WO2021142671A1

WO2021142671A1 - Monoclonal antibody for echovirus 30

Info

Publication number: WO2021142671A1
Application number: PCT/CN2020/072310
Authority: WO
Inventors: 张黎; 王康; 郑滨洋; 朱玲; 苏璇; 张倩; 陶焱炀; 崔仑标; 潘红星; 葛以跃; 吴涛; 王祥喜; 朱凤才
Original assignee: 江苏省疾病预防控制中心(江苏省公共卫生研究院)
Priority date: 2020-01-15
Filing date: 2020-01-15
Publication date: 2021-07-22

Abstract

Provided are a monoclonal antibody for echovirus 30, and corresponding nucleic acid molecule, expression vector, host cells, preparation method, immunoconjugate, bispecific molecule, drug composition, methods for detecting echovirus 30 and treating diseases.

Description

Monoclonal antibody to Echo virus type 30

Technical field

This application belongs to the field of biomedicine, and specifically relates to a monoclonal antibody against Echovirus type 30.

Background technique

Human Enteric Cytopathogenic Human Orphan Virus (ECHO), abbreviated as Echo virus, is a single-stranded positive-stranded RNA enterovirus with 34 serotypes. At first, its pathogenicity was not clear, but it was later proved that Echo virus is related to various diseases such as aseptic meningitis, infant diarrhea, hand, foot and mouth disease. A large number of studies and data analysis finally show that the disease is spread all over the world, causing widespread transmission or epidemic in the population, and its infection can cause aseptic meningitis, rash, gastrointestinal diseases, hepatitis and pneumonia and other human diseases. Among them, aseptic meningitis is the most common and can be spread through the respiratory and digestive tracts. Pregnant women can be transmitted to the fetus through the placenta after infection, which can cause fetal malformations and even stillbirth.

Echovirus 30 (Echo30) is a type of intestinal Echovirus. The virus spreads widely and is highly contagious. Most of the infections are children under 15 years of age. Infection can cause a variety of diseases, among which aseptic meningitis is a serious complication. Echo30 has become the main pathogen causing aseptic meningitis in children at home and abroad in recent years.

At present, there are relatively few studies on Echo30 virus, and there are no reports of specific antibodies. The purpose of this application is to prepare specific antibodies against Echo30 virus in order to detect Echo30 virus and related diseases caused by it.

Summary of the invention

The present invention provides isolated monoclonal antibodies that bind to Echo30 virus and exhibit many desired characteristics. The characteristics include high-affinity binding to Echo30 virus and neutralization of Echo30 virus activity.

In one aspect, the present invention relates to a monoclonal antibody or an antigen-binding portion thereof, the monoclonal antibody comprising one or more CDRs in the variable region of the heavy chain, and/or one or more CDRs in the variable region of the light chain Multiple CDRs;

Preferably,

The amino acid sequence of the CDR of the heavy chain variable region is selected from:

SEQ ID NO: the amino acid sequence shown in 1-3;

Or an amino acid sequence formed by substitution, deletion or addition of one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 1-3;

Or an amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 1-3, and has the same or similar function;

More preferably,

The heavy chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO:1, or has an amino acid sequence formed by substitution, deletion or addition of one or more amino acids based on the amino acid sequence shown in SEQ ID NO:1; or An amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO:1 and has the same or similar function;

The heavy chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 2, or has an amino acid sequence formed by substitution, deletion or addition of one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO: 2; or An amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 2 and has the same or similar function;

The heavy chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 3, or has an amino acid sequence formed by substitution, deletion or addition of one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 3; or An amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 3 and has the same or similar function;

Preferably,

The amino acid sequence of the CDR of the light chain variable region is selected from:

SEQ ID NO: the amino acid sequence shown in 5-7;

Or an amino acid sequence formed by substitution, deletion or addition of one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 5-7;

Or an amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 5-7 and has the same or similar function;

More preferably,

The light chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 5, or has an amino acid sequence formed by substitution, deletion or addition of one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 5; or An amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 5, and has the same or similar function;

The light chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 6, or has an amino acid sequence formed by substitution, deletion or addition of one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 6; or An amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 6 and has the same or similar function;

The light chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 7, or has an amino acid sequence formed by substitution, deletion or addition of one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO: 7; or An amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 7 and has the same or similar function.

Most preferably, the heavy chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 1; the heavy chain variable region CDR2 has SEQ ID NO: 2; the heavy chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 3. Amino acid sequence; light chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 5; light chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 6; light chain variable region CDR3 has SEQ ID NO: The amino acid sequence shown in 7.

Further, the monoclonal antibody includes a heavy chain variable region and/or a light chain variable region, wherein:

The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 4, or has an amino acid sequence formed by substitution, deletion or addition of one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO: 4; Or an amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 4 and has the same or similar function;

The light chain variable region has an amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence formed by substitution, deletion or addition of one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO: 8; Or an amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 8 and has the same or similar function.

Preferably, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 4; the light chain variable region has the amino acid sequence shown in SEQ ID NO: 8.

"At least 80% homology" used in the present invention means 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology.

The antigen-binding portion of the monoclonal antibody includes the Fab fragment, F(ab')2 fragment, and single-chain Fv fragment of the monoclonal antibody.

The monoclonal antibody disclosed in the present invention further includes a framework region. The framework region includes a mouse receptor framework region or a human receptor framework region.

The monoclonal antibody of the present invention may also be a CDR grafted antibody. Preferably, the CDR grafted antibody comprises one or more of the CDRs disclosed above.

Preferably, the CDR grafted antibody comprises an acceptor framework region.

The monoclonal antibody of the present invention may be a humanized antibody. Preferably, the humanized antibody comprises one or more of the CDRs disclosed above. More preferably, the humanized antibody contains three or more of the CDRs disclosed above. Most preferably, the humanized antibody contains 6 CDRs as disclosed above. In a specific embodiment, CDRs are inserted into the human acceptor framework region of the variable region of a human antibody. Preferably, the human antibody variable region is a shared human variable region. More preferably, the human acceptor framework region contains at least one framework region amino acid substitution at a key residue, wherein the key residue is selected from residues adjacent to the CDR; glycosylation site residues; rare residues; (20-42) Residues that have influence on the globulomer; residues that can have an influence on CDR; canonical residues; contact residues between the variable region of the heavy chain and the variable region of the light chain; the Vernier region Residues; and residues in the overlapping region between the variable heavy chain CDR1 defined by Chothia and the framework region of the first heavy chain defined by Kabat. Preferably, the human acceptor framework region comprises at least one framework region amino acid substitution, wherein the amino acid sequence of the framework region is at least 65% identical to the sequence of the human acceptor framework region and contains at least 70 identical to the human acceptor framework region The amino acid residues.

The monoclonal antibody disclosed in the present invention further comprises a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, and IgE constant regions.

The monoclonal antibodies disclosed in the present invention may be glycosylated antibodies. Preferably, the glycosylation type is a human glycosylation type or a glycosylation type produced by any of the eukaryotic cells disclosed herein, especially CHO cells.

The present invention also relates to the antigen binding portion of the monoclonal antibody of the present invention. The aforementioned antigen-binding portion includes, but is not limited to, antibody Fab fragments, F(ab')2 fragments, and single-chain Fv fragments. More antigen-binding parts are Fab' fragments, Fv fragments, and disulfide-linked Fv fragments.

The invention also provides an isolated nucleic acid encoding any of the antibodies disclosed herein. A further embodiment provides a vector comprising the isolated nucleic acid disclosed herein. The vector may be particularly selected from pcDNA; pTT (Durocher et al., Nucleic Acids Research 2002, Vol 30, No. 2); pTT3 (pTT with additional multiple cloning sites); pEFBOS (Mizushima, S. and Nagata, S. ., (1990) Nucleic Acids Research Vol 18, No. 17); pBV; pJV and pBJ.

The host cell is transformed with the vector disclosed herein. Preferably, the host cell is a prokaryotic cell. More preferably, the host cell is E. coli. In a related embodiment, the host cell is a eukaryotic cell. Preferably, the eukaryotic cell is selected from the group consisting of protist cells, animal cells (such as mammalian cells, avian cells and insect cells), plant cells and fungal cells. More preferably, the host cell is a mammalian cell, including but not limited to CHO and COS; or a fungal cell, such as a yeast cell, such as Saccharomyces cerevisiae; or an insect cell such as Sf9.

In a specific embodiment of the present invention, the nucleic acid encoding the heavy chain variable region CDR1 has the sequence shown in SEQ ID NO: 9; the nucleic acid encoding the heavy chain variable region CDR2 has the sequence shown in SEQ ID NO: 10; encoding The nucleic acid of the heavy chain variable region CDR3 has the sequence shown in SEQ ID NO: 11; the nucleic acid that encodes the light chain variable region CDR1 has the sequence shown in SEQ ID NO: 13; the nucleic acid that encodes the light chain variable region CDR2 has SEQ ID NO: 14; the nucleic acid encoding the light chain variable region CDR3 has the sequence shown in SEQ ID NO: 15.

In a specific embodiment of the present invention, the nucleic acid encoding the variable region of the heavy chain has the sequence shown in SEQ ID NO: 12; the nucleic acid encoding the variable region of the light chain has the sequence shown in SEQ ID NO: 16.

The present invention also provides a method for producing the antibody of the present invention, which can be obtained by methods known in the art.

Standard hybridoma technology enables the production of antibodies with a single specificity against the antigen of interest. It can be achieved by using standard techniques such as Kohler and Milstein (1975, Nature 256: 495-497) (see also Brown et al. (1981) J. Immunol 127: 539-46; Brown et al. (1980) J Biol Chem 255: 4980). -83; Yeh et al. (1976) PNAS 76: 2927-31; and Yeh et al. (1982) Int. J. Cancer 29: 269-75) described hybridoma technology to produce monoclonal antibodies. The technology for producing monoclonal antibody hybridomas is well known (usually see RHKenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York (1980); EALerner (1981) Yale J . Biol. Med., 54: 387-402; MLGefter et al. (1977) Somatic Cell Genet., 3: 231-36). In short, the immortal cell line (usually myeloma) is fused with the lymphocytes (usually spleen cells or lymph node cells or peripheral blood lymphocytes) of mammals immunized with Echo virus type 30, and the obtained The culture supernatant of the hybridoma cells in order to identify the hybridoma producing the monoclonal antibody of the present invention. Any of the many well-known methods for fusing lymphocytes and immortal cell lines can be used for this purpose (see also G. Galfre et al. (1977) Nature 266: 550-52; Gefter et al. Somatic Cell Genet., in Previously cited; Lerner, Yale J. Biol. Med., previously cited; Kenneth, Hybridoma, previously cited). In addition, those skilled in the art should understand that there are different variations of the above methods, which are also useful. Typically, immortal cell lines (such as myeloma cell lines) are derived from the same mammalian species as lymphocytes. For example, murine hybridomas can be established by fusing lymphocytes from mice immunized with the immunogenic preparation of the present invention with an immortalized mouse cell line. A preferred immortal cell line is a mouse myeloma cell line sensitive to a medium containing hypoxanthine, aminopterin and thymidine (HAT medium). Any of a variety of myeloma cell lines can be used as a fusion partner by default, such as P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma cell line. These myeloma cell lines are available from the American Type Culture Collection (ATCC), Rockville, MD. Typically, polyethylene glycol (PEG) is used to fuse HAT-sensitive mouse myeloma cells with mouse spleen cells. Then, HAT medium was used to select the hybridoma cells produced by the fusion, thereby killing the unfused and fused myeloma cells that did not produce the product (a few days later, the unfused spleen cells died because they were not transformed). The hybridoma cells producing the monoclonal antibody of the present invention are identified by screening the hybridoma culture supernatant with the aforementioned antibodies.

As an alternative to producing the antibodies of the present invention through immunization and selection, the recombinant combinatorial immunoglobulin library can be screened with Echovirus type 30, thereby isolating immunoglobulin library members with the desired binding affinity, thereby identifying and Isolate the antibodies of the invention. Kits for generating and screening display libraries are commercially available (eg, Pharmacia recombinant phage antibody system, catalog number 27-9400-01; and Stratagene phage display kit, catalog number 240612). In many embodiments, the display library is a scFv library or a Fab library. The phage display technology used to screen recombinant antibody libraries has been described in detail. Examples of methods and compounds that can be particularly advantageously used to generate and screen antibody display libraries can be found in, for example, WO 92/01047, US 5,969,108 and EP 589 877 by McCafferty et al. (scFv display is described in detail), and US 5,223,409 by Ladner et al. , US 5,403,484, US 5,571,698, US 5,837,500 and EP 436 597 (for example, describing pIII fusion); Dower et al.'s WO 91/17271, US 5,427,908, US 5,580,717 and EP 527 839 (detailed description of Fab display); International Published WO92/20791 and EP 368,684 (especially describing the sequence of the cloned immunoglobulin variable region); Griffiths et al. US 5,885,793 and EP 589 877 (detailed description of the isolation of human antibodies against human antigens by using recombinant libraries); Garrard WO 92/09690 (especially describing the phage expression technology); WO 97/08320 of Knappik et al. (describes the human recombinant antibody library HuCal); WO 97/29131 of Salfeld et al. (describes anti-human antigen (human tumor necrosis) Factor α) recombinant human antibody production and the in vitro affinity maturation of the recombinant antibody) and Salfeld et al.'s U.S. Provisional Application No. 60/126,603 and a patent application based thereon (similarly describes a human antigen (human interleukin-12 ) The production of recombinant human antibody, also described the in vitro affinity maturation of the recombinant antibody).

More descriptions of screening recombinant antibody libraries can be found in scientific publications, such as Fuchs et al. (1991) Bio/Technology 9: 1370-1372; Hay et al. (1992) Human Antibod Hybrid 3: 81-85; Huse et al. (1989) Science 246: 1275-1281; Griffiths et al. (1993) EMBO J 12: 725-734; Hawkins et al. (1992) J Mol Biol 226: 889-896; Clarkson et al. (1991) Nature 352: 624-628; Gram et al. (1992) PNAS89: 3576-3580; Garrard et al. (1991) Bio/Technology 9: 1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19: 4133-4137; Barbas et al. (1991) PNAS 88: 7978-7982; McCafferty et al. Nature (1990) 348:552-554 and Knappik et al. (2000) J. Mol. Biol. 296:57-86.

As an alternative to using a phage display system, recombinant antibody libraries can be expressed on the surface of yeast cells or bacterial cells. WO 99/36569 describes a method for preparing and screening a library expressed on the surface of yeast cells. WO 98/49286 describes a more detailed method of preparing and screening libraries expressed on the surface of bacterial cells.

Once the target antibody of the combinatorial library has been identified and fully characterized, the DNA sequences encoding the light and heavy chains of the antibody can be isolated by using standard molecular biology techniques, for example by using a display package (e.g., Phage) PCR amplification of DNA. The nucleotide sequences of genes encoding the light and heavy chains of antibodies that can be used to prepare PCR primers are known to those skilled in the art. A variety of the above-mentioned sequences are described in, for example, Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 and the ethnic sequence database VBASE.

The antibody or antibody portion of the present invention can be produced by recombinantly expressing genes encoding immunoglobulin light and heavy chains in host cells. In order to express the antibody recombinantly, a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody, thereby expressing the light and heavy chains in the host cell. They are preferably secreted into the medium in which the host cell is cultured. The antibody can be isolated from this medium. Standard recombinant DNA methods are used to obtain the genes encoding the antibody heavy and light chains, insert the genes into a recombinant expression vector and introduce the vector into a host cell. Such methods are described in, for example, Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, 2nd edition, Cold Spring Harbor, NY, (1989), Ausubel, FM, etc. (eds) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and US 4,816,397 of Boss et al.

Once the DNA fragments encoding the VH and VL segments of the antibody of interest are obtained, standard recombinant DNA techniques can be used to further manipulate the DNA fragments, for example, in order to convert the gene encoding the variable region into the gene encoding the full-length antibody chain, Fab fragment gene or scFv gene. These operations include operably linking a VL- or VH-encoding DNA fragment to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" should be understood here as meaning that two DNA fragments are connected in such a way that the amino acid sequence encoded by the two DNA fragments remains in frame.

By operably linking the VH-region encoding DNA to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3), the isolated DNA encoding the VH region can be converted into a gene encoding the full-length heavy chain . The sequence of the human heavy chain constant region gene is well known (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, USDepartment of Health and Human Services, NIH publication number 91-3242), And the DNA fragments spanning the region can be obtained by using standard PCR amplification. The heavy chain constant region may be a constant region derived from IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgE or IgD, preferably the constant region is derived from IgG, especially IgG1 or IgG4. In order to obtain the gene encoding the heavy chain Fab fragment, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain constant region CH1.

The VL-encoding DNA can be operably linked to another DNA molecule encoding the light chain constant region CL, thereby converting the isolated DNA encoding the VL region into a gene encoding the full-length light chain (and encoding the Fab light chain). Chain genes). The gene sequence of the human light chain constant region is well known (see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, USDepartment of Health and Human Services, NIH publication number 91-3242), and across the description The DNA fragments of the region can be obtained by using standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, preferably a kappa constant region.

In order to generate the scFv gene, the VH- and VL-encoding DNA fragments can be operably linked to another fragment encoding a flexible linker, such as amino acid sequence (Gly4-Ser) 3, so that the VH and VL sequences have VL and VH regions to each other. The continuous single-chain protein expression connected by the flexible linker (see Bird et al. (1988) Science 242: 423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al., Nature (1990) 348:552-554).

The single-domain VH and VL having the above-mentioned binding affinity can be isolated from the single-domain library by the above-mentioned method. As described herein, the pairing of two VH single-region chains (with or without CH1) or two VL chains or one VH chain and one VL chain with the desired binding affinity can be used in the antibody of the present invention.

In order to express the recombinant antibody or antibody portion of the present invention, DNAs encoding partial or full-length light and heavy chains can be inserted into an expression vector so that the gene can be operably linked to appropriate transcription or translation control sequences. In this context, the term "operably linked" should be understood as meaning that the antibody gene is linked in such a way that the transcription and translation control sequences in the vector fulfill its intended regulation of the transcription and translation of the antibody gene. In the carrier.

Preferably, the expression vector and expression control sequence are selected so as to be compatible with the expression host cell used. It is common to insert the gene encoding the light chain of the antibody and the gene encoding the heavy chain of the antibody into different vectors, or insert the two genes into the same expression vector. The antibody gene is inserted into the expression vector by using standard methods (for example, by connecting complementary restriction cleavage sites on the antibody gene fragment and the vector, or by joining blunt ends if no restriction cleavage sites exist). The expression vector may already carry the sequence encoding the constant region of the antibody before inserting the sequence encoding the light chain and the heavy chain. For example, one method is by inserting the VH and VL sequences into an expression vector that has encoded the heavy and light chain constant regions, respectively, thereby operably linking the VH segment and the CH segment in the vector and operably The VL segment and the CL segment are connected in the vector to convert the VH and VL sequences into full-length antibody genes.

Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates the secretion of the antibody chain from the host cell. The gene encoding the antibody chain can be cloned into a vector, thereby linking the signal peptide to the N-terminus of the gene encoding the antibody chain in frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein). In addition to the gene encoding the antibody chain, the expression vector of the present invention may have a regulatory sequence that controls the expression of the gene encoding the antibody chain in the host cell.

The term "regulatory sequence" is intended to include promoters, enhancers, and more expression control elements (such as polyadenylation signals) that control the transcription or translation of antibody chain-encoding genes. This type of regulatory sequence is described in, for example, Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Those skilled in the art should understand that the design of the expression vector including the selection regulatory sequence may depend on factors such as the selection of the host cell to be transformed, the desired protein expression intensity, and the like. Preferred regulatory sequences for expression in mammalian host cells include viral elements that cause strong and constitutive protein expression in mammalian cells, for example from cytomegalovirus (CMV) (such as CMV promoter/enhancer), simian Virus 40 (SV40) (such as SV40 promoter/enhancer), adenovirus (such as adenovirus major late promoter (AdMLP)) and polyoma virus promoter and/or enhancer. For a further description of viral regulatory elements and their sequences, see, for example, US 5,168,062 to Stinski, US 4,510,245 to Bell et al., and US 4,968,615 to Schaffner et al.

In addition to genes encoding antibody chains and regulatory sequences, the recombinant expression vector of the present invention may have additional sequences such as those that regulate the replication of the vector in host cells (such as the origin of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, for example, US Patent Nos. 4,399,216, 4,634,665, and 5,179,017 to Axel et al.). For example, the commonality of the selectable marker gene is that the host cell into which the vector has been inserted is resistant to cytotoxic drugs such as G418, hygromycin, or methotrexate. Preferred selectable marker genes include the gene encoding dihydrofolate reductase (DHFR) (for dhfr-host cells with methotrexate selection/amplification) and neo gene (for G418 selection).

In order to express the light chain and the heavy chain, an expression vector encoding the heavy chain and the light chain is transfected into the host cell by standard techniques. The various forms of the term "transfection" are intended to include a variety of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although it is theoretically possible to express the antibody of the present invention in prokaryotic or eukaryotic host cells, it is preferred to express the antibody in eukaryotic cells, especially mammalian host cells, because compared with prokaryotic cells, the above-mentioned true Nuclear cells, especially mammalian cells, are more likely to assemble and secrete correctly folded and immunologically active antibodies. It has been reported that the prokaryotic expression of antibody genes cannot effectively produce high-yield active antibodies (Boss, M.A. and Wood, C.R. (1985) Immunology Today 6: 12-13).

Preferred mammalian host cells for expressing the recombinant antibodies of the present invention include CHO cells (including the dhfr-CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, which are compatible with DHFR-selectable markers as described in RJ Kaufman and PASharp (1982) Mol. Biol. 159:601-621), NS0 myeloma cells, COS cells and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the host cell is cultured until the antibody is expressed in the host cell, thereby producing the antibody, or preferably, the antibody is secreted into the medium in which the host cell grows. The antibody can then be separated from the culture medium by using standard protein purification methods.

In order to produce a part of a complete antibody such as Fab fragments or scFv molecules, it is also possible to use host cells. Variations of the above method are of course included in the present invention. For example, it may be desirable to transfect a host cell with DNA encoding the light chain or the heavy chain (but not both) of the antibody of the invention. If the presence of a light chain or a heavy chain is not necessary for the target antigen binding, the DNA encoding the light chain or the heavy chain or both can be partially or completely removed by using recombinant DNA technology. The molecules expressed by the aforementioned truncated DNA molecules are also included in the antibody of the present invention. In addition, by using standard chemical methods to cross-link the antibody of the present invention with another antibody, it is possible to produce that one heavy chain and one light chain are the antibody of the present invention, and the other heavy chain and the other light chain have a A bifunctional antibody with the specificity of an antigen different from the target antigen.

In a preferred system for recombinant expression of the antibody of the present invention or an antigen-binding portion thereof, a recombinant expression vector encoding the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. In this recombinant expression vector, the genes encoding the antibody heavy and light chains are operably linked to the CMV enhancer/AdMLP-promoter control element in all cases to strongly influence the transcription of the genes. The recombinant expression vector also carries the DHFR gene, which can be used to select dhfr-CHO cells transfected with the vector by using methotrexate secretion/amplification. The selected transformed host cells are cultured so that the antibody heavy and light chains are expressed, and the intact antibody is re-isolated from the culture medium. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, cultivate the host cells, and obtain antibodies from the culture medium. Therefore, the present invention relates to a method for synthesizing the recombinant antibody of the present invention by culturing the host cell of the present invention in a suitable medium until the recombinant antibody of the present invention has been synthesized. In addition, the method may include isolating the recombinant antibody from the culture medium.

Another method for producing the antibodies of the invention includes a combination of in vivo and in vitro methods. For example, by immunizing an animal with an antigen in vivo and then screening a recombinant antibody library or a single-domain antibody library (e.g., containing heavy and/or light chains) prepared from lymphoid cells of the animal with the antigen in vitro, The antigen can act on the antibody library. According to another method, by immunizing an animal with an antigen in vivo and then subjecting a recombinant antibody library or a single-domain library produced from lymphoid cells of the animal to affinity maturation, the antigen can act on the antibody library. According to another method, by immunizing an animal with an antigen in vivo, then selecting individual antibody-producing cells that secrete the antibody of interest and obtaining heavy and light chain variable regions from the selected cell cDNAs (such as by using PCR), and The variable regions of the heavy and light chains are expressed in vitro in mammalian host cells (this is called the selected lymphocyte antibody method or SLAM), so that the selected antibody gene sequence can be further selected and manipulated to make the The antigen can act on the antibody library. In addition, monoclonal antibodies can be selected by expressing clones by expressing antibody genes encoding heavy and light chains in mammalian cells and selecting those mammalian cells that secrete antibodies with desired binding affinity.

In a specific embodiment of the present invention, the present invention discloses a method for producing antibodies: including culturing any of the host cells disclosed herein in a culture medium under conditions suitable for antibody production. The present invention also provides an antibody obtainable by the above method disclosed herein.

The method of the present invention for producing antibodies can be used to produce various types of antibodies. These include monoclonal, particularly recombinant antibodies, essentially human antibodies, chimeric antibodies, humanized antibodies and CDR grafted antibodies, and their antigen-binding portions.

The present invention further relates to a hybridoma capable of producing (secreting) the monoclonal antibody of the present invention.

The present invention also relates to a composition comprising the antibody of the present invention or an antigen-binding portion thereof as defined above.

According to a specific embodiment, the composition is a pharmaceutical composition comprising the antibody or antigen-binding portion of the invention and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., as long as they are physiologically compatible. Pharmaceutically acceptable carriers include, for example, water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof. In most cases, it is preferable to use isotonic agents such as sugars, polyols such as mannitol or sorbitol, or sodium chloride. The pharmaceutically suitable carrier may additionally contain relatively small amounts of auxiliary substances that increase the half-life or effectiveness of the antibody, such as wetting or emulsifying agents, preservatives or buffers.

For example, the pharmaceutical composition may be suitable for parenteral administration. Here, the antibody is preferably prepared as an injectable solution having an antibody content of 0.1-250 mg/ml. The injectable solution can be prepared in a liquid or lyophilized form, and the dosage form is a lead oxide glass bottle or vial, an ampoule or a drug-filled syringe. The buffer may contain L-histidine (1-50 mM, preferably 5-10 mM) and have a pH of 5.0-7.0, preferably 6.0. More suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate buffers. Sodium chloride can be used in order to adjust the solution tonicity to a concentration of 0-300 mM (for liquid dosage forms, 150 mM is preferred). For lyophilized dosage forms, cryoprotectants such as sucrose (such as 0-10%, preferably 0.5-1.0%) may also be included. Other suitable cryoprotectants are trehalose and lactose. For lyophilized dosage forms, fillers such as mannitol (such as 1-10%, preferably 2-4%) may also be included. Stabilizers such as L-methionine (e.g. 51-50 mM, preferably 5-10 mM) can be used in liquid and lyophilized dosage forms. More suitable fillers are glycine and arginine. Surfactants such as polysorbate 80 (e.g. 0-0.05%, preferably 0.005-0.01%) can also be used. More surfactants are polysorbate 20 and BRIJ surfactants.

The composition of the present invention can have a variety of dosage forms. These dosage forms include liquid, semi-solid and solid dosage forms, such as liquid solutions (such as injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred dosage form depends on the type of administration desired and the therapeutic application. Typically, the composition is preferably administered in the form of an injectable or infusible solution, such as a composition similar to other antibodies used for passive immunization of humans. The preferred route of administration is parenteral (such as intravenous, subcutaneous, intraperitoneal or intramuscular) administration. According to a preferred embodiment, the antibody is administered by intravenous infusion or injection. According to another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.

Under the conditions of preparation and storage, the therapeutic composition should generally be sterile and stable. The composition can be formulated as a solution, microemulsion, dispersion, liposome or other ordered structure suitable for high concentration of active substance. A sterile injectable solution can be prepared by introducing the required amount of the active substance (ie antibody) into a suitable solvent (using one of the above-mentioned ingredients or a combination thereof as appropriate), and then aseptically filtering the solution. Dispersions are usually prepared by introducing the active substance into a sterile carrier containing a basic dispersion medium and other necessary ingredients as appropriate. As for the sterile freeze-dried powder used for the preparation of sterile injectable solutions, vacuum drying and spray drying are the preferred preparation methods, which produce the active ingredient and more required ingredients from the previously sterile filtered solution. powder. For example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersant, or by using a surfactant, the proper fluidity of the solution can be maintained. Prolonged absorption of the injectable composition can be achieved by additionally incorporating formulations that delay absorption, such as monostearate and gelatin, into the composition.

Although the preferred type of administration for many therapeutic applications is subcutaneous injection, intravenous injection or infusion, the antibodies of the present invention can be administered by a variety of methods known to those skilled in the art. Those skilled in the art will understand that the route and/or type of administration depends on the desired result. According to specific embodiments, the active substance can be prepared with a carrier that protects the active substance from rapid release, for example, a sustained or controlled release formulation, which includes implants, transdermal creams, and microencapsulated release systems. Biologically degradable, biocompatible polymers such as ethylene glycol diacetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The methods for preparing the above dosage forms are well known to those skilled in the art; see, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

According to specific embodiments, the antibodies of the invention may be administered orally, for example in an inert diluent or a metabolizable edible carrier. Antibodies (and more ingredients, if needed) can also be encapsulated in hard or soft capsules, compressed into tablets, or added directly to food. For oral treatment, the antibody can be mixed with excipients and used in the form of oral tablets, buccal tablets, capsules, elixirs, suspensions, syrups, etc. If it is intended to administer the antibody of the present invention by a route other than parenteral, it may be necessary to select a coating from substances that prevent the inactivation of the antibody.

The present invention also provides a product for detecting the presence of Echo virus type 30, the product comprising the monoclonal antibody disclosed in the present invention or an antigen binding portion thereof.

The present invention also provides a product for diagnosing diseases caused by Echovirus type 30 infection. The product comprises the monoclonal antibody or its antigen binding part disclosed in the present invention.

The above-mentioned products of the present invention are tested or diagnosed according to immunological methods. In principle, it can be performed by using any analytical or diagnostic assay method in which antibodies are used, including agglutination and precipitation techniques, immunoassays, immunohistochemistry, and immunoblotting techniques, such as Western blotting, or preferably dot blotting. In vivo methods such as imaging methods are also included here.

It is advantageous to use immunoassays. Competitive immunoassay (i.e., an assay in which an antigen and a labeled antigen (tracer) compete for binding to an antibody) and sandwich immunoassay (i.e., an assay in which the binding of a specific antibody to the antigen is detected by a second antibody, usually a labeled antibody) ) Are all suitable. These assays can be homogeneous (i.e., do not separate into a solid phase and a liquid phase) or heterogeneous (i.e. separate the bound label from the unbound label, for example by a solid phase bound antibody). Depending on the labeling and measurement method, different multiphase and homogeneous immunoassays can be divided into specific categories, such as RIAs (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), FIA (fluorescence immunoassay), LIA (luminescence immunoassay) ), TRFIA (time-resolved FIA), IMAC (immune activation assay), EMIT (enzyme multiplication immunoassay test), TIA (turbidimetric immunoassay), I-PCR (immune-PCR).

Preferably, the product includes a kit comprising: a) the monoclonal antibody or antigen-binding portion of the present invention, and b) a conjugate comprising an antibody linked to a signal producing compound, wherein the conjugate The antibody is different from the monoclonal antibody isolated in the present invention.

The kit may also include a control or calibrator containing a reagent that binds to the antigen.

The kit may also include a container containing a pre-conditioned solid phase, such as a vial, bottle or strip, and other containers containing the corresponding conjugate. These kits may also contain vials or containers containing other reagents needed to perform the assay, such as washing, processing, and indicator reagents.

The antibody of the present invention or its antigen-binding portion can be coated on a solid phase (or exist in a liquid phase). Then the sample or biological sample (such as whole blood, cerebrospinal fluid, serum, etc.) is contacted with the solid phase. If an antigen (such as Echo virus type 30) is present in the sample, the above-mentioned antigen binds to the antibody on the solid phase and then detects by direct or indirect methods. The direct method involves simply detecting the presence of the complex itself and therefore the presence of the antigen. In the indirect method, the conjugate is added to the bound antigen. The conjugate includes a second antibody that binds to the bound antigen to which a signal generating compound or a label is attached. Once the second antibody binds to the bound antigen, the signal generating compound generates a measurable signal. The above signal then indicates the presence of antigen in the sample.

Examples of solid phases for immunoassays are porous and non-porous materials, latex particles, magnetic particles, microparticles (see U.S. Patent No. 5,705,330), beads, membranes, microtiter wells, and plastic test tubes. If necessary, the choice of the solid phase material and the method of labeling the antigen or antibody present in the conjugate depends on the performance characteristics of the desired assay format.

As mentioned above, the conjugate (or indicator) should contain an antibody (perhaps an anti-antibody, depending on the assay) to which a signal producing compound or label is attached. The signal generating compound or "label" is detectable by itself or can react with one or more other compounds to produce a detectable product. Examples of signal generating compounds include chromophores, radioisotopes (such as 125I, 131I, 32P, 3H, 35S, and 14C), chemiluminescent compounds (such as acridinium), particles (visible or fluorescent), nucleic acids, complexes Agents or catalysts such as enzymes (such as alkaline phosphatase, acid phosphatase, horseradish peroxidase, β-galactosidase and ribonuclease). In the case of using enzymes (such as alkaline phosphatase or horseradish peroxidase), the addition of a color-developing, fluorescent or luminescent substrate can cause the generation of a detectable signal. Other detection systems such as time-resolved fluorescence measurement, internal reflection fluorescence measurement, amplification (such as polymerase chain reaction) and Raman spectroscopy are also useful.

Examples of biological samples that can be tested by the above immunoassay include plasma, whole blood, dried whole blood, serum, cerebrospinal fluid, or water or organic-water extracts of tissues and cells.

"Monoclonal antibody" means an antibody molecule preparation in which antibodies share the same heavy chain and the same light chain amino acid sequence. It can be achieved through new technologies such as phage, bacteria, yeast or ribosome display and through hybridoma-derived antibodies as an example (e.g. through hybridoma technology such as the standard Kohler and Milstein hybridoma methodology ((1975) Nature 256: 495). -497) monoclonal antibodies are produced by traditional methods of preparing antibodies secreted by hybridomas. Therefore, non-hybridoma-derived antibodies with the same sequence are also referred to herein as monoclonal antibodies, although they can be obtained by non-classical methodology, the term "monoclonal" is not limited to hybridoma-derived antibodies, but is used Refers to all antibodies derived from a nucleic acid clone.

Therefore, the monoclonal antibodies of the present invention include recombinant antibodies. Here, the term "recombination" refers to any artificial combination of two different phase-separated sequence segments obtained by chemical synthesis or manipulation of isolated nucleic acid segments by genetic engineering techniques, for example. In particular, the term "recombinant antibody" refers to an antibody produced, expressed, produced or isolated by recombinant means, such as an antibody expressed using a recombinant expression vector transfected into a host cell; an antibody isolated from a recombinant combinatorial antibody library; Antibodies of transgenic animals (such as mice) that incorporate human immunoglobulin genes (see, for example, Taylor, LD, et al. (1992) Nucl. Acids Res. 20: 6287-6295); or use specific immunoglobulin gene sequences (E.g., human immunoglobulin gene sequence) and other DNA sequences are assembled, expressed, produced or isolated by any other method. Recombinant antibodies include, for example, chimeric antibodies, CDR grafted antibodies, and humanized antibodies. Those skilled in the art should be aware that the expression of conventional hybridoma-derived monoclonal antibodies in a heterologous system requires the production of recombinant antibodies, even if the amino acid sequence of the resulting antibody protein has not changed or is intended to be changed.

According to various embodiments, the antibody may comprise an amino acid sequence derived entirely from a single species, such as a human antibody or a mouse antibody. According to other embodiments, the antibody may be a chimeric antibody or a CDR grafted antibody or a different type of humanized antibody.

The present invention also provides a method for treating or preventing Echovirus type 30 infection, which method comprises administering the aforementioned monoclonal antibody or antigen-binding portion thereof to an individual.

Further, the monoclonal antibody or the antigen binding portion thereof administered to the individual is used for passive immunization.

The present invention also provides a method for treating or preventing diseases caused by Echovirus type 30 infection, the method comprising administering the aforementioned monoclonal antibody or antigen-binding portion thereof to an individual.

The present invention also provides a method for detecting Echo virus type 30, the method comprising the following steps:

(1) Provide samples suspected of Echo virus type 30;

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

(3) Detect the formation of a complex containing the monoclonal antibody or antigen-binding portion and the antigen. The presence of the complex indicates that the sample contains Echovirus type 30.

The present invention also provides a method for diagnosing diseases caused by Echovirus 30 infection, and the method includes the following steps:

(1) Provide samples from individuals suspected of suffering from diseases caused by Echovirus type 30 infection;

(3) Detect the formation of a complex containing the monoclonal antibody or antigen-binding portion and the antigen. The presence of the complex indicates that the individual has a disease caused by Echovirus type 30 infection.

Specifically, the method includes the following steps: 1) separating a biological sample from a patient; 2) contacting the biological sample with the monoclonal antibody or antigen-binding portion thereof of the present invention for a period of time and under conditions sufficient to form an antigen/antibody complex; And 3) detecting the presence of the antigen/antibody complex in the sample, and the presence of the complex indicates the diagnosis of the disease caused by the Echovirus type 30 infection of the patient. The antigen may be Echo virus type 30 or its protein.

In addition, the method includes the following steps: 1) separating the biological sample from the patient; 2) contacting the biological sample with the antigen for a period of time and under conditions sufficient to form an antibody/antigen complex; 3) adding a conjugate to the obtained antibody /Antigen complex for a period of time and under conditions sufficient to allow the conjugate to bind to the bound antibody, wherein the conjugate comprises the aforementioned antibody linked to a signal producing compound capable of producing a detectable signal; and 4) through detection The signal produced by the signal producing compound detects the presence of antibodies that may be present in the biological sample, and the signal indicates the diagnosis of the disease caused by the Echovirus 30 infection of the patient. The antigen may be Echo virus type 30 or its protein.

The present invention also provides the application of the aforementioned monoclonal antibody or its antigen-binding portion in the preparation of products for detecting Echo virus type 30.

The present invention also provides the application of the aforementioned monoclonal antibody or its antigen binding part in the preparation of products for diagnosing diseases caused by Echovirus type 30 infection.

The present invention also provides the application of the aforementioned monoclonal antibody or its antigen binding part in the preparation of a medicine for treating or preventing Echo virus type 30 infection.

The present invention also provides the application of the aforementioned monoclonal antibody or its antigen-binding portion in the preparation of medicines for the treatment or prevention of diseases caused by Echo virus type 30 infection.

The diseases caused by Echovirus type 30 infection disclosed in the present invention include but are not limited to meningitis, upper respiratory tract infection, myocarditis, skin rash, vomiting, fever, and headache.

The upper respiratory tract infection includes flu-like symptoms such as cough and sore throat.

The meningitis includes aseptic meningitis, and aseptic meningitis includes childhood aseptic meningitis.

The term "antibody" means an immunoglobulin molecule composed of 4 polypeptide chains (two heavy (H) chains and two light (L) chains). The chains are usually connected to each other by disulfide bonds. Each heavy chain is composed of the variable region of the heavy chain (herein referred to as HCVR or VH for short) and the constant region of the heavy chain. The heavy chain constant region is composed of three regions CH1, CH2 and CH3. Each light chain is composed of the variable region of the light chain (herein referred to as LCVR or VL for short) and the constant region of the light chain. The light chain constant region consists of the CL region. The VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDRs) and alternately distributed conserved regions called framework regions (FR). Therefore, each VH and VL region consists of three CDRs and four FRs arranged in the following order from N-terminal to C-terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well known to those skilled in the art.

The term "antigen-binding portion" (or simply "antibody portion") of an antibody refers to one or more fragments of the antibody of the present invention that still have the binding affinity as defined above. Fragments of complete antibodies have been shown to achieve the antigen-binding function of antibodies. According to the term "antigen-binding portion" of an antibody, examples of binding fragments include (i) Fab fragments, ie, monovalent fragments composed of VL, VH, CL and CH1 regions; (ii) F(ab')2 fragments, ie containing two A bivalent fragment of Fab fragments connected to each other in the hinge region by disulfide bridges; (iii) Fd fragments composed of VH and CH1 regions; (iv) Fv fragments composed of FL and VH regions of a single arm of an antibody; ( v) dAb fragment composed of VH region or VH, CH1, CH2, DH3, or VH, CH2, CH3 (Ward et al., (1989) Nature 341:544-546); and (vi) isolated complementarity determining region (CDR) ). Although the two regions of the Fv fragment (ie VL and VH) are encoded by separate genes, they can be further joined together using synthetic linkers such as poly-G4S amino acid sequences and recombination methods, so that they can be used as VL and VH. The regions combine to form a monovalent molecule (referred to as single-chain Fv (ScFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879- 5883) in a single protein chain. The term "antigen-binding portion" of an antibody is also intended to include such single-chain antibodies. Other forms of single chain antibodies such as "diabodies" are also included here. Diabodies are bivalent, bispecific antibodies, in which the VH and VL regions are expressed on a single polypeptide chain, but the linker peptide used is too short for the two regions that can bind to the same chain, thereby forcing The regions pair with complementary regions of different chains and form two antigen binding sites (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ, et al. ( 1994) Structure 2: 1121-1123). An immunoglobulin constant region refers to a heavy chain or light chain constant region.

The term "human antibody" refers to the variable as described by Kabat et al. (see Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242) The regions and constant regions correspond to or are derived from antibodies of human germline immunoglobulin sequences. However, the human antibody of the present invention may contain, for example, amino acid residues not encoded by human germline immunoglobulin sequences in CDR, and especially in CDR3 (for example, by random or site-specific mutagenesis in vitro or in vivo Mutations introduced by somatic mutations). The recombinant human antibody of the present invention has variable regions and can also include constant regions derived from human germline immunoglobulin sequences (see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). However, according to a specific embodiment, the above-mentioned recombinant human antibody is subjected to in vitro mutagenesis (or in vivo somatic mutagenesis if an animal transformed with human Ig sequence genes is used), so that the amino acid sequence of the VH and VL regions of the recombinant antibody is Although related to or derived from human germline VH and VL sequences, sequences that do not naturally exist in the human antibody germline repertoire in vivo. According to specific embodiments, this type of recombinant antibody is the result of selective mutagenesis or back mutation or both. Preferably, mutagenesis results in greater affinity for the target and/or less affinity for non-target structures than the parent antibody.

The term "chimeric antibody" refers to an antibody containing heavy and light chain variable region sequences from one species and constant region sequences from another species, such as murine heavy and light chain variable regions linked to human constant regions. Area of antibodies.

The term "CDR grafted antibody" refers to an antibody comprising heavy and light chain variable region sequences from one species, but one or more CDR regions of VH and/or VL are replaced by CDR sequences of another species, For example, an antibody having murine heavy chain and light chain variable regions in which one or more murine CDRs (such as CDR3) have been replaced by human CDR sequences.

The term "humanized antibody" refers to sequences containing heavy and light chain variable regions from non-human species (such as mouse, rat, rabbit, chicken, camel, sheep, or goat), but at least a portion of VH and/ Or the VL sequence has been changed to be more "human-like", that is, an antibody that more closely resembles the human germline variable region sequence. One type of humanized antibody is a CDR grafted antibody in which human CDR sequences have been inserted into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. Specifically, the term "humanized antibody" specifically refers to a framework region (FR) that immunospecifically binds to an antigen of interest and includes a framework region (FR) that basically has the amino acid sequence of a human antibody and a complementarity that basically has the amino acid sequence of a non-human antibody. Determining region (CDR) antibodies or variants, derivatives, analogs or fragments thereof. As used herein, the term "substantially" in the CDR context means that the CDR has at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the CDR of a non-human antibody. Amino acid sequence of amino acid sequence. The humanized antibody contains substantially all of at least one, and especially two variable regions (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to Those CDR regions of non-human immunoglobulins (ie, donor antibodies) and all or substantially all framework regions are consensus sequences of those framework regions of human immunoglobulins. Preferably, the humanized antibody also contains at least a part of an immunoglobulin constant region (Fc), usually that of a human immunoglobulin. In certain embodiments, a humanized antibody contains a light chain and at least the variable region of a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In certain embodiments, humanized antibodies contain only humanized light chains. In certain embodiments, a humanized antibody contains only a humanized heavy chain. In a specific embodiment, a humanized antibody only contains a humanized light chain variable region and/or a humanized heavy chain.

The humanized antibody can be selected from any type of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any subclass of immunoglobulin, including but not limited to IgG1, IgG2, IgG3, and IgG4.

As used herein, the term "CDR" refers to the complementarity determining region within the variable sequence of an antibody. There are three CDRs in each heavy chain and light chain variable region, which are called CDR1, CDR2, and CDR3 for each variable region. As used herein, the term "CDR set" refers to a set of three CDRs present in a single variable region capable of binding antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a clear residue numbering system that can be applied to any antibody variable region It also provides the precise residue boundaries that define the three CDRs. These CDRs can be called Kabat CDRs. Chothia and colleagues (Chothia & Lesk, J. Mol. Biol. 196: 901-917 (1987) and Chothia et al., Nature 342: 877-883 (1989)) found that some sub-positions in Kabat CDR, despite the large differences in the amino acid sequence level, adopted almost the same peptide backbone conformation. These sub-positions were named L1, L2, and L3. Or H1, H2, and H3, where "L" and "H" refer to light chain and heavy chain regions, respectively. These regions can be called Chothia CDRs, which have boundaries that overlap with Kabat CDR. Others are defined by boundaries that overlap with Kabat CDR CDRs have been described by Padlan (FASEB J.9: 133-139 (1995)) and MacCallum (J Mol Biol 262(5): 732-45 (1996)). However, other CDR boundary definitions do not need to be strictly followed. Any of the above systems still overlaps with Kabat CDR, although they can be shorter or longer according to the experimental conclusion that predicted or specific residues or residue groups or even the entire CDR does not significantly affect antigen binding. The methods used herein can utilize CDRs defined according to any of these systems, although preferred embodiments utilize CDRs defined by Kabat or Chothia.

As used herein, the term "framework region" or "framework region sequence" refers to the remaining sequence of the variable region minus the CDR. Because different systems can be used to determine the exact definition of CDR sequences, the meaning of framework region sequences can be interpreted differently accordingly. The 6 CDRs (CDR-L1, -L2 and -L3 of the light chain and CDR-H1, -H2 and -H3 of the heavy chain) also divide the framework on each chain of the light and heavy chains into four subregions ( FR1, FR2, FR3 and FR4), where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. It is not specified that the specific subregions are FR1, FR2, FR3, or FR4. The framework regions mentioned by others represent FR's combined in a single variable region of a naturally-occurring immunoglobulin chain. As used herein, FR represents one of the four subregions, and FR represents two or more of the four subregions constituting the framework region.

The term "acceptor" refers to an antibody amino acid or nucleic acid sequence that provides or encodes one or more framework regions and constant regions. In a specific embodiment, the term "acceptor" refers to an amino acid sequence that provides or encodes at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98%, or 100% of one or more framework regions The amino acid or nucleic acid sequence of the human antibody. According to this embodiment, the receptor may contain at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 amino acid residues that are not present in one or more specific positions of the human antibody. The receptor framework region and/or receptor constant region can be derived or obtained, for example, from germline antibody genes, mature antibody genes, functional antibodies (such as antibodies well known in the art, antibodies in development, or commercially available antibodies).

As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid. One type of vector is a "plasmid", which refers to circular double-stranded DNA in which additional DNA segments can be connected. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors can replicate autonomously in the host cell into which they are introduced (such as bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (such as non-episomal mammalian vectors) can be incorporated into the genome of the host cell as soon as they are introduced into the host cell, thereby replicating together with the host genome. In addition, certain vectors can direct the expression of operably linked genes. The above-mentioned vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally speaking, in recombinant DNA technology, expression vectors are usually used in the form of plasmids. In the specification of the present invention, "plasmid" and "vector" are used interchangeably, because plasmid is the most commonly used form of vector. However, the present invention is intended to include the above-mentioned other forms of expression vectors such as viral vectors (such as replication defective retroviruses, adenoviruses, and adeno-associated viruses), which provide the same effect.

The term "operably linked" refers to an adjacent relationship in which the described components exist in a relationship that allows them to function in their intended manner. The control sequence is "operably linked" to the coding sequence in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequence. The "operably linked" sequence includes the expression control sequence adjacent to the gene of interest and the expression control sequence that acts in trans or remotely controls the gene of interest. As used herein, the term "expression control sequence" refers to a polynucleotide sequence necessary to affect the expression and processing of the coding sequence to which they are linked. Expression control sequences include appropriate transcription start, end, promoter and enhancer sequences; effective RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (ie Kozak consensus sequence); Sequences that increase protein stability; and when needed, sequences that increase protein secretion. Depending on the characteristics of the host organisms, the above control sequences are different; in prokaryotes, the above control sequences usually include promoters, ribosome binding sites and transcription termination sequences; in eukaryotes, the above control sequences usually include promoters And transcription termination sequence. The term "control sequence" is intended to include elements whose presence is essential for expression and processing, and may also include additional elements whose presence is advantageous, such as leader sequences and fusion partner sequences.

As defined herein, "transformation" refers to any process by which foreign DNA enters a host cell. The transformation can occur under natural or artificial conditions using a variety of methods well known in the art. Transformation can rely on any known method for inserting exogenous nucleic acid sequences into prokaryotic or eukaryotic host cells. The method is selected based on the host cell to be transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. The aforementioned "transformed" cells include stably transformed cells in which the inserted DNA can replicate as an autonomously replicating plasmid or as part of a host chromosome. They also include cells that transiently express inserted DNA or RNA for a limited period of time.

As used herein, the term "recombinant host cell" (or simply "host cell") means a cell into which foreign DNA has been introduced. It should be understood that the aforementioned terms not only refer to a specific subject cell, but also refer to the progeny of such a cell. Due to mutations or environmental influences, certain modifications can occur in the offspring, so in fact the above-mentioned offspring may be different from the parent cell, but are still included in the scope of the term "host cell" as used herein. Preferably, the host cell includes prokaryotic and eukaryotic cells selected from any life world. Preferred eukaryotic cells include protist, fungal, plant and animal cells. The most preferred host cells include, but are not limited to, the prokaryotic cell line Escherichia coli; the mammalian cell lines CHO, HEK293 and COS; the insect cell line Sf9 and the fungal cell Saccharomyces cerevisiae.

Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (such as electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as commonly practiced in the art or as described herein. The foregoing techniques and methods can generally be performed according to conventional methods well known in the art and as described in various commonly used and more specific references cited and discussed throughout the specification of the present invention. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein as a reference for any purpose.

Here, "4B10", "4B10 antibody", "4B10 monoclonal antibody", and "4B10-IgG" can be used interchangeably, and all refer to the full-length 4B10 monoclonal antibody.

Description of the drawings

Figure 1 shows the SDS-PAGE chart of the antibody of the present invention;

Figure 2 shows the affinity activity curve of the antibody of the present invention, where A: 4B10-IgG, B: 4B10-Fab, C: 6C5-IgG, and D: 6C5-Fab;

Figure 3 shows the binding affinity curve of the virus of the present invention to the receptor, where A: FcRn, B: CD55;

Figure 4 shows the inhibition curve of the antibody of the present invention on the binding of the virus to its receptor, where A: 6C5 antibody is added first and then the receptor is added, B: receptor is added first and then 6C5 antibody is added; C: 4B10 antibody is added first and then the receptor is added Body, D: Add the receptor first and then add the 4B10 antibody;

Figure 5 shows the specificity determination diagram of the affinity activity of the antibody of the present invention;

Figure 6 shows a statistical diagram of the neutralizing activity of the antibody of the present invention, where A: 6C5, B: 4B10;

Figure 7 shows the specificity determination diagram of the neutralizing activity of the antibody of the present invention;

Figure 8 shows the results of the effect of antibodies on virus activity before or after the virus and cells contact, where A: before contact, B: after contact;

Figure 9 shows a statistical graph of serum titer after immunized mice with inactivated E30 particles;

Figure 10 shows a graph showing the effect of the binding of the antibody of the present invention to the virus on the epitope;

Figure 11 shows the results of the effect of the antibody and virus binding of the present invention on virus stability, in which: A: pH=5.5 buffer, B: pH=7.4 buffer, C: derivative value of A, D: derivative value of B .

Detailed ways

The following examples are intended to illustrate the invention without limiting its scope.

Example 1 Monoclonal antibody preparation

1. Immunogen (antigen): Echo30.

Each mouse is immunized 5 times, with 100 μl of antigen each time, and a total of 3-3.5 ml of virus is required.

2. Animal immunity

Two adjuvants were used for immunization, modified Freund's adjuvant and aqueous adjuvant respectively. Two groups were immunized with virus, with 3 mice each as a group, and 6 mice were immunized with one adjuvant. All immunizations were performed in the SPF animal room.

Select 6 Balb/c mice (female mice) aged 5-8 weeks. The immunogen and adjuvant are mixed and then emulsified separately for immunization. The first injection uses Freund's complete adjuvant, and the next 4 booster injections use Freund's adjuvant. Incomplete adjuvant, mix well with equal volume of antigen and then inject.

The immunization method is multi-point injection on the back. The main immunization amount is 100μl antigen/mouse, and the booster injection is 100μl antigen/mouse. The immunization cycle is 6-8 weeks.

3. Monoclonal antibody preparation process

Antiserum detection: The virus is coated with an enzyme-labeled plate, and the antiserum titer is detected by the indirect ELISA method. Serum titer is greater than 1:50K before proceeding to the next step of fusion. If there are multiple mice whose titer exceeds 1:50K, select the mouse with the highest titer for fusion.

Myeloma cell preparation: One week before fusion, resuscitate SP2/0 cells and culture them to logarithmic phase normally.

Spleen cell preparation: select the mice to be fused, sacrifice them by cervical dislocation on the day of fusion, take the spleen, collect and count the spleen cells in the standard procedure.

Cell fusion: mix myeloma cells and spleen cells in a ratio of 1:3-1:10, perform cell fusion operations in standard procedures, and then culture them in HAT DMEM complete medium. Hybridoma cells can be seen 3 days after fusion. Change the 1/2HAT complete medium on the 7th day, and change the 1/2HT medium on the 8th day. The screening test began about 10 days after the fusion.

Cell fusion results: After fusion, cultured with HAT selective medium, observed under a microscope, and saw multiple growing hybridoma cells, which proved that the fusion operation was successful.

Fusion screening: draw 100μl/well of cell supernatant for indirect ELISA detection. According to the ELISA results, the positive wells are judged. Use a single-channel pipette to pick the positive wells detected in the entire plate, and perform a second re-examination to further confirm the positive wells.

Subcloning: Perform two rounds of subcloning on the re-screened positive well cells. (Because the positive cell line obtained from the first subcloning is not stable and may contain multiple hybridoma cells, it is generally believed that the hybridoma cells are a single cell line after the second subcloning and are determined to be positive).

4. Virus neutralization experiment: inoculate cells with antibody-containing culture supernatant (culture supernatant prepared after the first subcloning, all positive for antigen ELISA) and a certain concentration of antibody, and plaques are reduced or not occurred The hole is the neutralizing antibody positive hole.

5. Ascites preparation and antibody purification

5.1 Ascites preparation

The above-mentioned positive cells were expanded and cultured and injected into the abdominal cavity of Balb/C mice (sensitized by Freund's incomplete adjuvant). Generally, the bulge of the mouse abdomen was seen in 7-10 days, which represented the production of ascites. When the mouse has obvious ascites, draw the ascites in time.

5.2 Ascites purification

The ascites of the above cells was purified with Protein A/G, and the purity of the antibody after purification was greater than 90%. After checking the purity of the concentration, adjust the concentration to 2mg/ml.

6. Antibody identification

SDS-PAGE was used to check the expression and purification of the antibody. The results are shown in Figure 1. It is confirmed that a relatively pure protein is obtained. The light and heavy chains of the antibody can be clearly observed after melting. Note: The lane M in the figure represents the protein Marker.

7. Antibody sequence determination

After sequencing, the identified amino acid sequence and nucleotide sequence of the monoclonal antibody named 4B10 are shown in Table 1 below.

The amino acid sequence and nucleotide sequence of the heavy chain variable region are shown in SEQ ID NO: 4 and SEQ ID NO: 12.

The amino acid sequence and nucleotide sequence of the light chain variable region are shown in SEQ ID NO: 8 and SEQ ID NO: 16.

Table 1 Antibody sequence

Example 2 Antibody function

1. Antigen antibody affinity determination

1.1 Surface Plasmon Resonance (SPR)

1.1.1 Steps:

Use Pierce Fab Preparation Kit (Thermo) to cut and purify 4B10-IgG (full-length antibody) and 6C5-IgG (full-length antibody) to obtain 4B10-Fab (Fab fragment of antibody) and 6C5-Fab (Fab fragment of antibody) .

The SPR experiment was performed using BIAcore T100 (Biacore, GE Healthcare), and the buffer was PBS containing 0.05% Tween-20. Use the NHS/EDC method to fix the purified E30 whole virus particles on the surface of the CM5 sensor chip until the RU value reaches 740. Then, gradient concentrations of IgG or Fab flow through the chip at a rate of 20 μl/min, and use 10 mM glycine-hydrochloric acid (pH 1.7) to regenerate the chip after each injection cycle. The binding affinity was obtained by using the software BIAevaluation (version 4.1) to fit the curve globally.

1.1.2 Results

The results are shown in Figure 2. The affinity of 4B10-IgG (full-length antibody), 4B10-Fab (Fab fragment of antibody), 6C5-IgG (full-length antibody), 6C5-Fab (Fab fragment of antibody) and E30 respectively reached : 2.88nM, 67.70nM, 1.51nM, 3.68nM.

1.2 Competitive SPR

1.2.1 Steps

The competitive SPR procedure is similar to the above-mentioned SPR, and the E30 virus particles are immobilized on the CM5 chip until the RU value reaches 740. The receptor or antibody is added to the first stitch until the fitting curve is saturated, and then the competing antibody or receptor to be tested is added to the second stitch. The binding competition relationship between the two substances before and after the E30 virus particles on the chip is reflected by the change trend of the fitted curve.

1.2.2 Results

The results showed that the affinity between the virus and the receptor (FcRn and CD55) reached 2.38μM (Figure 3A) and 2.11μM (Figure 3B), which were much smaller than the affinity between the virus and the antibody, and compared to the control group (the first injection was added 1A1, a specific antibody against E6 virus), the addition of E30 antibody can strongly inhibit the binding of FcRn to CD55 and E30 (the control is established, ICAM5 is a specific cell receptor for EVD68) (Figure 4A and Figure 4C). On the contrary, adding cell receptors first cannot effectively inhibit the binding of antibodies to the virus (Figure 4B and Figure 4D).

2. Determination of antigen-antibody binding specificity

The ELISA experiment was used for the determination.

2.1 steps

The purified virus particles (E30, E6, E3, E11 or CVB3) were coated on an ELISA plate (Costar, Corning, USA) at 30 ng/well, and then incubated overnight at 4°C. The coated plate was blocked with 1% BSA-containing PBST solution (PBS plus 0.1% Tween20) at 37°C for 2 hours. After that, the plate was washed five times with PBST, and 4B10-IgG or 6C5-IgG was added as the primary antibody to each well at a dilution of 1:1000 at 37°C for 1 hour. The plate was washed again with PBST five times, and HRP-conjugated goat anti-mouse IgG H&L (1:3000 dilution) (Sigma-Aldrich, St. Louis, USA) was added as a secondary antibody. Place at 37°C for 0.5 hour. The plate was washed five times with PBST, and 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Biyuntian, Shanghai, China) was added to each well for 5 minutes at room temperature. Finally, 2M H ₂ SO _{4 was} added to the plate to stop the reaction, and the absorbance value of each well at 450 nm was read.

2.2 Results

The results are shown in Figure 5. Both 4B10 and 6C5 can specifically recognize E30, but do not react with E3, E6, E11 and CVB3. It shows that the two are E30 specific.

3. Detection of antibody neutralization activity

3.1 Plaque reduction neutralization test (PRNT)

3.1.1 Steps

Dilute 4B10/6C5-IgG, 4B10/6C5-Fab or mouse serum in DMEM medium to achieve a 2-fold serial dilution, with the highest concentration being 128nM, 128nM, 384nM or 64nM, respectively. In addition, a control group without any antibodies, Fab or serum was established. The same amount of E30 virus (PFU ranging from 50 to 100) was mixed with each dilution, incubated at 37°C for 1 hour, and then added to the confluent monolayer of RD cells seeded in a 6-well plate. Return the plate to the cell incubator for 1 hour, shaking gently every 20 minutes, and then rinse with DMEM (pH 7.4) 3 times. Cover with 2% FBS-added agarose cover (1% (mass to volume ratio) AGAROSE Ⅱ (Amresco), solvent is double distilled water) (2mL/well), and place it in a 5% CO ₂ cell incubator at 37°C Incubate for 3 days. Then use 2.5% crystal violet staining to visualize the plaques, and the percentage of inhibition is calculated as (Ncontrol-Ntest)/Ncontrol x 100%, where Ncontrol and Ntest represent the average of the plaque counts observed in the control and test groups, respectively value. All experiments were repeated three times.

3.1.2 Results

The results are shown in Figure 6, the Neut50 of 4B10-IgG, 4B10-Fab, 6C5-IgG, and 6C5-Fab to E30 reached 0.3nM, 25nM, 1.2nM and 6.8nM, respectively.

3.2 Cross-neutralization experiment

3.2.1 Steps

50 nM or 500 nM of 4B10 or 6C5 was mixed with a certain amount of E30 (PFU=~50) at 37° C. for 1 hour, and then added to the monolayer of RD cells. After incubation and rinsing, cover the cells with agarose cover and incubate at 37°C for another three days, then stain with crystal violet. The calculation formula for the inhibition rate is: (Ncontrol-Ntest)/Ncontrol x 100%, where Ncontrol and Ntest represents the average of the plaque counts observed in the control group and the test group, respectively. All experiments were repeated three times.

3.2.2 Results

The results are shown in Figure 7. Both 50nM and 500nM 4B10-IgG and 6C5-IgG can neutralize the infectious activity of E30 100%, while the same amount of these two antibodies has no effect on E3, E6, E11 and CVB3. The neutralizing effect of E30 indicates that the two have extremely high specificity for the neutralizing activity of E30.

3.3 Pre/post attachment experiment

3.3.1 Steps

Preattachment

The antibody solution (final concentration of 0nM, 3nM, 30nM, 300nM) and virus (MOI=1) were placed on ice for 30min, the cells cultured overnight in the 12-well plate were digested into the EP tube, centrifuged at 1000rpm, 5min, and pipetted. Remove the supernatant, add 1ml DMEM, suspend the cells, centrifuge again, repeat 3 times, remove the supernatant, add the liquid after the antibody and virus action to suspend the cells, let it sit on ice for 30 minutes, and then wash it 5 times as above. Remove the supernatant, and finally add 200μl of lysis buffer for lysis.

Post attachment:

Digest the cells cultured overnight in a 12-well plate into an EP tube, wash 3 times, remove the supernatant, add virus solution (MOI=1) to resuspend the cells, incubate on ice for 30 min, wash 5 times according to the above steps, Remove the supernatant, add antibody solution (final concentration of 0 nM, 3 nM, 30 nM, 300 nM) to resuspend the cells, incubate on ice for 30 min, wash 5 times according to the above steps, remove the supernatant, and finally add 200 μl of lysis buffer for lysis.

Extract nucleic acid from the lysate obtained from Pre/post attachment and perform QPCR quantitative analysis.

QPCR step: quantify E30 remaining on the surface of RD cells after 4B10/6C5 treatment by QPCR. In short, before and after the MOI of the virus attaching to the cell is 1, E30 is mixed with various concentrations of 6C5 at 4°C. Then the cells were washed 3 times, and total RNA was extracted by RNeasy mini kit (Qiagen, Hilden, Germany), and SuperScrip III Platinum SYBR Green One was used on QuantStudio Dx Real-Time PCR Instrument (Applied Biosystems, Foster City, USA) -Step qRT-PCR Kit (Invitrogen, Carlsbad, USA) for QPCR. 20μL reaction system contains 0.2μL SuperScript III RT/Platinum Taq Mix, 5μL 2X SYBR Green Reaction Mix, 10μm forward primer (5'-AACAGCAGCGTTGCCCGCGTCTA-3') and reverse primer (5'-ACCCTGTAGTTCCCTACATA-3') 0.2μL each , 2μL total RNA, 2.4μL RNase-free H ₂ O. The QPCR amplification program is: 42°C, 5 minutes for reverse transcription, 95°C, 5 minutes for reverse transcription inactivation. Then it was denatured at 95°C for 15s, a total of 40 cycles, annealed at 60°C and extended for 30s. The endogenous housekeeping gene β-actin (forward primer: 5'-GCCCTGAGGCACTCTTCCA-3', reverse primer: 5'-CGGATGTCCACGTCACACTT-3') was used as an internal control to normalize the sample. Different samples were analyzed relative levels E30RNA by ² -ΔΔCt method.

3.3.2 Results

The results are shown in Figure 8: Antibodies (4B10 and 6C5) can effectively inhibit the activity of the virus before the virus contacts the cell (Figure 8A) or after the contact (Figure 8B), indicating that the antibody can competitively inhibit the recognition of the virus and the virus receptor The virus that binds and has bound the receptor can also be competed by antibodies (4B10 and 6C5).

4. Determination of antibody titer and immunodominance of antibody binding epitope

The cultured E30 virus was inactivated with formaldehyde, concentrated, and sucrose density gradient centrifugation. Hollow and solid virus particles with higher purity were obtained in different sucrose gradient layers; the hollow and solid particles were injected into the immunized mice according to the steps in Example 1. , And then take blood to get antiserum.

Use epitope competition assay

step:

In order to test whether the epitope bound to antibody 4B10 or 6C5 has immunodominance, the purified E30 whole particles are first coated on the Elisa plate, and then the plate is blocked according to the above Elisa method. Then add serum against E30 solid particles adjuvanted with aluminum hydroxide (anti-F-par sera), serum against virus hollow particles adjuvanted with aluminum hydroxide (anti-E-par sera) or aluminum hydroxide adjuvant Serum (anti-adj sera). After incubation at 37°C for 0.5 hour, all wells were washed five times, and HRP-conjugated 4B10/6C5-IgG was added for further incubation at 37°C for 0.5 hour. The wells were then washed, TMB substrate, 2M H ₂ SO _{4 was added} , and read at A450 as described above. The calculation formula of the inhibition percentage is (OD negative control-OD serum)/OD negative control×100%.

result:

The inactivated E30 solid particle and hollow particle antiserum can effectively neutralize the virus E30's infection of cells, with titers reaching 1:12 and 1:11 respectively (Figure 9), and the combination of the two and the virus can block off About 60-80% of 4B10 and 6C5 epitopes. It shows that the epitopes recognized by 4B10 and 6C5 are immunodominant epitopes (Figure 10).

6. The influence of antibodies on virus stability

Thermofluor experiment steps:

MX3005 qPCR instrument (Agilent, Santa Clara, USA) was used, and SYTO9 and SYPROred (Invitrogen, Carlsbad, USA) were used as fluorescent probes to detect the hydrophobic residues of RNA and protein. Establish a 50μl reaction system, which contains 2μg target protein, 5μM SYTO9 and 3X SYPROred, and raise the temperature from 25°C to 99°C. Fluorescence was recorded in triplicate at 1°C intervals.

Results: The binding of 4B10-IgG, 6C5-IgG and E30 can weakly destroy the stability of E30, suggesting that the binding interval of the two may be related to the binding area of the virus and the receptor (Figure 11), in the figure E30 F- particle.

The description of the above-mentioned embodiments is only used to understand the method of the present invention and its core idea. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

A monoclonal antibody or an antigen-binding portion thereof, wherein the monoclonal antibody includes one or more CDRs in the variable region of the heavy chain, and/or one or more CDRs in the variable region of the light chain ；

The amino acid sequence of the CDR of the heavy chain variable region is selected from:

SEQ ID NO: the amino acid sequence shown in 1-3;

Or an amino acid sequence formed by substitution, deletion or addition of one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 1-3;

Or an amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 1-3, and has the same or similar function;

The amino acid sequence of the CDR of the light chain variable region is selected from:

SEQ ID NO: the amino acid sequence shown in 5-7;

Or an amino acid sequence formed by substitution, deletion or addition of one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 5-7;

Or an amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 5-7 and has the same or similar function.
The monoclonal antibody or antigen-binding portion thereof according to claim 1, wherein the heavy chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO:1, or has the amino acid sequence shown in SEQ ID NO:1 An amino acid sequence formed by substitution, deletion or addition of one or more amino acids on the basis; or an amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO:1 and has the same or similar function;

The heavy chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 2, or has an amino acid sequence formed by substitution, deletion or addition of one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO: 2; or An amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 2 and has the same or similar function;

The heavy chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 3, or has an amino acid sequence formed by substitution, deletion or addition of one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 3; or An amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 3 and has the same or similar function;

The light chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 5, or has an amino acid sequence formed by substitution, deletion or addition of one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 5; or An amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 5, and has the same or similar function;

The light chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 6, or has an amino acid sequence formed by substitution, deletion or addition of one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 6; or An amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 6 and has the same or similar function;

The light chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 7, or has an amino acid sequence formed by substitution, deletion or addition of one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO: 7; or An amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 7 and has the same or similar function.
The monoclonal antibody or its antigen-binding portion of claim 1 or 2, wherein at least 80% homology means 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology.
The monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 3, wherein the heavy chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO:1; the heavy chain variable region CDR2 has SEQ ID NO: 2; heavy chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 3; light chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 5; light chain variable region CDR2 has SEQ ID NO: the amino acid sequence shown in 6; the light chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 7.
The monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 4, wherein the monoclonal antibody comprises a heavy chain variable region and/or a light chain variable region, wherein:

The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 4, or has an amino acid sequence formed by substitution, deletion or addition of one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO: 4; Or an amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 4 and has the same or similar function;

The light chain variable region has an amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence formed by substitution, deletion or addition of one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO: 8; Or an amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 8 and has the same or similar function.
The monoclonal antibody or antigen-binding portion thereof according to claim 5, wherein at least 80% homology means 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology.
The monoclonal antibody or antigen-binding portion thereof according to claim 5 or 6, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 4; the light chain variable region has SEQ ID NO: 8 amino acid sequence.
The monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 6, wherein the antigen-binding portion of the monoclonal antibody comprises the Fab fragment and F(ab')2 fragment of the monoclonal antibody , Single-chain Fv fragments.
An isolated nucleic acid encoding the amino acid sequence of any one of claims 1-8.
The nucleic acid of claim 9, wherein the nucleic acid sequence is as shown in SEQ ID NO: 9-16.
A vector comprising the nucleic acid of claim 9 or 10.
The vector of claim 11, wherein the vector comprises: pcDNA, pTT, pTT3, pEFBOS, pBV, pJV or pBJ.
A host cell comprising the vector of claim 11 or 12.
The host cell of claim 13, wherein the host cell comprises a prokaryotic cell and a eukaryotic cell.
The host cell of claim 14, wherein the prokaryotic cell comprises Escherichia coli.
The host cell of claim 14, wherein the eukaryotic cell comprises a protist cell, an animal cell, a plant cell or a fungal cell.
The host cell of claim 16, wherein the animal cell is mammalian cell, avian cell and insect cell.
A product for detecting Echo virus type 30, the product comprising the monoclonal antibody or an antigen binding portion thereof according to any one of claims 1-8.
A product for diagnosing diseases caused by Echovirus type 30 infection, the product comprising the monoclonal antibody or antigen binding portion thereof according to any one of claims 1-8.
The product of claim 19, wherein the disease includes meningitis, upper respiratory tract infection, myocarditis, skin rash, vomiting, fever, and headache.
The product of any one of claims 18-20, wherein the product comprises a kit.
The product of claim 21, wherein the kit comprises a conjugate of an antibody to which a signal producing compound is connected.
A composition comprising the monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-8.
The composition of claim 23, wherein the composition further comprises a pharmaceutically acceptable carrier.
A method for producing an antibody, characterized in that the method comprises culturing the host cell according to any one of claims 13-17 in a medium under conditions suitable for producing the antibody.
An antibody produced by the method of claim 25.
A method for treating or preventing Echovirus type 30 infection, characterized in that the method comprises administering the monoclonal antibody or antigen binding portion thereof according to any one of claims 1 to 8 to an individual.
The method of claim 27, wherein the monoclonal antibody or antigen binding portion thereof administered to the individual is used for passive immunization.
[Corrected according to Rule 26 06.03.2020]
A method for treating or preventing diseases caused by Echovirus type 30 infection, characterized in that the method comprises administering to an individual the monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-8.
The method of claim 29, wherein the monoclonal antibody or antigen binding portion thereof administered to the individual is used for passive immunization.
The method of claim 30, wherein the disease includes meningitis, upper respiratory tract infection, myocarditis, rash, vomiting, fever, and headache.
The method for detecting Echo virus type 30 is characterized in that the method comprises the following steps:

(1) Provide samples suspected of Echo virus type 30;

(2) Contacting the sample with the monoclonal antibody or antigen-binding portion of any one of claims 1-8;

(3) Detect the formation of a complex containing the monoclonal antibody or antigen-binding portion and the antigen. The presence of the complex indicates that the sample contains Echovirus type 30.
The method for diagnosing diseases caused by Echovirus type 30 infection is characterized in that the method comprises the following steps:

(1) Provide samples from individuals suspected of suffering from diseases caused by Echovirus type 30 infection;

(2) Contacting the sample with the monoclonal antibody or antigen-binding portion of any one of claims 1-8;

(3) Detect the formation of a complex containing the monoclonal antibody or antigen-binding portion and the antigen. The presence of the complex indicates that the individual has a disease caused by Echovirus type 30 infection.
The method of claim 33, wherein the disease includes meningitis, upper respiratory tract infection, myocarditis, skin rash, vomiting, fever, and headache.
The use of the monoclonal antibody or its antigen binding part according to any one of claims 1-8 in the preparation of a product for detecting Echo virus type 30.
The use of the monoclonal antibody or its antigen binding portion according to any one of claims 1-8 in the preparation of a product for diagnosing diseases caused by Echovirus type 30 infection.
Use of the monoclonal antibody or its antigen binding portion according to any one of claims 1-8 in the preparation of a medicine for treating or preventing Echovirus type 30 infection.
The use of the monoclonal antibody or its antigen binding portion according to any one of claims 1-8 in the preparation of a medicine for the treatment or prevention of diseases caused by Echovirus type 30 infection.
The method of claim 38, wherein the disease includes meningitis, upper respiratory tract infection, myocarditis, rash, vomiting, fever, and headache.