WO2009088159A1

WO2009088159A1 - Antibodies to respiratory syncytial virus

Info

Publication number: WO2009088159A1
Application number: PCT/KR2008/007328
Authority: WO
Inventors: Keun-Soo Kim; Choon Ju Jeon; Nam-Kyu Lim; Seyeon Kim; Sangyoon Lee; Hye Kyung Ju
Original assignee: Aprogen Inc.
Priority date: 2008-01-08
Filing date: 2008-12-11
Publication date: 2009-07-16
Also published as: KR100938998B1; KR20090076213A

Abstract

The present invention relates to an antibody or its antigen-binding fragment against respiratory syncytial virus (RSV) with a specific amino acid sequence, and a nucleic acid encoding the same, a recombinant vector and a transformant having the nucleic acid, and a pharmaceutical composition with the antibody for preventing and treating the respiratory syncytial virus infection, and a diagnostic kit with the antibody for detecting the respiratory syncytial virus. The present invention has much more excellent efficacy on a binding affinity to RSV, a neutralizing activity to RSV infection and a prevention of in vivo RSV infection than a conventional antibody- therapeutic agent (Synagis) commercialized.

Description

ANTIBODIES TO RESPIRATORY SYNCYTIAL VIRUS

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to an antibody against a surface antigen F of respiratory syncytial virus (RSV), and a gene encoding the antibody, a recombinant vector and a transformant having the same, and a method for preparing a human monoclonal antibody thereof.

BACKGROUND OF TECHNIQUE

RSV (respiratory syncytial virus) is a major cause of lower respiratory tract infection, inducing pneumonia and bronchitis (Brandt et al., Pediatrics 52:56(1973); Selwyn et al., Rev Infect Dis, 12:s870(1990)). In particular, RSV is a high risk group capable of causing severe infections in infants having pulmonary dysplasia, heart malformation, cystic fibrosis, cancer or various immune deficiency diseases, and in adults with some type of immunocompromised condition before bone marrow transplant (Chandwani et al., J Pediatr, 117:251(1990); Bruhn et al., J Pediatr, 90:382(1977)). It has been reported that infection rate of RSV in senior is similar to that of an influenza virus and excess mortality in RSV epidemic is higher than in influenza epidemic.

In USA, there are high-risk patients ranging of from 100,000 to 200,000 and is required hospitalization of no less than 90,000 in annual due to RSV infections, reported that 4,500 of them is eventually died. In domestic, RSV is also prevalent every year and 60% of all viral lower respiratory tract infections identified at Seoul national University children's hospital is caused by RSV infection, supposing that hospitalization and death by RSV infection may be very frequent in Korea.

Regardless of severe situation, current useful vaccines or specific therapeutic agents have been not provided yet. Formalin-killed vaccine used clinically in the past resulted in severe lower respiratory tract disorders by RSV infection after vaccine inoculation.

Administration of antibody has been attempted as a strategy to prevent RSV infection, and polyclonal antibody (Respigam) and monoclonal antibody (Synagis) developed in Medlmmune, Inc. (USA) has been currently utilized. The strategy using antibodies is deduced from the following evidences in that antibody from the mother can help prevent diseases: (a) severe RSV disorders are rarely generated until 5-6 weeks when antibody from the mother is abundant; (b) neutralizing antibody is deficient in some infants having bronchiolitis at an early stage; (c) there is an inverse relationship between antibody titer of umbilical cord blood to RSV and severity of RSV diseases; and (d) RSV-neutralizing antibody is administrated into experimental animals or infants, preventing severe RSV diseases.

On the other hand, RSV is a non-segmented RNA virus of the family paramyxoviridae. Ten proteins (NSl, NS2, P, N, M, SH, G, F, M2 and L protein) are synthesized in infected host cells and three proteins (F, G and SH protein) function as a surface antigen. Of them, G and F protein which have relative high molecular weight and are present in outer membrane of virus are heavy glycosylated proteins playing a very essential role in immunity of RSV infection and variation of antigenicity. G protein is related to attachment to infected host cells and F protein is associated with formation of cytomegalovirus, and invasion and attachment of virus. Both proteins are related to induction of neutralizing antibodies. Recently, RSV is classified into A and B subgroup depending on reactivity to monoclonal antibody, and difference of antigenicity is mainly caused from variation of G protein. Meanwhile, preventable antigen region of F protein has not significant differences between subgroups and is not rapidly changed. F protein is highly conserved in different RSV strains in amino acid level of about 89% (Johnson et al., J. Gen. Virol, 69:2623(1988); Johnson et al., J. VmI. 61:3163(1987)). F protein of RSV is considered as an optimized target of neutralizing antibody to prevent RSV infection since it does not exhibit minor variation of antigen observed in influenza A virus and enables to induce neutralizing antibodies and to inhibit proliferation after infection.

Medlmmune, Inc. has developed a humanized antibody against F protein called 'Synagis' (US Pat. Appln. Pub. No.: US20000724396) which is currently commercialized. In addition, it was demonstrated that the antibody has in vivo and in vitro neutralizing activity and reduces hospitalization period on a practical clinic. However, there is a problem in the senses that immune responses may be induced by repeated administration because Synagis as a humanized antibody includes amino acid sequences of rat antibody.

In addition to Synagis, human monoclonal antibodies to neutralize RSV have been made for preventing or treating RSV infection. In Scripps Research Institute, Fabl9 antibody was prepared using a phage display assay (US Pat. No. 6,685,942), and a human monoclonal antibody against both RF-I and RF-2 was developed by preparation of B cell tumor cell line using SCID mouse in IDEC pharmaceutical (US Appln. Pub. Pat. No. 2004/076631). However, since antibody preparation for preventing and treating RSV diseases is very insufficient in the view of severity of RSV infection, development of excellent monoclonal antibody with high reliability has been urgently demanded.

Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have made intensive studies to develop an antibody for preventing and treating a respiratory syncytial virus (RSV) infection. As results, we have discovered that a novel antibody has a specific binding affinity to RSV A and B subgroup, and exhibits not only an excellent neutralizing activity to RSV infection but also a remarkable efficacy on prevention of in vivo RSV infection.

Accordingly, it is an object of this invention to provide an antibody or its binding fragment against a respiratory syncytial virus. It is another object of this invention to provide a nucleic acid molecule encoding the antibody against the respiratory syncytial virus.

It is still another object of this invention to provide a recombinant vector, comprising the nucleic acid molecule encoding the antibody against the respiratory syncytial virus. It is further object of this invention to provide a host cell transformed with the recombinant vector.

It is still further object of this invention to provide a method for preparing an antibody against a respiratory syncytial virus.

It is another object of this invention to provide a pharmaceutical composition for preventing or treating a respiratory syncytial virus infection.

It is still another object of this invention to provide a diagnostic kit for detecting a respiratory syncytial virus.

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

In one aspect of this invention, there is provided an antibody or its binding fragment against a respiratory syncytial virus, comprising a heavy chain variable region having the following heavy chain complementarity determining region (CDR) amino acid sequences: CDR_Hi comprising the amino acid sequence of SEQ ID NO:31, CDR_H2 comprising the amino acid sequence of SEQ ID NO:32, and CDR_H3 comprising the amino acid sequence of SEQ ID NO:33. The present inventors have made intensive studies to develop an antibody for preventing and treating a respiratory syncytial virus (RSV) infection. As results, we have discovered that a novel antibody has a specific binding affinity to RSV A and B subgroups, and exhibits not only an excellent neutralizing activity to RSV infection but also a remarkable efficacy on prevention of in vivo RSV infection.

The antibody of this invention has a specific binding affinity to RSV. Particularly, the antibody of this invention specifically binds to RSV F protein.

By "antibody" referred in this specification is meant an antibody which is capable of specifically binding RSV. Antibody is meant to include the entire antibody as well as any antibody fragments.

The entire antibody includes two full-length light chains and two full-length heavy chains, and each light chain is linked to the heavy chain by disulfide bond. The heavy chain constant region includes five different isotypes (y, μ, α, δ and ε) of which the subclass is classified into γl, γ2, γ3, γ4, αl and α2. The light chain constant region includes two different isotypes (K and λ) (Cellular and Molecular Immunology, Wonsiewicz, M. J., Ed., Chapter 45, pp. 41-50, W. B. Saunders Co. Philadelphia, PA(1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, sinauer Associates, Inc., Sunderland, MA (1984)). Antigen binding fragment refers to any antibody fragment capable of binding antigen including Fab, F^b^*), F(ab% Fv and so on. Fab has one antigen binding site which is composed of one variable domain from each heavy and light chain of the antibody, one constant region of light chain and the first constant region (C_Hi) of heavy chain. Fab' is different to Fab in the senses that there is a hinge region containing one or more cysteine residues at C-terminal of C_Hi domain of heavy chain. F(abθ₂ antibody is produced by forming a disulfide bond between cysteine residues of hinge region of Fab'. Fv is a minimal antibody fragment including one variable region from each heavy and light chain and recombinant technique to prepare a Fv fragment is disclosed in PCT WO 88/10649, PCT WO 88/106630, PCT WO 88/07085, PCT WO 88/07086 and PCT WO 88/09344.

Two-chain Fv is linked by non-covalent bond between one variable region of each heavy and light chain, and single-chain Fv is generally linked by covalent bond via a peptide linker between one variable region of each heavy and light chain or is directly linked to each other at C-terminal, forming a dimer such as two-chain Fv. Such antibody fragments may be obtained using a proteolytic enzymes {e.g., a whole antibody is digested with papain to produce Fab fragments, and pepsin treatment results in the production of F(abO₂ fragments), and may be preferably prepared by genetic recombination techniques.

Preferably, the antibody in this invention is a form of Fab or entire antibody. In addition, the heavy chain constant region is selected from the isotypes consisting of Y, μ, α, δ or ε. Preferably, the heavy chain constant region includes γl (IgGl), γ3 (IgG3) and γ4 (IgG4) isotype, and most preferably γl (IgGl) isotype. The light chain constant region includes K and λ isotype and preferably K isotype. Therefore, it could be appreciated that the preferable antibody of the present invention is a form of Fab or IgGl having K light chain and γl heavy chain.

The term "heavy chain" refers to both a full-length heavy chain and its part, which includes variable domain (V_H) containing the amino acid sequence with a variable region sequence for specifically binding to antigen and three constant domains (C_H1, C_H2 and C_H3). The term "light chain" refers to both a full-length light chain and its part, which includes variable domain (V_L) containing the amino acid sequence with a variable region sequence for specifically binding to antigen and three constant domains (C_L). The antibody of the present invention includes the heavy chain CDRs containing CDR_Hi comprising the amino acid sequence of SEQ ID NO:31, CDR_H2 comprising the amino acid sequence of SEQ ID NO:32, and CDR_H3 comprising the amino acid sequence of SEQ ID NO:33. The ^ΛΛCDR (complementarity determining region)" means an amino acid sequence of hypervariable region of immunoglobulin heavy and light chain (Kabat et a/., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)). Three CDRs are involved in heavy chain (Cm, C_H2 and C_H3) and light chain (CDR_Li, CDR_L2 and CDR_L3), respectively. CDR provides a main contacting residue to combine antibody with antigen or epitope.

RSV antibody or its antigen-binding fragment may include analogs of amino acid sequences set forth in the appended Sequence Listing, which are capable of specifically recognizing F protein of RSV. For example, amino acid sequence of antibody may be altered to improve binding affinity and/or other biological characteristics of antibody, for example including the alterations prepared by deletion, insertion and/or substitution of amino acid residues of antibody.

Such amino acid variations may be provided on the basis of a relative similarity of amino acid side chains, e.g., hydrophobicity, hydrophilicity, charge and size. By the analysis for size, shape and type of the amino acid side chains, it could be clear that all of arginine, lysine and histidine residues are those having positive charge; alanine, glysine and serine have a similar size; phenylalanine, tryptophan and tylosin have a similar shape. Accordingly, based on these considerable factors, arginine, lysine and histidine; alanine, glysine and serine; and phenylalanine, tryptophane and tylosin may be considered to be biologically functional equivalents.

For introducing mutation, a hydropathic index of amino acids may be considered. Based on the hydrophobicity and the charge, the hydropathic index is given to each amino acid: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glysine (- 0.4); threonine (-0.7); serine (-0.8); tryptophane (-0.9); tylosin (-1.3); proline (- 1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagin (-3.5); lysine (-3.9); and arginine (-4.5). For providing an interactive biological function of proteins, the hydropathic index of the amino acid is very important. It is well known to one of skill in the art that variations can possess a similar biological activity only where proteins are replaced with amino acids having similar hydropathic index. Where variations are intended to introduce based on the hydropathic index, the substitution is preferably performed between amino acid residues having no more than ±2 difference in hydropathic index values more preferably within ±1, much more preferably within ±0.5.

It would be also obvious to those of skill in the art that substitutions of amino acids with other amino acids having similar hydrophilicity values may result in the generation of variants having biologically equivalent activities. As disclosed in U.S.

Pat. No. 4,554,101, each amino acid residue is assigned the following hydrophilicity values: arginine (+3.0); lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine

(+0.3); asparagin (+0.2); glutamine (+0.2); glysine (0); threonine (-0.4); proline (- 0.5); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-

1.5); leucine (-1.8); isoleucine (-1.8); tylosin (-2.3); phenylalanine (-2.5); tryptophane (-3.4).

Where variations are intended to introduce based on the hydrophilicity values, the substitution is preferably performed between amino acid residues having no more than ±2 difference in hydropathic index values more preferably within ±1, much more preferably within ±0.5.

The alteration of amino acid residues not to substantially impair protein activity is well known to one skilled in the art (H. Neurath, R. L. Hill, The Proteins,

Academic Press, New York, 1979). Such amino acid alteration includes Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe,

Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, but not limited to.

Considering the afore-mentioned variations having biologically equivalent activities, it could be understood that either antibody of this invention or the nucleic acid encoding the same includes substantially identical sequences to the sequences set forth in the appended Sequence Listing. The substantially identical sequences refers to those showing preferably at least 61%, more preferably at least 70%, still more preferably at least 80%, most preferably at least 90% nucleotide similarity to the sequences of the appended Sequence Listing, as measured using one of the sequence comparison algorithms. Methods of alignment of sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482(1981); Needleman and Wunsch, J. MoI. Bio. 48:443(1970); Pearson and Lipman, Methods in MoI. 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. MoI. Biol. 24:307- 31(1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. MoI. Biol. 215: 403-10(1990)) is available from several sources, including the National Center for Biological Information (NBCI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blasm, blastx, tblastn and tblastx. It can be accessed at http://www.ncbi.nlm.nih.gov/BLAST/. A description of how to determine sequence identity using this program is available at http://www.ncbi.nlm.nih.gov/BI-AST/blast help.html.

According to a preferable embodiment, the heavy chain variable region includes the amino acid sequence of SEQ ID NO:4.

According to a preferable embodiment, the antibody against RSV of the present invention further includes a light chain variable region having the following light chain CDR amino acid sequences: CDR_L1 comprising the amino acid sequence of SEQ ID NO:34, CDR_L2 comprising the amino acid sequence of SEQ ID NO:35, and CDR_L3 comprising the amino acid sequence of SEQ ID NO:36. More preferably, the light chain variable region of the present invention includes the amino acid sequence 0f SEQ ID N0:2.

The antibody of this invention includes, but not limited to, monoclonal antibody, polyclonal antibody, human antibody, humanized antibody, chimeric antibody, single-chain Fvs (scFV), single-chain antibody, Fab fragment, F(ab') fragment, disulfide-linked Fvs (sdFV) and anti-idiotype (anti-Id) antibody, and epitope-binding fragment thereof.

According to a preferable embodiment, the antibody of the present invention is a human antibody. The term "human antibody" refers to an antibody of which variable and constant region sequence of each heavy and light chain are derived from a human. As described in the examples below, the present inventors prepared anti-RSV human antibody using a genetic recombinant technique and cell technology. The human antibody has some advantages compared with non-human and chimeric antibody: (i) strong interaction between effector region of human antibody and other parts of human immune system (e.g., a targeted cell is more effectively disrupted by complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC)); and (ii) reduction of immune response against antibodies introduced into the body compared with total foreign non-human antibodies or partial foreign chimeric antibodies since human immune system don^'t recognize human antibody as foreign substances. It was also reported that a biological half life of injected non-human antibody is much shorter than that of human antibody in a human circulatory system. On the contrary, it is more preferable that amount and number of dose administrated is reduced because the half life of human antibody introduced into the body is substantially equivalent to that of natural-occurring human antibody.

According to this invention, the present antibody may be prepared to be various types of antibody. For example, as described in the Examples below, the present antibody is prepared as a form such as Fab antibody or is prepared as a whole antibody by recombination of Fab antibody with a human-derived constant region using variable regions of heavy and light chain obtained.

According to a preferable embodiment, the present antibody is a monoclonal antibody. The term "monoclonal antibody" refers to an antibody with single molecular composition obtained from a substantially equivalent antibody population and the monoclonal antibody has single binding specificity and affinity to a specific epitope. As described in the examples below, the present human antibody to be prepared by a genetic recombination technique could be called as a monoclonal antibody because its nucleic acid sequence is the same to its amino acid sequence.

The antibody of this invention combines with F protein of RSV to block RSV infection. The antibody provided according to the present invention is a neutralizing antibody which induces a neutralizing immune response. The term "neutralizing antibody" means an antibody molecule that induces a neutralizing immune response by removing or significantly reducing the biological activities or effector functions of a bound target antigen. Therefore, the antibody of this invention is a neutralizing antibody which removes or significantly reduces effector functions of F antigen {e.g., participating in the formation of macrophage and the invasion and attachment of viruses). The term "significant reducing" means that effector function of a target antigen is reduced not less than about 50%, preferably about 70% and more preferably about 90%. For example, the method to measure abilities of a candidate antibody for neutralizing the biological activities of an antigen is disclosed in Kawade, J. Interferon Res. 1:61-70(1980), which is herein incorporated by reference.

In another aspect of this invention, there is provided a nucleic acid molecule encoding a heavy chain variable region of an antibody against the respiratory syncytial virus comprising the amino acid sequence of SEQ ID NO:4. In still another aspect of this invention, there is provided a nucleic acid molecule encoding a light chain variable region of an antibody against the respiratory syncytial virus comprising the amino acid sequence of SEQ ID NO:2.

The term "nucleic acid molecule" comprehensively refers to a deoxyribonucleotide (gDNA and cDNA) or ribonucleotide polymer, and the basic nucleotides of nucleic acid molecule also include analogues with modified sugar or base as well as natural nucleotides (Scheit, Nucleotide Analogs, John Wiley, New

York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)). The sequence of the present nucleic acid molecule encoding the variable region of heavy and light chain could be modified. Such modification includes addition, deletion or non-conservative or conservative substitution of nucleotide.

According to a preferable embodiment, the nucleic acid molecule encoding the variable region of heavy chain includes the nucleotide sequence of SEQ ID NO:3.

According to a preferable embodiment, the nucleic acid molecule encoding the variable region of light chain includes the nucleotide sequence of SEQ ID NO:1.

The nucleic acid molecule of this invention encoding an anti-RSV antibody also includes a nucleotide sequence sharing substantial homology with the above nucleotide sequence. The substantial homology means the nucleotide sequence sharing homology of at least 80%, more preferably 90% and most preferable 95% by sequence alignment analysis using maximal alignment between the nucleotide sequence of this invention and other random sequences and algorithm ordinarily known to those skilled in the art.

In still further aspect of this invention, there is provided a recombinant vector, comprising the nucleic acid molecule encoding the light chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and the nucleic acid molecule encoding the heavy chain variable region comprising the amino acid sequence of

SEQ ID NO:4. The term "vector" is a tool for expressing a target gene in a host cell, including a plasmid vector; a cosmid vector; and a virus vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector and an adeno- associated virus vector, and preferably a plasmid vector. According to a preferable embodiment, the nucleic acid molecules encoding the variable region of light and heavy chain are operatively linked to a promoter.

The term "operatively linked" refers to functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence affects transcription and/or translation of the nucleic acid corresponding to the second sequence.

The vector system of this invention may be performed by various methods known to those skilled in the art and its practical method is described in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is herein incorporated by reference.

Typically, the vector of this invention may be constructed as cloning or expression vector. In addition, the vector of this invention may be constructed using a prokaryotic or eukaryotic cell as a host cell.

For instance, it is common to include a strong promoter for transcription (e.g., tac promoter, lac promoter, lac UV5 promoter, lpp promoter, p_L ^λ promoter, p_R ^λ promoter, raco promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, and so on), a ribosomal binding site for translation initiation, and a transcription/translation termination sequence where each a vector of this invention and a prokaryotic cell is used in an expression vector and the host cell. E col/ (e.g., HBlOl, BL21, DH5α, etc.) as a host cell may utilize a promoter and operator region for tryptophan biosynthesis pathway (Yanofsky, C, J. Bacteriol.,

158:1018-1024 (1984)), and p_L ^λ promoter (Herskowitz, I. and Hagen, D., Ann. Rev.

Genet, 14:399-445 (1980)) as a regulatory region. Bacillus as the host cell may use the promoter of a toxic protein gene of Bacillus thuringiensis (Appl. Environ. Microbiol. 64:3932-3938(1998); MoI. Gen. Genet. 250:734-741(1996)), or any promoter enabling to be expressed in Bacillus as the regulatory region.

On the other hand, the suitable vector used in this invention might be constructed by manipulating a plasmid (example: pSClOl, pGV1106, pACYC177, CoIEl, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFRl, pHV14, pGEX series, pET series and pUC19), a phage (example: λgt4'λB, λ-Charon, λΔzl and M 13) or a virus (example: SV40) commonly used by one ordinarily skilled in the art. In each a vector of this invention and an eukaryotic cell used as an expression vector and the host cell, the promoter derived from genome of animal cell (example: methallothionein promoter, β-actin promoter, human hemoglobin promoter and human muscle creatine promoter) or mammalian virus (example: adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of moloney virus, Epstein barr virus (EBV) and Rous sarcoma virus (RSV)) might be used, and polyadenylated sequence might be commonly used as the transcription termination sequence.

The vector of this invention could be fused with other sequences to purify an antibody expressed from it. For example, a fused sequence includes glutathione-S- transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA) and 6xHis (hexahistidine; Quiagen, USA) and so on. Since the protein expressed in the vector of the present invention is antibody, expressed antibody could be also purified throughout protein A column in an easy manner without additive sequences for purification.

On the other hand, the expression vector of this invention includes an antibiotics-resistance gene known to those ordinarily skilled in the art as a selection marker, for example resistant genes against ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

In the vector expressing the antibody or its part of the present invention, it is possible to utilize one vector system co-expressing the light and heavy chain in single vector or the other vector system expressing each light and heavy chain in independent vector. In latter system, both vectors are introduced into the host cell by co-transformation or targeted transformation. Co-tansformation is a method in which each vector DNA encoding a light and heavy chain gene is simultaneously introduced into the host cells and then the vectors expressing both light and heavy chains are selected. In targeted transformation, cells transformed with a vector containing a light chain (or heavy chain) gene are selected, and the selected cells expressing the light chain (or heavy chain) are again transformed with a vector containing a heavy chain (light chain) gene to finally select cells expressing both light and heavy chains. As described in the examples below, the antibody of this invention was provided by the vector system co-expressing the light and heavy chains in single vector.

According to a preferable embodiment, the nucleic acid molecule encoding the light chain variable region comprises the nucleotide sequence of SEQ ID NO:1, and the nucleic acid molecule encoding the heavy chain variable region comprises the nucleotide sequence of SEQ ID NO:3.

In still another aspect of this invention, there is provided a host cell transformed with the above-described recombinant vector.

The host cells in which the present vector is stably and successively cloned and expressed, also utilize any one known to those skilled in the art, for example prokaryotic host cells including Escherichia coll, Bacillus sp. strains such as Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas {e.g., Pseudomonas put/da, Proteus mirabilis) or Staphylococcus {e.g., Staphylocus carnosus), but not limited to. The host cell is preferably E coli and more preferably E coli ER2738, E coli XL-I Blue, E coli BL21(DE3), E α?//JM109, E coli DH series, E coli TOPlO, E co/7 TGl and £ rø// HBlOl. Most preferably, the host cell is E α>// ER2738 or E coli TGl.

The suitable eukaryotic host cell of the above vector includes fungi {e.g., Aspergillus species), yeasts {e.g., Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces and Neurospora crassa), other lower eukaryotic cells and cell derived from higher eukaryotic cells such as insect cells. In addition, mammalian- derived cells might be used as the host cells. Preferably, the host cells include, but not limited to, COS7 cell (monkey kidney cell), NSO cell, SP2/0, CHO (Chinese hamster ovary) cell, W138, BHK (baby hamster kidney) cell, MDCK, myeloma cell line, HuT 78 cell and 293 cell. More preferably, the host cell is C0S7 cell.

The method using microorganisms such as E α?//has higher productivity than that using animal cell, but it is not suitable to produce an intact Ig antibody due to glycosylation. However, the method could be used in the production of Fab and Fv. In this specification, "transformation" and/or "conversion" introduced into the host cells also includes any one of methods by which the nucleic acid is introduced into organisms, cells, tissues or organs and may be performed by selecting a suitable standard technique according to the host cells, as known to those skilled in the art.

These standard techniques include, but not limited to, electroporation, protoplast fusion, CaPO₄ precipitation, CaCI₂ precipitation, agitation with silicon carbide fiber, Agrobacteira-mediated transformation, and PEG-, dextran sulfate-, lipopectamine- and dry/inhibition-mediated transformation. For example, CaCI₂ precipitation and electroporation is generally used for prokaryotic cells (Sambrook et a/., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Haror Laboratory Press) (1989)). The infection of Agrobacterium tumefaciens is fit for transformation of some plant cells (Shaw et al., Gene, 23:315 (1983), WO 89/05859). Calcium phosphate precipitation is fit for mammalian cells (Graham et al., Virology, 52:456-457(1978)). General method and feature for transforming into mammalian host cells is disclosed in US Pat. No. 4,399,216. Detailed techniques on yeast cell conversion can be seen in Van Solingen et a/., J. Bact, 130:946 (1977) and Hsiao eta/., Proc. Natl. Acad. Sd. USA₁ 76:3829 (1979).

In still further aspect of this invention, there is provided a method for preparing an antibody to a respiratory syncytial virus, comprising the steps of:

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

(b) expressing the antibody to the respiratory syncytial virus in the host cell. The culture of transformed host cells in the antibody preparation may be carried out according to suitable media and culture conditions well-known in the art. The culture process may be feasible manipulated according to selected strains known to those skilled in the art. Various culture processes are disclosed in various references (for example, James M. Lee, Biochemical Engineering, Prentice-Hall International Editions, 138-176). Cell culture is divided into suspension and adhesion culture method according to cell growth pattern and into batch, fermentation and continuous culture according to culture method. The medium used in the culture has to satisfy required conditions of particular strain.

The medium for animal cell culture includes various carbon sources, nitrogen sources and trace elements. The example of carbon sources to be used includes a carbohydrate such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, a lipid such as soybean, sunflower, castor and coconut oil, a fatty acid such as palmitic acid, stearic acid and linoleic acid, an alcohol such as glycerol and ethanol, and an organic acid such as acetate. These carbon sources may be used either alone or in combination with each other. The example of nitrogen sources to be used includes an organic nitrogen source such as peptone, yeast extract, malt extract, corn steep liquid (CSL) and soybean-wheat, and an inorganic nitrogen source such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. These nitrogen sources may be used either alone or in combination with each other. The medium may include not only KH₂PO₄, K₂HPO₄ and sodium-containing salts thereof as a phosphate source but also metal salt such as magnesium sulfate and iron sulfate. In addition, the medium may include amino acids, Vitamins and suitable precursors.

During culture, pH of culture solution may be adjusted by adding chemical compounds such as ammonium hydrate, potassium hydrate, ammonia, phosphate and sulfate in a predetermined manner. Bubble production may be also inhibited using an antifoaming agent such as polyglycol ester during culture. Meanwhile, oxygen or oxygen-containing gas {e.g., air) is introduced into culture to maintain aerobic state of culture. The temperature of culture is maintained at a range of from 20⁰C to 45°C, and preferably from 25°C to 40⁰C.

Antibodies obtained by culturing of transformed host cells may be used in unpurified condition and may be used through purification with high-purity according to further various conventional methods, for example dialysis, salt precipitation and chromatography. Among them, chromatography is used as the most useful method and kinds and orders of column may be selected from ion-exchange chromatography, size-exclusion chromatography and affinity chromatography according to characteristics of antibody, culture methods, and so on.

In another aspect of this invention, there is provided a pharmaceutical composition for preventing or treating a respiratory syncytial virus infection, comprising: (a) a therapeutically effective amount of human antibody to the respiratory syncytial virus; and (b) a pharmaceutically acceptable carrier. Antibody may be used alone or in combination with conventional pharmaceutically acceptable carrier to prevent or treat the RSV infection since human monoclonal antibody prepared as described above has RSV infection- neutralizing ability. In the pharmaceutical compositions of this invention, the pharmaceutically acceptable carrier may be conventional one for formulation, including lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils, but not limited to. The pharmaceutical composition according to the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.

The pharmaceutical composition according to the present invention may be administered via the oral or parenterally. When the pharmaceutical composition of the present invention is administered parenterally, it can be done by intravenous, subcutaneous, intramuscular, intraperitoneal, endothelial, local, spleen, lung or rectal administration. For oral administration, active ingredients of oral compositions can be coated or formulated to be protected from hydrolysis in stomach. In addition, the pharmaceutical compositions can be administrated by random device in which active ingredients are moved into targeted cells.

A suitable dose of the pharmaceutical composition of the present invention may vary depending on pharmaceutical formulation methods, administration methods, the patient's age, body weight, sex, severity of diseases, diet, administration time, administration route, an excretion rate and sensitivity for a used pharmaceutical composition. Preferably, the pharmaceutical composition of the present invention is administered with a daily dose of 0.001-100 mg/kg (body weight). The term "pharmaceutically effective amount" refers to an amount suitable to prevent or treat a RSV infection.

According to the conventional techniques known to those skilled in the art, the pharmaceutical composition may be formulated with pharmaceutically acceptable carrier and/or vehicle as described above, finally providing several forms including a unit dose form and a multi-dose form. Formulation may be oil or aqueous media, resuspension or emulsion, extract, powder, granule, tablet and capsule and further comprise dispersant or stabilizer.

The antibody compositions of this invention may be independently administrated as a therapeutic agent or be sequentially or simultaneously administrated with a conventional therapeutic agent.

Antibody may be utilized in treatment of virus infection by biological administration with a form of antibody-therapeutic agent complex. The therapeutic agent includes chemotherapeutic agent, radionuclide, immunotherapeutic agent, cytokine, chemokine, toxin, biological agent and enzyme inhibitor. For example, the method to combine antibiotics with antibody is disclosed in G. Gregoriadies, ed., Academic Press London, (1979); Arnon eta/., Recent Results in Cancer Res., 75: 236 (1980); and Moolton et al., Immunolog. Res., 62:47(1982).

The suitable medicament to be coupled with the antibody or its part of the present invention includes anti-bacteria, insecticide, antifungal and related drugs, for example sulfonamide, penicillin, cephalosporin, aminoglycoside, tetracycline, chlorampenicol, piperazine, chloroquine, diaminopyridine, metroniazide, isoniazide, rifampin, streptomycin, sulfon, erythromycin, polymixin, nystatin, amphotericin, 5- fluorocytosine, 5-iode-2'-deoxyuridine, 1-adamantamine, adenine arabinoside, ammanitine, ribavarin and azidothimidine (AZT), and preferably ribavarin. Various conditions to target drug by specific targeted are disclosed in Trouet et al., Plenum Press, New York and London, 19-30 (1982). Pathogen is selectively killed by directly targeting antibody with high specificity prepared as an effective therapeutic agent to microorganism antigen to infection lesion, solving various problems generated from treatment of drug-resistance infection. In addition, drugs targeted to infection lesion may enhance efficacies at high concentration in infection lesions.

The immune regulatory agent used for therapeutic agent in antibody- therapeutic agent complex includes limpokine and cytokine, but not limited to.

In another aspect of this invention, there is provided a diagnostic kit for detecting a respiratory syncytial virus, comprising the antibody to the respiratory syncytial virus as described above.

For diagnosing RSV infection, the present antibody to F antigen protein of RSV may is used by applying it to a biological sample.

The term "biological sample" refers to tissue, cell, whole blood, serum, plasma, tissue autopsy sample (brain, skin, lymph node, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic cell and bacteria expression tissue, but not limited to. These biological samples may be incubated with the antibody of the present invention to detect RSV infection.

The formation of the above antibody-therapeutic agent complex may be detected using a colormetric method, an electrochemical method, a fluorimetric method, a luminometry, a particle counting method, a visual assessment or a scintillation counting method. The "detection" of this specification may be carried out using various labels to detect antibody-therapeutic agent complex. The illustrative examples of labels include enzymes, fluorescent substances, ligands, luminescent substances, microparticles or radioactive isotopes.

Examples of enzyme available as a detection label include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase and β-latamase. Examples of fluorescent substance include fluorescin,

Eu³⁺, Eu³⁺ chelator or crγptate. Ligand used as a detecting label includes biotin derivatives and luminescent substances include acridinium esters and isoluminol derivatives. Examples of microparticles include colloidal gold and colored latex, and radioactive isotope includes ⁵⁷Co, ³H, ¹²⁵I and ¹²⁵I-Bonton Hunter drug.

Preferably, antigen-antibody complex may be detected by ELISA. There are various types of ELISA, for example direct ELISA using labeled antibody recognizing antigen to be adhesive on solid support material, indirect ELISA using labeled secondary antibody recognizing capture antibody in complex of antibody which detects antigen to be adhesive on solid support material, direct sandwich ELISA using labeled or other antibody recognizing antibody in antigen-antibody complex to be adhesive on solid support material, and indirect sandwich ELISA detecting labeled secondary antibody which recognizes other antibody detecting antigen in antigen- antibody complex to be adhesive on solid support material. The antibody of this invention may include a detection marker, and otherwise the present antibody may be captured and then be detected using other antibody with detection marker.

The features and advantages of this invention are summarized as follows:

(a) The antibody of this invention is bound to a surface antigen of RSV, F protein with high specificity. Interestingly, the antibody of this invention has more excellent binding ability to RSV than Synagis as a commercial antibody-therapeutic agent. (b) The present antibody has more remarkable infection-neutralizing ability than Synagis.

(c) The present antibody is used to prevent or treat a RSV infection. As described as the examples below, the antibody of this invention has more notable in vivo efficacy than Synagis to prevent RSV infection.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 represents colony lift analysis to select Fab variant clonies with high affinity to antigens performed in the example of this invention. Fig. 2 schematically represents the procedure for preparing pdCMV-dhfrC- RSV13-9K.

Fig. 3 schematically represents the procedure for preparing pdCMV-dhfrC- RSV13-9 expressing an entire antibody. Fig. 4 is a graph to represent binding ability to RSV A and B subgroup of each

RSV13-9 antibody with intact IgG and synagis as a control.

Figs. 5-6 are graphs to represent extracellular activity to RSV A and B subgroup of each RSV13-9 antibody with intact IgG and synagis as a control.

Fig. 7 is a graph to represent intracellular activity to RSV of each RSV13-9 antibody and synagis as a control.

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

EXAMPLES EXAMPLE I: Construction of Human Antibody Library

The present inventors constructed human antibody library from PBL (peripheral blood lymphocytes) of infants restored after RSV infection to isolate a human antibody enabling to neutralize RSV infection.

1-1: Preparation of Human Fab cDNA Library

The cDNA library to Fd region of human light and heavy chains was prepared to construct human Fab cDNA library.

In detail, blood was taken form 5 patients and medical team and then lymphocytes were separated using Ficoll gradient. Total RNA was extracted from the above PBL and used as a template. Each cDNA corresponding to Fd region of human light and heavy chains was selectively synthesized using reverse transcriptase (Superscript II, Gibco BRL) and primer sets (CKId primer of SEQ ID NO: 5 and CGId primer of SEQ ID NO:6). The human light chain cDNA was used as a template and human light chain cDNA library was selectively synthesized by PCR in which each pair of 5'-specific primers (VKl of SEQ ID NO:7, VK2/4 of SEQ ID NO:8, VK3 of SEQ ID I\IO:9 and VK5 of SEQ ID NO: 10) of kappa variable region and CKId primer of SEQ ID NO:5 were used.

In addition, cDNA of human heavy chain Fd region was used as a template and human heavy chain Fd region cDNA library was selectively synthesized by PCR in which each pair of 5'-specific primers (VH135 of SEQ ID NO: 11, VH2 of SEQ ID NO: 12, VH4 of SEQ ID NO: 13, VH4b of SEQ ID NO: 14, VH6b of SEQ ID NO: 15, VH4gs of SEQ ID NO: 16) of human heavy chain Fd region and CGId primer of SEQ ID NO: 6 were used.

To enhance cloning efficiency of the above cDNA library, PCR was carried out using each primer sets (VKext of SEQ ID NO:17 and CKext of SEQ ID NO:18; VHext of SEQ ID NO: 19 and VH2ext of SEQ ID NO:20; VH4bext of SEQ ID NO:21 and CGldext of SEQ ID NO:22). PCR reaction was carried out as follows: predenaturing at 95°C for 5 min followed by 30 cycles of denaturing at 95°C for 50 sec, annealing at 55°C for 50 sec and elongating at 72°C for 1 min using Taq DNA polymerase.

1-2: Cloning and Transformation

Human light chain cDNA library obtained from the above example 1-1 was subcloned into pC3-Q-AKA/HzK (KR Pat No. 0318761) and transformed into E. coli.

In detail, each human light chain cDNA fragments amplified by PCR in the above example 1-1 and pComb3HSS vector (Scripps institute, USA) was purified after restriction with Sac I and Xba I. Restricted DNA fragments were incubated at 16°C overnight for ligation and ligases were inactivated by heating at 70°C for 10 min.

The glycogen and 3 M sodium acetate were added to the resulting products and then DNA was precipitated overnight by adding ethanol at 20⁰C. After washing with 70% ethanol, precipitated DNA was dried and resuspended with 20 μl distilled water. The above library DNA was transformated into E coli, ER2738 (Stratagene) using electroporation. Competent cells were prepared for transformation. ER2738 cells were grown in 500 ml 2xYT at 37°C with shaking until 0.5-0.7 OD (optical density) and then incubated on ice for 30 min. The supernatant was removed by centrifuging at 5000 rpm at 4⁰C for 15 min and the precipitated cells were resuspended at 500 ml of 10% glycerol. After removing the supernatant again by centrifugation at 5000 rpm at 4°C for 15 min, the precipitated cells were resuspended at 20 ml of 10% glycerol. The supernatant was removed by centrifuging at 4000 rpm at 4°C for 15 min and the cells were resuspended at 1-2 ml of 10% glycerol. The competent cells were divided into aliquote of 300 μl at 1.5 ml tube and stored at -70⁰C. 300 μl of ER2738 was well mixed with the library DNA and incubated on ice for 1 min. And then, the mixture was transferred to Gene Pulser cuvette (Biorad) and transformed with 2.5 kV at 200 Ω using an electroporator (Biorad), preparing transformed cells.

1-3: Binding of Light Chain Gene and Heavy Chain Fd Gene

Cells transformed in the above example 1-2 were incubated in 2xYT with shaking overnight and then plasmid DNA was isolated. Each plasmid DNA from human light chain library and human heavy chain gene fragment amplified by PCR were purified by restriction with Xho I and Spe I and ligated by the same method of the above example 1-2. Finally, the ligation products were transformed into ER2738 cells.

EXAMPLE II: Selection of Specific Human Antibody to RSV The present inventors carried out a colony lift assay in human antibody Fab library cells prepared in the above example I to isolate a Fab antibody bound to RSV (Radosevic, K eta/., J. Immunol. Methods, 272:219-233 (2003)).

The cells of about 10⁶ were smeared on nitrocellulose membrane directly placed on 2^χ YTA plate and then incubated overnight. The membrane was called as a master membrane. Meanwhile, the capture membrane to find the cells with strong antigen binding affinity was coated with purified RSV F protein (10 μg/ml in PBS) by incubating it at 37°C for 6 hrs. F protein was purified by affinity chromatography using mice monoclonal antibody (Biodesign, C65064M) to F protein from lysate of HEp-2 cell (ATCC) according to the method of Walsh et a/., J. Gen. Virol., 66:409- 415 (1985).

The membrane coated with F protein was washed 2 times with PBS and incubated at 37°C for 2 hrs after addition of 5% skim milk. After removing skim milk, the membrane was immersed in 2x YT media containing 100 μg/ml ampicillin and 1 ITiM IPTG (isopropyl-β-D-thiogalactopyranoside). This capture membrane was placed on 2x YT media containing 100 μg/ml ampicillin and 1 mM IPTG (isopropyl-β-D- thiogalactopyranoside) and then the master membrane on which the library cells were smeared, was stacked on the capture membrane, incubating at room temperature for 16-24 hrs. The above capture membrane was washed 5 times with PBS adding 0.05%

Tween 20 (PBST) and then kept to stand in PBS containing skim milk at 37°C for 6 hrs. To isolate clones bound to F protein (antigen), anti-human F(ab')₂ antibody conjugated with horseradish peroxidase was added to the capture membrane and incubated at 37⁰C for 1 hr. To exclude unbound antibodies, the membrane was washed 5 times with PBS and antigen binding ability was detected by ECL (enhanced chemiluminescence, Intron).

The cells exhibiting antigen binding ability was selected from the same position of master membrane and incubated in 2^χ YT media at 37°C until OD of 0.7. Finally, colony lift assay was carried out using the same procedure described above. The antigen binding ability to F protein of Fab clones obtained throughout 1^st, 2^nd, 3^rd and 4^th screening was analyzed by ELISA and clone with excellent binding affinity was selected, designated as pC3-13-9. This clone was further analyzed about nucleic acid sequence and binding ability to RSV.

EXAMPLE III: Nucleic Acid Sequence of Selected Fab Clones

The nucleic acid sequences of variable region of light and heavy chains in the above clones were sequenced using a T7 sequenase V2.0 DNA sequencing kit. The variable region of each light and heavy chain present in pC3-13-9 vector among cloning vectors prepared in the above example 1-2 is described in SEQ ID NO:1 and NO:3. In the light chain, the selected clone had high homology with the amino acid sequence of DPK9/O12 of VKl family, demonstrating that it was derived from DPK9/O12. However, the selected clone in the heavy chain was derived from human heavy chain germ cell line gene of DP28.

EXAMPLE IV: Analysis of Antiαen Binding Ability of Selected Fab Clones

To analyze the antigen binding ability to RSV of each Fab clone, £ coli ER2738 transformed with pC3-13-9 vector was cultured at 37°C with shaking until ODgoo_nm between 0.5 and 1.0 and then grown at 30⁰C overnight to add 1 mM IPTG for inducing Fab expression. Cells were harvested and soluble Fab antibody was obtained from periplasmic extract prepared by osmotic shock using TES buffer (0.2 M Tris-HCI, pH 8.0, 0.5 mM EDTA and 0.5 M sucrose).

Indirect ELISA was carried out to determine antigen binding ability of each Fab clones. Each well of ELISA plate was coated with 0.2 μg of purified RSV particle overnight and blocked with 2% BSA, followed by washing 4 times with TBS-T. Fab antibody present in periplasmic extract was added and incubated at 37°C for 1 hr. The antibodies unbound to antigen were removed using TBS-T. After dilution with TBS-T, anti-human F(ab')₂ IgG-HRP as a secondary antibody was used and the absorbance was measured at 492 nm by colometric reaction using OPD (o- phenylenediamine, Sigma) and H₂O₂. Mice monoclonal antibody (C65064M, Biodesign) to F protein and periplasmic extract unexpressing Fab protein was served as a positive control and a negative control, respectively.

As a result, the absorbance of Fabl3-9 was 0.82 and that of each entire IgG antibody to F protein (positive control) and E coli periplasmic extract unexpressing Fab protein was 0.90 and 0.05, suggesting that the Fab antibody of the present invention has the binding ability to purified RSV particle.

EXAMPLE V: Expression Plasmid Construction for Expressing Entire IqG

To prepare entire IgG antibody with binding ability to RSV, the signal sequence of antibody gene and the variable region of Fab heavy and light chain of C3-13-9 clones were synthesized using a recombinant PCR method.

V-I: Preparation of Expression Vector Containing the Light Chain

To prepare entire IgG antibody of anti-RSV Fab, the signal sequence of light chain gene and the sequence corresponding to light chain variable region of anti- RSV Fab were synthesized by the recombinant PCR method and subcloned into a Hindlll-BsiWl site of pdCMV-dhfrC-AKA/HzK (KR Pat. No. 318761).

To synthesize the signal sequence of light chain gene, PCR was performed using pdCMV-dhfr-AKA/HzK as a template and primer pairs (Ryu86 primer of SEQ ID NO:23 and KR-I (leader) primer of SEQ ID NO:24). In addition, PCR was performed using each primer (KF primer of SEQ ID NO:25 and KR primer of SEQ ID NO:26) to synthesize human light chain variable region gene from the pC3-13-9 clone. To link the signal sequence to light chain variable region, recombinant PCR was carried out using primer pairs (Ryu86 primer of SEQ ID NO:23 and KR primer of SEQ ID NO:26). PCR reaction was carried out as follows: predenaturing at 95°C for 5 min, 30 cycles (denaturing at 95°C for 1 min; annealing at 55⁰C for 30 sec; elongating at 72°C for 30 sec) using Taq DNA polymerase. Both end of DNA fragments was cut with Hind lll-BsiWl and then inserted into the Hind lll-BsiWl site of pdCMV-dhfrC-AKA/HzK, preparing pdCMV-dhfrC-RSV13-9K (Fig. 2).

V-2: Preparation of Expression Vector Containing the Light and Heavy Chain

The signal sequence of heavy chain gene and the sequence corresponding to heavy chain variable region of anti-RSV Fab were synthesized by the recombinant PCR method.

To synthesize the signal sequence of heavy chain gene, PCR was performed using pdCMV-dhfr-AKA/HzK as a template and primer pairs (Ryu94 primer of SEQ ID NO:27 and HRl (leader) primer of SEQ ID NO:28). In addition, PCR was performed using each primer (HF2 primer of SEQ ID NO:29 and HR2 primer of SEQ ID NO:30) to synthesize human heavy chain variable region gene of Fab described above. To link the signal sequence to light chain variable region, recombinant PCR was carried out using primer pairs (Ryu94 primer of SEQ ID NO:27 and HR2 primer of SEQ ID NO:30). PCR reaction was carried out as follows: predenaturing at 95°C for 5 min followed by 30 cycles of denaturing at 95°C for 1 min, annealing at 55°C for 30 sec and elongating at 72⁰C for 30 sec using Taq DNA polymerase.

Both ends of DNA fragments were cut with restriction enzyme EccR l-Apa I and then inserted into the EccR l-Apa I site of pdCMV-dhfrC-RSV13-9K in which anti RSV light chain was cloned, preparing the expression plasmid of this invention designated as pdCMV-dhfrC-RSV13-9 (Fig. 3). The pdCMV-dhfrC-RSV13-9 vector described above was deposited at KRIBB (Korea Research Institute Bioscience & Biotechnology) Genebank on 27 July 2007 (accession number: KCTC 11161BP).

EXAMPLE VI: Expression and Purification of Anti-RSV Antibody in Animal Cells

VI-I: Expression of Anti-RSV Antibody in Animal Cells

293E cells (Invitrogen) were inoculated on DMEM media (GIBCO) supplemented with 10% FBS and subcultured at 37°C in 5% CO₂ incubator. The cells were inoculated on 100 mm culture dish at a concentration of 4xlO⁵ cells/ml. After culturing at 37°C overnight, the cells were washed 3 times with OPTI-MEM I (GIBCO) solution. Meanwhile, 10 μg of antibody expression vector (pdCMV-dhfrC- RSV13-9) prepared above and 25 μl of Lipofectamine (GIBCO) was independently diluted with 500 μl of OPTI-MEM I. DNA-Lipofectamine mixture was prepared by mixing the expression vector and Lipofectamine-diluted solution in 15 ml tube and kept to stand at room temperature for above 15 min. 5 ml of OPTI-MEM I was added to each DNA-Lipofectamine mixture and mixed with washed 293E cells. And then, the cells were incubated at 37°C in 5% CO₂ incubator for 48 hrs, expressing anti-RSV antibody of this invention.

VI-2: Purification of Anti-RSV Antibody

The supernatant of the 293E cell culture was harvested by filtration with a set of filters to exclude particular substances and finally passed through a 0.2 mm filter. Based on the amount of total antibodies, the supernatant was subjected to a predetermined protein A column. After washing, the antibody was eluted with neutralization buffer (1 M Tris/HCI, pH 7.4) from the column (0.1 M glycine/HCI, pH 2.8). Buffers were exchanged by ultrafiltration using 2 L of sterile PBS and sterilized by filtration through a 0.2 nm filter. After purification, the antibody was stored in a refrigerator ice box until ready to use.

EXAMPLE VII: RSV Binding Ability of Anti-RSV Antibody

To evaluate efficiency on RSV binding ability of purified antibody, RSV binding ELISA using Synagis as a control together was performed. HEp-2 cells (ATCC) infected with RSV were cultured for 3 days and the culture solution was removed. The cells were fixed with 3.7% formaldehyde at 4⁰C for 30 min and incubated with 2% BSA for 2 hrs. After blocking, RSV13-9 and Synagis sequentially diluted every two-fold was added to plate and incubated for 1 hr. And then, secondary antibody conjugated with HRP bound to anti-human IgG-Fc was added and further incubated for 1 hr. Colometric reaction was induced by OPD and OD value was measured using ELISA reader to evaluate the binding ability to RSV. As shown in Rg. 4, concentration increase of RSV13-9 antibody was in accordance with final signal increase of ELISA. It could be appreciated that RSV13-9 antibody of the present invention specifically combines with RSV. It was also evident that RSV13-9 antibody of the present invention has specific binding ability to RSV A and B subgroup. Surprisingly, the antibody of the present invention has more excellent binding ability to RSV than Synagis as a commercial antibody therapeutic agent (Fig. 4).

EXAMPLE VIII: Extracellular Functional Activity of Anti-RSV Antibody

Infection neutralization assay was carried out to determine whether the antibody of this invention has a virus-neutralizing extracellular capacity. Neutralization assay was performed by preincubating purified monoclonal antibody with virus before virus was applied to cells, whereby it is possible to examine the ability of monoclonal antibody preventing virus infectivity.

After HEp-2 cells were inoculated on 24-well plate at 10⁴ of cell density 1 day before experiment, the same amount of RSV was added to each 1.5 ml tube in which the antibodies to RSV13-9 and Synagis were sequentially diluted at a concentration-dependent manner, and then incubated at 37°C for 30 min in CO₂ incubator. The media inoculated on 24-well plate the other day were removed. The plate was washed one times with PBS and 200 μl of each antibody and RSV per well was added three times, incubating at 37°C for 1 hr in CO₂ incubator. After 1 hr incubation, all media were eliminated in the well and 1 ml of media containing 2% FBS and 5% methyl cellulose was added to each well. The cells were grown at 37°C for 6 days in CO₂ incubator and the plaque grown on each well of plate were colored. The colorimetric method is as follows: The methyl cellulose media of each well were removed. 3.7% formaldehyde solution diluted with PBS was added to the well and kept to stand at 4⁰C for 30 min. The solution of each well were removed and 5% skim milk solution was added to the well. After incubating with shaker at room temperature for 2 hrs, mouse anti-RSV F antibody diluted was added to each well and then further reacted in a shaker for 1 hr at room temperature. Each well was washed three times with PBS. Goat anti-mouse IgG Fc-HRP (Pierce) diluted was added to each well and then further reacted in a shaker for 1 hr at room temperature. After washing three times with PBS, each well was colored using DAB (diaminobenzidine, Sigma) solution and the plaque number was counted to measure neutralization capacity (Figs. 5-6).

As shown in Figs. 5-6, concentration increase of RSV13-9 antibody of this invention was in accordance with decrease in the number of formed plaque. In addition, the RSV13-9 antibody of the present invention enables to reduce the number of formed plaque of both RSV A and B subgroup. Consequently, it could be appreciated that the RSV13-9 antibody of the present invention has significant neutralization capacity to neutralize RSV infection. The antibody of the present invention also has more remarkable neutralization capacity to RSV than Synagis as a commercial antibody therapeutic agent.

EXAMPLE IX: In Vivo Functional Activity of Anti-RSV Antibody IX-I: Establishment of Animal Test Model of Anti-RSV Antibody Each group administering Synagis, RSV13-9 and no treatment to 3 Balb/c mice (Orient) was used to confirm in vivo neutralization capacity. To evaluate the effect by observing RSV infection and proliferation in lung, preliminary study was performed to determine the amount of suitable antibody and virus. The administration amount of antibody was used in a range of 0.15 or 3 mg/kg and that of virus was used in a range of 5 x 10⁵, 5 x 10⁶ or 5 x 10⁷ plaque forming unit/individual. In vivo experiment was carried out as follows: 100 μl of antibody was intramuscularly injected 1 day before RSV infection and mice were anesthetized with evertin. Various concentrations of RSV were parenterally administered. After 4 days RSV infection, lung was extracted and its weight was measured. Lung was smashed using a crusher containing suitable HBSS. The crushed lung was centrifugated at 4,000 rpm for 30 min at 4°C and RSV amounts of its supernatant were titered. RSV titration was carried out using a plaque formation assay. The plaque formation assay was performed according to the following method. After HEp-2 cells were inoculated on 24-well plate with 1 x 10⁵ cells per well 1 day before experiment, the supernatant prepared by extracting lung was sequentially diluted. The media inoculated on 24-well plate the other day was removed. The wells were washed one times with PBS and each 200 μl per well was added to the well three times, incubating at 37°C for 1 hr in CO₂ incubator. After 1 hr incubation, all media were eliminated in the well and 1 ml of media containing 2% FBS and 5% methyl cellulose was added to each well. The cells were grown at 37°C for 6 days in CO₂ incubator and the plaque grown on each well of plate were colored. The colorimetric method is as follows: The methyl cellulose media of each well were removed. 3.7% formaldehyde solution diluted with PBS was added to the well and kept to stand at 4°C for 30 min. The solution of each well was removed and 5% skim milk solution was added to the well. After incubating with shaker at room temperature for 2 hrs, mouse anti-F antibody diluted was added to each well and then further reacted in a shaker for 1 hr at room temperature. Each well was washed three times with PBS. Goat anti-mouse IgG Fc-HRP (Pierce) diluted was added to each well and then further reacted in a shaker for 1 hr at room temperature. After washing three times with PBS, each well was colored using DAB solution and the plaque number was counted to determine the plaque titration. As a result, the animal test model was obtained from evaluating effectiveness of each test factors.

IX-2: Evaluation of In Vivo Function of Anti-RSV Antibody The efficacy for preventing in vivo RSV infection was determined using the above animal model. The experiments were carried out using each group administering RSV13-9 antibody, Synagis and injection solution to 3 Balb/c mice (Orient), and no treatment group. Based on the predetermined amount described above, the administration amount of antibody was used at 0.15 or 3 mg/kg and that of virus was used in the amount of 5 x 10⁶ plaque forming unit. 100 μl of antibody was intramuscularly injected 1 day before RSV infection and mice were anesthetized with evertin. RSV with concentration of 5 x 10⁵ plaque forming unit were infected by administration through the nasal cavity. After 4 days RSV infection, lung was extracted and its weight was measured. Lung was smashed using a crusher containing HBSS. The crushed lung was centrifugated at 4,000 rpm for 30 min at 4°C and RSV amounts of its supernatant were titered. RSV titration was carried out using a plaque formation assay. The following method was performed according to the same method as described above.

As shown in Fig. 7, the concentration increase of RSV13-9 antibody of this invention was in accordance with decrease in RSV titration of mouse lung. Therefore, it could be appreciated that the RSV13-9 antibody of the present invention has remarkable in vivo efficacy to prevent RSV infection. Interestingly, the antibody of the present invention also has more excellent in vivo capacity to prevent RSV infection than Synagis as a commercial antibody therapeutic agent.

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

BUDAl¹EST TREATi' OK THE INTERN¹ATfOKAL RECOGKlTtON OF THE DEPOSIT Or MICROORGANISM^ FOK THE PUNI¹OSE OF [¹ATENT

INTERNATIONAL FOEM

RECEIPT IN TIIE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1

IO : KIM, Jae-Seob

E- 18, Korea Advanced Institute of Science and Technology 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-703 Republic of Korea

1 . E⁾ENTIFICATION OF THE MICROORGANISM

Identification reference given by the Accession number given by the DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:

Escherichia coli »H5@/pdCMV-dhf rC-RS V 13-9 KCTC 11161BP

II . SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I above was accompanied by:

[ X ] a scientific description

I ] a proposed laxonomic designation

(Mark with a cross where applicable⁾

DI. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the miαrarganism identified under I above, which was received by it on July 27, 2007.

IV₁ RECEIPT OF REQUEST FOR CONVERSION

The microorganism identified under I above was received by this International Depositary Authority on and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on

Y. INTERNATIONAL DEPOSITARY AUTHORITY

Name: Korean Collection for Type Cultures Signature(s) of person(s) having the power to represent the International Depositary Authority of authorized offiάal(s):

Address: Korea Research Institute of Bioscience and Biotechnology (KRIBB)

#52, Oun-dong, Yusαng-ku, Taejon 305-333, OH, Hee-Mock, Director Republic of Korea Date: August 1, 2007

Form BP-Q CKCTC Ftim 17⁾ s^lc* paut;

Claims

What is claimed is:

1. An antibody or its binding fragment against a respiratory syncytial virus, comprising a heavy chain variable region having the following heavy chain complementarity determining region (CDR) amino acid sequences: CDRm comprising the amino acid sequence of SEQ ID NO:31, CDR_H2 comprising the amino acid sequence of SEQ ID NO:32, and CDR_H3 comprising the amino acid sequence of SEQ ID NO:33.

2. The antibody or its binding fragment according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:4.

3. The antibody or its binding fragment according to claim 1, wherein the antibody against the respiratory syncytial virus further comprises a light chain variable region having the following light chain CDR amino acid sequences: CDR_Li comprising the amino acid sequence of SEQ ID NO:34, CDR_L2 comprising the amino acid sequence of SEQ ID NO:35, and CDR_L3 comprising the amino acid sequence of SEQ ID NO:36.

4. The antibody or its binding fragment according to claim 3, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:2.

5. The antibody or its binding fragment according to claim 1, wherein the antibody is a human antibody.

6. A nucleic acid molecule encoding a heavy chain variable region of an antibody against the respiratory syncytial virus comprising the amino acid sequence of SEQ ID NO:4.

7. The nucleic acid molecule according to claim 6, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:3.

8. A nucleic acid molecule encoding a light chain variable region of an antibody against the respiratory syncytial virus comprising the amino acid sequence of SEQ ID NO:2.

9. The nucleic acid molecule according to claim 8, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:1.

10. A recombinant vector, comprising a nucleic acid molecule encoding a light chain variable region comprising the amino acid sequence of SEQ ID NO:2; and a nucleic acid molecule encoding a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4.

11. The recombinant vector according to claim 10, wherein the nucleic acid molecule encoding the light chain variable region comprises the nucleotide sequence of SEQ ID NO:1, and the nucleic acid molecule encoding the heavy chain variable region comprises the nucleotide sequence of SEQ ID NO:3.

12. A host cell transformed with the recombinant vector of claim 10 or 11.

13. A method for preparing an antibody against a respiratory syncytial virus, comprising the steps of:

(a) culturing the host cell of claim 12; and (b) expressing the antibody against the respiratory syncytial virus in the host cell.

14. A pharmaceutical composition for preventing or treating a respiratory syncytial virus infection, comprising: (a) a therapeutically effective amount of the antibody against the respiratory syncytial virus according to any one of claims 1-5; and (b) a pharmaceutically acceptable carrier.

15. A diagnostic kit for detecting a respiratory syncytial virus, comprising the antibody against the respiratory syncytial virus according to any one of claims 1-5.