KR100938998B1 - Antibodies to Respiratory Syncytial Virus - Google Patents

Abstract

The present invention provides an antibody or antigen-binding fragment thereof having a specific amino acid sequence, a nucleic acid molecule encoding the same, a recombinant vector and a transformant comprising the nucleic acid molecule, and a respiratory synstium comprising the antibody. The present invention relates to a pharmaceutical composition for preventing and treating a viral infection and a diagnostic kit for detecting respiratory syntium virus. Compared to the conventional commercial antibody of the present invention, Synagis, the binding ability to RSV, neutralizing ability to RSV, and in vivo infection prevention effect against RSV are much better.

Respiratory Synstium Virus, Antibody, Monoclonal Antibody, Fab

Description

Antibodies to Respiratory Syncytial Virus

The present invention relates to antibodies against surface antigen F of respiratory syncytial virus (RSV), genes encoding them, recombinant vectors, transformants and methods for preparing human monoclonal antibodies therefrom.

RSV (respiratory syncytial virus) is a major pathogen of lower respiratory tract infections and is the leading cause of pneumonia and bronchitis (Brandt et al., Pediatrics) . 52:56 (1973); Selwyn et al., Rev Infect Dis , 12: s870 (1990), a high-risk group that can cause fatal infections, especially in pulmonary dysplasia, congenital heart disease, cystic fibrosis cancer, infants with various immunodeficiencies, and adults with preimmune immunodeficiency. Chandwani et al., J Pediatr , 117: 251 (1990); Bruhn et al., J Pediatr , 90: 382 (1977). This is similar to the influenza virus in the infection rate in the elderly, and the excess mortality is reported to be higher in the RSV epidemic than in the influenza epidemic.

In the United States, there are between 100,000 and 200,000 high-risk patients, more than 90,000 hospitalizations per year due to RSV infection, and about 4,500 reported deaths. RSV is prevalent every year in Korea, and it is estimated that 60% of all viral lower airway infections isolated from Seoul National University Children's Hospital are caused by RSV.

Despite this serious situation, no vaccine or specific therapeutic agent is currently available. In the past, formalin-killed vaccines have been developed and clinical trials have been conducted, but severe lower respiratory tract infections occurred after RSV infection.

As a preventive strategy against RSV, the method of administering antibodies was devised, and MedImmune of USA developed and developed a polyclonal antibody called Respigam and a monoclonal antibody called Synagis. The strategy using these antibodies is based on the following evidence that antibodies from the parent help prevent disease. (1) Relatively less severe RSV disease until 5-6 weeks of age, with the most antibodies from the mother; (2) some infants with capillary bronchitis initially lack neutralizing antibodies; (3) there is an inverse relationship between antibody titers to umbilical cord blood and RSV disease; (4) The administration of RSV neutralizing antibodies to experimental animals or infants can prevent severe RSV disease.

RSV is a genome of non-segmented RNA and belongs to paramyxoviridae. In the infected host cells, 10 proteins, such as NS1, NS2, P, N, M, SH, G, F, M2 and L, are synthesized. The surface antigens include F protein (fusion protein), G protein, and SH protein. The G and F proteins, which are present in the outer membrane of the virus with relatively high molecular weight, are glycosylated glycosylated and play the most important role in the immune and antigenic variation of RSV infection. G protein is attached to the host cell to be infected, F protein is involved in the formation of giant cells and invasion and attachment of the virus, F protein and G protein is involved in the induction of neutralizing antibodies. In recent years, RSV is classified into subgroup A and subgroup B by reactivity with various monoclonal antibodies. This antigenic difference is mainly due to G protein mutation. On the other hand, the prophylactic antigenic sites of the F protein do not differ much among the subgroups and do not change quickly. F proteins are very well conserved across different RSV strains of about 89%, in the amino acid levels (Johnson et al, J. Gen Virol , 69:.... 2623 (1988); Johnson et al, J. Vrol 61: 3163 (1987)). In other words, the F protein of RSV is an optimized target of neutralizing antibody for suppression of infection because it does not show the urine of antigen seen in influenza A virus and can induce neutralizing antibody and inhibit proliferation after infection. have.

MedImmune of the United States has developed and commercialized a humanized antibody against the F protein described above, Synagis (US Patent Application Publication No. US20000724396), which has in vivo and ex vivo neutralization ability and is hospitalized for use in actual clinical use. It has been proven to be effective in reducing duration. However, Synagis is a humanized antibody that contains the amino acid sequence of the mouse antibody has a problem that can be induced immune response to repeated administration.

Meanwhile, human monoclonal antibodies have been developed that can neutralize RSV to prevent and treat RSV infection. US Scripps Research Institute (US Pat. No. 6685942) has developed Fab19 antibodies by phage display method, and in IDEC pharmaceutical (US Patent Application Publication No. 2004076631), RF-1 and B-1 tumor cell lines were prepared by SCID mice. RF-2 Two human monoclonal antibodies have been developed. However, compared to the severity of the disease caused by RSV, the production of antibodies for the purpose of prevention and treatment is not made much, and the development of excellent monoclonal antibodies with high reliability is still required.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have tried to develop antibodies that can prevent and treat respiratory syncytial virus (RSV) infection, and thus have specific binding ability to RSV A and B subgroups, and have excellent neutralization ability against RSV. In addition, the present invention has been completed by developing a novel antibody having excellent efficacy in preventing infection in vivo against RSV.

An object of the present invention is to provide an antibody or antigen-binding fragment thereof against respiratory synstium virus.

Another object of the present invention is to provide a nucleic acid molecule encoding an antibody against respiratory synstium virus.

It is another object of the present invention to provide a recombinant vector comprising a nucleic acid molecule encoding an antibody against respiratory synstium virus.

Another object of the present invention to provide a host cell transformed with the recombinant vector.

Another object of the present invention is to provide a method for producing an antibody against respiratory synstium virus.

Another object of the present invention to provide a pharmaceutical composition for the prevention and treatment of respiratory synstium virus infection.

Yet another object of the present invention is to provide a diagnostic kit for detecting respiratory syntium virus.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention provides an antibody or antigen-binding fragment thereof against a respiratory synstium virus comprising a heavy chain variable region having the following heavy chain CDR region (complementarity determining region) amino acid sequence: CDRH1 comprising the 31 sequence, CDRH2 comprising the SEQ ID NO: 32 sequence and CDRH3 comprising the SEQ ID NO: 33 sequence.

The present inventors have tried to develop antibodies that can prevent and treat respiratory syncytial virus (RSV) infection, and thus have specific binding ability to RSV A and B subgroups, and have excellent neutralization ability against RSV. In addition, new antibodies have been developed that have excellent efficacy in preventing infection in vivo against RSV.

Antibodies of the invention have a specific binding capacity to RSV. In particular, the antibodies of the invention specifically bind to the F protein of RSV.

As used herein, the term “antibody” as used to refer to RSV is a specific antibody to RSV, which specifically binds to the F protein of RSV and includes antigen-binding fragments of antibody molecules as well as complete antibody forms. do.

A complete antibody is a structure having two full length light chains and two full length heavy chains, each of which is linked by heavy and disulfide bonds. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types and subclasses gamma 1 (γ1), gamma 2 (γ2), and gamma 3 (γ3). ), Gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant regions of the light chains have kappa (κ) and lambda (λ) types (Cellular and Molecular Immunology, Wonsiewicz, MJ, Ed., Chapter 45, pp. 41-50, WB Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, sinauer Associates, Inc., Sunderland, MA (1984)).

An antigen binding fragment of an antibody molecule means a fragment having an antigen binding function and includes Fab, F (ab '), F (ab') ₂ and Fv. Fab in the antibody fragment has a structure having a variable region of the light and heavy chains, a constant region of the light chain and the first constant region of the heavy chain (C _H1 ) having one antigen binding site. Fab 'differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C-terminus of the heavy chain C _H1 domain. F (ab ') ₂ antibodies are produced when the cysteine residues of the hinge region of Fab' form disulfide bonds. Recombinant techniques for generating Fv fragments with minimal antibody fragments in which Fv has only a heavy chain variable region and a light chain variable region are described in PCT International Patent Applications WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086 and It is disclosed in WO 88/09344. Double-chain Fv is a non-covalent bond in which the heavy chain variable region and the light chain variable region are linked, and the single-chain Fv is generally shared by the variable region of the heavy chain and the short chain variable region through a peptide linker. It may be linked by bond or directly at the C-terminus to form a dimer-like structure such as a double chain Fv. Such antibody fragments can be obtained using proteolytic enzymes (for example, restriction digestion of the entire antibody with papain can yield Fab and cleavage with pepsin can yield F (ab ') ₂ fragment). Can be produced through genetic recombination techniques.

In the present invention, the antibody is preferably in the form of Fab or in the form of a complete antibody. In addition, the heavy chain constant region may be selected from any one isotype of gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε). Preferably, the constant regions are gamma 1 (IgG1), gamma 3 (IgG3) and gamma 4 (IgG4), most preferably the gamma 1 (IgG1) isotype. The light chain constant region may be kappa or lambda type, preferably kappa type. Therefore, it can be seen that the preferred antibody of the present invention is Fab form or IgG1 form having a kappa (κ) light chain and a gamma 1 (γ1) heavy chain.

As used herein, the term “heavy chain” refers to an entirety comprising a variable region domain V _H and three constant region domains C _H1 , C _H2 and C _H3 comprising an amino acid sequence having sufficient variable region sequence to confer specificity to the antigen. It means both length heavy chains and fragments thereof. The term “light chain” herein also refers to both the full-length light chain and fragments thereof comprising the variable region domain V _L and the constant region domain C _L comprising an amino acid sequence having sufficient variable region sequence to confer specificity to the antigen. do.

The antibody of the present invention comprises a heavy chain variable region comprising CDRH1 comprising SEQ ID NO: 31, CDRH2 comprising SEQ ID NO: 32, and CDRH3 comprising SEQ ID NO: 33.

As used herein, the term “complementarity determining region” refers to the amino acid sequences of the hypervariable regions of immunoglobulin heavy and light chains (Kabat et al., Sequences of Proteins of Immunological Interest , 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987)). The heavy chains (CDRH1, CDRH2 and CDRH3) and light chains (CDRL1, CDRL2 and CDRL3) each contain three CDRs. CDRs provide key contact residues for the antibody to bind antigen or epitope.

The RSV antibody or antigen-binding fragment thereof of the present invention may include a variant of the amino acid sequence described in the attached sequence list within a range capable of specifically recognizing the F protein of RSV. For example, changes can be made to the amino acid sequence of the antibody to improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions and / or substitutions of amino acid sequence residues of the antibody.

Such amino acid variations are made based on the relative similarity of amino acid side chain substituents such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have a similar shape. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; Phenylalanine, tryptophan and tyrosine are biologically equivalent functions.

In introducing variants, hydropathic idex of amino acids can be considered. Each amino acid is assigned a hydrophobicity index depending on its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).

The hydrophobic amino acid index is very important in conferring the interactive biological function of proteins. It is known that substitution with amino acids having similar hydrophobicity indexes can retain similar biological activity. When introducing mutations with reference to the hydrophobicity index, substitutions are made between amino acids which exhibit a hydrophobicity index difference of preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

On the other hand, it is also well known that substitutions between amino acids having similar hydrophilicity values result in proteins with equivalent biological activity. As disclosed in US Pat. No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Asphaltate (+ 3.0 ± 1); Glutamate (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).

When introducing mutations with reference to hydrophilicity values, substitutions are made between amino acids which exhibit a hydrophilicity value difference of preferably within ± 2, more preferably within ± 1 and even more preferably within ± 0.5.

Amino acid exchange in proteins that do not alter the activity of the molecule as a whole is known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.

Considering the above-described variations with biologically equivalent activity, an antibody of the present invention or a nucleic acid molecule encoding the same is also interpreted to include sequences that exhibit substantial identity with the sequences listed in the Sequence Listing. The above substantial identity is at least 61% when the sequence of the present invention is aligned as closely as possible with any other sequence, and the aligned sequence is analyzed using algorithms commonly used in the art. By a sequence that shows homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv . Appl . Math . 2: 482 (1981) ; Needleman and Wunsch, J. Mol . Bio . 48: 443 (1970); Pearson and Lipman, Methods in Mol . Biol . 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc . Acids Res . 16: 10881-90 (1988); Huang et al., Comp . Appl . BioSci . 8: 155-65 (1992) and Pearson et al., Meth . Mol . Biol. 24: 307-31 (1994). NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol . Biol . 215: 403-10 (1990)) is accessible from the National Center for Biological Information (NBCI), etc. It can be used in conjunction with sequencing programs such as blastx, tblastn and tblastx. BLSAT is accessible at http://www.ncbi.nlm.nih.gov/BLAST/. Sequence homology comparison methods using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

According to a preferred embodiment of the present invention, the heavy chain variable region of the present invention comprises the amino acid sequence of SEQ ID NO: 4 sequence.

According to a preferred embodiment of the invention, the antibody of the invention against RSV further comprises a light chain variable region having the following light chain CDR amino acid sequence: CDRL1 comprising SEQ ID NO: 34, SEQ ID NO: 35 CDRL2 comprising the CDRL3 and SEQ ID NO: 36 sequence. More preferably, the light chain variable region of the antibody of the present invention comprises the amino acid sequence of SEQ ID NO: 2 sequence.

Antibodies of the invention include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFV), single chain antibodies, Fab fragments, F (ab ') fragments, disulfide-binding Fvs (sdFV) And anti-idiotype (anti-Id) antibodies, epitope-binding fragments of the antibodies, and the like.

According to a preferred embodiment of the present invention, the antibody of the present invention is a human antibody.

As used herein, the term “human antibody” refers to an antibody from which the sequences of the variable and constant regions of the heavy and light chains are derived from humans. Anti-RSV human antibodies were prepared using cell engineering techniques. Human antibodies have advantages over non-human and chimeric antibodies: the effector portion of the human antibody interacts better with other parts of the human immune system (eg, complementarity-dependent cytotoxicity (CDC) or antibody-dependent cells). Cytotoxicity (ADCC) destroys target cells more efficiently), and the immune response to antibodies introduced into these organisms is not exogenous due to the human immune system recognizing human antibodies as foreign substances, either as a whole foreign non-human antibody or a partial foreign chimeric antibody. Less than on the back It has also been reported that injected non-human antibodies have a much shorter half-life in the human circulation than the half-life of human antibodies. In contrast, human antibodies introduced into vivo are more useful because they have substantially the same half-life as naturally occurring human antibodies, thereby reducing the dose and frequency administered.

According to the invention, the antibodies of the invention can be prepared in various forms of antibodies. For example, as described in the Examples below, the antibodies of the present invention can be prepared with Fab antibodies, and are also complete by recombination with human constant regions using light and heavy chain variable regions obtained from Fab antibodies. It is also possible to provide antibodies in the form).

According to a preferred embodiment of the invention, the antibody of the invention is a monoclonal antibody. The term “monoclonal antibody” refers to antibody molecules of a single molecular composition obtained from substantially the same antibody population, which monoclonal antibodies exhibit single binding specificity and affinity for specific epitopes. As described in the following examples, the human antibody of the present invention, which can be provided by genetic recombination methods, may be referred to as a monoclonal antibody because the nucleic acid sequence and the amino acid sequence are identical.

Antibodies of the invention can bind to the F protein of RSV and block RSV infection. Thus, the antibodies provided according to the invention are neutralizing antibodies that induce a neutralizing immune response. The term “neutralizing antibody” refers to an antibody molecule capable of eliminating or significantly reducing the biological activity or effector function of a binding target antigen. Thus, the antibodies of the present invention are neutralizing antibodies that can eliminate or significantly reduce the effector function of antigen F (eg, involved in the formation of giant cells and the penetration and adhesion of viruses). By “significant reduction” is meant reducing the effector function of the target antigen (eg, F protein) by at least about 500%, preferably at least about 700%, more preferably at least about 90%. Methods for determining the ability of candidate antibodies to neutralize the biological activity of an antigen are described, for example, in Kawade, J. Interferon. Res . 1: 61-70 (1980), which is incorporated herein by reference.

According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding a heavy chain variable region of an antibody against respiratory synstium virus comprising the amino acid sequence of SEQ ID NO: 4.

According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding a light chain variable region of an antibody against respiratory synstium virus comprising the amino acid sequence of SEQ ID NO: 2 sequence.

As used herein, the term “nucleic acid molecule” is meant to encompass DNA (gDNA and cDNA) and RNA molecules inclusively, and the nucleotides that are the basic structural units in nucleic acid molecules are naturally modified nucleotides, as well as modified sugar or base sites. It also includes analogues (Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990). Nucleic acid molecular sequences encoding the heavy and light chain variable regions of the present invention can be modified. Such modifications include addition, deletion or non-conservative substitutions or conservative substitutions of nucleotides.

According to a preferred embodiment of the present invention, the nucleic acid molecule encoding the heavy chain variable region comprises the nucleotide sequence of SEQ ID NO: 3 sequence.

According to a preferred embodiment of the present invention, the nucleic acid molecule encoding the light chain variable region comprises the nucleotide sequence of SEQ ID NO: 1.

Nucleic acid molecules of the invention encoding anti-RSV antibodies are also construed to include nucleotide sequences that exhibit substantial identity to the nucleotide sequences described above. This substantial identity is at least 80% when the nucleotide sequence of the present invention is aligned with the nucleotide sequence of the present invention to the maximum correspondence, and the aligned sequence is analyzed using algorithms commonly used in the art. A nucleotide sequence that exhibits homology, more preferably at least 90% homology, most preferably at least 95% homology.

According to another aspect of the invention, the invention is a nucleic acid molecule encoding a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2 sequence; And it provides a recombinant vector comprising a nucleic acid molecule encoding a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 sequence.

As used herein, the term “vector” refers to a plasmid vector as a means for expressing a gene of interest in a host cell; Cosmid vector; And viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors, and the like, and are preferably plasmid vectors.

According to a preferred embodiment of the present invention, the nucleic acid molecule encoding the light chain variable region and the nucleic acid molecule encoding the heavy chain variable region in the vector of the present invention are operatively linked with the promoter.

As used herein, the term “operably linked” refers to the functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and other nucleic acid sequences, thereby The regulatory sequence will control the transcription and / or translation of said other nucleic acid sequence.

The recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are described in Sambrook et al., Molecular Cloning , A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

Vectors of the present invention can typically be constructed as vectors for cloning or vectors for expression. In addition, the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts.

For example, when the vector of the present invention is an expression vector and the prokaryotic cell is a host, a strong promoter capable of promoting transcription (for example, the tac promoter, lac Promoter, lac UV5 promoter, lpp promoter, p _L ^λ promoter, p _R ^λ promoter, rac 5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), ribosomal binding site for initiation of detoxification and It is common to include transcription / detox termination sequences. E. coli (eg, as a host cell) HB101, BL21, DH5α, etc.) is used, E. coli Promoter and operator sites of the tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol . , 158: 1018-1024 (1984)) and the leftward promoter of phage λ (p _L ^λ promoter, Herskowitz, I. and Hagen, D., . Ann Rev Genet, 14:. . 399-445 (1980)) may be used as a control region. When Bacillus bacteria are used as host cells, the promoter of the Bacillus Chuo toxin protein gene of ringen sheath (Appl Environ Microbiol 64: 3932-3938 ( 1998); Mol Gen Genet 250:...... 734-741 (1996) ) Or any promoter expressible in Bacillus may be used as a regulatory site.

On the other hand, vectors that can be used in the present invention are plasmids often used in the art (eg, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14). , pGEX series, pET series and pUC19, etc.), phages (eg, λgt4.λB, λ-Charon, λΔz1 and M13, etc.) or viruses (eg SV40, etc.).

On the other hand, when the vector of the present invention is an expression vector and the eukaryotic cell is used as a host, promoters derived from the genome of mammalian cells (for example, metallothionine promoter, β-actin promoter, human heroglobin promoter and human muscle) Creatine promoter) or a promoter derived from a mammalian virus (eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk of HSV) Promoter, mouse breast tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of moroninivirus, promoter of Epstein bar virus (EBV) and promoter of Loews sacoma virus (RSV))) It generally has a polyadenylation sequence as a transcription termination sequence.

Vectors of the invention may be fused with other sequences to facilitate purification of the antibody expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).

In addition, since the protein expressed by the vector of the present invention is an antibody, the expressed antibody can be easily purified through a Protein A column or the like without additional sequences for purification.

On the other hand, the expression vector of the present invention as an optional marker, and includes antibiotic resistance genes commonly used in the art, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neo There are genes resistant to mycin and tetracycline.

The vector expressing the antibody or antibody fragment of the present invention can be either a vector system in which the light and heavy chains are simultaneously expressed in one vector or a system in which the light and heavy chains are expressed in separate vectors. In the latter case, both vectors are introduced into the host cell via co-transfomation and targeted transformation. Simultaneous transformation is a method of introducing each of the vector DNAs encoding the light and heavy chains into the host cell at the same time and then selecting the cells expressing both the light and heavy chains. Target transformation involves selecting the cells transformed with the vector containing the light (or heavy) chain and transforming the selected cells expressing the light chain with the vector containing the heavy (or light) chain to express both the light and heavy chains. This is the method of finally selecting the cells. In the following examples, antibodies were provided using a vector system in which the light and heavy chains are simultaneously expressed in one vector.

According to a preferred embodiment of the present invention, the nucleic acid molecule encoding the light chain variable region comprises a nucleotide sequence of SEQ ID NO: 1 sequence, the nucleic acid molecule encoding the heavy chain variable region is a nucleotide sequence of SEQ ID NO: 3 Include.

According to another aspect of the present invention, the present invention provides a host cell transformed with the recombinant vector.

Host cells capable of stably and continuously cloning and expressing the vectors of the present invention are known in the art and can be used with any host cell, for example Escherichia coli), Bacillus sp, Streptomyces, such as Bacillus subtilis and Bacillus Chuo ringen sheath (Streptomyces), Pseudomonas (Pseudomonas) (e.g., Pseudomonas footage is (Pseudomonas putida)), Proteus Billy's Mum (Proteus mirabilis ) or Staphylococcus (e.g., Staphylocus prokaryotic host cells such as carnosus )), but are not limited to these. The host cell is preferably E. coli and more preferably E. coli ER2738, E. coli XL-1 Blue, E. coli BL21 (DE3), E. coli JM109, E. coli DH series, E. coli TOP10, E. coli TG1 and E. coli HB101. Most preferably the host cell is E. coli ER2738 or E. coli TG1.

Suitable eukaryotic host cells of the vector are of the genus Aspergillus. fungi, such as species, Pichia pastoris ), Saccharomyces cerevisiae ), yeasts such as Schizosaccharomyces and Neurospora crassa , other lower eukaryotic cells, and cells of higher eukaryotes such as insect-derived cells. In addition, cells derived from plants or mammals can also be used as host cells. Preferably, the host cell is monkey kidney cells (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) Cells, MDCK, myeloma cell line, HuT 78 cells and 293 cells. More preferably, the host cell is a COS7 cell.

When using microorganisms such as E. coli , productivity is higher than that of animal cells, but it is not preferable for the production of intact Ig antibodies due to glycosylation problems, but it can be used for the production of Fab and Fv. have.

As used herein, “transformation” and / or “transfection” into a host cell includes any method of introducing a nucleic acid into an organism, cell, tissue, or organ and is a standard suitable for a host cell as is known in the art. The technique can be selected and performed. These methods include electroporation, plasma fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, agro bacteria mediated transformation, PEG, dextran Sulfate, lipofectamine and dry / inhibitory mediated transformation methods and the like. Calcium treatment or electroporation, for example using the calcium chloride method, is commonly used for prokaryotic cells (Sambrook et al., Molecular Cloning : A Laboratory Manual (New York: Cold Spring Haror Laboratory Press) (1989) Agrobacterium tumefaciens Transfection with tumefaciens ) is used for the transformation of certain plant cells (Shaw et. al, Gene , 23: 315 (1983), International Publication WO 89/05859). For mammalian cells without cell walls, Calcium phosphate precipitation can be used (Graham et. Al, Virology , 52: 456-457 (1978)). General methods and features of transformation into mammalian host cells are described in US Pat. No. 4,399,216. Transformation into yeast is generally described by Van Solingen et al . ( J. Bact ., 130: 946 (1977)) and Hsiao et al . ( Proc . Natl . Acad. Sci . ( USA ) , 76: 3829 (1979)).

According to another aspect of the present invention, the present invention provides a method for producing a human antibody against respiratory syntium virus, comprising the following steps:

(a) a nucleic acid molecule encoding a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2; And culturing the host cell transformed with the recombinant vector comprising a nucleic acid molecule encoding a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; And

(b) expressing a human antibody against respiratory synthium virus in said host cell.

Cultivation of the transformed host cell in the antibody preparation can be made according to the appropriate medium and culture conditions known in the art. This culture process can be used by those skilled in the art can be easily adjusted according to the strain selected. Such various culture methods are disclosed in various documents (eg, James M. Lee, Biochemical Engineering , Prentice-Hall International Editions, 138-176). Cell culture is divided into a batch culture, a fed-batch, and a continuous culture depending on the suspension culture and adhesion culture, the culture method according to the growth method of the cell. The medium used for culturing must adequately meet the requirements of the particular strain.

In animal cell culture, the medium contains various carbon sources, nitrogen sources and trace element components. Examples of carbon sources that can be used are glucose, sucrose, lactose, fructose, maltose, carbohydrates such as starch and cellulose, fats such as soybean oil, sunflower oil, castor oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, Alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These carbon sources may be used alone or in combination. Examples of nitrogen sources that can be used include organic nitrogen sources and urea, such as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL) and soybean wheat, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate Inorganic nitrogen sources are included. These nitrogen sources may be used alone or in combination. The medium may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate and the corresponding sodium-containing salts as a person. It may also include metal salts such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, and appropriate precursors may be included.

During the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. In addition, during the culture, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble generation. In addition, to maintain the aerobic state of the culture, oxygen or oxygen-containing gas (eg, air) is injected into the culture. The temperature of the culture is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C.

Antibodies obtained by culturing the transformed host cell can be used without purification and can be further purified and purified using various conventional methods, for example, dialysis, salt precipitation and chromatography. Among them, a method using chromatography is most used, and the type and order of columns can be selected from ion exchange chromatography, size exclusion chromatography, affinity chromatography, and the like according to the characteristics of the antibody and the culture method.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising a pharmaceutically effective amount of a human antibody against respiratory synstium virus; And (b) provides a pharmaceutical composition for the prevention and treatment of respiratory synstium virus infection comprising a pharmaceutically acceptable carrier.

Since the human monoclonal antibody prepared by the above method exhibits RSV neutralizing ability, it can be used as a prophylactic and therapeutic composition against RSV infection alone or in combination with a conventional pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and agents are Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration and rectal administration Or the like. In oral administration, because proteins or peptides are digested, oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach. The pharmaceutical composition may also be administered by any device in which the active agent may migrate to the target cell.

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to reaction, Usually a skilled practitioner can easily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.001-100 mg / kg. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to prevent or treat infection of RSV.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media or in the form of extracts, powders, suppositories, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

The antibody composition of the present invention may be administered as a separate therapeutic agent or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents.

Antibodies can be introduced in vivo in the form of antibody-therapeutic conjugates to be used for the treatment of viral infections. Therapeutic agents include chemotherapeutic agents, radionuclides, immunotherapeutics, cytokines, chemokines, toxins, biological agents and enzyme inhibitors. Methods for binding antibiotics to antibodies are described, for example, in G. Gregoriadies, ed., Academic Press London , (1979); Arnon et al., Recent Results in Cancer Res . 75: 236 (1980); And Moolton et al., Immunolog . Res ., 62:47 (1982).

Preferred agents for coupling with the antibodies or antibody fragments of the invention are antibacterial, antiparasitic, antifungal and related agents, for example sulfonamides, penicillins and cephalosporins, aminoglycosides, tetracyclines, chloramphenicols, piperazine, chloroquine , Diaminopyridin, metroniazide, isoniazid, rifampin, streptomycin, sulfone, erythromycin, polymyxin, nystatin, amphotericin, 5-fluorocytosine, 5-iodine-2'-deoxyuridine , 1-adamantaamine, adenine arabinoside, ammannitine, ribovarin and azithrothymidine (AZT), preferably ribovarin. Various conditions suitable and desirable for targeting an agent to specific target sites are described, for example, in Trouet et al., Plenum Press , New York and London, 19-30 (1982). By using a highly specific antibody prepared against microbial antigens, an effective therapeutic agent can be targeted directly to infectious lesions to selectively kill the infecting bacteria, thereby solving many problems that arise when treating infections resistant to drugs. In addition, drugs targeted as lesions may increase the efficacy at high concentrations in the infected area.

Immunomodulators that can be used as therapeutic agents in the antibody-therapeutic conjugates include, but are not limited to, lymphokines and cytokines.

According to another aspect of the present invention, the present invention provides a diagnostic kit for respiratory syntium virus detection comprising the antibody of the present invention to the respiratory syntium virus described above.

Antibodies of the present invention against the F antigen protein of RSV can be applied to biological samples to diagnose RSV infection.

As used herein, the term “biological sample” includes tissue, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatants, ruptured eukaryotic cells, and bacterial expression systems. It is possible, but not limited to. RSV infection can be confirmed by reacting these biological samples with or without manipulation of the antibody of the present invention.

The formation of the antigen-antibody complexes described above can be performed by colormetric method, electrochemical method, fluorimetric method, luminometry, particle counting method, visual assessment or visual assessment. It can be detected by scintillation counting method.

“Detection” herein is for detecting antigen-antibody complexes and can be carried out using various labels. Specific examples of the label include enzymes, fluorescent, ligands, luminescent, microparticles or radioisotopes.

Enzymes used as detection markers include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase and β-latamase and the like. Phosphorus, Eu³ ⁺ , Eu³ ⁺ chelate or cryptate, and the like, and ligands include biotin derivatives and the like, luminescent materials include acridinium esters and isoluminol derivatives, etc., microparticles such as colloidal gold and Pigmented latexes, and the like, and radioisotopes include ⁵⁷ Co, ³ H, ¹²⁵ I and ¹²⁵ I-Bolton Hunter reagents, and the like.

Preferably, the antigen-antibody complex can be detected using enzyme immunosorbent adsorption (ELISA). Enzyme immunosorbent methods (ELISA) include direct ELISA using labeled antibodies that recognize antigens attached to a solid support, and indirect ELISAs using labeled secondary antibodies that recognize captured antibodies in a complex of antibodies that recognize antigens attached to a solid support. Direct sandwich ELISA using another labeled antibody that recognizes the antigen in the complex of the antibody and the antibody attached to the solid support, followed by reaction with another antibody that recognizes the antigen in the complex of the antibody and the antigen attached to the solid support. Various ELISA methods include indirect sandwich ELISAs using labeled secondary antibodies that recognize the antibody. The antibody of the present invention may have a detection label, and if it does not have a detection label, the antibody of the present invention may be captured and may be identified by treating another antibody having the detection label.

The features and advantages of the present invention are summarized as follows:

(a) The antibody of the present invention binds to the surface antigen F protein of RSV with high specificity. Interestingly, the antibodies of the present invention have much better binding to RSV than Synagis, a conventional commercial antibody therapeutic.

(b) The antibody of the present invention is superior to the neutralizing ability against RSV over Synagis, a conventional commercial antibody therapeutic.

(c) The antibodies of the present invention can be used for the prevention or treatment of RSV infection. As described in the Examples below, the antibodies of the present invention are superior to the in vivo infection prevention effect against RSV over Synagis, a conventional commercial antibody therapeutic.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Example  I: Preparation of Human Antibody Library

In order to find human antibodies that can neutralize RSV infection, we first constructed human antibody libraries from infants' peripheral blood lymphocytes (PBLs) recovered after RSV infection.

Ⅰ-1: Human Fab cDNA  Preparation of the Library

CDNAs for human light and human heavy chain Fd regions were prepared to prepare human Fab antibody libraries.

Specifically, blood is taken from 5 patients and medical staff to separate Lymphocytes using Ficoll concentration gradient, and then total RNA is separated from the PBL, and the reverse transcriptase (Superscript II, Gibco) is used as a template. BD) and CK1d primers of SEQ ID NO: 5 and CG1d primers of SEQ ID NO: 6 were used to selectively synthesize cDNAs corresponding to the Fd regions of the human light and human heavy chains, respectively. This human light chain cDNA was used as a template for the 5 'specific primers of the kappa variable region of the human light chain (VK1 of SEQ ID NO: 7, VK2 / 4 of SEQ ID NO: 8, VK3 of SEQ ID NO: 9) Human antibody light chain cDNA libraries were selectively synthesized using PCR using VK5) of SEQ ID NO: 10 sequence and CK1d primers of SEQ ID NO: 5, respectively, in pairs.

In addition, 5D specific primers of the variable region of the human heavy chain (VH135 of SEQ ID NO: 11, VH2 of SEQ ID NO: 12, VH4 of SEQ ID NO: 13, SEQ ID NO: 13) of the human heavy chain FD region cDNA library of human antibody heavy chain was selected by PCR using VH4b of SEQ ID NO: 14, VH6 of SEQ ID NO: 15, VH4gs of SEQ ID NO: 16), and CG1d primers of SEQ ID NO: 6, respectively, in pairs Synthesized.

In addition, in order to increase the cloning efficiency of the cDNA library, a pair of the VKext primer of SEQ ID NO: 17 sequence and the CKext primer of SEQ ID NO: 18 sequence, VHext of SEQ ID NO: 19 sequence, VH2ext of SEQ ID NO: 20 sequence, sequence catalog PCR was performed using the VH4bext of the 21st sequence and the CG1dext primer of the 22nd sequence. At this time, these PCR reaction conditions were performed 30 times using Taq DNA polymerase (preliminary modification at 95 ° C. for 5 minutes, 50 seconds at 95 ° C., 50 seconds at 55 ° C., and 1 minute at 72 ° C.).

Ⅰ-2: Cloning  And transformation

We cloned the human light chain cDNA library obtained in Example I-1 into pC3-Q-AKA / HzK (see Korean Patent No. 0318761) to transform E. coli .

Specifically, the human light chain DNA fragments obtained by PCR amplification in Example I-1 and the pComb3HSS vector (Scripps Laboratories, USA) were digested with restriction enzymes Sac I and X ba I, respectively, and purified. After ligation overnight at l ° C., the enzyme was inactivated by heating at 70 ° C. for 10 min. Thereafter, glycogen and 3M sodium acetate were added to the ligation reaction product, and ethanol was added at −20 ° C. to precipitate DNA overnight. The precipitated DNA was washed with 70% ethanol, dried and suspended in 20 μl of distilled water, and the library DNA was transformed with E. coli ER2738 (Stratagene) by electroporation.

First, competent cells were prepared for transformation. Escherichia coli ER2738 cells were shaken at 2 × YT 500 ml, 37 ° C. at an optical density (0.5-0.7) and left on ice for 30 minutes. After removing the supernatant by centrifugation for 15 minutes at 4 ℃, 4000 rpm, precipitated cells were suspended with 500 ml of 10% glycerol. After removing the supernatant by centrifugation again at 4 ° C. and 5000 rpm for 15 minutes, the precipitated cells were suspended with 250 ml of 10% glycerol and centrifuged again at 4 ° C. and 5000 rpm for 15 minutes. The supernatant was removed, and the precipitated cells were suspended in 20 ml of 10% glycerol, followed by centrifugation at 4 ° C and 4000 rpm for 15 minutes to remove the supernatant, and then the cells were suspended in 1-2 ml of 10% glycerol. 300 μl aliquots of 1.5 ml tubes were stored at −70 ° C. until use. The library DNA was added to 300 μl of the E. coli ER2738, which is the competent cells, followed by well mixing. After standing on ice for 1 minute, the cells were transferred to a Gene Pulser cuvette (Biorad), which was transferred to an electroporator (Biorad). Transformed cells were prepared by applying pulses at 2.5 kV and 200 Hz.

Ⅰ-3: Light chain  Gene and Heavy chain Fd  Gene binding

After adding 2 × YT medium to the transformed cells in Example I-2, the cells were shaken at 37 ° C. overnight, and plasmid DNA was isolated from the cells. The human light chain library plasmid DNA and the human heavy chain gene fragment amplified by PCR were cut and isolated by restriction enzymes Xho I and Spe I, and then bound in the same manner as in Example I-2, and transformed into E. coli ER2738 cells. It was.

Example  II: RSV Screening for human antibodies

The present inventors have used colony lift assay method (Radosevic, K et al . , J. Immunol . Methods 272: 219-233 (2003) on human antibody Fab library cells prepared in Example I above to find Fab antibodies that bind to RSV . ) Was performed.

To this end, first, a nitrocellulose membrane was directly placed on a 2 × YTA plate, and about 1,000,000 cells were plated thereon and incubated overnight. The membrane at this time is called a master membrane. In the meantime, the capture membrane (capture membrane) to find a strong antigen-binding capacity was coated on the purified RSV F protein (10 ㎍ / ml in PBS) for 6 hours at 37 ℃. F protein, such as Walsh (J. Gen Virol 66:.. 409-415 (1985)) mouse monoclonal antibody (Biodesign C65064M) for the F protein by the method from the lysate (lysate) in the HEp-2 cells (ATCC) of Purification by affinity chromatography.

The membrane coated with the F protein was washed twice with PBS, and then left at 37 ° C. for 2 hours by adding 5% skim milk. After removing the skim milk, it was soaked in 2X YT medium containing 100 μg / ml ampicillin and 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside). The capture membrane was placed on a 2X YT plate to which 100 μg / ml ampicillin and 1 mM IPTG was added, and then the library cells were placed on the master membrane plated thereon and left at room temperature for 16-24 hours.

The capture membrane was washed five times with PBS (PBST) added with 0.05% Tween 20, and then left at 37 ° C. for 6 hours with PBS containing skim milk. To find clones bound to the antigen F protein, an anti-human F (ab ') ₂ antibody (Pierce) with horseradish peroxidase attached to the capture membrane was diluted 1: 1000 and then added. It was left to stand at 37 degreeC for 1 hour. To remove the unbound antibody was washed 5 times with PBST, the reaction was confirmed by ECL (enhanced chemiluminescence, Intron) to confirm the antigen binding capacity.

The antigen-binding ability cells were obtained from the master membrane at the same position, 2 × YTA was added thereto, and then cultured at 37 ° C. until O.D was about 0.7, followed by colony lift assay. The antigen-binding ability of the Fab clones obtained after the 1st, 2nd, 3rd and 4th screening to the F protein was analyzed by ELISA to select clones having good binding capacity among Fab clones having binding ability to RSV, and then pC3-13-9. It was named. The clone's nucleic acid sequence and RSV binding capacity were further analyzed.

Example  III: Selected Fab  Clones Nucleic acid sequence  analysis

Nucleic acid sequences for the variable regions of the light and heavy chains of the clones were determined by the T7 Sequence V2.0 DNA Sequencing Kit (Amersham). As a result, pC3-13-9 was selected from the cloning vectors prepared in Example I-2. The variable regions of the light and heavy chains in the vector had nucleic acid sequences described in SEQ ID NO: 1 and SEQ ID NO: 3. For the light chain, the selected clone was very similar to the amino acid sequence of DPK9 / O12 of the VK1 family in the human light chain germline gene, indicating that the human light chain was derived from DPK9 / O12, whereas for heavy chain, DP28 It was found that the human heavy chain germ line gene of.

Example  Ⅳ: Selected Fab  Clones Antigen binding ability  analysis

In order to analyze the antigen-binding capacity of RSC of each clone Fab selected above, the present inventors shaken the E. coli ER2738 transformed with the vector pC3-13-9 at 37 ° C. while the OD value at 600 nm was 0.5 to 1.0. After incubation, the cells were incubated overnight at 30 ° C. while inducing Fab expression with 1 mM IPTG solution. Cells were harvested and subjected to osmotic shock using TES (0.2 M Tris-HCl, pH 8.0; 0.5 mM EDTA, 0.5 M sucrose) buffer to give soluble in the periplasmic extract. ) Fab antibodies were obtained.

Indirect ELISA was performed to determine the antigen binding capacity of each Fab clone. That is, each well of the ELISA plate was coated with 0.2 μg of purified RSV particles overnight, blocked with 2% BSA and washed four times with TBS-T. The Fab antibody present in the periplasm extract was added and reacted at 37 ° C. for 1 hour, and the antibody that did not bind to the antigen was removed with TBS-T buffer. Anti-human F (ab ') ₂ IgG-HRP was diluted with TBS-T as a secondary antibody, bound with OPD ( o- phenylenediamine, Sigma) and H ₂ O ₂ , and the absorbance was measured at 492 nm. In this case, a mouse monoclonal antibody (C65064M) against Biodesign's F protein was used as a positive control, and a periplasm extract of a protein not expressing Fab was used as a negative control.

As a result, the absorbance of Fab13-9 was 0.82, and the absorbance of the complete IgG antibody against F protein used as the positive control was 0.90, whereas the periplasmic extract of E. coli, which did not express Fab as the negative control, had the absorbance. 0.05. This result means that the Fab antibody of the present invention has the ability to bind to purified RSV particles.

Example  Ⅴ: complete IgG  Preparation of Expression Plasmids for Expression

In order to prepare a fully IgG-type antibody having binding capacity to RSV, the signal sequence of the antibody gene, the Fab heavy chain and the light chain variable region of the C3-13-9 clone were synthesized using recombinant PCR.

Ⅴ-1: Light chain  Preparation of Expression Vectors Including

In order to prepare an antibody of the complete IgG form of the anti-RSV Fab, first, the signal sequence of the light chain gene and the sequence corresponding to the light chain variable region of the anti-RSV Fab are linked by recombinant PCR, followed by pdCMV-dhfrC-AKA / HzK (Korean patent). Sub-cloned to Hind III- BsiW I position of US Pat .

Specifically, in order to synthesize the signal sequence of the light chain gene, PCR is carried out by pairing pdCMV-dhfr-AKA / HzK as a template and Ryu86 primer of SEQ ID NO: 23 and KR-1 (leader) primer of SEQ ID NO: 24 Was performed. In addition, PCR was performed using a pair of KF primers of SEQ ID NO: 25 and KR primers of SEQ ID NO: 26 in pairs to synthesize human light chain variable region genes from the pC3-13-9 clone. In order to connect the signal sequence and the light chain variable region, recombinant PCR was performed using a pair of Ryu86 primers of SEQ ID NO: 23 and KR primers of SEQ ID NO: 26. PCR reaction conditions were premodified at 95 ° C. for 5 minutes, followed by 30 times using Taq DNA polymerase for 1 minute at 94 ° C., 30 seconds at 55 ° C., and 30 seconds at 72 ° C. Both ends of the DNA fragment were digested with restriction enzymes Hind III and BsiW I and inserted at the Hind III- BsiW I position of pdCMV-dhfrC-AKA / HzK to prepare pdCMV-dhfrC-RSV13-9K (FIG. 2).

Ⅴ-2 Light chain  And Heavy chain  Preparation of Expression Vectors Including

In the case of the heavy chain, the signal sequence of the heavy chain gene and the heavy chain variable region of the anti-RSV Fab were synthesized using recombinant PCR.

Specifically, in order to synthesize the signal sequence of the antibody gene, PCR is performed by pairing pdCMV-dhfr-AKA / HzK with a Ryu94 primer of SEQ ID NO: 27 and HR1 (leader) primer of SEQ ID NO: 28 It was. In addition, PCR was performed using a pair of HF2 primer of SEQ ID NO: 29 sequence and HR2 primer of SEQ ID NO: 30 sequence to synthesize heavy chain variable region gene of the Fab. Again, recombinant PCR was performed using the Ryu94 primer of SEQ ID NO: 27 and the HR2 primer of SEQ ID NO: 30 to link the signal sequence and the heavy chain variable region. PCR reaction conditions were performed 30 times using Taq DNA polymerase for 1 minute at 94 ° C, 30 seconds at 55 ° C, and 30 seconds at 72 ° C after premodification at 95 ° C for 5 minutes.

After cutting both ends of the DNA fragments with restriction enzymes EcoR I and Apa I, the anti-RSV light chain was inserted into the EcoR I- Apa I position of the cloned pdCMV-dhfrC-RSV13-9K, thereby expressing the expression plasmid of the present invention. It was prepared, it was named pdCMV-dhfrC-RSV13-9 (Fig. 3). The vector pdCMV-dhfrC-RSV13-9 was deposited with the Korea Biotechnology Research Institute Gene Bank on July 27, 2007 (Accession Number: KCTC 11161BP).

Example  Ⅵ: Terms RSV  Expression and Purification of Antibodies in Animal Cells

Ⅵ-1: Clause RSV  Expression of Antibodies in Animal Cells

293E cells (Invitrogen) were seeded in DMEM culture medium (GIBCO) containing 10% fetal calf serum and passaged at 37 ° C. and 5% CO ₂ incubator. The cells were seeded at a concentration of 4 × 10 ⁵ cells / ml in a 100 mm culture dish and incubated overnight at 37 ° C. and washed three times with OPTI-MEM I (GIBCO) solution. On the other hand, 10 μg of the antibody expression vector pdCMV-dhfrC-RSV13-9 prepared above was taken and diluted with 500 μl of OPTI-MEM I. 25 μl of lipofectamine (GIBCO) was also applied to 500 μl of OPTI-MEM I. Diluted with. The expression vector and the lipofectamine dilution were mixed in a 15 ml tube to prepare a DNA-lipopeptamine mixture, which was left at room temperature for at least 15 minutes. 5 ml of OPTI-MEM I was added to each of the DNA-lipopeptamine mixtures, and the mixture was evenly mixed with cleanly washed 293E cells, followed by incubation for 48 hours at 37 ° C and 5% CO ₂ incubator. Was expressed.

Ⅵ-2: Item RSV  Purification of Antibodies

The supernatant of the 29E cell culture was collected, filtered through a set of filters to remove specific material, and finished with a 0.2 nm filter. The supernatant was flowed out through a preliminarily prepared Protein A column based on the total amount of antibody. After washing, the antibody was eluted from the column (0.1 M glycine / HCl, pH 2.8) with neutral buffer (1 M Tris / HCl, pH 7.4). The buffer was exchanged by ultrafiltration using 2 L of sterile PBS and sterilized using filtration through a 0.2 nm filter. The antibody was purified and stored on refrigerated ice until use.

Example  Ⅶ: anti- RSV  Antibody RSV Binding capacity

RSV binding ELISA was performed with the control group Synagis to analyze the efficacy of RSV binding ability of the purified antibody. After RSV infection to HEV-2 cells (ATCC), the cells were cultured for 3 days to form HEV2 cells infected with RSV. After removal of the culture solution and the addition of 3.7% formaldehyde, the cells were fixed at 4 ° C for 30 minutes, followed by 2% BSA. Addition was blocked for 2 hours. After blocking, the purified RSV13-9 and the control Synagis were serially diluted two times, added to the plate, reacted for 1 hour, and then HRP-coupled secondary antibody (Pierce) was added to the anti-human IgG-Fc. After reacting with time, color development with OPD was performed, followed by analysis of binding efficacy to RSV by measuring OD values using an ELISA reader. As can be seen in Figure 4, as the concentration of the RSV13-9 antibody of the present invention increased the final signal of the ELISA increased. Therefore, it can be seen that the RSV13-9 antibody of the present invention specifically binds to RSV. In addition, the RSV13-9 antibody of the present invention showed specific binding ability to both RSV A and B subgroups. Surprisingly, the RSV13-9 antibody of the present invention was much better in binding capacity to RSV than Synagis, a conventional commercial antibody therapeutic (FIG. 4).

Example  Ⅷ: anti- RSV  Functional Activity of Antibodies in Vitro

In order to determine whether the antibody of the present invention has a virus neutralizing effect in vitro, infection neutralization assay was performed. Neutralization assays are performed by pre-reacting purified monoclonal antibodies with the virus prior to treating the cells with the virus, and as a result, the ability of the monoclonal antibodies to inhibit viral infectivity can be investigated.

One day prior to the experiment, HEp-2 cells were inoculated into 24-well plates at 10 ⁴ cell density, and the next day, RSV was transferred to a 1.5 ml tube of RSV13-9 and the control Synagis antibody serially diluted by concentration. The same amount was added and reacted for 30 minutes at 37 ℃, CO ₂ incubator. After removing the medium of the 24-well plate inoculated the day before and washing once with PBS, the reacted antibody and RSV were repeatedly added three times at 200 μl per well and reacted for 1 hour in a 37 ° C. CO ₂ incubator. . After 1 hour all medium was removed from the wells and 2% FBS, 5% methyl cellulose medium was added 1 ml per well. 37 ° C., CO ₂ for 6 days After incubation in the incubator, the plate was developed when plaque appeared in each well. Color development method is as follows. After removing the methyl cellulose medium of each well, 3.7% formaldehyde solution diluted with PBS was added to the wells and left at 4 ° C. for 30 minutes. The solution was removed from the wells, a 5% skim milk powder solution was added to the wells, reacted for 2 hours in a room temperature stirrer, and diluted with mouse anti-RSV F antibody to each well, and reacted for 1 hour in a room temperature stirrer. After the reaction, the wells were washed three times with PBS, and then goat-anti mouse IgG Fc-HRP (Pierce) was diluted and added and reacted for 1 hour in a room temperature stirrer. After washing each well with PBS three times, the wells were developed using DAB (diaminobenzidine, Sigma) solution, and the number of plaques was measured to confirm the neutralization ability (FIGS. 5A-5B).

As can be seen in Figures 5a and 5b, as the concentration of the RSV13-9 antibody of the present invention increased, the number of plaque formation was reduced. In addition, the RSV13-9 antibody of the present invention reduced the number of plaque formation for both RSV A and B subgroups. As a result, it can be seen that the RSV13-9 antibody of the present invention has a neutralizing ability to neutralize the infection of RSV. In addition, the RSV13-9 antibody of the present invention was superior to RSV neutralizing ability over RSV than Synagis, a conventional commercial antibody therapeutic.

Example  Ⅸ: anti- RSV  Antibody In vivo  Functional activity

Ⅸ-1: anti- RSV  Establishment of animal test model of antibody

In order to confirm the neutralizing ability in vivo, divided into three groups, one administered by Synagis, RSV13-9 and the other by no treatment, was performed using three Balb / c mice (orients). Preliminary studies were conducted to ascertain the amount of antibodies and the amount of virus appropriate for assessing efficacy by observing RSV lung infection and lung proliferation. Antibody doses were used in amounts of 0.15 or 3 mg / kg and viruses were performed in 5 x 10 ⁵ , 5 x 10 ⁶ or 5 x 10 ⁷ plaque forming units / individuals. In vivo experiments were carried out in the following manner. First, the mice were injected intramuscularly with a volume of 100 μl of the antibody one day before RSV infection, and then the mice were anesthetized with Evertin the next day, and then RSV was infected by administration of various concentrations of RSV in the nasal route. Four days after RSV infection, the lungs were removed, weighed, placed in a grinder containing an appropriate amount of HBSS, and the lungs were crushed. The pulmonary lungs were centrifuged at 4 ° C, 4000 rpm for 30 minutes, and the supernatant was then subjected to RSV titration. RSV titration was confirmed through plaque formation assay. Plaque formation analysis was performed in the following manner. One day prior to the experiment, HEp-2 cells were inoculated into 24-well plates at 1 x 10 ⁵ cells per well, and the supernatant prepared by lung extraction the next day was serially diluted. After washing once with PBS, the diluted supernatant was repeatedly added three times, 200 μl per well, and reacted in a 37 ° C. CO ₂ incubator for 1 hour. After 1 hour all medium was removed from the wells and 2% FBS, 5% methyl cellulose medium was added 1 ml per well. After incubation in a CO ₂ incubator at 37 ° C. for 6 days, plaques appeared in each well and the plates developed. Color development method is as follows. After removing the methyl cellulose medium of each well, 3.7% formaldehyde solution diluted with PBS was added to the wells and left at 4 ° C. for 30 minutes. The solution was removed from the wells, and 5% skim milk powder solution was added to the wells, followed by reaction for 2 hours in a room temperature stirrer, and diluted with mouse anti-F antibody to each well, followed by reaction for 1 hour in the room temperature stirrer. After the reaction, the wells were washed three times with PBS, and then goat-anti mouse IgG Fc-HRP (Pierce) was diluted and added and reacted for 1 hour in a room temperature stirrer. Each well was washed three times with PBS, and then the wells were developed using DAB solution and plaque titers were determined by measuring the plaque number to examine the appropriateness of each test factor setting to establish an animal test model.

Ⅸ-2: anti- RSV  Antibody In vivo  Establish function

The established animal model was used to analyze the efficacy of RSV infection prevention in vivo. RSV13-9 antibody, Synagis administration group, injection solution administration group consisting of RSV-treated test group and RSV-free control group was performed using three Balb / c mice, respectively, the amount of antibody and the amount of virus based on the above determination Antibody doses were used in amounts of 0.15, 0.8 and 3 mg / kg and virus was used for 5 x 10 ⁶ plaque forming units. First, the mice were injected intramuscularly with a volume of 100 μl of antibody one day before RSV infection, and the next day mice were anesthetized with Evertin, and RSV was infected by nasal administration of RSV at a concentration of 5 × 10 ⁶ plaque forming units. Four days after RSV infection, the lungs were removed, placed in a grinder containing HBSS, and the lungs were ground. The pulverized lung was centrifuged at 4 ° C., 4000 rpm for 30 minutes, and the supernatant was then subjected to RSV titration. RSV titration was confirmed through plaque formation assay. The subsequent method was carried out in the same manner as described above.

As can be seen in Figure 6, as the amount of RSV13-9 antibody of the present invention administered to the mouse increased the titer of RSV in the lung of the mouse. Therefore, it can be seen that the RSV13-9 antibody of the present invention is effective in preventing RSV infection in vivo. Interestingly, the RSV13-9 antibody of the present invention was superior to the in vivo infection prevention effect against RSV than Synagis, a conventional commercial antibody therapeutic.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Figure 1 shows a colony lift assay for screening Fab variant clones with high affinity for antigens carried out in one embodiment of the present invention.

Figure 2 is a production diagram showing the manufacturing process of pdCMV-dhfrC-RSV13-9K.

Figure 3 is a production diagram showing the manufacturing process of pdCMV-dhfrC-RSV13-9 expressing the full form of the antibody.

FIG. 4 shows the results of binding of RSV13-9 antibodies in the complete IgG form and their binding to RSV A and B subgroups of the control group, synagis.

5A and 5B show the ex vivo activity of synagis, RSV13-9 and control for RSV A and B subgroups, respectively.

6 shows the in vivo activity of RSV13-9 and control of synagis against RSV.

<110> Aprogen, Inc. <120> Antibodies to Repiratory Syncytial Virus <160> 36 <170> KopatentIn 1.71 <210> 1 <211> 321 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (321) <223> light chain variable region of RSV13-9 <400> 1 gac atc ctg atg acc cag tct cca tct tcc ctg tct gca tct gta gga 48 Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 gac aga gtc acc atc act tgc cgg gca agt cag agc att agc agc tat 96 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr              20 25 30 tta aat tgg tat cag cag aaa cca ggg aaa gcc cct aag ctc ctg atc 144 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile          35 40 45 tat gct gca tcc agt ttg caa agt ggg gtc cca tca agg ttc agt ggc 192 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 agt gga tct ggg aca gat ttc act ctc acc atc agc agt ctg caa cct 240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 gaa gat ttt gca act tac tac tgt caa cag agt tac agt acc ccg ctc 288 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu                  85 90 95 act ttc ggc gga ggg acc aaa gtg gag att aaa 321 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 2 <211> 107 <212> PRT <213> Homo sapiens <400> 2 Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr              20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile          35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 3 <211> 378 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (378) <223> heavy chain variable region of RSV13-9 <400> 3 cag gtg acc ttg aag gag tct ggt cct gtg ctg gtg aaa ccc aca gag 48 Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu   1 5 10 15 acc ctc acg ctg acc tgc acc gtc tct ggg ttc tca ctc agc aat gct 96 Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Ala              20 25 30 aga atg ggt gtg agc tgg atc cgt cag ccc cca ggg aag gcc ctg gag 144 Arg Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu          35 40 45 tgg ctt gca cgc att gat tgg gat gat gat aaa tac tac agc aca tct 192 Trp Leu Ala Arg Ile Asp Trp Asp Asp Asp Lys Tyr Tyr Ser Thr Ser      50 55 60 ctg aag acc agg ctc acc atc tcc aag gac acc ccc aaa aac cag gtg 240 Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Pro Lys Asn Gln Val  65 70 75 80 gtc ctt aca atg acc aac atg gac cct gtg gac aca gcc acg tat tac 288 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr                  85 90 95 tgt gca cgg aat agc tac tat gat agt agt ggt tat tac ctc ctc tac 336 Cys Ala Arg Asn Ser Tyr Tyr Asp Ser Ser Gly Tyr Tyr Leu Leu Tyr             100 105 110 ttt gac tac tgg ggc cag gga acc ctg gtc acc gtc tcc tca 378 Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 125 <210> 4 <211> 126 <212> PRT <213> Homo sapiens <400> 4 Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu   1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Ala              20 25 30 Arg Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu          35 40 45 Trp Leu Ala Arg Ile Asp Trp Asp Asp Asp Lys Tyr Tyr Ser Thr Ser      50 55 60 Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Pro Lys Asn Gln Val  65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr                  85 90 95 Cys Ala Arg Asn Ser Tyr Tyr Asp Ser Ser Gly Tyr Tyr Leu Leu Tyr             100 105 110 Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 125 <210> 5 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Ck1d primer <400> 5 ccgtctagaa ttaacactct cccctgttga agctctttgt gacgggcgaa ctcag 55 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CG1d primer <400> 6 tgtactagtt ttgtcacaag atttgg 26 <210> 7 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> VK1 primer <400> 7 gagccgcacg agcccgagct ccagatgacc cagtctcc 38 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> VK2 / 4 primer <400> 8 ccgcacgagc ccgagctcgt gatgacycag tctcc 35 <210> 9 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> VK3 primer <400> 9 ccgcacgagc ccgagctcgt gwtgacrcag tctcc 35 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> VK5 primer <400> 10 ccgcacgagc ccgagctcac actcacgcag tctcc 35 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VH135 primer <400> 11 gtgcagctgc tcgagtctgg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VH2 primer <400> 12 rtcaccttgc tcgagtctgg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VH4 primer <400> 13 gtgcagctgc tcgagtcggg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VH4b primer <400> 14 gtgcagctac tcgagtgggg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VH6 primer <400> 15 gtacagctgc tcgagtcagg 20 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> VH4gs primer <400> 16 gtgcagctac tcgagtgggg c 21 <210> 17 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> VKext <400> 17 gaggaggagg aggaggagcc gcacgagccc ga 32 <210> 18 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> CKext primer <400> 18 gacgacgacg acgacgcgcc gtctagaatt aacactctc 39 <210> 19 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> VHext primer <400> 19 gtcgtcgtcg tcgtcgtcgt cgtaggtgca gctgctcgag tc 42 <210> 20 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> VH2ext primer <400> 20 gtcgtcgtcg tcgtcgtcgt agcagrtcac cttgctcgag tc 42 <210> 21 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> VH4bext primer <400> 21 gtcgtcgtcg tcgtcgtcgt aggtgcagct actcgagtg 39 <210> 22 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> CG1dext primer <400> 22 agagagagag agagagaggc atgtactagt tttgtca 37 <210> 23 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Ryu86 <400> 23 gctgcaaagc ttggaagcaa gatggattca 30 <210> 24 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> KR-1 <400> 24 gattccccac aggtaccaga tacccat 27 <210> 25 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> KF <400> 25 acctgtgggg acatcctgat gacccagtct c 31 <210> 26 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> KR <400> 26 ctccgtacgt ttaatctcca ctttggtccc tccg 34 <210> 27 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Ryu94 <400> 27 gagaattcac attcacgatg tacttg 26 <210> 28 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> HR1 <400> 28 aggtcacctg actctggaca ccatttaag 29 <210> 29 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> HF2 <400> 29 cagagtcagg tgaccttgaa ggagtctggt cctgtgctg 39 <210> 30 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> HR2 <400> 30 gatgggccct tggtggaggc tgaggag 27 <210> 31 <211> 7 <212> PRT <213> Homo sapiens <220> <221> CHAIN (222) (1) .. (7) <223> CDRH1 of RSV13-9 heavy chain varable region <400> 31 Asn Ala Arg Met Gly Val Ser   1 5 <210> 32 <211> 16 <212> PRT <213> Homo sapiens <220> <221> CHAIN (222) (1) .. (16) <223> CDRH2 of RSV13-9 heavy chain varable region <400> 32 Arg Ile Asp Trp Asp Asp Asp Lys Tyr Tyr Ser Thr Ser Leu Lys Thr   1 5 10 15 <210> 33 <211> 16 <212> PRT <213> Homo sapiens <220> <221> CHAIN (222) (1) .. (16) <223> CDRH3 of RSV13-9 heavy chain varable region <400> 33 Asn Ser Tyr Tyr Asp Ser Ser Gly Tyr Tyr Leu Leu Tyr Phe Asp Tyr   1 5 10 15 <210> 34 <211> 11 <212> PRT <213> Homo sapiens <220> <221> CHAIN (222) (1) .. (11) <223> CDRL1 of RSV13-9 light chain variable region <400> 34 Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn   1 5 10 <210> 35 <211> 7 <212> PRT <213> Homo sapiens <220> <221> CHAIN (222) (1) .. (7) <223> CDRL2 of RSV13-9 light chain variable region <400> 35 Ala Ala Ser Ser Leu Gln Ser   1 5 <210> 36 <211> 9 <212> PRT <213> Homo sapiens <220> <221> CHAIN (222) (1) .. (9) <223> CDRL3 of RSV13-9 light chain variable region <400> 36 Gln Gln Ser Tyr Ser Thr Pro Leu Thr   1 5  

Claims (15)

CDRH1 consisting of CDRH1 consisting of SEQ ID NO: 31, CDRH2 consisting of SEQ ID NO: 32, and heavy chain variable region having a heavy chain CDR (complementarity determining region) amino acid sequence of CDRH3 consisting of SEQ ID NO: 33, and CDRL1 consisting of SEQ ID NO: 34 Or an antibody or antigen-binding fragment thereof comprising a light chain variable region having a light chain CDR amino acid sequence of CDRL2 consisting of SEQ ID NO: 35 and CDRL3 consisting of SEQ ID NO: 36. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region is composed of the amino acid sequence of SEQ ID NO: 4. delete The antibody or antigen-binding fragment thereof of claim 1, wherein the light chain variable region consists of an amino acid sequence of SEQ ID NO: 2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a human antibody. A nucleic acid molecule encoding a heavy chain variable region of an antibody against respiratory synstium virus consisting of the amino acid sequence of SEQ ID NO: 4 sequence. 7. The nucleic acid molecule according to claim 6, wherein the nucleic acid molecule consists of a nucleotide sequence of SEQ ID NO: 3. delete delete A nucleic acid molecule encoding a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2; And a nucleic acid molecule encoding a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 4. The nucleic acid molecule of claim 10, wherein the nucleic acid molecule encoding the light chain variable region consists of a nucleotide sequence of SEQ ID NO: 1, and the nucleic acid molecule encoding the heavy chain variable region consists of a nucleotide sequence of SEQ ID NO: 3 Characterized by a recombinant vector. A host cell transformed with the recombinant vector of claim 10. A method for preparing an antibody against respiratory synstium virus, comprising the following steps: (a) culturing the host cell of claim 12; And (b) expressing an antibody against respiratory synstium virus in said host cell. (a) a pharmaceutically effective amount of an antibody against the respiratory synstium virus of any one of claims 1, 2, 4 and 5; And (b) a pharmaceutical composition for preventing and treating respiratory synstium virus infection comprising a pharmaceutically acceptable carrier. A diagnostic kit for detecting respiratory syntium virus, comprising the antibody against any one of claims 1, 2, 4, and 5 of the respiratory synstium virus.

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