal (e.g., a non-human mammal such as a mouse, rabbit, sheep, etc.) immunized with the CVB1 virus, which contains polyclonal antibodies against CVB 1.

In certain embodiments, the methods may be used for diagnostic purposes, e.g., whether a subject is infected with a CVB1 virus may be diagnosed based on the presence or level of the CVB1 virus in a sample. In such embodiments, the sample may be a blood sample (e.g., whole blood, plasma, or serum), fecal matter, oral or nasal secretions, or alveolar lavage from a subject (e.g., a mammal, preferably a human).

In certain embodiments, the methods may be used for non-diagnostic purposes, e.g., the sample is not a sample from a subject, e.g., an oncolytic virus sample or a vaccine sample.

In certain embodiments, the subject is a mammal, e.g., a human.

In another aspect, there is provided the use of a monoclonal antibody or antigen binding fragment thereof of the invention in the manufacture 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.

In certain embodiments, the kit detects the presence or level of CVB1 virus in a sample by a detection method as described above and optionally diagnoses whether the subject is infected with CVB1 virus based on the detection result.

In certain embodiments, the sample is a blood sample (e.g., whole blood, plasma, or serum), fecal matter, oral or nasal secretions, or alveolar lavage from a subject (e.g., a mammal, preferably a human).

Therapeutic methods and pharmaceutical compositions

The monoclonal antibodies or antigen binding fragments thereof of the invention are useful for neutralizing CVB1 virus in vitro or in a subject, blocking or inhibiting infection of cells by CVB1 virus, and for preventing and/or treating CVB1 virus infection or a disease associated with CVB1 virus infection in a subject.

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

In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as an additional antiviral agent.

In certain embodiments, in the pharmaceutical composition, the monoclonal antibody or antigen-binding fragment thereof of the invention and the additional pharmaceutically active agent are provided as separate components or as components of a single composition. Thus, the antibody or antigen binding fragment thereof of the invention and the additional pharmaceutically active agent may be administered simultaneously, separately or sequentially.

In certain exemplary embodiments, the pharmaceutically acceptable carrier and/or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), dextrose solutions (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

In another aspect, the invention provides a method for neutralizing coxsackievirus B1 (CVB 1), comprising using a monoclonal antibody or antigen binding fragment thereof or a pharmaceutical composition of the invention. The methods can be used to neutralize CVB1 virus in vitro or in a subject (e.g., human).

In certain embodiments, the methods are used to neutralize the virulence of coxsackievirus B1 (CVB 1) in a sample. In certain embodiments, the method comprises: a sample comprising CVB1 virus is contacted with a monoclonal antibody or antigen-binding fragment thereof or a pharmaceutical composition of the invention.

In certain embodiments, the monoclonal antibody or antigen binding fragment thereof is used alone or in combination with another pharmaceutically active agent (e.g., another antiviral agent).

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

In certain embodiments, the monoclonal antibody or antigen binding fragment thereof is used alone or in combination with another pharmaceutically active agent (e.g., another antiviral agent). The antibody or antigen binding fragment thereof of the invention and the additional pharmaceutically active agent may be administered simultaneously, separately or sequentially.

In certain embodiments, the disease associated with CVB1 viral infection includes aseptic meningitis, myocarditis, meningoepithymitis, hand-foot-and-mouth disease, chest pain, or neonatal fulminant hepatitis with coagulation disorders.

In certain embodiments, the subject is a mammal, e.g., a human.

In another aspect, the invention relates to the use of a monoclonal antibody or antigen binding fragment thereof of the invention in the manufacture of a medicament for:

(1) Neutralizing the CVB1 virus in vitro or in a subject (e.g., human); and/or

(2) For preventing or treating a CVB1 viral infection or a disease associated with a CVB1 viral infection in a subject.

In certain embodiments, the subject is a mammal, e.g., a human.

The monoclonal antibody of the present invention or antigen-binding fragment thereof, or the pharmaceutical composition of the present invention may be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection and injectable concentrated solutions), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical compositions of the present invention should be sterile and stable under the conditions of manufacture and storage. One preferred dosage form is an injection. Such injections may be sterile injectable solutions. For example, sterile injectable solutions can be prepared by the following methods: the recombinant proteins of the present invention are incorporated in the necessary amount in a suitable solvent, and optionally, simultaneously with other desired ingredients (including, but not limited to, pH modifiers, surfactants, adjuvants, ionic strength enhancers, isotonicity agents, preservatives, diluents, or any combination thereof), followed by filter sterilization. In addition, the sterile injectable solutions may be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for convenient storage and use. Such sterile lyophilized powders may be dispersed in a suitable carrier prior to use, such as water for injection (WFI), water for bacteriostatic injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), dextrose solutions (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

The monoclonal antibodies, or antigen-binding fragments thereof, or pharmaceutical compositions of the invention may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powder, ointment, or drops), or nasal route. However, for many therapeutic uses, the preferred route/mode of administration is parenteral (e.g., intravenous injection or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the intended purpose. In a preferred embodiment, the monoclonal antibody or antigen-binding fragment thereof, or pharmaceutical composition of the invention is administered by intravenous injection or bolus injection.

The pharmaceutical compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of a monoclonal antibody of the invention, or an antigen-binding fragment thereof. "prophylactically effective amount" means an amount sufficient to prevent, arrest, or delay the onset of a disease. By "therapeutically effective amount" is meant an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of a monoclonal antibody or antigen binding fragment thereof of the invention may vary depending on the factors: the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

In the present invention, the dosing regimen may be adjusted to achieve the optimal target response (e.g., therapeutic or prophylactic response). For example, the dosage may be administered in a single dose, may be administered multiple times over a period of time, or may be proportionally reduced or increased as the degree of urgency of the treatment situation.

In the present invention, the subject may be a mammal, such as a human.

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the procedures of cell culture, biochemistry, nucleic acid chemistry, immunology laboratories and the like as used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "coxsackievirus B1 (CVB 1)" refers to one of the picornaviridae (Picomaviridae), enteroviruses (enteroviruses), coxsackievirus (coxsackievirus) group B whose genome is single-stranded positive strand RNA. Genomic or cDNA sequences of CVB1 are well known in the art and can be found in various public databases (e.g., genBank database accession number: MG 780414). There are two types of viral particles for CVB 1: hollow particles (no RNA, no infectivity), solid particles (RNA, infectivity), both viral particles are immunogenic.

As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having one Light Chain (LC) and one Heavy Chain (HC). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 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 3 domains (CH 1, CH2 and CH 3). 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. The constant domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as may mediate binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). VH and VL regions can also be subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each V is _H And V _L By the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable regions (VH and VL) of each heavy/light chain pair form antigen binding sites, respectively. The allocation of amino acids in various regions or domains may follow Kabat, sequences of Proteins ofImmunological 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.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. The exact boundaries of these amino acid residues may be defined according to various numbering systems known in the art, for example as in 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, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the 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).

In the present invention, the CDRs contained in the antibodies or antigen binding fragments thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained by an antibody or antigen binding fragment thereof of the invention are preferably determined by Kabat, chothia or IMGT numbering system. In certain embodiments, the CDRs contained in an antibody or antigen binding fragment thereof of the invention are preferably determined by the IMGT numbering system.

As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies may be of different isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.

As used herein, surgeryThe term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to an antigen, also referred to as an "antigen-binding portion. See generally Fundamental Immunology, ch.7 (Paul, W., ed., 2 nd edition, raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes, antigen binding fragments of antibodies may be generated by recombinant DNA techniques 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, diabodies (diabodies), linear antibodies (linear antibodies), nanobodies (technology from domanis), domain antibodies (technology from Ablynx) and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding capacity to the polypeptide. Engineered antibody variants are reviewed in Holliger et al, 2005; nat Biotechnol, 23:1126-1136.

As used herein, the term "full length antibody" means an antibody consisting of two "full length heavy chains" and two "full length light chains". Wherein "full length heavy chain" refers to 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-to C-terminal direction; and, when the full length antibody is an IgE isotype, optionally further comprises a heavy chain constant region CH4 domain. Preferably, a "full length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the N-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-to C-terminal direction. The two pairs of full length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between HR of the two full length heavy chains. The full length antibodies of the invention may be from a single species, e.g., human; chimeric or humanized antibodies are also possible. The full length antibodies of the invention comprise two antigen binding sites formed by VH and VL pairs, respectively, which specifically recognize/bind the same antigen.

As used herein, the term "Fd fragment" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al Nature 341:544 546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F (ab') ₂ Fragment "means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab 'fragment" means a reduction-linked F (ab') ₂ The resulting fragment after disulfide bonding of the two heavy chain fragments in the fragment consists of one complete light and heavy chain Fd fragment (consisting of VH and CH1 domains).

As used herein, the term "Fv fragment" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragment that forms the complete antigen binding site. It is believed that the six CDRs confer antigen binding specificity to the antibody. However, even one variable region (e.g., fd fragment, which contains only three CDRs specific for an antigen) is able to recognize and bind antigen, although its affinity may be lower than the complete binding site.

As used herein, the term "Fc fragment" means an antibody fragment formed by disulfide bonding of the second and third constant regions of a first heavy chain of an antibody with the second and third constant regions of a second heavy chain. The Fc fragment of an antibody has a number of different functions, but does not participate in antigen binding.

As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH domains are linked by a linker (linker) (see, e.g., bird et al, science 242:423-426 (1988); huston et al, proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckaphun, the Pharmacology of Monoclonal Antibodies, volume 113, roseburg and Moore, springer-Verlag, new York, pages 269-315 (1994)). Such scFv molecules may have the general structure: NH (NH) ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers are composed of repeated GGGGSAmino acid sequence or variant thereof. For example, a polypeptide having an amino acid sequence (GGGGS) can be used ₄ Variants thereof may be used (Holliger et al (1993), proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present 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, disulfide bonds may also exist between VH and VL of scFv. As used herein, the term "di-scFv" refers to an antibody fragment formed by the ligation of two scFv.

As used herein, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but uses a linker that is too short to allow pairing between two domains of the same chain, forcing the domains to pair with complementary domains of the other chain and creating two antigen binding sites (see, e.g., holliger p. Et al, proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. Et al, structures 2:1121-1123 (1994)).

Each of the above antibody fragments retains the ability to specifically bind to the same antigen to which the full-length antibody binds and/or competes with the full-length antibody for specific binding to the antigen.

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

In this context, unless the context clearly indicates otherwise, when referring to the term "antibody" it includes not only whole antibodies, but also antigen-binding fragments of antibodies.

As used herein, the terms "monoclonal antibody," "mAb," and "mAb" have the same meaning and are used interchangeably to refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have a high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies, which typically recognize different epitopes on an antigen. Furthermore, the modifier "monoclonal" merely indicates the character of the antibody as being obtained from a population of highly homologous antibodies, and is not to be construed as requiring preparation of the antibody by any particular method.

As used herein, the term "chimeric antibody (Chimeric antibody)" refers to an antibody in which a portion of the light chain or/and heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular class or subclass of antibody) and another portion of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or a different species or belong to the same or a different class or subclass of antibody), but which nevertheless retains binding activity for the antigen of interest (u.s.p 4,816,567to Cabilly et al.; morrison et al, proc.Natl. Acad.Sci.USA,81:6851 6855 (1984)). For example, the term "chimeric antibody" may include antibodies (e.g., human murine chimeric antibodies) in which the heavy and light chain variable regions of the antibody are from a first antibody (e.g., murine antibody) and the heavy and light chain constant regions of the antibody are from a second antibody (e.g., human antibody).

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

The chimeric or humanized antibody of the present invention can be prepared according to the sequence of the murine monoclonal antibody prepared as described above. DNA encoding the heavy and light chains can be obtained from a murine hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques.

To prepare chimeric antibodies, the murine immunoglobulin variable region can be linked to a human immunoglobulin constant region using methods known in the art (see, e.g., U.S. Pat. No.4,816,567 to Capilli et al). For example, the DNA encoding VH is operably linked to another DNA molecule encoding a 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, e.g., kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest, fifth Edition, U.S. device of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments comprising these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, igG2, igG3, igG4, igA, igE, igM or IgD constant region, but is generally preferably an IgG1 or IgG4 constant region. For example, DNA encoding VL is operably linked to another DNA molecule encoding a light chain constant region CL to obtain a full length light chain gene (as well as Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, e.g., kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest, fifth Edition, U.S. device of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments comprising these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region, but is generally preferred.

To prepare humanized antibodies, murine CDR regions can be inserted into a human framework sequence using methods known in the art (see U.S. Pat. No.5,225,539 to Winter; U.S. Pat. No.5,530,101 to Queen et al; U.S. Pat. Nos.5,585,089; 5,693,762 and 6,180,370; and Lo, benny, K.C., editor, in Antibody Engineering: methods and Protocols, volume 248,Humana Press,New Jersey,2004).

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction can be determined by the equilibrium dissociation constant (K _D ) And (3) representing. In the present invention, the term "K _D "refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen. Specific binding properties between two molecules can be determined using methods well known in the art, for example, using Surface Plasmon Resonance (SPR) in a BIACORE instrument.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as e.g. escherichia coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or human cells.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (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), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be conveniently performed using, for example, a computer program such as the Align program (DNAstar, inc.) Needleman et al (1970) j.mol.biol.48: 443-453. The percent identity between two amino acid sequences can also be determined using the algorithms of E.Meyers and W.Miller (Comput. Appl biosci.,4:11-17 (1988)) which have been integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), the gap length penalty of 12 and the gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (JMoI biol.48:444-453 (1970)) algorithm that has been incorporated into the GAP program of the GCG software package (available on www.gcg.com), using the Blossum 62 matrix or PAM250 matrix, and GAP weights (GAP weights) of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the desired properties of a protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, such as substitutions with residues that are physically or functionally similar (e.g., of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. 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, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., 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).

The twenty conventional amino acids referred to herein are written following conventional usage. See, e.g., immunology-a Synthesis (2nd Edition,E.S.Golub and D.R.Gren,Eds, sinauer Associates, sundland, mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally indicated by single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active ingredient, which is well known in the art (see, e.g., remington's Pharmaceutical sciences. Mediated by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and includes, but is not limited to: pH modifiers, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, 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, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugar, naCl, and the like. Agents that delay absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning commonly understood by those skilled in the art and are capable of stabilizing the desired activity of the active ingredient in a medicament, including but not limited to sodium glutamate, gelatin, SPGA, saccharides (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin or casein) or degradation products thereof (e.g., lactalbumin hydrolysate), and the like. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), dextrose solutions (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

As used herein, the term "preventing" refers to a method that is performed in order to prevent or delay the occurrence of a disease or disorder or symptom in a subject. As used herein, the term "treatment" refers to a method that is performed in order to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., no longer worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and diminishment of symptoms (whether partial or total), whether detectable or undetectable. Furthermore, "treatment" may also refer to an extension of survival compared to the expected survival (if not treated).

As used herein, the term "subject" refers to a mammal, such as a primate mammal, e.g., a human.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, a disease-preventing effective amount refers to an amount sufficient to prevent, or delay the onset of a disease; a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determination of such effective amounts is well within the ability of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

As used herein, the term "neutralizing activity" refers to the functional activity of an antibody or antibody fragment that binds to an antigenic protein on a virus, thereby preventing the maturation of virus-infected cells and/or virus progeny and/or the release of virus progeny, and an antibody or antibody fragment having neutralizing activity may prevent the amplification of a virus, thereby inhibiting or eliminating the infection by a virus.

Advantageous effects of the invention

The monoclonal antibody or antigen binding fragment provided by the invention has good affinity to CVB1 solid particles and/or hollow particles, and can specifically detect CVB1 viruses or virus particles. In addition, the monoclonal antibodies or antigen binding fragments of the invention are also capable of neutralizing the virulence of CVB1, thereby being useful in the prevention and treatment of CVB1 infection and diseases associated with CVB1 infection. Thus, the monoclonal antibodies or antigen binding fragments of the invention are of great clinical value.

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Drawings

FIG. 1 shows the results of transmission electron microscopy analysis of CVB1 different types of viral particles separated using sucrose gradient density centrifugation.

FIG. 2 shows the binding activity of different monoclonal antibodies to CVB1 virus solid particles and hollow particles.

FIG. 3 shows the results of detection of CVB1 virus particles by a double antibody sandwich ELISA method based on monoclonal antibody 5F 5.

FIG. 4 shows the results of immunofluorescence assays based on monoclonal antibodies 5F5, 8A10 and 9A3 on CVB1 virus infected cells.

Figure 5 shows the evaluation of the prophylactic activity of monoclonal antibodies 5F5, 8a10 and 9A3 on CVB1 infection in a mouse model.

Figure 6 shows the evaluation of the therapeutic activity of monoclonal antibodies 5F5, 8a10 and 9A3 on CVB1 infection in a mouse model.

Sequence information

The information of the partial sequences to which the present invention relates is provided in table 1 below.

Table 1: description of the sequence

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, molecular biology experimental methods and immunoassays used in the present invention are basically described in j.sambrook et al, molecular cloning: laboratory Manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, fine-compiled guidelines for molecular biology experiments, 3 rd edition, john Wiley & Sons, inc., 1995; the use of restriction enzymes was in accordance with the conditions recommended by the manufacturer of the product. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.

Example 1: preparation of CVB1 Virus particles

1.1 cultivation of viruses:

the CVB1 strain used in this example was 301 (GenBank No. MT129657), and the cultured virus cells were human rhabdomyoma cells RD @CCL-136 ^TM ). RD cells are first cultured in 10cm dishes (NEST) in MEM medium (GIBCO) containing 10% fetal bovine serum (PAA). When the cell confluency reached 80%, the medium was changed to serum-free MEM medium and the virus was inoculated in an amount of moi=0.1. After culturing at 37℃for 3 days, the virus was harvested after the cells had been completely diseased. The virus harvesting method comprises the following steps: scraping the cells, freeze thawing for 3 times, centrifuging to remove cell fragments, collecting cell lysis supernatant, filtering with 0.22 μm filter membrane to obtain virus stock solution, and freezing at-80deg.C.

1.2 sucrose Density gradient centrifugation to obtain viral particles of different natures:

the virus stock obtained above was concentrated and precipitated by PEG8000 at 6% PEG6000,0.3mol/L NaCl overnight at 4deg.C. The pellet was then centrifuged at 10000g and resuspended in PBS. Then sucrose gradient centrifugation is carried out, the sucrose gradient is 15% -50%, SW41 Ti is rotated by 120000g for centrifugation for 2.5h, and samples are respectively taken for electron microscope negative staining observation. As shown in FIG. 1, sucrose gradient centrifugation can effectively separate solid particles and hollow particles of CVB1 virus.

Example 2: preparation of monoclonal antibodies

The CVB1 virus stock solution prepared in example 1 is mixed with Freund's complete adjuvant and emulsified uniformly, and BALB/c female mice with 6-8 weeks of age are subjected to multi-point injection, wherein the immunogen is injected by back subcutaneous injection, inguinal subcutaneous injection, foot pad injection, limb intramuscular injection and other routes, and the injection dose is 500 mu L/time. Boosting was then performed in the same manner, with each 2 week-old boost, and the immunogen was a mixture of CVB1 virus stock prepared in example 1 and Freund's incomplete adjuvant, and 20. Mu.L tail venous blood or 200. Mu.L eyeball venous blood was collected for titer determination prior to each boost. Serum titers were determined by indirect ELISA, and when the mice reached the plateau, the mice stopped immunization and were allowed to rest for two months before fusion. 72h before fusion, 100 mu L of CVB1 virus stock solution (without adjuvant) is directly injected into the spleen of the mouse for immunization enhancement. After 72h, mice were sacrificed to collect mouse antisera (mouse polyclonal antisera) while the spleens of the mice were taken as a cell suspension (suspended in RPMI 1640 medium) and counted by cell counting plate. The cells were mixed with mouse myeloma cells Sp2/0 in an amount of 1/6 of the spleen cells, followed by cell fusion with 50% polyethylene glycol (PEG). Mixing the cell suspension with equal volume of feeder cells (BALB/c mouse macrophage and thymus cells), placing into 96-well cell culture plate (200 μL/well), 5% CO ₂ 37 ℃. After 3 days, medium half-changes were made with 1640 medium containing hypoxanthine, aminopterin and thymidine. Seven days later, ELISA plates were coated with CVB1 virus stock and hybridoma cell culture supernatants were assayed by indirect ELISA. For ELISA positive cell wells, cloning was performed using limiting dilution method.

Purification of monoclonal antibodies: healthy BALB/c mice of 10 weeks of age were given an intraperitoneal injection of paraffin oil, 0.5mL each. After 2-7 days, the cloned hybridoma cells were collected, centrifuged to remove the supernatant, and the serum-free 1640HT medium was added to adjust the cell density to 2X 10 ⁵ -2×10 ⁶ Each mouse was injected with 0.5 mL/mL. After 7-10 days, the abdomen of the mice was enlarged and ascites was collected. Centrifuge at 3000rpm for 15min, aspirate the intermediate clear liquid and preserve at-20 ℃. And (5) purifying the antibody after the ascites is collected. The ascites was diluted twice with 0.02mol/L PBS pH7.4, and an equal volume of saturated ammonium sulfate was slowly added with stirring overnight at 4 ℃. Centrifuge at 12000rpm for 15min at 4℃and discard supernatant. Dissolving the precipitate in PBS, placing into dialysis bag, placing into 0.02mol/L PB (pH 7.4) of 50-100 times volume, and desalting at 4deg.C under stirring for about 12 hr, and changing dialysate for 3 times. Purifying the dialyzed antibody by using a Protein A column (GE) under an AKTA purification system, carrying out affinity column hanging under the condition of 0.02mol/L PB (pH 7.4), eluting by using 0.1M citric acid, and obtaining the eluent which is the purified antibody. The eluate was dialyzed into 0.02mol/L PB (pH 7.4) and stored at-20 ℃.

Example 3: screening of monoclonal antibodies specifically binding to CVB1 Virus particles

ELISA assay (indirect): CVB1 virus solid particle antigen and hollow particle antigen prepared in example 1 were diluted 100-fold with 20mM PB7.4, respectively, coated with 96-well ELISA plates, 100. Mu.L/well, coated 2h at 37℃and washed 1 time with PBST, and non-specific binding sites blocked with relevant blocking solution (20 mM PB7.4 containing 150mM NaCL,0.5% casein, 0.002% gelatin), 200. Mu.L/well, blocked overnight at 4 ℃. A sample of the monoclonal antibody to be tested (0.1 mg/mL) prepared in example 2 was taken and 100. Mu.L was added to the ELISA plate and incubated at 37℃for 1h. PBST plates were washed 5 times, GAM-HRP (horseradish peroxidase-labeled goat anti-mouse antibody, available from bio-rad, USA, diluent formulation with blocking solution) was added, and incubated at 37℃for 30min. PBST plates were washed 5 times, developed for 15min with TMB developing solution, stopped with stop solution, and read with a microplate reader (TECAN sunrise). And finally screening more than 30 monoclonal antibodies to obtain 3 monoclonal antibodies 5F5, 8A10 and 9A3 capable of specifically binding CVB1 virus particles, wherein the screening results of part of antibodies are shown in figure 2, 5F5 can specifically bind CVB1 solid particles without obvious reaction with hollow particles, 8A10 and 9A3 can specifically bind CVB1 virus solid particles and hollow particles, and the binding activity is obviously better than that of other monoclonal antibodies.

Variable region sequence analysis was further performed on mab 5F5, 8a10 and 9A3, whose VH and VL sequences are shown in the following table. Further, CDR sequences of three monoclonal antibodies were determined based on the IMGT database (http:// www.imgt.org/IMGT_vquest/analysis).

Table 2: monoclonal antibody variable region sequences

Example 4: establishment of CVB1 solid particle antigen detection method

In the embodiment, 5F5 is used as the coating monoclonal antibody, and a double-antibody sandwich ELISA detection method is established to detect CVB1 solid particle antigen.

4.1 preparation of horseradish peroxidase-labeled antibodies:

taking HRP and NaIO ₄ 1mg each of the above-mentioned materials are dissolved by adding ultrapure water; then drop wise adding NaIO to HRP solution ₄ Shaking and mixing the solution while adding the solution, and placing the mixed solution at 4 ℃ for 30min in a dark place; dissolving 1 mu L of ethylene glycol in ultrapure water, dropwise adding, shaking and mixing uniformly while adding, and standing at room temperature in a dark place for 30min, so that the enzyme oxidation process is completed. During oxidation of HRP, purified antibodies were dialyzed against 50mM CB buffer, ph 9.6. After the oxidation of HRP, mixing the dialyzed antibody with HRP according to the required proportion, and dialyzing in 50mM CB buffer for more than 6 hours; then using freshly prepared NaBH ₄ Solution termination, naBH ₄ The amount was 0.2mg; shaking up and standing for 2h at 4 ℃. Then dialyzed overnight against 10mM PBS pH 7.2.

Three HRP-labeled antibodies were obtained by the above method: polyclonal-HRP, murine polyclonal antibody conjugated to CVB1 virus particles (mouse polyclonal antisera prepared in example 2); 8A10-HRP, monoclonal antibody binding to CVB1 viral particles (8A 10 selected in example 3); 9A3-HRP, monoclonal antibody binding to CVB1 viral particles (9A 3 selected in example 3).

4.2ELISA experiments (double antibody sandwich ELISA)

The 5F5 mab was diluted to a concentration of 0.1. Mu.g/mL with 20mM PB7.4, coated on 96-well ELISA plates, 100. Mu.L/well, and coated for 2h at 37 ℃. Plates were washed 1 time with PBST and non-specific binding sites were blocked with relevant blocking solution (20 mM PB7.4 with 150mM NaCl,0.5% casein, 0.002% gelatin), 200. Mu.L/well, blocked overnight at 4 ℃. Different CVB1 virus samples were diluted 100-fold with blocking solution, and ELISA plates were added at 100. Mu.L/well and incubated for 1h at 37 ℃. The PBST plates were washed 5 times, and the obtained HRP-labeled antibodies (polyclonal-HRP, 8A10-HRP or 9A 3-HRP) were prepared in 4.1 and incubated at 37℃for 30min. PBST plates were washed 5 times, developed for 15min with TMB developing solution, stopped with stop solution, and read with a microplate reader (TECAN sunrise). As a result, as shown in FIG. 3, 5F5 was able to detect CVB1 solid particle antigen, but not hollow particle antigen, regardless of whether it was paired with polyclonal antibody or monoclonal antibody. The results show that the double-antibody sandwich ELISA method using 5F5 as the coating antibody can be used for detecting CVB1 solid particle antigen.

Example 5: identification of antibody subtypes

CVB1 virus solid particle antigen prepared in example 1 was diluted 100-fold with 20mM PB7.4, coated with 96-well ELISA plate, 100. Mu.L/well, coated 2h at 37℃and washed 1 time with PBST, and the non-specific binding sites blocked with relevant blocking solution (20 mM PB7.4 containing 150mM NaCL,0.5% casein, 0.002% gelatin), 200. Mu.L/well, blocked overnight at 4 ℃. 100 mu L of the monoclonal antibody diluted by 500 times is added to an ELISA plate, and incubated for 1h at 37 ℃. PBST plates were washed 5 times, and goat anti-mouse secondary antibodies (Thermo) labeled HRP, anti-IgG 1, igG2a, igG2b, igG3, and IgM were added, respectively. Incubate at 37℃for 30min. PBST plates were washed 5 times, developed for 15min with TMB developing solution, stopped with stop solution, and read with a microplate reader (TECAN sunrise). The results showed that mab 5F5, 8a10 and 9A3 were all of the IgG2a subtype.

Example 6: monoclonal antibodies 5F5, 8A10 and 9A3 for detection of CVB1 infected cells

RD cells were plated in 24-well cell culture plates (500. Mu.L, 5X 10) ⁴ Well), a circular cover slip is laid in the well in advance. After the cells are attached, the CVB1 virus is inoculated with 5000TCID ₅₀ Well, cell blank control was also set. After 12h, the supernatant was aspirated with a gun tip,wash once with PBS wash, 1 mL/well. The PBS was removed by suction, 4% paraformaldehyde was added, 1 mL/well, and the mixture was fixed at room temperature in a dark place for 15min. 0.5% Triton X-100 (PBS) was added and the mixture was allowed to permeate, 1 mL/well, at room temperature for 10min. PBS was washed three times, each for 3min. A sealing film is leveled and fixed on a 24-hole cell culture plate cover, and 50 mu L of goat serum is dripped on the sealing film corresponding to each hole. After addition, the cover glass with the cells laid is lifted by a curved needle, the edge is clamped by forceps, the cells face down on the membrane, and the wet box is placed at 37 ℃ for 1h. Coverslips were washed three times with PBS for 3min each in an original Kong Fanghui well plate with the cell side facing upwards. Antibody (1 mg/mL) was diluted 200-fold with 2% BSA and added to the cells (procedure and blocking procedure), blocking was performed at 37℃for 1h. Coverslips were washed three times with PBS for 3min each in an original Kong Fanghui well plate with the cell side facing upwards. Secondary antibody GAM-FITC (Sigma) was diluted with 2% BSA, 1:500-fold dilution, and the wet cassette incubated at 37℃for 30min in the dark (procedure same as blocking). Coverslips were washed three times with PBS for 3min each in an original Kong Fanghui well plate with the cell side facing upwards. DAPI (Invitrogen) was diluted 1:2000 with PBS and applied to the cells (procedure with blocking procedure), and protected from light at room temperature for 5min. Coverslips were washed three times with PBS for 3min each in an original Kong Fanghui well plate with the cell side facing upwards. And (5) light shielding. Marking on clean glass slide, dripping about 30 μl of sealing tablet (70% glycerol, 2.5% anti-killing agent) on the cleaned glass slide, covering with cover glass with cells, sealing edge of nail oil, and air drying in dark. Observed under a fluorescence microscope and photographed. As a result, as shown in FIG. 4, the cells infected with CVB1 virus showed green fluorescence, while the blank control showed no green fluorescence. The above results indicate that mab 5F5, 8a10 and 9A3 can be used to detect CVB1 infected cells.

Example 7: neutralizing titers of monoclonal antibodies 5F5, 8a10 and 9A3

Neutralization experiment: the neutralizing activity of hybridoma cell culture supernatants or crude pure antibodies was identified by conventional neutralization assay methods. Human rhabdomyosarcoma cells (RD) were plated in 96-well cell culture plates (5X 10) ³ /hole). After 10h, the samples to be tested were diluted with serum-free MEM medium (starting from 8-fold dilution, 2-fold dilution, 10 gradients) and at least 4 wells were repeated for each sample. With serum-freeMEM dilutes CVB1 Virus to 100TCID ₅₀ 50. Mu.L was added to the gradient diluted mAb sample wells. After incubation at 37℃for 1h, 100. Mu.L of the mab-virus mixture was added to a 96-well cell culture plate pre-plated with RD cells. 37 ℃,5% CO ₂ Cultures were observed for 7 consecutive days and cytopathic effects were recorded. The maximum dilution factor that can inhibit cell pore lesions by more than 50% is taken as the neutralization titer of the sample. The results show that the monoclonal antibodies 5F5, 8A10 and 9A3 have higher in-vitro neutralization activity, and the neutralization titers are respectively about 8192, 131072 and 2048.

Example 8: monoclonal antibodies 5F5, 8A10 and 9A3 experiments to prevent CVB1 infection in mice

The experimental neonates were grouped as follows: one day-old BALB/c mice were selected, and an antibody-preventive group and a PBS control group were set up, each group consisting of 15 animals. After the newborn mice are starved for 4 hours, monoclonal antibodies (5F 5, 8A10 and 9A 3) are injected into the abdominal cavity, and the dosage is 30 mug/mouse; the PBS control group was intraperitoneally injected with the same volume of PBS. After 4h, the newborn mice of the antibody preventive group and the PBS control group were intraperitoneally infected with CVB1 virus at a challenge dose of 100TCID ₅₀ . All animals were observed continuously for 20 days.

The experimental results are shown in fig. 5, and the PBS control mice mostly die 7 days after challenge; while 5F5, 8A10 and 9A3 were normal in preventing the group symptoms, no abnormality was seen. It was demonstrated that mab 5F5, 8a10 and 9A3 all have the effect of preventing CVB1 infection.

Example 9: mouse in vivo therapeutic experiments with monoclonal antibodies 5F5, 8A10 and 9A3

Because the monoclonal antibodies 5F5, 8A10 and 9A3 have higher neutralizing activity on CVB1 viruses, the monoclonal antibodies can protect CVB1 infected mice from morbidity when used in animal model tests for treating CVB1 infection.

The experimental neonates were grouped as follows: one day-old BALB/c mice were selected, and an antibody protection group and a PBS control group were set up, each group being 15. After 4h of hunger of the newborn mice, the newborn mice of the antibody protection group and the PBS control group are injected into the abdominal cavity to infect the CVB1 virus, and the attacking dose is 100TCID ₅₀ . The antibody protection group is subjected to intraperitoneal injection of monoclonal antibodies (5F 5, 8A10 and 9A 3) 24 hours after toxin attack, and the dosage is 30 mug/patient; same volume of PBS control group intraperitoneal injectionIs not shown). All animals were observed continuously for 20 days.

The experimental results are shown in fig. 6, wherein the PBS control group mice have emaciation symptoms, hind limb paralysis symptoms occur 4 days after toxin expelling, and most of the mice die 7 days after toxin expelling; in contrast, mab 5F5, 8a10 and 9A3 protected the group symptoms were all normal, no abnormalities were seen. It was demonstrated that mab 5F5, 8a10 and 9A3 were effective in treating mice infected with CVB 1.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims

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

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof comprises: as set forth in SEQ ID NO:21 and a heavy chain variable region (VH) as set forth in SEQ ID NO:22, and a light chain variable region (VL).

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the monoclonal antibody is selected from the group consisting of murine antibodies, chimeric antibodies, humanized antibodies, or multispecific antibodies; and/or the antigen binding fragment is selected from Fab, fab ', F (ab') 2, fd, fv, dAb, a complementarity determining region fragment, or a single chain antibody (e.g., scFv).

4. The monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 1-3, wherein the antibody is an IgG antibody (e.g., an IgG1, igG2, igG3, or IgG4 antibody).

5. The monoclonal antibody or antigen binding fragment thereof according to any one of claims 1-4, wherein the monoclonal antibody or antigen binding fragment thereof carries a detectable label, such as an enzyme (e.g. horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g. an acridine ester compound), a fluorescent dye, a radionuclide or biotin.

6. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-4, or a heavy chain variable region and a light chain variable region thereof.

7. A vector comprising the nucleic acid molecule of claim 6; preferably, the vector is a cloning vector or an expression vector.

8. A host cell comprising the nucleic acid molecule of claim 6 or the vector of claim 7.

9. A method of making the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4, comprising culturing the host cell of claim 8 under conditions that allow expression of the monoclonal antibody or antigen-binding fragment thereof, and recovering the monoclonal antibody or antigen-binding fragment thereof from the cultured host cell culture.

10. A kit comprising the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-5;

preferably, the monoclonal antibody or antigen binding fragment thereof comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound), a fluorescent dye, a radionuclide, or biotin; alternatively, the kit further comprises a secondary antibody that specifically recognizes the monoclonal antibody or antigen-binding fragment thereof; optionally, the secondary antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound), a fluorescent dye, a radionuclide, or biotin.

11. A method for detecting the presence or level of coxsackievirus B1 (CVB 1) in a sample for non-diagnostic purposes comprising using the monoclonal antibody or antigen binding fragment thereof of any one of claims 1-5;

preferably, the assay is an immunological assay, such as an enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay;

Preferably, the method is used to detect solid particles and hollow particles of CVB1 virus;

preferably, the monoclonal antibody or antigen binding fragment thereof further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound), a fluorescent dye, a radionuclide, or biotin; alternatively, the method further comprises detecting the monoclonal antibody or antigen binding fragment thereof using a secondary antibody bearing a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound), a fluorescent dye, a radionuclide, or biotin).

12. Use of the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-5 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;

preferably, the sample is a blood sample (e.g., whole blood, plasma or serum), fecal matter, oral or nasal secretions, or alveolar lavage from a subject (e.g., a mammal, preferably a human).

13. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4, and a pharmaceutically acceptable carrier and/or excipient;

preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as an additional antiviral agent.

14. Use of the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4 for the preparation of a medicament for neutralizing virulence of CVB1 virus in a sample, or for preventing or treating a CVB1 viral infection or a disease associated with a CVB1 viral infection in a subject;

preferably, the monoclonal antibody or antigen binding fragment thereof is used alone or in combination with another pharmaceutically active agent (e.g., another antiviral agent);

preferably, the disease associated with CVB1 viral infection includes aseptic meningitis, myocarditis, meningoepithymitis, hand-foot-and-mouth disease, chest pain, or neonatal fulminant hepatitis with coagulation disorders;

preferably, the subject is a mammal, such as a human.