CN107674123B - Anti-idiotype antibody and application thereof - Google Patents

Abstract

The present invention relates to an anti-idiotype antibody and its application. Specifically, the present invention relates to an anti-idiotypic antibody specific for broad-spectrum neutralizing monoclonal antibodies against influenza B virus (eg, monoclonal antibody 11B10), a cell line producing the anti-idiotype antibody, the anti-idiotype antibody Methods of preparing anti-idiotypic antibodies, and compositions (eg, pharmaceutical compositions or vaccines) comprising the anti-idiotypic antibodies. Furthermore, the present invention also relates to the use of said anti-idiotypic antibodies. The anti-idiotypic antibodies of the invention can be used to prevent and/or treat influenza virus infection and/or diseases caused by such infection (eg, influenza).

Description

Anti-idiotype antibody and application thereof

Technical Field

The present invention relates to the fields of immunology and molecular virology, in particular the field of the prevention and treatment of influenza viruses. In particular, the present invention relates to an anti-idiotype antibody specific for a broadly neutralizing monoclonal antibody to influenza B virus (e.g., monoclonal antibody 11B10), a cell strain producing the anti-idiotype antibody, a method of making the anti-idiotype antibody, and a composition (e.g., a pharmaceutical composition or a vaccine) comprising the anti-idiotype antibody. Furthermore, the invention relates to the use of said anti-idiotype antibody. The anti-idiotype antibodies of the invention are useful for the prevention and/or treatment of infection by influenza virus and/or diseases caused by said infection (e.g. influenza).

Background

The influenza virus is an enveloped, single-stranded negative-sense RNA virus of the family Orthomyxoviridae (Orthomyxoviridae), genus influenza. Its genome is composed of 8 segmentalized RNAs and codes more than eleven kinds of virus proteins. Influenza viruses are classified into types A (A), B (B) and C (C) (Horimoto T. et al, Nat Rev Microbiol,2005,3(8):591-600) according to the difference in antigenicity and genetic characteristics of the viral Nucleoprotein (NP) and matrix protein (M). Influenza a Virus (Flu a) has a wide range of hosts and rapid variation, and can cause a pandemic worldwide. Further, influenza A viruses are classified into 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11) according to the antigenicity of the virus envelope glycoproteins Hemagglutinin (HA) and Neuraminidase (NA), wherein the subtypes other than the H1N1 and H3N2 subtypes that are seasonal in human, are influenza viruses that infect animals (Huang SY. et al, epidemic and infection,2015,143(14): 2965-74). Influenza B Virus (Flu B) can be classified into two sublines Yamagata and Victoria (Y subline and V subline for short) according to its HA antigenicity difference. Influenza b only infects humans and seals, with slower variation compared to influenza a, but the viruses of both sublines can alternate seasonally or co-circulate among people, causing a large number of cases of infection worldwide each year (Yasugi m. et al, PLoS vaccines, 2013; 9(2): e 1003150). Influenza C Virus (Flu C) has the slowest mutation and weak pathogenicity, and can only infect pregnant women and children with low resistance.

Influenza viruses are a major threat to human health, and their constant and rapid antigenic drift has led to widespread seasonal influenza among populations. Seasonal influenza annually causes at least 250,000-. In addition, influenza outbreaks remain a significant threat to mankind. Since the discovery of influenza viruses, humans historically have had five worldwide influenza pandemics totaling tens of millions of deaths, with the 1918 major outbreak of Spanish influenza causing about 2000-. Other outbreaks of influenza (H1N1) that occurred in the 20 th century also included the asian influenza (H2N2) outbreak in 1957 and the hong kong influenza (H3N2) in 1968, both of which posed a serious public health threat and panic in human society (Xu r. et al, science 2010,328: 357-. In the 21 st century, the outbreak of flu still did not stop. The new stream of nails (H1N1) that broke out in mexico in 2009 and spread rapidly worldwide again is a pandemic that rings human society. According to WHO statistics, up to 8/6/2010, there are a total of 18449 cases of confirmed death reported in 200 countries and regions worldwide (WHO pandemics (h1n1)2009-update 112.6Aug, 2010). When influenza pandemics are over, influenza viruses tend to evolve into seasonal influenza, which continues to be epidemic and continue to harm human health through antigenic drift during the course of the epidemic. In recent years, avian influenza viruses such as H5N1, H7N9, H9N2 and H10N8 have broken through The species barrier to cause human infection, which poses a serious threat to human life health (Takayama I., et al, understanding infection Diseases,2016, 22(3): 557-9; Lam TT. et al, Nature,2013, 502(7470): 241-4; Choi YK. et al, Journal of virology,2004, 78(16): 8609-14; Chen H et al, The Lancet, 2014; and (9918): 714-21). Studies have predicted that avian influenza viruses may be converted, by sustained mutation, into stable "human-borne" pandemic influenza virus strains (Imai m. et al, Nature, 2012, 486(7403): 420-8). This does not prohibit people from worrying about fatal attacks to the human society once the virus is spread among people. In summary, influenza caused by influenza virus is a major infectious disease facing humans.

Vaccination with influenza vaccine is the most effective means of preventing and controlling influenza (http:// www.who.int/influenza/vacines/en /). However, current influenza vaccines rely primarily on the virus surface antigen Hemagglutinin (HA) protein to induce neutralizing antibodies for protection. Because of the differences in HA antigenicity among different types of influenza viruses, one type of influenza vaccine strain generally cannot prevent the other types of influenza. At present, the classical trivalent influenza vaccine can only be used for preventing the infection of two influenza a viruses (H1N1 and H3N2) and one influenza b virus, but can not prevent the infection of other types of influenza viruses. Besides a large number of types, influenza viruses have the characteristic of rapid variation, particularly the fastest variation of HA. When the HA gene of an epidemic virus strain is mutated to cause a large difference between the antigenicity of the epidemic virus strain and the antigenicity of a vaccine virus strain, the protective effect of an influenza vaccine is lost or reduced. Therefore, the world health organization needs to develop time-consuming and labor-consuming influenza monitoring work in a global scope every year, periodically evaluate the effectiveness of the influenza vaccine strain, and timely update the influenza vaccine strain. Nevertheless, mismatching of vaccine strains frequently occurs (Vesikari T. et al, N Engl J Med, 2011, 365(15): 1406-16). Therefore, the development of a universal influenza vaccine that can prevent different types of influenza viruses without being disabled by influenza virus variation has become an important development direction in the field of influenza virus research and the field of vaccinology research.

Several proteins encoded by the influenza virus genome have been shown to induce immune protection: for example, both conserved viral Nucleoprotein (NP) and matrix protein (M1) induce the body to generate T cell immune responses that protect against influenza virus infection (Berthoud TK. et al, Clinical infection diseases, 2011; 52(1): 1-7); the highly conserved ion channel protein (M2) can eliminate influenza virus by inducing the body to produce antibodies with ADCC activity; the more conserved Neuraminidase (NA) can exert antiviral effects by inducing the body to produce an immune protective response that inhibits viral Neuraminidase activity (Kramer F. et al, Nature reviews Drug discovery, 2015,14 (3): 167-82). Among the most potent proteins is the surface membrane protein HA, which induces the production of neutralizing antibodies, directly preventing influenza infection and replication. Studies have found that HA can be divided into a head region and a stem region. The HA stem region is embedded in the virus envelope, so that the epitope is not easily exposed; and the HA head region is exposed outside the virus envelope, so that a plurality of immunodominant epitopes exist, a neutralizing antibody can be induced to generate, the virus is directly prevented from infecting cells, and the HA head region is a first-choice target for developing influenza vaccines. However, immunodominant epitopes in the HA head region are highly variable, do not induce long-term effective neutralizing antibodies, and do not meet the research requirements of universal influenza vaccines, which is also the main reason that Current whole virus influenza vaccine strains need to be updated regularly (Mallajosya VV, et al, Current opinion in virology, 2015, 11: 103-12). Fortunately, new studies have found that rare broad-spectrum neutralizing mabs that recognize the head and stem regions of HA are able to combat infection by a wide variety of influenza virus types (Ekiert DC. et al, Nature, 2012, 489(7417): 526-32; Ekiert DC. et al, Science, 2011, 333(6044): 843-50; Dreyfus C. et al, Science, 2012, 337(6100): 1343-8; Wu Y. et al, Nature communications, 2015, 6: 7708). These findings indicate that highly conserved, broad-spectrum neutralizing epitopes are hidden in the highly variable HA protein.

Because these broad-spectrum neutralizing epitopes are rare non-immunodominant epitopes, and they do not easily induce the body to produce a large amount of broad-spectrum neutralizing antibodies after the whole virus vaccine is immunized, it is necessary to display these non-dominant broad-spectrum epitopes as epitope vaccines by applying in vitro expression technology to enhance the immune response effect. However, structural studies show that most of the broad-spectrum neutralizing epitopes in HA are conformational epitopes, and the reconstruction and expression of the conformational epitopes are always a scientific problem, and no effective technical means exists. One possible strategy is a molecular vaccine design method based on structural biology and bioinformatics, which applies bioinformatics software (e.g. PISCES, MAMMOTH, Meta-server, etc.), finds a framework protein with a structural contour that more closely matches the epitope to be reconstructed as a carrier by structural comparison and energy optimization calculation of large databases, so that conformational epitopes more naturally get reconstructed expression on the carrier protein (He l. et al, Current opinion in virology, 2015, 11: 103-12; office g. et al, Proceedings of the National Academy of Sciences, 2010, 107(42): 17880-7). This method has been used for the reconstitution expression of conformational neutralizing epitopes of RSV viruses and the constructed epitopes were able to successfully induce RSV neutralizing antibodies in rhesus monkey animal models (Norlen L. et al, Journal of controlled release, 2000, 63(1): 213-26). However, there are still many problems to be perfected in this method, such as the limitation of protein backbone database, the probability of bioinformatics calculation data and the response of protein backbone in different immune systems, etc., which still cannot meet the molecular design requirement of influenza, HIV and other virus vaccines. Therefore, there is a need to explore new strategies and methods for more convenient and efficient reconstitution of hemagglutinin conformational epitope expression.

Anti-idiotype antibody vaccines are a new vaccine strategy developed in the later 70 s of the 20 th century. In this strategy, animals were immunized with a neutralizing antibody (Ab 1) recognizing a viral antigen as an antigen; thus, the presence of an idiotypic determinant specifically recognizing an epitope of a viral antigen in the variable region of Ab1 stimulates the production of anti-idiotypic antibodies (AId antibody, Ab 2). The Ab2 can mimic/reconstruct the conformation of the viral epitope, and further induce the body to produce neutralizing antibody with the function similar to that of Ab1, and play the role of vaccine (Foon KA et al, Journal of Clinical Investigation, 1995, 96(1): 334). Anti-idiotype antibody vaccines have gained better application in tumor vaccine research. In 2014, the first non-small cell lung cancer vaccine containing anti-idiotypic antibodies was successfully marketed, which mimics the reconstituted tumor glycolipid epitope and can induce high titer and high affinity antibodies with significantly better efficacy than the native tumor antigen (Hern a ndez AM. et al, Expert review of vaccines, 2015,14 (1): 9-20; Gajdosik z. et al, Drugs Today (Barc), 2014, 4: 301-7). In addition, anti-idiotype antibody vaccines have also been used in influenza vaccine research (Schulman JL. et al, monograms in allergy, 1986, 22: 143-9). However, this study did not find the possibility of anti-idiotype antibodies to develop into a universal influenza vaccine.

The inventors of the present application have previously discovered a broad-spectrum neutralizing mab against influenza B virus (11B10, see chinese patent application 201610382361.6) that is capable of specifically binding to the hemagglutinin protein of influenza B viruses of the Yamagata and Victoria sublines across the HA subline, exhibiting broad-spectrum virus binding reactivity and broad-spectrum virus-neutralizing capacity. Based on these findings, the inventors further developed anti-idiotype antibodies specific to the broadly neutralizing mab (11B10), which are capable of eliciting broadly neutralizing antibodies against influenza B viruses of different sublines and exerting a broad spectrum immunoprotection in mice, and thus are useful as a universal influenza vaccine.

Disclosure of Invention

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory procedures, as used herein, are conventional procedures that are widely used in the relevant art. Also, for a better understanding of the present invention, the following definitions and explanations of the main relevant terms are provided.

As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (V)_H) And heavy chain constant region (C)_H) And (4) forming. The heavy chain constant region consists of 3 domains (C)_H1、C _H2 and C_H3) And (4) forming. Each light chain is composed of a light chain variable region (V)_L) And light chain constant region (C)_L) And (4) forming. The light chain constant region consists of a domain C_LAnd (4) forming. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). V_HAnd V_LRegions may also be subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each V_HAnd V_LBy the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are composed of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. Variable region (V) of each heavy/light chain pair_HAnd V_L) Antibody binding sites were formed separately. The allocation of amino acids to the various regions or domains follows Kabat Sequences of Proteins of Immunological Interest (National I)nstitutes of Health, Bethesda, Md. (1987and1991)), or Chothia&Lesk (1987) J.mol.biol.196: 901-917; chothia et al (1989) Nature 342: 878-883. 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 antibody may be of a different isotype, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

As used herein, the term "variable region" with respect to an immunoglobulin molecule has the meaning commonly understood by those of ordinary skill in immunology. Both antibody heavy and antibody light chains can be divided into "variable regions" and "constant regions". The separation points of the variable and constant regions can be readily determined by one of ordinary skill in the art based on general rules describing antibody structures, such as Kabat et al, "Sequences of Proteins of Immunological Interest:5th Edition" U.S. department of Health and Human Services, U.S. Govern Printing Office (1991).

As used herein, the 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 the antigen, which is 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 purposes₂Fd, Fv, dAb, and Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies (diabodies), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding capability on the polypeptide.

As used herein, the term "Fd fragment" means a fragment consisting of V_HAnd C _H1 domain; the term "Fv fragment" means a V consisting of a single arm of an antibody_LAnd V_HAntibody fragments consisting of domains; the term "dAb fragment" means a fragment consisting of V_HAntibody fragments consisting of domains (Ward et al, Nature 341:544-546 (1989)); the term "Fab fragment" means a fragment consisting of V_L、V_H、C_LAnd C _H1 domain; the term "F (ab')₂By fragment "is meant an antibody fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region.

In some cases, the antigen-binding fragment of an antibody is a single chain antibody (e.g., scFv), where V_LAnd V_HThe domains form monovalent molecules by pairing linkers that enable them to be generated as a single polypeptide chain (see, e.g., Bird et al, Science 242:423-426(1988) and Huston et al, Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such scFv molecules can have the general structure: NH (NH)₂-V_L-linker-V_H-COOH or NH₂-V_H-linker-V_L-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a polypeptide having an amino acid sequence (GGGGS)₄But variants thereof can also be used (Holliger et al (1993), Proc. Natl. Acad. Sci. USA 90: 6444-. Other linkers useful in the present invention are described by Alfthan et al (1995), Protein Eng.8: 725-.

In some cases, the antigen-binding fragment of an antibody is a diabody, i.e., a diabody, in which V is_HAnd V_LThe domains are expressed on a single polypeptide chain, but a linker that is too short to allow pairing between two domains of the same chain, thereby forcing the domains to pair with complementary domains of another chain and generating two antigen binding sites (see, e.g., Holliger P. et al, Proc. Natl. Acad. Sci. USA 90: 6444-.

Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., monoclonal antibody 1C12 provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of antibodies are specifically screened for specificity in the same manner as for intact antibodies.

Herein, when the term "antibody" is referred to, it includes not only intact antibodies, but also antigen-binding fragments of antibodies, unless the context clearly indicates otherwise.

As used herein, the terms "monoclonal antibody" and "monoclonal antibody" 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 high specificity for a single epitope on the antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are generally obtained using hybridoma technology first reported by Kohler et al (Nature,256:495,1975), but can also be obtained using recombinant DNA technology (see, e.g., U.S. P4, 816, 567).

For example, monoclonal antibodies can be prepared as follows. Mice or other suitable host animals are first immunized with the immunogen (with adjuvant added if necessary). The mode of injection of the immunogen or adjuvant is usually subcutaneous multi-site injection or intraperitoneal injection. Immunogens can be pre-conjugated to certain known proteins, such as serum albumin or soybean pancreatin inhibitors, to enhance the immunogenicity of the antigen in the host. The adjuvant may be Freund's adjuvant or MPL-TDM, etc. After the animal is immunized, lymphocytes that secrete antibodies that specifically bind the immunogen will be produced in vivo. Alternatively, lymphocytes can be obtained by in vitro immunization. The lymphocytes of interest are collected and fused with myeloma cells using a suitable fusing agent such as PEG to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1996). The hybridoma cells prepared as described above may be grown by inoculating into a suitable culture medium, preferably containing one or more substances capable of inhibiting the growth of unfused, maternal myeloma cells. For example, for parental myeloma cells that lack hypoxanthine guanine phosphotransferase (HGPRT or HPRT), the addition of hypoxanthine, aminopterin, and thymidine (HAT medium) to the culture broth will inhibit the growth of HGPRT-deficient cells. Preferred myeloma cells should have high fusion rate, stable antibody secretion ability, sensitivity to HAT culture solution, and the like. Among them, THE myeloma cells are preferably derived from murine myelomas such as MOP-21 or MC-11 mouse tumor-derived strains (THE salt Institute Cell Distribution Center, San Diego, Calif. USA), and SP-2/0 or X63-Ag8-653 Cell strain (American Type Culture Collection, Rockville, Md.USA). In addition, human Monoclonal antibodies have been reported to be prepared using human myeloma and human murine allogeneic myeloma cell lines (Kozbor, J.Immunol.,133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp.51-63, Marcel Dekker, Inc., New York, 1987). The culture medium of the growing hybridoma cells was used to detect the production of monoclonal antibodies against specific antigens. Methods for determining the binding specificity of a monoclonal antibody produced by a hybridoma cell include, for example, immunoprecipitation or in vitro binding assays, such as Radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA). For example, the affinity of a mAb can be determined using the Scatchard assay described by Munson et al, anal. biochem.107:220 (1980). After determining the specificity, affinity and reactivity of the Antibodies produced by the hybridomas, the cell lines of interest can be subcloned by standard limiting dilution methods as described in (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1996). Suitable culture medium may be DMEM or RPMI-1640 or the like. In addition, hybridoma cells can also be grown in animals as ascites tumors. The monoclonal antibodies secreted by the subcloned cells can be isolated from the cell culture fluid, ascites fluid, or serum using conventional immunoglobulin purification methods, such as protein a sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Monoclonal antibodies can also be obtained by genetic engineering recombination techniques. The DNA molecules encoding the monoclonal antibody heavy chain and light chain genes can be isolated from the hybridoma cells by PCR amplification using nucleic acid primers that specifically bind to the monoclonal antibody heavy chain and light chain genes. The resulting DNA molecule is inserted into an expression vector, and then host cells (e.g., E.coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulin) are transfected and cultured under appropriate conditions to obtain a recombinantly expressed antibody of interest.

As used herein, the term "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 belonging to a particular antibody class or subclass) and another portion of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belonging to the same or different antibody class or subclass), but which nevertheless retains binding activity to an antigen of interest (u.s.p 4,816,567to Cabilly et al; Morrison et al, proc.natl.acad.sci.usa,81: 6851-.

As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or a portion of the CDR regions of a human immunoglobulin (recipient antibody) with the CDR regions of a non-human antibody (donor antibody), which may be a non-human (e.g., mouse, rat, or rabbit) antibody of the desired specificity, affinity, or reactivity. In addition, some amino acid residues of the Framework Region (FR) of the acceptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further refine or optimize the performance of the antibody. For more details on humanized antibodies, see, e.g., Jones et al, Nature,321:522-525 (1986); reichmann et al, Nature,332: 323-; presta, curr, Op, struct, biol.,2: 593-; and Clark, immunological. today 21:397- & 402 (2000).

As used herein, "neutralizing antibody" refers to an antibody or antibody fragment that eliminates or significantly reduces the virulence (e.g., the ability to infect cells) of a target virus.

As used herein, the term "idiotype" refers to the unique antigenicity of the variable region of an immunoglobulin molecule itself. The idiotype is located in the Fab region and is usually involved in the variable regions of the heavy and light chains that make up the antigen binding site. The idiotype of an antibody is determined primarily by the amino acid differences in the hypervariable regions.

As used herein, the term "anti-idiotypic antibody" refers to an antibody that specifically recognizes/binds to the idiotype of the antibody used to make them (i.e., having as an epitope the idiotype of the variable region of the antibody used to make them), which is capable of mimicking/reconstituting the epitope recognized by the antibody used to make them.

As used herein, the term "epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. An "epitope" is also referred to in the art as an "antigenic determinant". Epitopes or antigenic determinants usually consist of chemically active surface groups of molecules such as amino acids or carbohydrates or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. For example, an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation, which can be "linear" or "conformational". See, e.g., epitopic Mapping Protocols in Methods in Molecular Biology, vol 66, g.e. morris, Ed. (1996). In a linear epitope, the points of all interactions between a protein and an interacting molecule (e.g., an antibody) are linearly present along the primary amino acid sequence of the protein. In conformational epitopes, the point of interaction exists across protein amino acid residues that are separated from each other.

As used herein, the term "epitope peptide" refers to a peptide fragment on an antigen that can serve as an epitope. In some cases, the epitope peptide alone is capable of being specifically recognized/bound by an antibody directed against the epitope. In other cases, it may be desirable to fuse the epitope peptide to a carrier protein so that the epitope peptide can be recognized by a specific antibody. As used herein, the term "carrier protein" refers to a protein that can act as a carrier for an epitope peptide, i.e., it can be inserted into an epitope peptide at a specific location (e.g., inside the protein, N-terminal or C-terminal) so that the epitope peptide can be presented and thus the epitopeThe peptide can be recognized by an antibody or by the immune system. Such carrier proteins are well known to those skilled in the art and include, for example, the HPV L1 protein (epitope peptides can be inserted between amino acids 130-131 or 426-427 of said protein, see Slupetzky, K. et al, molecular plasmid expression a for expression on epitope surface loops [ J]J Gen Virol,2001,82: 2799-; varsani, A. et al, Chimeric human papillomavir type 16(HPV-16) L1particles presenting the common neutral hepatitis for the L2minor capsid protein of HPV-6and HPV-16[ J]J Virol,2003,77:8386-8393), HBV core antigen (amino acids 79-81 of the protein may be replaced by epitope peptides, see Koletzki, D., et al, HBV core particles antigen of the insertion and surface exposure of the enzyme pore protective region of viral nucleic acid protein [ J Virus]Biol Chem,1999,380:325-333), the core protein of the woodchuck hepatitis virus (amino acids 79-81 of said protein can be replaced by epitope peptides, see Sabine

Gertrud Beterams and Michael Nassal, J.Virol.1998,72(6):4997), CRM197 protein (epitope peptides can be attached to the N-terminus or C-terminus of the protein or fragment thereof). Optionally, a linker (e.g., a flexible or rigid linker) may be used between the epitope peptide and the carrier protein to facilitate folding of each.

Antibodies can be screened for binding competition with the same epitope using conventional techniques known to those skilled in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete for binding to the antigen. A high throughput method for obtaining antibodies binding to the same epitope based on their cross-competition is described in international patent application WO 03/48731. Accordingly, antibodies and antigen-binding fragments (i.e., antigen-binding portions) thereof that compete with the monoclonal antibody of the present invention (e.g., monoclonal antibody 1C12) for binding to the same epitope on monoclonal antibody 11B10 used as an antigen can be obtained using conventional techniques known to those skilled in the art.

As used herein, the term "isolated" or "isolated" refers to a product obtained from a natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be altered from its natural environment, or it may be isolated from its natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and a polynucleotide or polypeptide that is the same in high purity and that is isolated from such a natural state is said to be isolated. The term "isolated" or "isolated" does not exclude the presence of substances mixed artificially or synthetically or other impurities which do not affect the activity of the substance.

As used herein, the term "e.coli expression system" refers to an expression system consisting of e.coli (strain) derived from commercially available strains such as, but not limited to: GI698, ER2566, BL21(DE3), B834(DE3), BLR (DE 3).

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing 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, and 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; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (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 contain a replication initiation site.

As used herein, the term "host cell" refers to a cell that can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

As used herein, the term "immunogenicity" refers to the ability of a body to be stimulated to form specific antibodies or to sensitize lymphocytes. It refers to the characteristic that an antigen can stimulate specific immune cells to activate, proliferate and differentiate the immune cells and finally produce immune effector substances such as antibodies and sensitized lymphocytes, and also refers to the characteristic that the immune system of the organism can form specific immune response of the antibodies or sensitized T lymphocytes after the antigen stimulates the organism. Immunogenicity is the most important property of an antigen, and the success of an antigen in inducing an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host and the mode of immunization.

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. In certain embodiments, an antibody that specifically binds to (or is specific for) an antigen means that the antibody is present in an amount less than about 10^-5M, e.g. less than about 10^-6M、10^-7M、10^-8M、10^-9M or 10^-10M or less affinity (K)_D) Binding the antigen.

As used herein, the term "K_D"refers to the dissociation equilibrium constant for 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 more tight the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, the antibody (e.g., monoclonal antibody 1C12 of the invention) is administered at less than about 10^-5M, e.g. less than about 10^-6M、10^-7M、10^-8M、10^-9M or 10^-10Dissociation equilibrium constant (K) of M or less_D) Binding to the antigen, e.g., as determined in a BIACORE instrument using Surface Plasmon Resonance (SPR).

As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations as is well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the terms "hybridoma" and "hybridoma cell line" are used interchangeably, and when referring to the terms "hybridoma" and "hybridoma cell line," it also includes subclones and progeny cells of the hybridoma. For example, when reference is made to hybridoma cell line 1C12 or 11B10, it also refers to subclones and progeny cells of hybridoma cell line 1C12 or 11B 10.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical sciences. edited by geno AR,19th ed. pennsylvania: mach Publishing Company,1995), and include, but are not limited to: pH regulator, surfactant, adjuvant, and ionic strength enhancer. For example, pH adjusting agents 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.

As used herein, the term "adjuvant" refers to a non-specific immunopotentiator which, when delivered with or prior to an antigen into the body, enhances the body's immune response to the antigen or alters the type of immune response. Adjuvants are of various types, including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, and the like. Freund's adjuvant is currently the most commonly used adjuvant in animal testing. Aluminum hydroxide adjuvants are used more often in clinical trials.

As used herein, the term "protein vaccine" refers to a polypeptide-based vaccine, which optionally further comprises an adjuvant. The polypeptide in the vaccine can be obtained by genetic engineering technology or chemical synthesis method. As used herein, the term "nucleic acid vaccine" refers to a DNA or RNA (e.g., plasmid, such as an expression plasmid) based vaccine, which optionally further comprises an adjuvant.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, a desired effect. For example, an amount effective to prevent a disease (e.g., an influenza virus infection or a disease associated with an influenza virus infection) refers to an amount sufficient to prevent, or delay the onset of a disease (e.g., an influenza virus infection or a disease associated with an influenza virus infection); a therapeutically effective amount for a disease is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts. For example, an 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, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.

As used herein, the terms "Yamagata subline" and "Yamagata subline of influenza B virus" refer to a subline of influenza B virus whose hemagglutinin protein is antigenic and evolutionarily related to and in the same evolutionary branch as representative strain B/Yamagata/16/1988 of influenza B virus; it includes the following exemplary strains: B/Harbin/7/1994, B/Florida/4/2006, B/Xiamen/891/2006, B/Xiamen/756/2007, B/Xiamen/1147/2008, B/Xiamen/N697/2012. The terms "Yamagata subline" and "Yamagata subline influenza b virus" have the same meaning and are used interchangeably.

As used herein, the terms "Victoria subline" and "Victoria subline influenza B virus" refer to an influenza B virus subline whose hemagglutinin protein is antigenic and evolutionarily related to and in the same evolutionary branch as representative strain B/Victoria/2/1987; these include, but are not limited to, the following exemplary strains: B/Hong Kong/330/2001, B/Malaysia/2506/2004, B/Xiaomen/3043/2006, B/Xiaomen/165/2007, B/Brisbane/60/2008, B/Brisbane/33/2008, B/Xiaomen/1346/2008, B/Xiaomen/N639/2010, B/Xiaomen/N678/2012. The terms "Victoria subline" and "Victoria subline influenza b virus" have the same meaning and are used interchangeably.

As used herein, the terms "hemagglutinin protein" and "HA protein" refer to the antigenic glycoproteins encoded by fragment 4 of the influenza virus genome, which are present on the surface of the viral membrane and are synthesized in the endoplasmic reticulum of the cell, and have a molecular weight of about 76 kD. HA proteins can be hydrolyzed to HA1 polypeptide (molecular weight 47kD, also referred to herein as the "HA 1 domain") and HA2 polypeptide (molecular weight 29kD, also referred to herein as the "HA 2 domain") which are linked by disulfide bonds to form an HA molecule with a typical type i membrane protein structure. The hemagglutinin protein is immunogenic and anti-hemagglutinin antibodies can be used to neutralize influenza virus. Hemagglutinin proteins are well known to those skilled in the art and the amino acid sequences can be found in various public databases, such as NCBI. The HA1 polypeptide/domain is located at the head of the HA protein and is in a globular structure that contains the receptor binding site of the virus and is capable of binding to sialic acid receptors on the host cell membrane, thereby mediating viral entry into the cell. The HA2 polypeptide/domain can then assist in the fusion of the viral envelope with the host cell membrane, which plays an important role in the process of virus entry into the host cell.

As used herein, the term "hemagglutination-inhibiting activity" refers to the functional activity of antisera, antibodies or antigen-binding fragments thereof to inhibit the coagulation phenomenon caused by the binding of the influenza virus HA protein to sialic acid receptors on the surface of erythrocytes. Antisera, antibodies or antigen-binding fragments with hemagglutination-inhibiting activity are capable of inhibiting the binding of a virus to a cellular receptor.

As used herein, the term "neutralizing activity" refers to the functional activity of antisera, antibodies, or antigen-binding fragments thereof, to bind to an antigenic protein on a virus and thereby reduce or inhibit the virulence (e.g., the ability to infect cells) of the target virus. Antisera, antibodies or antigen-binding fragments with neutralizing activity are capable of preventing maturation of virus-infected cells and/or progeny virus and/or release of progeny virus.

The inventors of the present application have previously discovered a broad-spectrum neutralizing mab against influenza B virus (11B10, see chinese patent application 201610382361.6) that is capable of specifically binding to the hemagglutinin protein of influenza B viruses of the Yamagata and Victoria sublines across the HA subline, exhibiting broad-spectrum virus binding reactivity and broad-spectrum virus-neutralizing capacity. Based on these findings, the inventors further developed anti-idiotypic antibodies specific for the idiotype of this broadly neutralizing mab (11B10), which are capable of eliciting broadly neutralizing antibodies against influenza B viruses of different sublines and exerting a broad spectrum immunoprotection in mice, and thus are useful as universal influenza vaccines for preventing influenza B virus infection of different types/sublines, providing cross-protective immunity against influenza B viruses of different types/sublines.

Accordingly, in one aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof comprising: CDR1, CDR2 and CDR3 of the heavy chain variable region of SEQ ID NO 1-3 respectively; and/or, the amino acid sequences are respectively CDR1, CDR2 and CDR3 of the light chain variable region of SEQ ID NO. 4-6.

In certain preferred embodiments, the monoclonal antibody comprises CDR1, CDR2, and CDR3 having the amino acid sequences of the heavy chain variable region of SEQ ID NOS: 1-3, respectively; and CDR1, CDR2 and CDR3 of the light chain variable region having the amino acid sequences of SEQ ID Nos. 4-6, respectively.

In certain preferred embodiments, the monoclonal antibody comprises the heavy chain variable region as set forth in SEQ ID NO. 7.

In certain preferred embodiments, the monoclonal antibody comprises the light chain variable region as set forth in SEQ ID NO 8.

In certain preferred embodiments, the monoclonal antibody comprises a heavy chain variable region as set forth in SEQ ID NO. 7and a light chain variable region as set forth in SEQ ID NO. 8.

In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab ', F (ab')₂Fd, Fv, dAb, complementarity determining region fragment, single chain antibody (e.g., scFv), mouse antibody, rabbit antibody, humanized antibody, fully human antibody, chimeric antibody (e.g., human murine chimeric antibody), or bispecific or multispecific antibody.

In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a non-CDR region, and the non-CDR region is from a species that is not murine, e.g., from a human antibody.

In certain preferred embodiments, the monoclonal antibody is a monoclonal antibody produced by hybridoma cell line 1C12, which hybridoma cell line 1C12 is deposited with the China Center for Type Culture Collection (CCTCC) and has a collection number of CCTCC NO: C201673.

In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof is capable of specifically binding to an idiotype (or antigen-binding site) of a second monoclonal antibody comprising: the amino acid sequences are CDR1, CDR2 and CDR3 of the heavy chain variable region of SEQ ID NO. 11-13, respectively, and the amino acid sequences are CDR1, CDR2 and CDR3 of the light chain variable region of SEQ ID NO. 14-16, respectively. In certain preferred embodiments, the second monoclonal antibody comprises: the heavy chain variable region shown as SEQ ID NO. 17 and the light chain variable region shown as SEQ ID NO. 18. In certain preferred embodiments, the second monoclonal antibody is monoclonal antibody 11B10, and the monoclonal antibody 11B10 is produced by hybridoma cell line 11B10 deposited at the China Center for Type Culture Collection (CCTCC) with a collection number of CCTCC NO: C201432.

In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof is capable of eliciting an antiserum having hemagglutination-inhibiting activity and/or neutralizing activity in an animal (e.g., a mouse or a human). In certain preferred embodiments, the antisera have hemagglutination-inhibiting activity and/or neutralizing activity against at least 2 sublines of influenza b virus. In certain preferred embodiments, the antiserum has hemagglutination-inhibiting activity and/or neutralizing activity against influenza b viruses of the Yamagata subline and Victoria subline.

In another aspect, the present invention provides an anti-idiotype antibody or antigen binding fragment thereof, which is capable of blocking/blocking at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% or preferably at least 99% of the binding between the idiotype of monoclonal antibody 11B10 and monoclonal antibody 1C12, wherein the monoclonal antibody 11B10 is produced by hybridoma cell line 11B10 deposited in the chinese typical culture collection (CCTCC) and having a deposition number of CCTCC NO: C201432; the monoclonal antibody 1C12 is produced by hybridoma cell strain 1C12 which is preserved in China Center for Type Culture Collection (CCTCC) and has the preservation number of CCTCC NO: C201673.

Such anti-idiotypic antibodies recognize the same epitope on mab 11B10 as monoclonal antibody 1C12, or there is spatial overlap such that such anti-idiotypic antibodies are capable of reducing the binding of mab 1C12 to the idiotype of mab 11B10 by at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, or preferably at least 99%.

A given monoclonal antibody can be assayed for its ability to reduce the binding of a given monoclonal antibody to an antigen (e.g., the idiotype of monoclonal antibody 11B10) using conventional methods such as those described in Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). One exemplary method comprises: pre-coating antigen on a microplate, then adding a series of diluted unlabelled antibodies to be detected and labeled known monoclonal antibodies with specific concentrations into the pre-coated microplate together for incubation, and then determining the number of known antibodies bound to the plate under different dilutions of the antibodies to be detected after washing. The stronger the test antibody competes for the known antibody for binding to the antigen, the weaker the known antibody binds to the antigen, and the fewer the known antibody binds to the plate. Typically, the antigen is pre-coated on a 96-well microplate and the ability of the monoclonal antibody to be tested to block the labeled known monoclonal antibody is determined using radiolabeling or enzymatic labeling.

The invention also provides an isolated nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment thereof of the invention or an anti-idiotype antibody or antigen-binding fragment thereof of the invention. Such nucleic acid molecules can be isolated from hybridoma cells, or can be obtained by genetic engineering recombinant techniques or chemical synthesis methods.

Thus, in another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding an antibody heavy chain variable region comprising CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs 1-3, respectively.

In certain preferred embodiments, the antibody heavy chain variable region has the amino acid sequence shown as SEQ ID NO 7.

In certain preferred embodiments, the nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO 9.

In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding an antibody light chain variable region comprising CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs 4-6, respectively.

In certain preferred embodiments, the antibody light chain variable region has the amino acid sequence shown as SEQ ID NO 8.

In certain preferred embodiments, the nucleic acid molecule has the nucleotide sequence shown as SEQ ID NO. 10.

In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding an antibody heavy chain variable region as defined above, and a nucleic acid sequence capable of encoding an antibody light chain variable region as defined above.

In another aspect, the invention provides an isolated nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment thereof of the invention as defined above.

In another aspect, the invention provides a vector comprising an isolated nucleic acid molecule as defined above. The vector of the present invention may be a cloning vector or an expression vector.

In certain preferred embodiments, the vectors of the invention are, for example, plasmids, cosmids, phages and the like.

In another aspect, host cells comprising the isolated nucleic acid molecules or vectors of the invention are also provided. 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.). The cell of the invention may also be a cell line, such as 293T cells.

In another aspect, there is also provided a method of making a monoclonal antibody or antigen-binding fragment thereof of the invention, comprising culturing a host cell of the invention under suitable conditions, and recovering the monoclonal antibody or antigen-binding fragment thereof of the invention from the cell culture.

In another aspect, the present invention provides hybridoma cell line 1C12, which is deposited in China Center for Type Culture Collection (CCTCC) and has a preservation number of CCTCC NO: C201673.

As demonstrated herein, the amino acid sequence of the heavy chain variable region of monoclonal antibody 1C12 is set forth in SEQ ID NO:7 (an exemplary nucleotide sequence is set forth in SEQ ID NO: 9), and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:8 (an exemplary nucleotide sequence is set forth in SEQ ID NO: 10).

The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of monoclonal antibody 1C12 are respectively SEQ ID NO. 1-3; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are SEQ ID NO. 4-6, respectively.

In another aspect, the invention provides a composition comprising a monoclonal antibody or antigen-binding fragment thereof, an anti-idiotype antibody or antigen-binding fragment thereof, an isolated nucleic acid molecule, a vector or a host cell as described above.

In another aspect, the invention provides a kit comprising a monoclonal antibody of the invention, or an antigen-binding fragment thereof. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof of the invention further comprises a detectable label. In certain preferred embodiments, the kit further comprises a second antibody that specifically recognizes the monoclonal antibody of the invention, or an antigen-binding fragment thereof. Preferably, the second antibody further comprises a detectable label.

Such detectable labels are well known to those skilled in the art and include, but are not limited to, radioisotopes, fluorescent materials, luminescent materials, colored materials and enzymes (e.g., horseradish peroxidase), and the like. For example, the detectable label may be a radioisotope, an enzyme, a substrate for an enzyme, a luminescent substance such as isoluminol and acridinium ester, a fluorescent substance such as fluorescein and rhodamine, a colored substance such as latex particles and colloidal gold, and the like. For example, enzymes for labeling include, but are not limited to, peroxidase (e.g., horseradish peroxidase (HRP)), alkaline phosphatase, beta-galactosidase, acetylcholinesterase, and glucose oxidase. Suitable enzyme substrates include, for example, 2,2 '-azino-bis (3-ethylbenzothiopyrroline-6 sulfonic acid), luminol-hydrogen peroxide, o-phenylenediamine-hydrogen peroxide (for peroxidase), p-nitrophenylphosphate, 4-methyl umbelliferyl phosphate, 3- (2' -spiroadamantane) -4-methoxy-4- (3 "-phosphoryl) phenyl-1, 2-diethoxyalkane (for alkaline phosphatase), p-nitrophenyl- β -D-galactose and methyl umbelliferyl- β -D-galactose (for β -galactosidase). Fluorescent substances for labeling include, but are not limited to, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, eosin, green fluorescent protein, phycoerythrin, coumarin, methylcoumarin, pyrene, malachite green, stilbene, fluorescein, Cascade blue, dichlorotriazinyl fluorescein, dansyl chloride, phycoerythrin, fluorescent lanthanide complexes, Cy3, Cy5, and the like. The radioactive isotopes include, but are not limited to,¹⁴C、¹²³I、¹²⁴I、¹³¹I、³⁵s or³H。

Other detectable labels include, but are not limited to, quantum dot labels, chromophore labels, affinity ligand labels, electromagnetic spin labels, heavy atom labels, epitope labels (such as FLAG or HA epitopes), and binding pairs capable of forming complexes (e.g., streptavidin/biotin, avidin/biotin, or antigen/antibody complexes (such as rabbit IgG and anti-rabbit IgG)).

Methods of binding a detectable label to an antigen or antibody are known in the art and include, but are not limited to, maleimide (j.biochem. (1976),79,233), biotin activation (j.am.chem.soc. (1978),100,3585), hydrophobic binding, ester activation, or isocyanate.

In another aspect, the present invention provides a method of detecting the presence or level of an antibody against influenza b virus hemagglutinin protein in a sample, comprising using the monoclonal antibody or antigen binding fragment thereof of the present invention. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof of the invention further comprises a detectable label. In another preferred embodiment, the method further comprises detecting the monoclonal antibody or antigen-binding fragment thereof of the invention using a second antibody carrying a detectable label. The methods may be used for diagnostic purposes (e.g., the sample is a sample from a patient), or for non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient). In certain preferred embodiments, the antibody against influenza b virus hemagglutinin protein specifically binds to the HA1 domain of the hemagglutinin protein of influenza b viruses of the Yamagata subfamily and Victoria subfamily. In certain preferred embodiments, the anti-influenza B virus hemagglutinin protein antibody recognizes the same epitope as the monoclonal antibody 11B10, or is adjacent in spatial position, or overlaps with each other in sequence or in spatial position. In certain preferred embodiments, the antibody against influenza b virus hemagglutinin protein comprises: the amino acid sequences are CDR1, CDR2 and CDR3 of the heavy chain variable region of SEQ ID NO. 11-13, respectively, and the amino acid sequences are CDR1, CDR2 and CDR3 of the light chain variable region of SEQ ID NO. 14-16, respectively. In certain preferred embodiments, the antibody against influenza b virus hemagglutinin protein comprises: the heavy chain variable region shown as SEQ ID NO. 17 and the light chain variable region shown as SEQ ID NO. 18. In certain preferred embodiments, the antibody against influenza B virus hemagglutinin protein is mab 11B 10.

In another aspect, the invention provides a method of determining whether a subject produces antibodies against an influenza b virus hemagglutinin protein, comprising: detecting the presence of an antibody against influenza b virus hemagglutinin protein in a sample from said subject using the monoclonal antibody or antigen binding fragment thereof of the invention. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof of the invention further comprises a detectable label. In another preferred embodiment, the method further comprises detecting the monoclonal antibody or antigen-binding fragment thereof of the invention using a second antibody carrying a detectable label. In certain preferred embodiments, the antibody against influenza b virus hemagglutinin protein is as defined above.

In another aspect, there is provided the use of a monoclonal antibody of the invention, or an antigen-binding fragment thereof, in the preparation of a kit for detecting the presence or level of an antibody against influenza b virus hemagglutinin protein in a sample, or for determining whether a subject produces antibodies against influenza b virus hemagglutinin protein. In certain preferred embodiments, the antibody against influenza b virus hemagglutinin protein is as defined above.

In certain preferred embodiments, the sample includes, but is not limited to, fecal matter, oral and nasal secretions, and the like, from a subject (e.g., a mammal, preferably a human).

In certain preferred embodiments, the monoclonal antibody is an antibody comprising: CDR1, CDR2 and CDR3 of the heavy chain variable region having amino acid sequences shown in SEQ ID NO. 1-3, respectively, and/or CDR1, CDR2 and CDR3 of the light chain variable region having amino acid sequences shown in SEQ ID NO. 4-6, respectively. In certain preferred embodiments, the monoclonal antibodies comprise: the heavy chain variable region shown as SEQ ID NO. 7 and/or the light chain variable region shown as SEQ ID NO. 8. In certain preferred embodiments, the monoclonal antibody is mab 1C 12.

General methods for using antibodies or antigen-binding fragments thereof to detect the presence or level of a virus or antigen of interest in a sample are well known to those skilled in the art. In certain preferred embodiments, the detection method may use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, competition, and the like.

In another aspect, the present invention provides a pharmaceutical composition comprising a monoclonal antibody of the invention, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier and/or excipient. In certain preferred embodiments, the monoclonal antibodies comprise: VH CDR1-3 with amino acid sequences shown as SEQ ID NO. 1-3, and/or VL CDR1-3 with amino acid sequences shown as SEQ ID NO. 4-6. In certain preferred embodiments, the monoclonal antibodies comprise: VH shown as SEQ ID NO. 7 and/or VL shown as SEQ ID NO. 8. In certain preferred embodiments, the monoclonal antibody is a monoclonal antibody produced by hybridoma cell line 1C12, which hybridoma cell line 1C12 is deposited with the China Center for Type Culture Collection (CCTCC) and has a collection number of CCTCC NO: C201673.

In certain preferred embodiments, the pharmaceutical composition is a vaccine. In certain preferred embodiments, the pharmaceutical composition further comprises other pharmaceutically active agents (e.g., anti-influenza drugs, such as M2 protein ion channel inhibitors (e.g., amantadine and rimantadine) and neuraminidase inhibitors (e.g., oseltamivir)). In certain preferred embodiments, the pharmaceutical composition further comprises an additional vaccine, such as an influenza virus vaccine (e.g., brida).

In another aspect, there is provided the use of a monoclonal antibody or antigen-binding fragment thereof or an anti-idiotype antibody or antigen-binding fragment thereof of the invention in the manufacture of a pharmaceutical composition for the prevention or treatment of influenza b virus infection or a disease associated with influenza b virus infection (e.g., influenza) in a subject. In certain preferred embodiments, the pharmaceutical composition is a vaccine.

In another aspect, the invention provides a monoclonal antibody or antigen binding fragment thereof or an anti-idiotype antibody or antigen binding fragment thereof as described above for use in the prevention or treatment of influenza b virus infection or a disease associated with influenza b virus infection (e.g. influenza) in a subject.

In another aspect, the present invention provides a method for preventing or treating influenza b virus infection or a disease associated with influenza b virus infection (e.g., influenza) in a subject, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a monoclonal antibody or antigen-binding fragment thereof or an anti-idiotype antibody or antigen-binding fragment thereof of the present invention, or a pharmaceutical composition of the present invention.

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

The monoclonal antibody or antigen-binding fragment thereof or the anti-idiotype antibody or antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention can be administered to a subject by any suitable route of administration. Such routes of administration include, but are not limited to, oral, buccal, sublingual, topical, parenteral, rectal, intrathecal, or nasal routes.

In certain preferred embodiments, the monoclonal antibody is an antibody comprising: VH CDR1-3 with amino acid sequences shown as SEQ ID NO. 1-3, and/or VL CDR1-3 with amino acid sequences shown as SEQ ID NO. 4-6. In certain preferred embodiments, the monoclonal antibodies comprise: VH shown as SEQ ID NO. 7 and/or VL shown as SEQ ID NO. 8. In certain preferred embodiments, the monoclonal antibody is mab 1C 12.

The drugs and pharmaceutical compositions provided by the present invention may be used alone or in combination, or in combination with other pharmaceutically active agents (e.g., anti-influenza drugs, such as M2 protein ion channel inhibitors (e.g., amantadine and rimantadine), neuraminidase inhibitors (e.g., oseltamivir), or other influenza vaccines (e.g., bridard)).

Advantageous effects of the invention

The invention provides an anti-idiotype antibody for specifically recognizing influenza virus hemagglutinin broad-spectrum neutralization monoclonal antibody 11B10, which simulates/reconstructs conformational conserved epitopes recognized by the broad-spectrum neutralization monoclonal antibody 11B10 and located in influenza virus hemagglutinin protein, and breaks through the limitation of the existing conformational epitopes for simulating/reconstructing influenza virus hemagglutinin protein. The anti-idiotype antibody aiming at the influenza virus hemagglutinin broad-spectrum neutralizing monoclonal antibody can be used for detecting and evaluating the existence and the concentration of the influenza virus hemagglutinin broad-spectrum neutralizing antibody. More importantly, the anti-idiotype antibodies of the invention are capable of inducing neutralizing antisera (broadly neutralizing antibodies) against influenza b viruses of different sublines in mice and exerting a broad spectrum of immunoprotection, and are therefore useful as universal influenza vaccines for the prevention of influenza b virus infections of different types/sublines, providing cross-protective immunity against influenza b viruses of different types/sublines.

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

Drawings

FIG. 1 shows the results of a competition ELISA for assessing the ability of the cell supernatant of a positive hybridoma cell line 1C12 to block the binding of mAb 11B10 to influenza B virus (B/Xiaomen/3043/2006); wherein the abscissa represents the sample to be tested: NC represents cell supernatants from unrelated hybridoma cell lines, used as negative controls; 1C12 represents cell supernatant from hybridoma cell line 1C 12; the ordinate represents the ability of the test sample to compete for the binding of mab 11B10 to influenza B virus (competition rate), which is calculated by the following formula: 1- (OD value of ELISA assay to detect binding of 11B10 to influenza B virus in the presence of excess test sample/OD value of ELISA assay to detect binding of 11B10 to influenza B virus in the absence of test sample) × 100%.

FIG. 2 shows the results of ELISA experiments for assessing the binding ability of immune sera induced in mice by anti-idiotype antibody 1C12 to Yamagata subfamily influenza B virus B/Florida/4/2006 (FIG. 2A) and Victoria subfamily influenza B virus B/Brisbane/60/2008 (FIG. 2B); wherein the abscissa represents the dilution factor of the immune serum; the ordinate represents the detection result (i.e.absorbance at 450 nm) of the ELISA experiment.

FIG. 3 shows the results of an ELISA assay for assessing the binding reactivity of anti-idiotype antibody 1C12 to monoclonal antibody 11B 10; wherein the abscissa represents the dilution factor of the antibody to be detected (initial concentration of 0.1mg/ml), and the ordinate represents the detection result of ELISA (i.e., absorbance at 450 nm). The results show that the anti-idiotype antibody 1C12 has a very strong binding reactivity to the monoclonal antibody 11B 10: when the monoclonal antibody 11B10 was diluted to a hundred thousand fold, the anti-idiotype antibody 1C12 was still able to bind to monoclonal antibody 11B 10; in contrast, the anti-idiotype antibody 1C12 did not have any reactivity towards HRP-labeled negative antibodies.

FIG. 4 shows the body weight change and survival rate of mice after infection with B/Florida/04/2006(FL04-MA) (FIGS. 4A and 4B) or B/Brisbane/60/2008(BR60-MA) (FIGS. 4C and 4D); wherein mice of the blank control group (labeled "PBS" in the figure) were not vaccinated and were not infected with virus; mice of the virus control group (labeled "FL 04 virus control" or "BRI 60 virus control" in the figure) were not vaccinated, but were infected with FL04-MA or BR 60-MA; mice of the negative antibody control group (labeled "negative antibody immunization group" in the figure) were vaccinated with irrelevant antibodies and infected with either FL04-MA or BR 60-MA; mice from the treatment group (labeled "1C 12 immunization group" in the figure) were vaccinated with antibody 1C12 and infected with the virus FL04-MA or BR 60-MA.

Sequence information

Information on the sequences to which the present invention relates is provided in table 1 below.

TABLE 1

Description of biological Material preservation

The present invention relates to the following biological materials which have been preserved in the China center for type culture Collection (CCTCC, China, Wuhan, university of Wuhan):

the hybridoma cell strain 11B10 has the preservation number of CCTCC NO of C201432 and the preservation time of 3 months and 26 days in 2014; and

the hybridoma cell strain 1C12 has a preservation number of CCTCC NO: C201673 and a preservation time of 2016, 5 and 10 days.

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, the molecular biological experimental methods and immunoassay methods used in the present invention are essentially described by reference to j.sambrook et al, molecular cloning: a laboratory manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, eds. molecular biology laboratory Manual, 3 rd edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes follows the conditions recommended by the product manufacturer. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed.

Example 1: preparation of anti-idiotype antibody against mAb 11B10

1. Preparation of Fab fragment of mAb 11B10

Mab 11B10 was prepared and purified according to the methods previously described (see chinese patent application 201610382361.6). The purified monoclonal antibody 11B10 was subjected to enzymatic hydrolysis with protease, followed by purification and recovery of the produced Fab fragment by ion exchange chromatography, and quantification of the protein concentration of the recovered Fab fragment. In the ion exchange chromatography method, the affinity column used was a 1ml Hitrip column (Pharmacia) to which an anti-Fab antibody (anti-Fab-AP, 1:15000 dilution, sigma) was coupled; the wash buffer used was PBS (pH 7.4) and the elution buffer was 0.2M glycine-HC 1(pH 2.4). The eluted product was immediately neutralized with Tris-HC1(1.0M, pH 8.0) and stored for further use.

2. Laboratory mouse

SPF-grade female Balb/C mice, 6 weeks old, were provided by the laboratory animal center, Xiamen university.

3. Preparation of hybridomas

Hybridoma cells secreting antibodies against the idiotype of mab 11B10 were prepared using standard in vivo immunization protocols and PEG fusion methods using Fab fragments of mab 11B10 as immunogens. For a detailed preparation of hybridoma cells, see Ed Harlow et al, "Antibodies A Laboratory Manual", Cold Spring Harbor Laboratory 1988. The brief procedure is as follows:

3.1 mouse immunization:

the Fab fragment of mab 11B10 prepared above was mixed and emulsified in equal volumes with Complete Freunds Adjuvant (CFA) for the first immunization of mice; the immunization protocol was performed by intramuscular injection through the extremities in a volume of 400. mu.l per mouse (containing 100. mu.g of emulsified Fab fragment). In addition, Fab fragments of monoclonal anti-11B 10 were mixed and emulsified with freund's incomplete adjuvant (IFA) and then used to boost mice 3 times (14 d, 28d, 42d after the first immunization, respectively); the immunization protocol was performed by intramuscular injection through the extremities in a volume of 400. mu.l per mouse (containing 100. mu.g of emulsified Fab fragment). Finally, at 56d after the first immunization, the mice were splenic boosted with the immunogen being the Fab fragment of mAb 11B10, and each mouse was injected with a volume of 100. mu.l containing 50. mu.g of the Fab fragment. 3 days after the completion of immunization, the spleen of the mouse was taken and used for the fusion experiment.

3.2 cell fusion:

taking the spleen of a mouse, and grinding to obtain a spleen cell suspension. Then, the spleen cell suspension was mixed with mouse myeloma cell SP2/0 in logarithmic growth phase, and cell fusion was performed under the action of PEG 1500. The fused cells were resuspended in 400ml of fusion medium and plated out into 20 96-well cell culture plates for culture. Fusion medium was RPMI1640 complete screening medium containing HAT and 20% FBS.

3.3 selection of hybridomas:

3.3.1 screening of Positive cell lines

The fused cells are cultured on a 96-well cell culture plate for 10 days, and then cell supernatants are extracted for ELISA detection to evaluate the reactivity of the cell supernatants with the Fab fragment of the monoclonal antibody 11B10 and determine candidate positive cell strains. Subsequently, the cell supernatant of the candidate positive cell strain is evaluated by a competition ELISA experiment to block the binding capacity of the monoclonal antibody 11B10 and the influenza B virus (B/Xiamen/3043/2006), so that the positive cell strain which can efficiently block the binding capacity of the monoclonal antibody 11B10 and the influenza B virus is screened. Cloning the screened positive cell strain for 3 times to obtain the hybridoma cell strain capable of stably secreting the antibody.

3.3.2 culture of hybridoma cell lines

And performing amplification culture on the screened hybridoma cell strain capable of stably secreting the antibody in a carbon dioxide incubator. Then, the obtained hybridoma cells were injected into the abdominal cavity of the mouse, respectively. After 7-10 days, ascites containing the monoclonal antibody was aspirated from the abdominal cavity of the mouse.

3.3.3 determination of Positive hybridoma cell lines

Recovering and purifying monoclonal antibody secreted by hybridoma cells from the obtained ascites. Mixing the purified monoclonal antibody with an aluminum adjuvant according to a volume ratio of 1:1, and using the mixture to immunize a mouse; the immunization protocol was intramuscular, with a total injection of 100. mu.l volume per mouse, containing 20. mu.g of emulsified mAb. 14d after immunization, mouse sera were collected and evaluated for specific binding to influenza B virus B/Florida/4/2006(Yamagata subfamily) and B/Brisbane/60/2008(Victoria subfamily).

Briefly, influenza B strains B/Floria/04/2006(Yamagata) and B/Brisbane/60/2008(Victoria) were inactivated with 0.03% formalin at 4 ℃. The inactivated virus was subjected to sucrose density gradient centrifugation at 25200rpm for 3 hours at 4 ℃ in an ultracentrifuge. The viral pellet was dissolved with 1 XPBS overnight at 4 ℃ to obtain a virus solution. Subsequently, the titer of the virus fluid was determined using the HA titer assay (see WHO flu virology laboratory Manual). The virus titer of the virus solution was adjusted to 128HA, which was then pre-coated on 96-well polystyrene microtiter plates at 200. mu.l per well. Subsequently, the 96-well plate was blocked with a blocking solution. The serum of the test mouse was diluted 100-fold as the initial concentration, and then diluted in a ten-fold gradient (2 times). The diluted mouse serum was added to the above microplate in a volume of 100ul per well and incubated at 37 ℃ for 30 minutes. The microplate was washed 5 times with ELISA wash (PBST), followed by addition of 100. mu.l of diluted HRP-labeled secondary antibody and incubation at 37 ℃ for 30 minutes. After washing the ELISA plate with PBST for 5 times, adding a color developing agent, and developing for 20 min. Subsequently, the a450 absorbance of each well of the microplate was read on a microplate reader.

A monoclonal antibody capable of eliciting in mice immune sera specifically binding to two sublines of influenza B virus was identified as the final positive anti-idiotype antibody. Accordingly, the hybridoma cell line secreting the positive anti-idiotype antibody is determined as the final positive hybridoma cell line.

4. Results and analysis

By the above method, a positive hybridoma cell line 1C12 was selected, which secretes a positive anti-idiotype antibody 1C 12.

FIG. 1 shows the results of a competition ELISA for assessing the ability of the cell supernatant of a positive hybridoma cell line 1C12 to block the binding of mAb 11B10 to influenza B virus (B/Xiaomen/3043/2006); wherein the abscissa represents the sample to be tested: NC represents cell supernatants from unrelated hybridoma cell lines, used as negative controls; 1C12 represents cell supernatant from hybridoma cell line 1C 12; the ordinate represents the ability of the test sample to compete for the binding of mab 11B10 to influenza B virus (competition rate), which is calculated by the following formula: 1- (OD value of ELISA assay to detect binding of 11B10 to influenza B virus in the presence of excess test sample/OD value of ELISA assay to detect binding of 11B10 to influenza B virus in the absence of test sample) × 100%. The experimental results in FIG. 1 show that the cell supernatant of hybridoma cell line 1C12 can efficiently block the binding of monoclonal antibody 11B10 and influenza B virus.

FIG. 2 shows the results of ELISA experiments for assessing the binding ability of immune sera induced in mice by anti-idiotype antibody 1C12 to Yamagata subfamily influenza B virus B/Florida/4/2006 (FIG. 2A) and Victoria subfamily influenza B virus B/Brisbane/60/2008 (FIG. 2B); wherein the abscissa represents the dilution factor of the immune serum; the ordinate represents the detection result (i.e.absorbance at 450 nm) of the ELISA experiment. The experimental results in FIG. 2 show that the anti-idiotype antibody 1C12 induced in mice in immune serum is capable of specifically recognizing and binding to influenza B viruses of two sublines (Yamagata subline and Victoria subline). This result indicates that the anti-idiotype antibody 1C12 induces antisera in animals that specifically react with at least two sublines of influenza B virus.

Example 2: binding reactivity of anti-idiotype antibody 1C12 with monoclonal antibody 11B10

1. Materials and methods

Monoclonal antibody 1C12 (200. mu.l, 100 ng/well) was pre-coated onto a 96-well polystyrene microplate. Subsequently, the 96-well plate was blocked with a blocking solution. Monoclonal antibody 11B10 was labeled with horseradish peroxidase HRP. The labeled mab 11B10 was diluted to 0.1mg/ml as the initial concentration and then further diluted in a tenfold gradient (5 times). The diluted mAb 11B10 was added to the above microplate in a volume of 100. mu.l per well and incubated at 37 ℃ for 30 minutes. After incubation, the microplate was washed 5 times with PBST, then the developer was added and developed for 20 min. Subsequently, the a450 absorbance of each well of the microplate was read on a microplate reader. In addition, a parallel experiment was also performed using an irrelevant antibody, which served as a negative control.

2. Results and analysis

The ELISA results are shown in FIG. 3. FIG. 3 shows the results of an ELISA assay for assessing the binding reactivity of anti-idiotype antibody 1C12 to monoclonal antibody 11B 10; wherein the abscissa represents the dilution factor of the antibody to be detected (initial concentration of 0.1mg/ml), and the ordinate represents the detection result of ELISA (i.e., absorbance at 450 nm). As shown in FIG. 3, the anti-idiotype antibody 1C12 has a very strong binding reactivity to mAb 11B 10: when the monoclonal antibody 11B10 was diluted to a hundred thousand fold, the anti-idiotype antibody 1C12 was still able to bind to monoclonal antibody 11B 10; in contrast, the anti-idiotype antibody 1C12 did not have any reactivity towards HRP-labeled negative antibodies.

The above results show that the anti-idiotype antibody 1C12 can specifically bind to the monoclonal antibody 11B10, and has extremely strong binding reactivity. This indicates that the anti-idiotype antibody 1C12 can be used for specific detection of broadly neutralizing monoclonal antibodies against influenza virus hemagglutinin.

Example 3 analysis of the functional Activity of anti-idiotype antibodies 1C12 induced immune sera

The cross-reactivity of antibody 1C 12-induced antisera against influenza b virus strains of different sublines was evaluated using representative strains of influenza b virus isolated at different times, in different regions, representing different types of variation, and representative strains of influenza a virus used as controls, using the hemagglutination inhibition assay (HI) and the microneutralization assay (MN).

1. Hemagglutination inhibition assay (HI)

Hemagglutination inhibition experiments (HI) were performed according to WHO guidelines. The results of the experiment are shown in table 2. The results show that the antiserum induced by antibody 1C12 in mice, except for B/Lee/1940, is specifically reactive against all influenza B strains tested, but not against influenza A viruses tested. This experimental result demonstrates that the antibody 1C 12-induced antiserum is capable of acting on a broad spectrum of influenza b virus strains (in particular strains of the Yamagata subline and the Victoria subline), with broad-spectrum reactivity across the HA subline; namely, has hemagglutination inhibitory activity against influenza B viruses of at least 2 sublines (Yamagata subline and Victoria subline).

Table 2: hemagglutination inhibition Activity of antiserum induced by antibody 1C12 against influenza Virus (HI titer)

Note: HI titer represents the maximum dilution of the virus that antisera can completely inhibit HA activity; wherein, the value is less than or equal to 10, which means no reaction.

2. Micro neutralization test (MN)

The neutralizing titer (neutralizing titer) is an important index for evaluating whether antiserum has the potential for preventing and treating diseases. In this experiment, neutralizing activity of antiserum induced by antibody 1C12 against representative strains of influenza B virus of each subline was examined by a microwell cell neutralization assay (Hulse-Post et al, PNAS.2005,102: 10682-7). The results of the experiment are shown in table 3. The results show that antibody 1C 12-induced antisera had broad-spectrum cross-neutralizing activity against most of the early-aged, non-subline influenza b viruses, all Yamagata sublines and Victoria sublines tested, and no reactivity against influenza a viruses. This experimental result demonstrates that the antibody 1C 12-induced antiserum is capable of acting on a broad spectrum of influenza b virus strains (in particular strains of the Yamagata subline and the Victoria subline), with broad-spectrum neutralizing activity across the HA subline; that is, it has neutralizing activity against influenza B viruses of at least 2 sublines (Yamagata subline and Victoria subline).

Table 3: neutralizing Activity of antibody 1C 12-induced antiserum against influenza Virus

Note: no more than 10, no reaction is indicated.

Example 4 isolation and sequence analysis of the light chain Gene and the heavy chain Gene of mAb 1C12

Semi-adherent culture of about 10⁷Individual hybridoma cells 1C 12. Adherent cells were suspended by blowing them up and transferred to a new 4ml centrifuge tube. The cells were centrifuged at 1500rpm for 3min and the cell pellet was collected. Subsequently, the cell pellet was resuspended in 100 μ l sterile PBS (pH 7.45) and transferred to a new 1.5ml centrifuge tube. Add 800. mu.l Trizol (Roche, Germany) to the centrifuge tube, mix by gentle inversion, and let stand for 10 min. Subsequently, 200. mu.l of chloroform was added to the centrifuge tube, vigorously shaken for 15s, left to stand for 10min, and then centrifuged at 12000rpm for 15min at 4 ℃. Transferring the supernatant liquid to a new 1.5ml centrifuge tube, adding equal volume of isopropanol, mixing, standing for 10min, and standingThen centrifuged at 12000rpm for 10min at 4 ℃. The supernatant was discarded, and the precipitate was washed with 600. mu.l of 75% ethanol. Subsequently, it was centrifuged again at 12000rpm for 5min at 4 ℃ and the supernatant was discarded. The precipitate was dried at 60 ℃ under vacuum for 5 min. The clear precipitate was dissolved in 70ul DEPC H₂O, and split charging into two tubes. Mu.l of reverse transcription primer is added into each tube, wherein the reverse transcription primer added into one tube is MVJkR (5'-CCG TTT GKA TYT CCA GCT TGG TSC C-3') (SEQ ID NO:19) and is used for amplifying the light chain variable region gene; the reverse transcription primer added in the other tube was MVDJhR (5'-CGG TGA CCG WGG TBC CTT GRC CCC A-3') (SEQ ID NO:20) for amplifying the heavy chain variable region gene. Mu.l dNTP (Shanghai, Ltd.) was added to each tube, incubated in a 72 ℃ water bath for 10min, and then immediately placed in an ice bath for 5 min. Subsequently, 10. mu.l of 5 Xreverse transcription buffer, 1. mu.l of AMV (10 u/. mu.l, Pormega), and 1. mu.l of Rnasin (40 u/. mu.l, Promega) were added to each tube and mixed well. Then, reverse transcription reaction was performed at 42 ℃ to reverse transcribe RNA into cDNA.

Subsequently, the variable regions of the antibody genes were isolated by the polymerase chain reaction method using the primers shown in Table 4 and the primers MVJkR (SEQ ID NO:19) and MVDJhR (SEQ ID NO:20) (synthesized by Shanghai Boya). Wherein, MuIgkVl5'-F4 and MVJkR are respectively an upstream primer and a downstream primer for amplifying a light chain variable region gene, and MuIgVh5' -B2 and MVDJhR are respectively an upstream primer and a downstream primer for amplifying a heavy chain variable region gene. The templates used were the two cDNAs obtained in the previous step. The PCR conditions were: 94 ℃ for 5min, 35 cycles (94 ℃ for 40s, 53 ℃ for 1min, 72 ℃ for 50s), 72 ℃ for 15 min. The PCR amplification product was recovered and cloned into pMD 18-T vector and then sent to Shanghai Boya for sequencing. From the sequencing results, the gene sequences encoding the heavy chain variable region and the light chain variable region of antibody 1C12 were determined, and further the amino acid sequences of the heavy chain variable region and the light chain variable region were determined. Sequencing results show that the nucleotide sequences of the heavy chain variable region and the light chain variable region of the antibody 1C12 are shown as SEQ ID NO 9 and 10, respectively, and the amino acid sequences are shown as SEQ ID NO 7and 8, respectively.

Further, the amino acid sequences of the 6 CDR regions of antibody 1C12 were determined by reference to the Kabat method (Kabat EA, Wu TT, Perry HM, Gottesman KS, Coeller K.sequences of proteins of immunological interest, U.S. Department of Health and Human Services, PHS, NIH, Bethesda, 1991). The results showed that CDR1, CDR2 and CDR3 of the heavy chain variable region of antibody 1C12 are shown in SEQ ID NO:1-3, respectively, and CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO:4-6, respectively.

Table 4: sequence of upstream primer for amplifying variable region gene of monoclonal antibody 1C12

Example 5 use of anti-idiotype antibody 1C12 for the prevention of influenza Virus infection

The previous examples have been confirmed by trace neutralization tests: anti-idiotype antibody 1C 12-induced immune sera had strong neutralizing activity against influenza B virus strains of at least two HA sublines (Yamagata subline and Victoria subline) both at different isolates and at different years of isolation. To further verify the effect of anti-idiotypic antibody 1C12 as a vaccine to induce immune protection against influenza virus in animals, the present inventors evaluated the in vivo antiviral effect of anti-idiotypic antibody 1C12 in a biosafety laboratory based on mouse animal models infected with influenza b virus of Yamagata sublines and Victoria sublines. The specific scheme is as follows:

(1) materials and methods

Animals: Balb/C mice, SPF, 6-8 weeks old, female, approximately 20g in weight.

Vaccine: fab fragments of anti-idiotype antibody 1C12

Immunization protocol: the Fab fragment of antibody 1C12 was mixed with aluminum adjuvant at a volume ratio of 1:1 for immunization of mice. The immunization was performed intramuscularly at an immunization dose of 20. mu.g protein/mouse and an injection volume of 100. mu.l/mouse. After immunization for 14d, mice were challenged. The influenza b strains used were as follows:

mouse adapted strains of Yamagata subline influenza b virus: B/Florida/04/2006, FL04-MA for short;

mouse adapted strains of Victoria subline influenza B virus: B/Brisbane/60/2008, BR60-MA for short.

Anesthetic agent: isoflurane (isoflurane).

Animal grouping: mice were sent to a biosafety laboratory one day in advance, grouped in 5 cages, labeled G1, G2, … …, and the body weight of each mouse was recorded. The detailed grouping scheme is shown in table 5.

Viral infection: the Yamagata subline virus B/Florida/04/2006 was pre-diluted to 10⁵TCID₅₀Ul, Victoria subline virus B/Brisbane/60/2008 was pre-diluted to 10⁶TCID₅₀Ul. The virus was inoculated in 50. mu.l/mouse. Prior to inoculation, mice were anesthetized with isofradecane and then vaccinated intranasally with virus.

And (4) observing and recording: mice were recorded daily for weight change, survival and corresponding behavioral characterization 1-14 days after viral infection.

Table 5: test protocol

(2) Results and analysis

14 days after immunization of mice intramuscularly with the Fab fragment of antibody 1C12, mice were infected with a lethal dose of one week each of Yamagata subline influenza B virus FL04-MA and Victoria subline influenza B virus BR 60-MA. The immunization effect of antibody 1C12 as a vaccine was judged by measuring the body weight of each group of mice and calculating the survival rate after virus infection. The results of the experiment are shown in fig. 4.

The experimental results of fig. 4 show that: during the whole experiment, no obvious weight fluctuation of the mice in the blank control group is observed; mice in the virus control group of both viruses showed significant weight loss. The experimental results of fig. 4 also show that mice of the Y subfamily virus control group all died 6 days after infection, and mice of the negative antibody control group of FL04-MA all died 8 days after infection; mice of the V subline virus control group all died 6 days after virus infection, and mice of the negative antibody control group of BR60-MA all died 6 days after infection. In contrast, 1C12 antibody at a dose of 20 μ g/mouse was able to recover body weight in mice infected with the virus FL04-MA or BR60-MA for both influenza B viruses tested (FIGS. 4A and 4C). In addition, the results of the experiment in FIG. 4 also show that 1C12 antibody at a dose of 20. mu.g/mouse can normally survive for 14 days in mice infected with the virus FL04-MA or BR60-MA with a therapeutic effect of 100% (FIGS. 4B and 4D).

The above experimental results show that the anti-idiotype antibody 1C12 can effectively prevent infection by influenza b viruses of two HA sublines and diseases caused by the same in animals, and thus can be used as an effective broad-spectrum vaccine against influenza b viruses of at least two sublines.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (60)

1. A monoclonal antibody or antigen-binding fragment thereof, comprising: CDR1, CDR2 and CDR3 of the heavy chain variable region of SEQ ID NO 1-3 respectively; and, CDR1, CDR2 and CDR3 of the light chain variable region having the amino acid sequences of SEQ ID Nos. 4-6, respectively.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein said monoclonal antibody comprises a heavy chain variable region as set forth in SEQ ID NO. 7 and/or a light chain variable region as set forth in SEQ ID NO. 8.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein said monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab ', F (ab')₂Fv, single chain antibody, mouse antibody, humanized antibody, chimeric antibody or multispecific antibody.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein the single chain antibody is an scFv.

5. The antibody or antigen-binding fragment thereof of claim 3, wherein the chimeric antibody is a human murine chimeric antibody; alternatively, the multispecific antibody is a bispecific antibody.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof comprises non-CDR regions from a species other than murine.

7. The antibody or antigen-binding fragment thereof of claim 6, wherein the non-CDR region is from a human antibody.

8. The antibody or antigen-binding fragment thereof of claim 1, wherein the monoclonal antibody is a monoclonal antibody produced by hybridoma cell line 1C12, wherein hybridoma cell line 1C12 is deposited with the China Center for Type Culture Collection (CCTCC) and has a preservation number of CCTCC NO: C201673.

9. The antibody or antigen-binding fragment thereof of claim 1, wherein said monoclonal antibody or antigen-binding fragment thereof is capable of specifically binding to the idiotype, or antigen-binding portion thereof, of a second monoclonal antibody comprising: the amino acid sequences are CDR1, CDR2 and CDR3 of the heavy chain variable region of SEQ ID NO. 11-13, respectively, and the amino acid sequences are CDR1, CDR2 and CDR3 of the light chain variable region of SEQ ID NO. 14-16, respectively.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein the second monoclonal antibody comprises: the heavy chain variable region shown as SEQ ID NO. 17 and the light chain variable region shown as SEQ ID NO. 18.

11. The antibody or antigen-binding fragment thereof of claim 9, wherein the second monoclonal antibody is monoclonal antibody 11B10, and the monoclonal antibody 11B10 is produced by hybridoma cell line 11B10 deposited at the chinese culture collection center (CCTCC) and having a collection number of CCTCC NO: C201432.

12. The antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein said monoclonal antibody or antigen-binding fragment thereof is capable of inducing an antiserum having hemagglutination-inhibiting activity and/or neutralizing activity against influenza b viruses of the Yamagata subfamily and Victoria subfamily in an animal.

13. The antibody or antigen-binding fragment thereof of claim 12, wherein said monoclonal antibody or antigen-binding fragment thereof is capable of inducing an antiserum having hemagglutination inhibitory activity and/or neutralizing activity against influenza b viruses of the Yamagata subfamily and Victoria subfamily in a mouse or a human.

14. An isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding an antibody heavy chain variable region comprising CDR1, CDR2 and CDR3 having the amino acid sequences SEQ ID NOs 1-3, respectively, and a nucleic acid sequence encoding an antibody light chain variable region comprising CDR1, CDR2 and CDR3 having the amino acid sequences SEQ ID NOs 4-6, respectively.

15. The isolated nucleic acid molecule of claim 14, wherein the amino acid sequence of the antibody heavy chain variable region is set forth in SEQ ID NO 7.

16. The isolated nucleic acid molecule of claim 14, wherein the amino acid sequence of the antibody light chain variable region is set forth in SEQ ID No. 8.

17. The isolated nucleic acid molecule of any one of claims 14-16, wherein the nucleic acid molecule has a nucleotide sequence as set forth in SEQ ID No. 9 and SEQ ID No. 10.

18. An isolated nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof of claim 1.

19. A vector comprising the isolated nucleic acid molecule of any one of claims 14-18.

20. A host cell comprising the isolated nucleic acid molecule of any one of claims 14-18 or the vector of claim 19.

21. A method of preparing the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-13, comprising culturing the host cell of claim 20 under suitable conditions, and recovering the monoclonal antibody or antigen-binding fragment thereof from the cell culture.

22. Hybridoma cell line 1C12, which is preserved in China Center for Type Culture Collection (CCTCC) and has a preservation number of CCTCC NO: C201673.

23. A composition comprising the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-13, or the isolated nucleic acid molecule of any one of claims 14-18, or the vector of claim 19, or the host cell of claim 20.

24. A kit comprising the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-13.

25. The kit of claim 24, wherein the monoclonal antibody or antigen-binding fragment thereof further comprises a detectable label.

26. The kit of claim 25, wherein said detectable label is selected from the group consisting of a radioisotope, a luminescent substance, a colored substance, and an enzyme.

27. The kit according to claim 26, wherein the luminescent material is a fluorescent material.

28. The kit of any one of claims 24-27, wherein the kit further comprises a second antibody that specifically recognizes the monoclonal antibody or antigen-binding fragment thereof.

29. The kit of claim 28, wherein said second antibody further comprises a detectable label.

30. The kit of claim 29, wherein said detectable label is selected from the group consisting of a radioisotope, a luminescent substance, a colored substance, and an enzyme.

31. The kit according to claim 30, wherein the luminescent material is a fluorescent material.

32. A method for non-diagnostic purposes of detecting the presence or level of an antibody against the hemagglutinin protein of influenza b virus in a sample, comprising the use of the monoclonal antibody or antigen binding fragment thereof according to any one of claims 1 to 13.

33. The method of claim 32, wherein the monoclonal antibody or antigen-binding fragment thereof further comprises a detectable label.

34. The method of claim 33, wherein said detectable label is selected from the group consisting of a radioisotope, a luminescent substance, a colored substance, and an enzyme.

35. The method of any one of claims 32-34, wherein the method further comprises detecting the monoclonal antibody or antigen-binding fragment thereof using a second antibody carrying a detectable label.

36. The method of claim 35, wherein said detectable label is selected from the group consisting of a radioisotope, a luminescent substance, a colored substance, and an enzyme.

37. The method of claim 34 or 36, wherein the luminescent material is a fluorescent material.

38. Use of the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-13 in the preparation of a kit for detecting the presence or level of an antibody against influenza b virus hemagglutinin protein in a sample, or for determining whether a subject has produced an antibody against influenza b virus hemagglutinin protein.

39. The use of claim 38, wherein the sample is fecal matter, oral or nasal secretions from a subject.

40. The use of claim 39, wherein the subject is a mammal.

41. The use of claim 39, wherein the subject is a human.

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

43. The pharmaceutical composition of claim 42, wherein the pharmaceutical composition further comprises an additional pharmaceutically active agent.

44. The pharmaceutical composition of claim 43, wherein the additional pharmaceutically active agent is an anti-influenza drug.

45. The pharmaceutical composition of claim 43, wherein the additional pharmaceutically active agent is an M2 protein ion channel inhibitor, a neuraminidase inhibitor, or an influenza vaccine.

46. The pharmaceutical composition of claim 45, wherein the M2 protein ion channel inhibitor is selected from the group consisting of amantadine and rimantadine.

47. The pharmaceutical composition of claim 45, wherein said neuraminidase inhibitor is oseltamivir.

48. The pharmaceutical composition of claim 45, wherein the influenza virus vaccine is brigda.

49. The pharmaceutical composition of any one of claims 42-48, wherein the pharmaceutical composition is a vaccine.

50. Use of the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-13 for the preparation of a pharmaceutical composition for preventing or treating an influenza b virus infection of a Yamagata subline or Victoria subline or a disease associated with an influenza b virus infection of a Yamagata subline or Victoria subline in a subject.

51. The use of claim 50, wherein the disease is influenza.

52. The use of claim 50, wherein the subject is a mammal.

53. The use of claim 50, wherein the subject is a human.

54. The use of claim 50, wherein the pharmaceutical composition is used alone or in combination with other pharmaceutically active agents.

55. The use of claim 54, wherein the other pharmaceutically active agent is an anti-influenza virus drug.

56. The use of claim 54, wherein the other pharmaceutically active agent is an M2 protein ion channel inhibitor, a neuraminidase inhibitor, or an influenza vaccine.

57. The use of claim 56, wherein the M2 protein ion channel inhibitor is selected from the group consisting of amantadine and rimantadine.

58. The use of claim 56, wherein said neuraminidase inhibitor is oseltamivir.

59. The use of claim 56, wherein the influenza virus vaccine is brigda.

60. The use of any one of claims 50-59, wherein the pharmaceutical composition is a vaccine.

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