JP2011050269A - Human antibody against rabies virus and pharmaceutical composition thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody having new epitope-recognizing specificity for attaining the diagnoses, prevention and/or treatments of symptoms caused by Rabies-related viruses. <P>SOLUTION: There is provided a rabies virus-resistant monoclonal antibody or its antigen-binding fragment which can specifically bind to Rabies virus (RV) to neutralize its biological activity and binds to an epitope containing the specific amino acid residue of G protein. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a glycoprotein of RV (hereinafter referred to as G protein) used for diagnosis, prevention and / or treatment of symptoms caused by rabies-related viruses such as Rabies virus (hereinafter referred to as RV). .) Relates to human monoclonal antibodies that specifically bind to the novel epitope present above, as well as antigen-binding fragments thereof.

  The rabies virus is a zoonotic virus infection that can infect all warm-blooded animals such as dogs, bats, cats, and raccoons. Generally, when bitten by an infected animal, the virus is transmitted and infected with saliva. It is a fatal viral infection that will die almost 100% when it occurs, and it is estimated that over 50,000 deaths from rabies worldwide occur worldwide. Over 90% of cases are concentrated in Asia and Africa, especially India, Bangladesh, Southeast Asia and so on.

Rabies virus is classified into the genus Lyssavirus of the Rhabdoviridae family, and the virus particles have a characteristic bullet-like morphology of 70-80 x 180 nm. The viral genome is a single-stranded negative-strand RNA of about 12,000 bases. Nucleoprotein (N protein) that forms capsid, Phosphoprotein (P protein) that is a phosphorylated protein, Matrix protein (M protein) that is involved in virus budding ), Glycoprotein (G protein), the glycoprotein that forms the envelope, and Large protein (L protein) that acts as an RNA-dependent RNA polymerase.
The Lissavirus genus is classified into the following seven genotypes from the analysis of genotype and serotype (Non-patent Document 1).
1) genotype 1: Ravies virus
2) genotype 2: Lagos bat virus
3) genotype 3: Mokola virus
4) genotype 4: Duvenhage virus
5) genotype 5: EBL1 (European bat lyssavirus type 1)
6) genotype 6: EBL2 (European bat lyssavirus type 2)
7) genotype 7: ABL (Australian bat lyssavirus)
Of these, Lissaviruses other than genotype 2 Lagosbad virus are known to cause rabies-like encephalitis in humans. In Australia, where no rabies has been reported so far, people bitten by bats affected by Lissavirus have recently died of the same symptoms as rabies. Moreover, it is thought that these can be protected by the rabies vaccine by crossing serologically with rabies virus (RV) except a mocola virus (nonpatent literature 1). Therefore, it is considered that the monoclonal antibody (MAb) against the G protein of rabies virus may bind to these Lissaviruses and neutralize the activity.
Therefore, in this specification, not only the rabies virus but also the Lissavirus that causes these rabies-like infections will be referred to as “rabies-related viruses”.

  The occurrence of rabies in humans is mainly due to being bitten by an infected dog, but there is a high risk if the infected dog licks the mucous membrane such as a wound, eyes, and lips. RV invading from a bite or the like is transported from the peripheral nerve to the center retrogradely from the peripheral nerve via acetylcholine receptor (Nicotinic Acetylcholine Receptor: nAChR) and the like. The incubation period varies depending on the bitten site, but the period until the virus reaches the cranial nerve tissue and develops is longer when the foot is bitten than when the face is bitten. It is said that it is about two weeks when it is close to the brain and several months when it is far away. The mortality rate after the onset is almost 100%, and treatment has not been established yet.

As a preventive method, if it is before infection (before exposure), it can be prevented by vaccination, and vaccination before traveling to a country where rabies occurs is effective. Active immunization with vaccines is the same for dogs. In Japan, the dogs are obligated to vaccinate once a year according to the Rabies Prevention Law.
As preventive measures after exposure, most of them can be killed by first washing the wounds with soap, etc., and disinfecting them with disinfectant or ethanol for disinfection. And it is desirable to start vaccination promptly.
WHO divides the severity of exposure into three levels when exposed to rabies animals. Of these, after severe RV exposure belonging to Category III, administration of anti-rabies immunoglobulin preparation (RIG) around wounds in addition to vaccination is recommended. The number of people who are subject to post-exposure prophylaxis (hereinafter referred to as PEP) after exposure to these vaccines and RIG is approximately 1,000 times the number of rabies deaths, reaching 50 million people worldwide every year. It is predicted.
Currently, equine serum (ERIG) and human immunoglobulin (HRIG) can be used as RIG. However, ERIG has many problems, such as the possibility of the appearance of antibodies against horses that are heterogeneous and the possibility of causing anaphylaxis. In addition, HRIG also has various problems such as extremely high single dose, difficult and expensive supply, and possible contamination with unknown pathogens derived from humans. It is done.

In recent years, neutralizing antibodies against the G protein of RV have been considered as an option for preventing rabies virus infection and the onset of rabies. However, RNA viruses such as rabies virus are prone to gene mutation, and not only various wild strains are observed, but also the formation of mutant strains is likely to occur during the incubation period after infecting a living body. Accordingly, there is a demand for the development of a monoclonal antibody against an epitope that hardly causes mutation (hereinafter referred to as MAb) or a monoclonal antibody against an epitope whose pathogenicity is remarkably reduced when mutated.
In addition, a report by Hanlon et al. (Non-Patent Document 2) suggests that human monoclonal antibodies against RV will replace HRIG in the future in the prevention of onset of post-exposure (PEP), as well as conventional MAbs. Have been reported to fail to prevent the growth of resistant virus mutants.

Five types of epitopes on the G protein of RV have been reported so far (see Table 1).

  On the other hand, in a recent study by Takayama-Ito et al. (Non-patent Document 6), the 242nd (Ala: alanine), 255th (Asp: aspartic acid), and 268th (Ile: isoleucine) sites on the G protein It has been reported that RV, which is involved in the pathogenicity of rabies virus, is mutated at one or more of its amino acid sites, and the pathogenicity is greatly reduced.

Source Nobuyuki, Rabies and Lissa (rabies related) virus. 2004; 54: p213-222 Hanlon, C.A., et al., Experimental utility of rabies virus-neutralizing human monoclonal antibodies in post-exposure prophylaxis., Vaccine. 2001; 19: p3834-3842 Marissen WE et al., Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: fine mapping and escape mutant analysis.J Virol. 2005; 79: p4672-4678. Identification and characterization of a human monoclonal antibody that potently neutralizes a broad panel of rabies virus isolates.Vaccine. 2007 Apr 12; 25 (15): 2800-10. Epub 2006 Dec 29. Prehaud C et al., Antigenic site II of the rabies virus glycoprotein: structure and role in viral virulence.J Virol. 1988; 62: p1-7. Takayama-Ito M et al., Multiple amino acids in the glycoprotein of rabies virus are responsible for pathogenicity in adult mice.Virus Res. 2006; 115: p169-175

  Under these circumstances, in order to make the diagnosis, prevention and / or treatment of symptoms caused by rabies-related viruses realistic, epitopes that are unlikely to be mutated or have reduced pathogenicity when mutated The discovery of antibodies with recognition specificity is highly desired.

  A human antibody or antigen-binding fragment that has a different epitope recognition specificity, binds specifically to the G protein of RV, and sufficiently inhibits its biological activity is a diagnostic of symptoms caused by a wide range of rabies-related viruses, In addition to being useful for prevention and / or treatment, it is considered particularly useful in the therapeutic strategy of post-exposure prophylaxis (PEP) of rabies virus.

  Currently, in addition to vaccination, anti-rabies immunoglobulin preparation (RIG) is recommended around wounds for prevention of post-exposure onset of rabies (PEP). Currently, equine serum (ERIG) and human immunoglobulin (HRIG) are used as RIG, but it is possible that antibodies against heterologous horses may appear and that anaphylaxis may occur. There are many problems. In addition, HRIG has a big problem because it is expensive and there is a risk of contamination with unknown pathogens derived from humans. Under such circumstances, a human-derived monoclonal antibody has been tried to be used in a cocktail of MAbs having different epitopes. Further proposals of useful human-derived monoclonal antibodies are desired.

  As a result of diligent efforts to obtain the antibody as described above, the present inventors succeeded in obtaining a human monoclonal antibody having a high neutralizing ability against RV, and the antibody is a glycoprotein of RV. (Hereinafter referred to as G protein) Confirm that the antibody specifically binds to an epitope (stereostructured epitope) containing the 242nd amino acid (alanine) involved in pathogenicity. The present invention has been completed.

  An escape virus from an antibody that recognizes the 242nd amino acid on the G protein as an epitope is predicted to be greatly reduced in pathogenicity. From this, human MAb that recognizes this novel epitope or its antigen binding Fragments are expected to be useful for the diagnosis, prevention and / or treatment of symptoms caused by rabies-related viruses. In particular, it is considered to be one of the promising candidates as a human antibody used for prevention of onset after exposure to rabies or as a MAbs cocktail preparation.

  The present invention is effective for a wide range of rabies-related viruses and neutralization-resistant mutants thereof, and is a human monoclonal antibody that specifically recognizes a novel epitope at a site involved in virulence on the G protein or a novel epitope thereof. A pharmaceutical composition that provides an antigen-binding fragment and is used for diagnosis, prevention, and / or treatment of symptoms caused by rabies-related viruses, particularly for preventing onset after exposure to rabies virus. It is to provide.

  That is, the present invention includes a human anti-RV monoclonal antibody that recognizes a novel epitope of rabies virus described below, or a binding fragment thereof, a DNA (polynucleotide) encoding the antibody or binding fragment, and a DNA containing the DNA And a host cell containing the vector.

(1) A monoclonal antibody or antigen-binding fragment thereof that specifically binds to rabies virus (RV) and can neutralize its biological activity, and binds to an epitope containing the 242nd amino acid (alanine) of G protein. Anti-rabies virus antibody or antigen-binding fragment thereof.
(2) (a) From the amino acid sequences of SEQ ID NOs: 7, 8 and 9, and amino acid sequences having deletion, substitution, insertion and / or addition mutations of one to several amino acid residues in those amino acid sequences The amino acid sequence of any one or more of light chain CDRs 1, 2 and 3 selected from the group consisting of:
(b) from the group consisting of the amino acid sequences of SEQ ID NOs: 10, 11 and 12, and amino acid sequences having deletion, substitution, insertion and / or addition mutations of one to several amino acid residues in those amino acid sequences The antibody or antigen-binding fragment thereof according to (1), comprising the amino acid sequence of any one or more of the selected heavy chain CDRs 1, 2 and 3.
(3) (a) From the amino acid sequences of SEQ ID NOs: 7, 8 and 9, and amino acid sequences having deletion, substitution, insertion and / or addition mutations of one to several amino acid residues in those amino acid sequences The amino acid sequences of light chain CDRs 1, 2 and 3 selected from the group consisting of:
(b) from the group consisting of the amino acid sequences of SEQ ID NOs: 10, 11 and 12, and amino acid sequences having deletion, substitution, insertion and / or addition mutations of one to several amino acid residues in those amino acid sequences The antibody or antigen-binding fragment thereof according to (1) above, comprising the amino acid sequences of selected heavy chain CDRs 1, 2 and 3.
(4) (a) the amino acid sequences of SEQ ID NOs: 7, 8 and 9 (light chain CDRs 1, 2 and 3) and
(b) The antibody or antigen-binding fragment thereof according to (1), which comprises the amino acid sequences of SEQ ID NOs: 10, 11, and 12 (heavy chain CDRs 1, 2 and 3).
(5) (a) the amino acid sequence of SEQ ID NO: 5; the amino acid sequence having a deletion, substitution, insertion and / or addition mutation of one to several amino acid residues in the amino acid sequence of SEQ ID NO: 5; or A light chain variable region (LCVR) represented by an amino acid sequence having 95% or more homology with the amino acid sequence of SEQ ID NO: 5,
(b) the amino acid sequence of SEQ ID NO: 6; the amino acid sequence having a deletion, substitution, insertion and / or addition mutation of one to several amino acid residues in the amino acid sequence of SEQ ID NO: 6; or SEQ ID NO: 6 The antibody or antigen-binding fragment thereof according to (1) above, comprising a heavy chain variable region (HCVR) represented by an amino acid sequence having 95% or more homology with the amino acid sequence of
(6) (a) the light chain variable region (LCVR) represented by the amino acid sequence of SEQ ID NO: 5, and
(b) The antibody or antigen-binding fragment thereof according to (1) above, comprising the heavy chain variable region (HCVR) represented by the amino acid sequence of SEQ ID NO: 6.
(7) (a) the amino acid sequence of SEQ ID NO: 2; the amino acid sequence having a deletion, substitution, insertion and / or addition mutation of one to several amino acid residues in the amino acid sequence of SEQ ID NO: 2; or A light chain (L chain) represented by an amino acid sequence having 95% or more homology with the amino acid sequence of SEQ ID NO: 2, and
(b) the amino acid sequence of SEQ ID NO: 4; the amino acid sequence having a deletion, substitution, insertion and / or addition mutation of one to several amino acid residues in the amino acid sequence of SEQ ID NO: 4; or SEQ ID NO: The antibody or antigen-binding fragment thereof according to (1) above, comprising a heavy chain (H chain) represented by an amino acid sequence having 95% or more homology with the amino acid sequence of 4.
(8) The antibody according to (1) 1 above, which comprises a light chain (L chain) represented by the amino acid sequence of SEQ ID NO: 2 and a heavy chain (H chain) represented by the amino acid sequence of SEQ ID NO: 4. Its antigen-binding fragment.
(9) The antibody or antigen-binding fragment thereof according to any one of (1) to (8), wherein the monoclonal antibody is a human monoclonal antibody.

(10) For diagnosis, prevention, and / or treatment of a symptom caused by a rabies-related virus, comprising the antibody or antigen-binding fragment thereof according to any one of (1) to (9) above and a pharmaceutically acceptable carrier. Pharmaceutical composition to be used (11) The pharmaceutical composition according to the above (10), which prevents post-exposure onset of infection caused by rabies virus.

(12) A DNA encoding an anti-RV monoclonal antibody or an antigen-binding fragment thereof that can specifically bind to RV and neutralize its biological activity, and is selected from the group consisting of SEQ ID NOs: 2 and 4-12 An isolated DNA selected from a DNA encoding an amino acid sequence, and a DNA that hybridizes with the DNA under highly stringent conditions.
(13) The DNA according to (12), which encodes the amino acid sequences of SEQ ID NOs: 2 and 4.
(14) The DNA according to (13), which encodes the amino acid sequences of SEQ ID NOs: 5 and 6.
(15) A vector incorporating the DNA according to any one of (12) to (14) above.
(16) A host cell into which the vector of (15) has been introduced.
(17) A method for producing the antibody or antigen-binding fragment of any one of (1) to (9), comprising a step of culturing the host cell according to (16).

  The anti-RV antibody or antigen-binding fragment thereof according to the present invention not only specifically binds to RV causing rabies and loses (neutralizes) its biological activity, but also an epitope recognized by conventional anti-RV antibodies Thus, neutralizing ability can be exhibited even for RVs that exhibit resistance in conventional anti-RV antibodies. In the RV virus that escapes from the anti-RV antibody or antigen-binding fragment thereof according to the present invention, the 242nd amino acid is mutated, suggesting that the pathogenicity is greatly reduced. Furthermore, human monoclonal antibodies do not cause an excessive immune response against humans. Thus, for example, when used alone or in a cocktail with other human MAbs with different epitopes for the prevention of post-exposure development (PEP) of rabies virus, the combination of the antibody or antigen-binding fragment may be present anti-rabies immunoglobulin preparations. Expected to improve efficacy compared to treatment with

  Based on the above properties, the anti-RV antibody or antigen-binding fragment thereof according to the present invention is useful as a pharmaceutical composition used for diagnosis, prevention and / or treatment of symptoms caused by rabies-related viruses.

It is a figure which shows the comparison with the epitope on the existing rabies virus G protein, and the epitope of EV5021. The nucleotide sequence obtained from Applied Biosystems 3130 genetic analyzer (Applied biosystems) was converted to an amino acid sequence and compared with the amino acid sequence of the parent strain CVS-11 using existing alignment software. The already reported antigen recognition sites (Antigenic sites I, II, III, IV and minor a) are also illustrated. The epitope site deduced from the amino acid sequence of the EV5021 escape virus was the 242nd Ala. It is the electrophoresis photograph which shows the result of having analyzed the recognition site of EV5021 rabies virus G protein by Western blotting. BHK-21 cells (1 × 10 ⁶ cells) were inoculated with CVS-11 strain (1 × 10 ⁷ FFU) and then cultured in a 37 ° C., 5% CO ₂ incubator for 2 days to collect infected cells. Infected and non-infected cells were Western blotted and reacted with EV5021 (200-fold diluted) at 4 ° C overnight, washed with TBS-T, and then secondary antibody (goat anti-human POD, 2000-fold diluted, Cappel # 55220 or goat anti-human IgA + G + M, 100-fold diluted, Funakoshi # 04-10-07) was reacted at 4 ° C. for 4 hours. No specific binding between the antibody and the denatured / reduced G protein was observed in any of the CVS-11 strain infected cells and non-infected cells. In a test to determine the 50% fluorescence focus inhibitory concentration (50% FR) of EV5021, the figure shows the relationship between the infection rate of rabies virus on cultured cells in vitro and the concentration-dependent infection inhibition by EV5021. A reaction solution obtained by reacting 100 FFU of CVS-11 and MAb for 1 hour was added to NA cells cultured in a monolayer, and the virus was adsorbed for 1 hour. The reaction solution was removed, washed with PBS, and cultured in a 1% methylcellulose overlay medium at 37 ° C. in a 5% CO ₂ incubator for 3 days. After fixing with paraformaldehyde, it was reacted with a FITC-labeled antibody against the N protein of RV, and the number of infection foci was measured. The antibody concentration that reduced the number of infection foci to 50% was expressed as 50% FR.

1. Antibody or antigen-binding fragment thereof according to the present invention In one embodiment, the present invention is directed to an anti-rabies virus monoclonal antibody capable of specifically binding to rabies virus (RV) and neutralizing its biological activity. An antigen-binding fragment (hereinafter referred to as “the antibody of the present invention”) is provided. Typically, the antibody of the present invention is an anti-rabies virus monoclonal antibody or an antigen-binding fragment thereof that binds to an epitope (stereostructured epitope) containing amino acid 242 (alanine) of the G protein of rabies virus.

  Information on the entire genome sequence of rabies virus can be obtained in publicly accessible sequence databases such as GenBank, for example by referring to the sequence of accession number M13215.1. In addition, information on the amino acid sequence of the rabies virus G protein (or glycoprotein: Glycoprotein) is obtained by referring to the sequence of GenBank, for example, accession numbers AAA47218.1, AAA47213.1, AAP81751.1, etc. can do. In addition, information on the nucleotide sequence encoding the rabies virus G protein can be obtained by referring to sequences such as M13215.1, M32751.1, and AF406694.1 in the GenBank sequence database. The 19 amino acid residues at the N-terminus of the amino acid sequence of the rabies virus G protein identified by the above accession number correspond to a signal peptide (for example, Prehaud C et al., Antigenic site II of the rabies virus glycoprotein: J Virol. 1988; 62: p1-7.; Marissen WE et al., Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: fine mapping and escape mutant analysis.J Virol. 2005; 79 : p4672-4678.).

  Preferred examples of the antibody of the present invention include, for example, a monoclonal antibody or antigen-binding fragment thereof that specifically binds to rabies virus (RV) and can neutralize its biological activity, and (a) SEQ ID NO: 7 , 8 and 9 and an amino acid sequence selected from the group consisting of amino acid sequences having deletion, substitution, insertion and / or addition mutations of one to several amino acid residues in those amino acid sequences At least one (preferably two or more, more preferably three) light chain CDRs, and (b) the amino acid sequences of SEQ ID NOs: 10, 11, and 12, and one to several amino acid residues in those amino acid sequences At least one (preferably two or more) having an amino acid sequence selected from the group consisting of amino acid sequences having group deletion, substitution, insertion and / or addition mutations More preferably contains the heavy chain CDR of three), anti-RV monoclonal antibody or antigen-binding fragment thereof can be mentioned.

A more preferred example of the antibody of the present invention is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to rabies virus (RV) and can neutralize its biological activity,
(a) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having a deletion, substitution, insertion and / or addition mutation of one to several amino acid residues in the amino acid sequence;
(b) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having a deletion, substitution, insertion and / or addition mutation of one to several amino acid residues in the amino acid sequence;
(c) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having a deletion, substitution, insertion and / or addition mutation of one to several amino acid residues in the amino acid sequence;
(d) a heavy chain CDR1, comprising the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having a deletion, substitution, insertion and / or addition mutation of one to several amino acid residues in the amino acid sequence;
(e) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having a deletion, substitution, insertion and / or addition mutation of one to several amino acid residues in the amino acid sequence; and
(f) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having a deletion, substitution, insertion and / or addition mutation of one to several amino acid residues in the amino acid sequence;
An anti-RV monoclonal antibody or an antigen-binding fragment thereof.

  An even more preferred example of the antibody of the present invention is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to rabies virus (RV) and can neutralize its biological activity, comprising: (a) SEQ ID NO: 7 , 8 and 9 amino acid sequences (light chain CDRs 1, 2 and 3) and (b) SEQ ID NO: 10, 11 and 12 amino acid sequences (heavy chain CDRs 1, 2 and 3) or an anti-RV monoclonal antibody thereof Examples include antigen-binding fragments.

  An even more preferred example of the antibody of the present invention is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to rabies virus (RV) and can neutralize its biological activity, comprising: (a) SEQ ID NO: 5 The amino acid sequence of SEQ ID NO: 5 and the amino acid sequence having a deletion, substitution, insertion and / or addition mutation of one to several amino acid residues, or 95% of the amino acid sequence of SEQ ID NO: 5 The light chain variable region (LCVR) represented by the amino acid sequence having the above homology, and (b) the amino acid sequence of SEQ ID NO: 6, lack of one to several amino acid residues in the amino acid sequence of SEQ ID NO: 6 Heavy chain variable region represented by an amino acid sequence having deletion, substitution, insertion and / or addition mutation, or an amino acid sequence having 95% or more homology with the amino acid sequence of SEQ ID NO: 6 Containing (HCVR), anti-RV monoclonal antibody or antigen-binding fragment thereof can be mentioned.

  An even more preferred example of the antibody of the present invention is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to rabies virus (RV) and can neutralize its biological activity, comprising: (a) SEQ ID NO: 5 An anti-RV monoclonal antibody or an antigen-binding fragment thereof containing a light chain variable region (LCVR) represented by the amino acid sequence of (b) and (b) a heavy chain variable region (HCVR) represented by the amino acid sequence of SEQ ID NO: 6. It is done.

  An even more preferred example of the antibody of the present invention is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to rabies virus (RV) and can neutralize its biological activity, comprising: (a) SEQ ID NO: 2 The amino acid sequence of SEQ ID NO: 2 and the amino acid sequence having deletion, substitution, insertion and / or addition mutation of one to several amino acid residues, or 95% of the amino acid sequence of SEQ ID NO: 2 In the light chain (L chain) represented by the amino acid sequence having the above homology, and (b) the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 4, lacking one to several amino acid residues A heavy chain (H chain) represented by an amino acid sequence having a deletion, substitution, insertion and / or addition mutation, or an amino acid sequence having 95% or more homology with the amino acid sequence of SEQ ID NO: 4 Having anti-RV monoclonal antibody or antigen-binding fragment thereof can be mentioned.

  An even more preferred example of the antibody of the present invention is a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to rabies virus (RV) and can neutralize its biological activity, the amino acid sequence of SEQ ID NO: 2 And a heavy chain (H chain) represented by the amino acid sequence of SEQ ID NO: 4, or an anti-RV monoclonal antibody or an antigen-binding fragment thereof.

  Preferably, the anti-RV monoclonal antibody is a human monoclonal antibody.

  The term “epitope” or “antigenic determinant” as used herein refers to the site of an antigen to which an immunoglobulin or antibody (or antigen-binding fragment thereof) specifically binds. Epitopes contain one or more amino acid residues and can be formed, for example, from both consecutive amino acids or non-contiguous amino acids that are juxtaposed by three-dimensional folding of the protein. The epitope on RV that is specifically recognized by the antibody of the present invention is a conformation-dependent epitope.

As used herein, the term “antibody” is four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. It shall refer to an immunoglobulin molecule consisting of things. The monoclonal antibody in the present invention also consists of immunoglobulin molecules each containing two light chains (L chains) and heavy chains (H chains). Each H chain is composed of an H chain variable region (sometimes referred to as “HCVR” or “V _H ”) and an H chain constant region (the H chain constant region is composed of three domains, “C _H 1”). , “C _H 2” and “C _H 3” (generic name: C _H )). Each L chain consists of an L chain variable region (sometimes referred to as “LCVR” or “V _L ”) and an L chain constant region (the L chain constant region is a single domain, referred to as “C _L ”). May be).

  HCVR and LCVR are particularly important in that they are involved in antibody binding specificity. Since antibodies interact with target antigens primarily through LCVR and HCVR amino acid residues, the amino acid sequences within the variable region are more different between individual antibodies than sequences outside the variable region. Furthermore, HCVR and LCVR can be further subdivided into a region called a framework region (FR) and a hypervariable region called a complementarity determining region (CDR) that are kept more constant between various antibodies. HCVR and LCVR are each composed of 3 CDRs and 4 FRs, which are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.

  The term “antigen-binding fragment” (or simply “antibody fragment”) of an antibody refers to a fragment of one or more antibodies capable of specifically binding to an antigen (eg, RV). The fragment includes a peptide having a minimum amino acid sequence that specifically binds to an antigen. Also included in the “antigen-binding fragment” are single-chain antibodies (scFV), bispecific antigens, multispecific antigens and the like having variable regions and complementarity determining regions of antibodies that specifically bind to antigens. In the present specification, for simplification, “antibody or antigen-binding fragment” is also simply referred to as “antibody”.

“Antibodies capable of neutralizing the biological activity of RV” are intended to refer to antibodies that inhibit the biological activity of RV by binding to RV.
As used herein, the terms “inhibitory effect”, “inhibition”, “suppression”, “can inhibit” and the like refer to a biological activity attributable to an antigen (RV) of about 5-100%, preferably 10 -100%, more preferably 20-100%, more preferably 30-100%, more preferably 40-100%, more preferably 50-100%, more preferably 60-100%, more preferably 70-100. %, More preferably 80 to 100%.

  Since the amino acid sequence of the variable region is responsible for most antibody-antigen interactions, the variable region sequence derived from a specific naturally occurring antibody or the sequence of the CDR portion is a different antibody having different properties. By constructing an expression vector such that it is contained in an invariant region or framework sequence derived from it, a recombinant antibody that mimics the properties of a particular naturally occurring antibody can be expressed.

  Therefore, it is not necessary to obtain the entire sequence of a particular antibody when recreating an intact recombinant antibody having binding properties similar to those of the original antibody. The antibody heavy and light chain variable region sequence or CDR portion sequences may be sufficient for this purpose.

  SEQ ID NOs: 7, 8, and 9 are amino acid sequences corresponding to light chain CDR1, CDR2, and CDR3, respectively. SEQ ID NOs: 10, 11 and 12 are amino acid sequences corresponding to CDR1, CDR2 and CDR3 of the heavy chain, respectively. Accordingly, preferred antibodies of the invention include all of SEQ ID NOs: 7-12 (light chain CDR1, CDR2 and CDR3, and heavy chain CDR1, CDR2 and CDR3). However, it is not necessary to include all amino acid sequences of SEQ ID NOs: 7 to 12 as long as they are monoclonal antibodies that can specifically bind to RV and neutralize its biological activity. Moreover, as long as it has the said neutralization activity, CDR sequence | arrangement WHEREIN: In the amino acid sequence of sequence number: 7-12, 1-several (specifically 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1) amino acid sequences having deletion, substitution, insertion and / or addition mutations of amino acid residues There may be.

  The amino acid sequence other than the CDR is not particularly limited, and so-called CDR-grafted antibodies in which the amino acid sequence other than the CDR is derived from other antibodies, particularly other types of antibodies, are also included in the antibody of the present invention. Among these, humanized antibodies in which amino acid sequences other than CDRs are also derived from humans are preferred, but one to several amino acid residues (specific numbers are the same as described above) in the framework region (FR) as necessary. There may be deletion, substitution, insertion and / or addition mutations. A known method can be used as a method for producing a humanized antibody (Riechmann L, et al., Reshaping human antibodies for therapy. Nature, 332: 323-327, 1988). In the present invention, of course, fully human antibodies are preferred.

  The deletion, substitution, insertion and / or addition of one or more amino acid residues in the amino acid sequence of the protein of the present invention means that one or more amino acid residues in one and a plurality of amino acid sequences in the same sequence It means that there are amino acid residue deletion, substitution, insertion and / or addition, and two or more of deletion, substitution, insertion and addition may occur simultaneously.

  Examples of amino acid residues that can be substituted with each other are shown below. Amino acid residues contained in the same group can be substituted for each other. Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine; Group B: aspartic acid, glutamic acid, isoaspartic acid , Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid; group C: asparagine, glutamine; group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid; group E : Proline, 3-hydroxyproline, 4-hydroxyproline; Group F: serine, threonine, homoserine; Group G: phenylalanine, tyrosine.

  In the present invention, preferred antibodies are: (a) the amino acid sequence of SEQ ID NO: 5; 1 to several (specifically, 1 to 9, 1 to 8, 1 to 8 in the amino acid sequence of SEQ ID NO: 5). (7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1) deletion, substitution, insertion and / or addition mutation of amino acid residues An amino acid sequence; or an amino acid sequence having a homology of 95% or more (preferably 96% or more, 97% or more, 98% or more, 99% or more or 99.5% or more) with the amino acid sequence of SEQ ID NO: 5 A light chain variable region (LCVR), and (b) amino acid sequence of SEQ ID NO: 6; deletion of 1 to several amino acid residues (specific numbers are the same as above) in the amino acid sequence of SEQ ID NO: 6 An amino acid sequence having a substitution, insertion and / or addition mutation; or SEQ ID NO: 6 Amino acid sequence and 95% (specific percentages similar to those described above) containing the heavy chain variable region represented by amino acid sequence homology to (HCVR).

  The identity of amino acid sequences and nucleotide sequences is determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc. Natl Acad Sci USA 90: 5873, 1993) by Carlin and Arthur. Can be determined. Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. When analyzing an amino acid sequence using BLASTX, parameters are set to score = 50 and wordlength = 3, for example. When using BLAST and Gapped BLAST programs, the default parameters of each program are used.

  In the present invention, more preferred antibodies are (a) a light chain variable region (LCVR) represented by the amino acid sequence of SEQ ID NO: 5 and (b) a heavy chain variable region (HCVR) represented by the amino acid sequence of SEQ ID NO: 6. ).

  In the present invention, further preferred antibodies are: (a) amino acid sequence of SEQ ID NO: 2; deletion of 1 to several amino acid residues (specific numbers are the same as above) in the amino acid sequence of SEQ ID NO: 2. A light chain represented by an amino acid sequence having a substitution, insertion and / or addition mutation; or an amino acid sequence having 95% or more homology with the amino acid sequence of SEQ ID NO: 2 (specific% is the same as described above) ( L chain), and (b) amino acid sequence of SEQ ID NO: 4; deletion, substitution, insertion of one to several amino acid residues (specific numbers are the same as above) in the amino acid sequence of SEQ ID NO: 4 And / or an amino acid sequence having an additional mutation; or a heavy chain (H chain) represented by an amino acid sequence having a homology of 95% or more (specific% is the same as described above) with the amino acid sequence of SEQ ID NO: 4 contains.

  The above-described anti-RV antibody or antigen-binding fragment thereof according to the present invention has an epitope different from that of a conventional anti-RV antibody, so that it specifically binds to RV causing rabies and only neutralizes its biological activity. It also has a neutralizing ability against RV that is resistant to conventional anti-RV antibodies.

Here, “specifically binds” refers to specifically recognizing and binding to a predetermined antigen with respect to any other antigen. The dissociation constant (Kd value) with RV (particularly human RV) in the antibody of the present invention is preferably 1 × 10 ⁻⁷ M or less, more preferably 2 × 10 ⁻⁸ M or less. A known method can be used to measure the dissociation constant between the antibody and RV. For example, it can be measured by a protein interaction analyzer such as BIACORE 3000 (registered trademark) using an anti-RV antibody immobilized on a chip.

  Regarding the neutralizing ability of anti-RV antibody, for example, rapid fluorescence focus suppression test (RFFIT) (reference: Smith J., Yager P., Baer G. 1973. A rapid reproducible test for determining rabies neutralizing antibody. Bull. World Health Organ. 48, 535-541) can be used. The neutralizing antibody titer is expressed in international units (IU / ml) by comparing the 50% fluorescence focus inhibitory concentration (50% FR) indicated by the antibody with the 50% FR of WHO standard serum. The neutralizing titer (RFFIT IU / mg) per unit protein of the anti-RV antibody or antigen-binding fragment thereof according to the present invention is preferably 10 IU / mg or more, more preferably 100 IU / mg or more. Have

  The antibody of the present invention is a full-length antibody or an antigen-binding fragment thereof, or SEQ ID NOs: 5 and 6 indicating variable region, or amino acid sequences of SEQ ID NOs: 7 to 12 indicating complementarity determining regions (CDRs). Based on the above, recombinant human monoclonal antibodies and monoclonal antibodies (including chimeric antibodies and humanized antibodies) that can specifically bind to RV and neutralize its biological activity by well-known techniques in the art. Or antigen-binding fragments thereof. Fab, F (ab '), F (ab') 2, Fv, dAb, Fd, complementarity determining region (CDR) fragment, single chain antibody (scFv), bivalent single chain antibody, single chain phage Antibodies, bispecific antibodies (diabodies), trispecific antibodies (triabodies), tetraspecific antibodies (tetrabodies), and sufficient immunoglobulin to form antigen binding specific to rabies virus or fragments thereof. The (poly) peptide containing at least a fragment belongs to the antigen-binding fragment of the present invention.

  Heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody, but to add missing sequences, for example, clone cDNA sequences can be ligated. Alternatively, it can be combined with synthetic oligonucleotides by PCR amplification. Alternatively, the entire variable region can be synthesized as a set of short, overlapping oligonucleotides and combined in a PCR amplification method to create a fully artificial variable region clone.

2. DNA according to the present invention
Another embodiment of the present invention encodes an anti-RV monoclonal antibody or antigen-binding fragment thereof that specifically binds to an epitope containing the 242nd amino acid (alanine) of the G protein of RV and can neutralize its biological activity. An isolated DNA selected from a DNA that encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4-12, and a DNA that hybridizes with the DNA under highly stringent conditions It relates to DNA. Any DNA encoding an anti-RV monoclonal antibody or antigen-binding fragment thereof capable of binding to RV and neutralizing its biological activity can be used in the present invention if the DNA has high homology with the DNA. included.

"High stringency conditions" are, for example, 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide at 50 ° C. (e.g., J. Sambrook et al Molecular Cloning, A Laboratory Manual 2 ^nd ed., Cold Spring Harbor Laboratory Press (1989), especially section 11.45 "Conditions for Hybridization of Oligonucleotide Probes"). Under these conditions, it can be expected that a polynucleotide having high homology (eg, DNA) can be efficiently obtained as the temperature is increased. However, multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration can be considered as factors that affect hybridization stringency. Those skilled in the art will select these factors as appropriate. It is possible to achieve similar stringency.

  Examples of DNA that hybridizes under highly stringent conditions include DNA encoding the amino acid sequence of SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10, 11 or 12, for example, 70% or more , 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more DNA having homology.

  The homology of the base sequence can be determined using the above-described homology search algorithm (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc Natl Acad Sci USA 90: 5873, 1993) .

  In addition, DNA more preferable in the present invention is DNA encoding the amino acid sequences of SEQ ID NOs: 5 and 6.

3. The Vector, Host Cell and Antibody Production Method According to the Present Invention The present invention also relates to a vector incorporating the above DNA, a host cell introduced with the vector, and an antibody production method using these.
The antibody of the present invention can also be prepared as a recombinant human antibody using a known method (see Nature, 312: 643, 1984, Nature, 321: 522, 1986, etc.). For example, the antibody of the present invention can be produced by culturing host cells into which the vector of the present invention has been introduced, and purifying the produced antibody from the culture supernatant or the like. More specifically, an expression vector for animal cells containing a gene encoding human antibody C _H and / or human antibody C _L produced from cDNA encoding V _H and V _L from the same cell or another human cell. It can be produced by constructing a human antibody expression vector by inserting each vector, introducing it into an animal cell and expressing it.

The vector into which the nucleic acid encoding the V _H or V _L of the antibody of the present invention is not necessarily limited, but a vector or a high expression vector that is generally used for expression of protein genes and the like and that is particularly suitable for expression of antibody genes is preferred. . Suitable examples include vectors containing the EF promoter and / or CMV enhancer. In addition, expression vectors that normally incorporate nucleic acids encoding _VH or _VL are prepared and cotransfected into host cells, but they may be incorporated into a single expression vector.
The host cell into which the expression vector is introduced is not necessarily limited, but a cell that is generally used for the expression of protein genes and the like and is particularly suitable for the expression of antibody genes is preferred. Examples include bacteria (such as E. coli), actinomycetes, yeast, insect cells (such as SF9), and mammalian cells (such as COS-1, CHO, and myeloma cells).

  In order to industrially produce a recombinant antibody, a recombinant animal cell line that stably produces the antibody, for example, a CHO cell line is generally used. Known methods can be used to generate, clone, and amplify and screen such recombinant cell lines (eg, Omasa T .: J. Biosci. Bioeng., 94, 600-605). , 2002 etc.).

The present invention includes an antigen-binding fragment of the antibody of the present invention in addition to an antibody composed of two heavy chains and two light chains. Examples of the antigen-binding fragment include Fab (fragment of antigen binding), Fab ′, and F (ab ′) _2. For example, a single chain antibody (single chain Fv: scFv) is obtained by binding an active fragment of an antibody with a linker or the like. ) And disulfide stabilized antibodies (disulfide stabilized Fv: dsFv). Examples of peptides containing active fragments of antibodies include peptides containing CDRs. These can be produced by a known method such as a method of treating the antibody of the present invention with an appropriate proteolytic enzyme or a gene recombination technique.
Purification of the antibody can be performed using a known purification means such as salting out, gel filtration, ion exchange chromatography or affinity chromatography.

In addition, recently developed scFv (synthetic _VH and _VL genes are shuffled by phage display antibody technology using recombinant DNA (recombinant antibody) expressed on the phage surface by utilizing genetic engineering technology. A specific antibody can also be obtained by expressing a single chain fragment of variable region antibody as a phage fusion protein. This technique is highly evaluated as a humanized antibody production technique that can avoid immunity and is replaced with a cell fusion method. Any specific antibody or antigen-binding fragment thereof produced using this technique with reference to the amino acid sequences of SEQ ID NOs: 2 and 4 to 12 in the present specification belongs to the technical scope of the present invention.
Furthermore, an antibody obtained by applying the Potentgent technology, which has been developed in recent years, to significantly improve the ADCC activity of an antibody by modifying the sugar chain portion of the antibody (reference: Niwa R. , et al, Clin. Cancer Res., 10, 6248-6255 (2004)) or an antibody obtained by applying the Complement technology for improving CDC activity to the antibody of the present invention (reference: Kanda S., et al, Glycobiology, 17, 104-118 (2007)) also belongs to the technical scope of the present invention.

  As a method for antibody production, polyclonal antibodies and monoclonal antibodies are usually obtained using laboratory animals such as mice, rabbits, goats, etc., but the antibodies thus obtained were used. Since it has a sequence characteristic to animal species, if it is administered to humans as it is, it is recognized as a foreign substance by the human immune system, and a human anti-animal antibody response may occur (that is, an antibody of an antibody is produced).

  The anti-RV monoclonal antibody or antigen-binding fragment thereof according to the present invention can be obtained, for example, from blood-derived antibody-producing cells such as healthy individuals who have been vaccinated with RV vaccine, and is a fully human antibody. Even if this fully human antibody is administered to the human body as an antibody drug, it is considered that it has no immunogenicity and no immune reaction is observed.

  In addition, since the anti-RV monoclonal antibody of the present invention has an epitope specificity different from that of the conventional anti-RV antibody, the anti-RV monoclonal antibody has a neutralizing ability even for RVs that are resistant to conventional anti-RV antibodies. It can be demonstrated.

4). Pharmaceutical composition containing antibody according to the present invention Next, the present invention relates to a pharmaceutical composition used for diagnosis, prevention and / or treatment of symptoms caused by rabies-related virus, in particular, prevention of onset after exposure to rabies virus. There is provided a pharmaceutical composition for use in which comprises the antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

  The anti-RV antibody or antigen-binding fragment thereof according to the present invention is different in epitope specificity from conventional anti-RV antibodies, and the epitope site is also involved in the pathogenicity of rabies virus. Efficacy can also be expected for RVs that are resistant to conventional anti-RV antibodies.

As used herein, “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersion media, coatings, isotonic and absorption delaying agents and the like.
Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. When used as an injection, the composition preferably contains a pH adjuster or isotonic agent such as sugar, polyalcohol such as mannitol or sorbitol, or sodium chloride. Pharmaceutically acceptable carriers may further contain minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, buffers, stabilizing agents and the like that increase the storage or effectiveness of the antibody or antigen-binding fragment. .

  The composition of the present invention can be made into various dosage forms. Such compositions include, for example, liquids, semi-solids such as solutions (eg, injectable and infusible solutions) and dispersions, suspensions, tablets, capsules, troches, pills, powders, liposomes, suppositories, etc. Solid dosage forms are included. The preferred form depends on the intended mode of administration and therapeutic application. Generally preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used to passively immunize humans with other antibodies. Preferred dosage forms are parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). In preferred embodiments, the antibody is administered by intravenous infusion or intravenous injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.

  The antibodies and antigen-binding fragments of the invention can be incorporated into pharmaceutical compositions suitable for parenteral administration. When an antibody or an antigen-binding fragment is used alone, it is preferably prepared as an injectable preparation containing 0.1 to 250 mg / mL of antibody. On the other hand, when a plurality of types of antibodies are mixed and used in the same preparation, they are preferably prepared as injectable preparations each containing 0.001 to 100 mg / mL of antibody. The mixing ratio of the plurality of types (cocktails) of antibodies can be set as appropriate.

  Injectable formulations may consist of the active ingredient dissolved in a liquid or lyophilized active ingredient in a flint or amber vial, ampoule or prefilled syringe. The buffer may be L-histidine (1-50 mM) at pH 5.0-7.0 (optimally pH 6.0), 5-10 mM L-histidine when optimal. Other suitable buffering agents include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate, or potassium phosphate. Sodium chloride can be used to change the osmotic pressure of a 0-300 mM concentration solution (150 mM, optimal for liquid dosage forms). The lyophilized dosage form can contain a cryoprotectant, mainly 0-10% (in the best case 0.5-5.0%) sucrose. Other suitable cryoprotectants include mannitol, trehalose and lactose. The lyophilized dosage form may contain a bulking agent, primarily 1-10% mannitol (2-4% when optimal). For both liquid and lyophilized dosage forms, stabilizers, primarily 1-50 mM (optimally 5-10 mM) L-methionine can be used. Other suitable stabilizers include glycine, arginine, polysorbate 80, and the like, and in the case of polysorbate 80, 0-0.05% (optimally 0.005-0.01%) can be included. Other surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactant.

  The pharmaceutical composition generally must be sterile or stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions are prepared by mixing the required amount of active compound (ie antibody or antigen-binding fragment) in a suitable solvent with one or a combination of the above components, if necessary, and then filter sterilized. Can be prepared. Generally, dispersions are prepared by mixing the active compound with a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of a sterile powder formulation for the preparation of a sterile injectable solution, the preferred method of preparation is freeze vacuum drying and spray drying of the sterile filtered solution as previously described, thereby adding to the active ingredient powder, A composition containing any other desired ingredients is obtained. The proper fluidity of the solution can be achieved, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. Can be maintained. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts or gelatin.

  Supplementary active compounds can also be incorporated into the pharmaceutical compositions. In certain embodiments, the antibodies or antigen-binding fragments of the invention are formulated with or administered concurrently with one or more other therapeutic agents effective for rabies PEP. For example, an anti-RV antibody or antigen-binding fragment of the invention can be administered as a cocktail formulation with one or more other anti-RV antibodies that bind to other epitopes, or concurrently with such other antibodies. . Furthermore, one or more antibodies of the present invention can be used in combination of two or more of the aforementioned therapeutic agents. Such combination therapy can greatly reduce the probability of the emergence of rabies escape viruses.

  When the antibody or antigen-binding fragment of the present invention or a pharmaceutical composition comprising the same is used for diagnosis, prevention and / or treatment of symptoms caused by a rabies-related virus, they can be used in vitro, in vitro, and live. It can be used in the body and may be used in combination with other active ingredients such as vaccines. It will be apparent to those skilled in the art that the subject patient may be before exposure, after exposure, or after onset.

  Next, the process of obtaining the anti-RV monoclonal antibody and the antigen-binding fragment thereof according to the present invention will be described. However, the technique for obtaining the antibody according to the present invention is not limited to these descriptions, and as described above. Needless to say, ordinary changes in the technical field can be made.

  The anti-RV monoclonal antibody and the antigen-binding fragment thereof according to the present invention are obtained by isolating a cell clone that produces the antibody from blood of a healthy person who has experience of vaccination for rabies through various steps, and The antibody obtained from the antibody-positive cell supernatant through the step of selecting antibody-positive cells from the cell library can be obtained by affinity purification.

Isolation of fully human antibody-producing cell clones against rabies virus (RV) Blood is collected from an adult who has undergone RV vaccination, B lymphocytes are isolated from the peripheral blood, and proliferation of the B lymphocytes is induced. Methods for inducing proliferation are known per se, for example, a transform method (D. Kozbor) using “Epstein-Barr virus” (hereinafter referred to as EBV), which is an inducer of cancer. Et al.).
That is, the above-mentioned B lymphocytes are infected with EBV to induce proliferation, and the proliferated cells are used as an antibody-producing cell library.

2) Recovery of monoclonal antibody from antibody-producing cell library A method for recovering a monoclonal antibody from a proliferation-induced cell can be performed by a well-known method commonly used in the production of monoclonal antibodies.
Lymphocytes that produce antibodies that bind to RV are selected from the antibody-producing cell library, and the antibodies are removed. That is, a cell population (clone) that produces an antibody that binds to RV is selected from the antibody-producing cell library by a limiting dilution method.
For detection of the fraction that binds to RV, it is preferable to employ ELISA using RV and a labeled mouse anti-human IgG antibody.
By culturing the selected antibody-positive cell population and repeating the screening, a cell population (clone) that produces only the target antibody can be obtained. A flowchart showing the steps up to the separation of the above antibody-producing cell clones As shown in FIG.

3) Affinity purification using Protein A or G To purify anti-RV antibodies, selected cells can be grown in roller bottles, 2 liter spinner flasks, or other culture systems.
The supernatant can be filtered and concentrated, and the protein can be purified by affinity chromatography using protein A or protein G-Sepharose (Piscataway, NJ, Pharmacia). The buffer solution can be exchanged into PBS and the concentration can be determined by OD ₂₈₀ or preferably by nephelometer analysis.
The isotype can be determined by a method specific to the isotype antigen.

Since the anti-RV antibody thus obtained is a fully human antibody prepared from B lymphocytes sensitized in the human body, the possibility of an immune reaction to the antibody is low.
Another feature of the production of antibody-producing cell clones is the use of an EB virus that has the activity of inducing proliferation by infecting B lymphocytes.
The advantage of the EB virus method is that a natural antibody produced in the human body can be produced and an antibody with high affinity can be obtained. For example, human antibodies against RV have been found to be about 10-100 times more affinity than antibodies made by artificially immunizing mice.
A population of B lymphocytes proliferated by EB virus infection becomes a library of antibody-producing cells.
A specific antibody-producing cell clone can be isolated from this library to obtain a human antibody.

As described above, the present invention has been specifically described with respect to specific embodiments, but changes and modifications can be made and these are within the technical scope of the present invention.
The antibodies or antigen-binding fragments thereof of the present invention also encompass nucleic acids, vectors and host cells for expressing the recombinant antibodies of the present invention or antibody-binding portions thereof.

In addition, the relationship between the nucleotide or amino acid sequence appearing in this specification and the SEQ ID NO is as follows.
SEQ ID NO: 1
Represents the anti-human rabies virus antibody (EV5021) light chain nucleotide sequence.
SEQ ID NO: 2
Represents the anti-human rabies virus antibody (EV5021) light chain amino acid sequence.
SEQ ID NO: 3
Represents the anti-human rabies virus antibody (EV5021) heavy chain nucleotide sequence.
SEQ ID NO: 4
Represents the anti-human rabies virus antibody (EV5021) heavy chain amino acid sequence.
SEQ ID NO: 5
Anti-human rabies virus antibody (EV5021) represents the light chain variable region amino acid sequence.
SEQ ID NO: 6
Anti-human rabies virus antibody (EV5021) represents the heavy chain variable region amino acid sequence.
SEQ ID NO: 7
Represents the anti-human rabies virus antibody (EV5021) light chain CDR1 amino acid sequence.
SEQ ID NO: 8
Represents the anti-human rabies virus antibody (EV5021) L chain CDR2 amino acid sequence.
SEQ ID NO: 9
Anti-human rabies virus antibody (EV5021) L chain CDR3 amino acid sequence.
SEQ ID NO: 10
Represents the anti-human rabies virus antibody (EV5021) heavy chain CDR1 amino acid sequence.
SEQ ID NO: 11
Represents the anti-human rabies virus antibody (EV5021) heavy chain CDR2 amino acid sequence.
SEQ ID NO: 12
Represents the anti-human rabies virus antibody (EV5021) heavy chain CDR3 amino acid sequence.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
Unless otherwise stated, procedures used in this example can refer to Molecular Cloning: A Laboratory Manual (Third Edition) (Sambrook et al., Cold Spring Harbor Laboratory Press, 2001).

1. Separation of fully human antibody-producing cell clones for RV In healthy males who have been vaccinated once a year for the first time after a blood rabies vaccine was added one month before blood collection, and a sufficient increase in antibody titer was confirmed Peripheral blood was collected. The isolated peripheral blood lymphocytes are infected with EB virus, seeded in a 96-well multiwell culture plate, cultured for 2-3 weeks, and neutralizing activity against rabies virus CVS-11 strain by rapid fluorescence focus inhibition test using culture supernatant And a positive clone was obtained. Furthermore, a cell clone producing the target antibody was obtained by repeating such a cloning operation. The CVS-11 strains used were all grown on BHK-21 cells in Eagle's minimum essential medium containing 2% fetal calf serum and stored at -80 ° C.

2. Cloning of DNA Encoding Anti-RV Antibody From total-RNA of antibody-producing cells, reverse transcription was performed using an Oligo-dT primer, and the antibody gene was amplified by PCR using the obtained cDNA as a template. Primers used for PCR were designed based on a cDNA database encoding the heavy and light chains of human IgM antibody. In order to amplify full-length H chain cDNA and L chain cDNA, the 5 ′ terminal primer has a translation initiation point and the 3 ′ terminal primer has a translation termination point.

3. Determination of antibody amino acid sequence based on nucleotide sequence
Antibody H chain and L chain cDNAs amplified by the PCR method were inserted into a plasmid vector, and each base sequence was confirmed by an ABI sequencer. The amino acid sequences of the antibody H chain and L chain and the antibody isotype were determined from the obtained base sequence. The obtained EV5021 was IgM · κ.
In addition, Kabat's method was used for the analysis of the complementarity determining region (CDR) of the antibody (see: www.bioinf.org.uk: Dr. andrew CR Martin's Group, Antibodies: General Information). Six CDRs of the obtained EV5021 are shown in SEQ ID NOs: 7-12. Specifically, the amino acid sequences of L chain CDR1, L chain CDR2, L chain CDR3, H chain CDR1, H chain CDR2, and H chain CDR3 are shown in SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively.

4). Confirmation that the obtained antibody gene encodes an anti-RV antibody Each of the obtained H chain and L chain cDNAs was inserted into an expression vector and simultaneously introduced into 293T cells. Gene transfer was performed under the conditions recommended by the manufacturer using Lipofectamine (Invitrogen) and Plus reagent (Invitrogen) (Invitrogen catalog: Cat.No.18324-111, Cat.No.18324-012, or Cat.No. 18324-020). After 2 days, the culture supernatant was collected and confirmed to have neutralizing activity against the CVS-11 strain by a rapid fluorescence focus inhibition test.

5). 5. Production of antibody protein 5.1 Introduction of human anti-RV antibody gene into CHO cells The resulting anti-RV antibody (EV5021) expression plasmid is introduced into CHO cells and cultured in the presence of an appropriate selection marker to produce antibodies. A CHO cell clone expressing constitutively was obtained.
CHO cells stably expressing the antibody were cultured in a serum-free medium, and the culture supernatant was collected.

5.2 Establishment of anti-rabies human MAb It was possible to establish anti-rabies human monoclonal antibody EV5021 by screening from serum obtained from healthy men. EV5021 was IgM type and neutralizing ability against CVS-11 strain was 125 IU / mg. As a result of the Western blot analysis, the MAb did not show specific binding to the denatured / reduced G protein in CVS-11 strain-infected cells. The same tendency was observed when compared with non-infected cells. That is, it was suggested that EV5021 recognizes the three-dimensional structural epitope of G protein of CVS-11 strain (FIG. 2).

6). Determination of recognition epitope of anti-rabies human MAb based on nucleotide sequence of neutralization resistant mutant 6.1 Selection of neutralization resistant mutant
0.5 ml each of CVS-11 strain and anti-rabies human MAb (1-3 IU) were mixed and reacted at 37 ° C for 1.5 hours. The reaction solution was inoculated into a Chick embryo-related cell (CER), adsorbed with virus at 37 ° C for 1 hour, and then the reaction solution was removed and washed with PBS. Thereto, 10% FCS-EMEM / 1% agarose gel was overlaid and cultured in a 5% CO ₂ incubator for 4 days. Each of the formed independent plaques was collected and expanded on BHK-21 for 3 days. Furthermore, a strain showing resistance to the neutralization reaction by each human antibody by RFFIT method was identified as a neutralization resistant mutant for the antibody.

6.2 Determination of amino acid sequence of neutralization-resistant mutant strain mRNA of the identified neutralization-resistant mutant strain was extracted by ISOGEN (Nippongene), cDNA was synthesized by OligogodT primer (Invitrogen), and G protein was extracted from the N-terminus. It was divided into two fragments and amplified by PCR. The following PCR and sequence primers were used [N terminal fragment; Sense primer: 5'-ATCACTTGTTTACCTCTGGAG-3 '(SEQ ID NO: 13) / Antisense primer: 5'-ACTTATACAGGCCTCTTTCATC-3' (SEQ ID NO: 14), Sense primer: 5'- TATAACTGGAAGATGGCCGGTG-3 '(SEQ ID NO: 15) / Antisense primer: 5'- ACTCCATATGTTGCTGGAG-3' (SEQ ID NO: 16), C terminal fragment; 5'- CCATCATGACCACCAAGTC-3 '(sequence) No: 17) / Antisense primer: 5′-CCATCATGACCACCAAGTC-3 ′ (SEQ ID NO: 18) / 5′-GGCCTTCACAGTCTGATCTC-3 ′ (SEQ ID NO: 19)]. The PCR product was purified using QIAquick PCR Purification Kit (QIAGEN). Each of the purified fragments was subjected to a cycle sequence reaction using BigDye ^(R) Terminator v3.1 Cycle Sequencing Kit (Applied biosystems), and the nucleotide sequence was analyzed by Applied Biosystems 3130 genetic analyzer (Applied biosystems). Then, the amino acid sequences of the neutralization-resistant mutant virus strain and the parent strain virus were compared to determine the site where the mutation was introduced, that is, the antibody recognition and binding site.

6.3 Determination of recognition epitope of anti-rabies human MAb When the nucleotide sequence of the G protein of the neutralization resistant mutant (EV5021es) was determined, the 242nd was mutated from Ala (GCG) to Val (GTG). The site has not been reported so far as an epitope (see Table 1 and FIG. 1).
From this result (Fig. 2), it was speculated that this anti-rabies human MAb recognizes the conformation of the RV G protein. If a linear epitope is recognized, it should be able to be detected even when the G protein is in a reduced or denatured state during analysis by Western blotting.

7). Evaluation of neutralization activity using anti-RV antibody rapid fluorescence focus inhibition test (RFFIT) 7.1 Rapid fluorescence focus inhibition test (RFFIT)
The neutralizing antibody titer against RV was measured by the RFFIT method. Each serum sample was diluted 2-fold in a 96-well plate using Eagle's minimum essential medium containing 2% fetal calf serum. All samples were measured in duplicate and serially diluted anti-rabies human MAb was inoculated with 30-100 TCID50 (50% tissue culture infectious dose) of CVS-11 in the presence of 37 ° C and 5% CO ₂ Incubated for 90 minutes. Thereafter, BHK-21 cells (1.0 × 10 ⁶ cells / ml) were added to each well and incubated again at 37 ° C. for 24 hours. Thereafter, the plate was fixed with 90% acetone, and reacted with FITC-labeled anti-RV virus antibody (Fujirebio Diagnostics, Malvern) at 37 ° C for 45 minutes. The 50% fluorescence focus suppression concentration was calculated by the Speerman-Karber method. Neutralizing antibody titer is determined in international units (IU / ml) by comparison with WHO standard serum. When neutralizing titer per unit weight is determined, neutralizing titer (IU) of EV5021 against CVS-11 strain is 125 IU / mg.

7.2 In vitro neutralization test of human anti-rabies MAb
CVS-11 strain and anti-rabies human MAb were reacted at 37 ° C for 90 minutes, and 1.5 x 10 ⁵ cells / well of C1300 (NA) cells pre-cultured in a 24-well plate for 3 days were prepared and washed twice with PBS. Thereafter, the reaction solution was inoculated and the virus was adsorbed at 37 ° C for 1 hour. After inoculation, the supernatant virus solution was aspirated, washed once with PBS, and further cultured in 10% FCS-EMEM medium containing 1% methylcellulose for 3 days. Add 4% paraformaldehyde and react at room temperature for 1 hour to fix the cells. After washing twice with PBS, add methanol and leave for 5 minutes, then wash with PBS, and then add FITC-labeled anti-rabies virus antibody and 45 ° C at 37 ° C. The reaction was performed for 50 minutes and expressed as 50% focus suppression concentration (50% FR). Infectious focus of about 100 FFU was confirmed in cells without MAb. Becomes approximately 80% inhibition in the EV5021 5 ^-4 dilution, the final 50% FR was 15.05 ng / ml (Figure 3).

  The anti-rabies virus antibody of the present invention and a pharmaceutical composition containing the antibody can be used for diagnosis, prevention and / or treatment of symptoms caused by a rabies-related virus. This antibody has a different epitope from conventional anti-RV antibodies and can be expected to be particularly useful for prevention of post-exposure onset (PEP) of rabies virus.

Claims (17)

  Anti-rabies virus that binds to an epitope containing the 242nd amino acid (alanine) of G protein, which is a monoclonal antibody or antigen-binding fragment thereof that can specifically bind to rabies virus (RV) and neutralize its biological activity An antibody or antigen-binding fragment thereof. (a) from the group consisting of the amino acid sequences of SEQ ID NOs: 7, 8 and 9, and amino acid sequences having deletion, substitution, insertion and / or addition mutations of one to several amino acid residues in those amino acid sequences The amino acid sequence of any one or more of the selected light chain CDRs 1, 2 and 3, and
(b) from the group consisting of the amino acid sequences of SEQ ID NOs: 10, 11 and 12, and amino acid sequences having deletion, substitution, insertion and / or addition mutations of one to several amino acid residues in those amino acid sequences The antibody or antigen-binding fragment thereof according to claim 1, comprising one or more amino acid sequences of selected heavy chain CDRs 1, 2 and 3. (a) from the group consisting of the amino acid sequences of SEQ ID NOs: 7, 8 and 9, and amino acid sequences having deletion, substitution, insertion and / or addition mutations of one to several amino acid residues in those amino acid sequences The amino acid sequences of selected light chain CDRs 1, 2 and 3, and
(b) from the group consisting of the amino acid sequences of SEQ ID NOs: 10, 11 and 12, and amino acid sequences having deletion, substitution, insertion and / or addition mutations of one to several amino acid residues in those amino acid sequences The antibody or antigen-binding fragment thereof according to claim 1, comprising the amino acid sequences of selected heavy chain CDRs 1, 2 and 3.   (a) contains the amino acid sequences of SEQ ID NOs: 7, 8 and 9 (light chain CDRs 1, 2 and 3) and (b) contains the amino acid sequences of SEQ ID NOs: 10, 11 and 12 (heavy chain CDRs 1, 2 and 3) The antibody or antigen-binding fragment thereof according to claim 1.   (a) the amino acid sequence of SEQ ID NO: 5; the amino acid sequence having a deletion, substitution, insertion and / or addition mutation of one to several amino acid residues in the amino acid sequence of SEQ ID NO: 5; or SEQ ID NO: A light chain variable region (LCVR) represented by an amino acid sequence having 95% or more homology with the amino acid sequence of 5, and (b) an amino acid sequence of SEQ ID NO: 6; one to several of the amino acid sequences of SEQ ID NO: 6 A heavy chain variable region (HCVR) represented by an amino acid sequence having a deletion, substitution, insertion and / or addition mutation of amino acid residues; or an amino acid sequence having 95% or more homology with the amino acid sequence of SEQ ID NO: 6 Or the antigen-binding fragment thereof.   The light chain variable region (LCVR) represented by (a) the amino acid sequence of SEQ ID NO: 5 and (b) the heavy chain variable region (HCVR) represented by the amino acid sequence of SEQ ID NO: 6 Or an antigen-binding fragment thereof.   (a) the amino acid sequence of SEQ ID NO: 2; the amino acid sequence having a deletion, substitution, insertion and / or addition mutation of one to several amino acid residues in the amino acid sequence of SEQ ID NO: 2; or SEQ ID NO: A light chain (L chain) represented by an amino acid sequence having 95% or more homology with the amino acid sequence of 2, and (b) the amino acid sequence of SEQ ID NO: 4; in the amino acid sequence of SEQ ID NO: 4, one to several An amino acid sequence having a deletion, substitution, insertion and / or addition mutation of the amino acid residues; or a heavy chain (H chain) represented by an amino acid sequence having 95% or more homology with the amino acid sequence of SEQ ID NO: 4 The antibody or antigen-binding fragment thereof according to claim 1, comprising   The antibody or antigen-binding fragment thereof according to claim 1, comprising a light chain (L chain) represented by the amino acid sequence of SEQ ID NO: 2 and a heavy chain (H chain) represented by the amino acid sequence of SEQ ID NO: 4.   The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the monoclonal antibody is a human monoclonal antibody.   A pharmaceutical composition used for diagnosis, prevention and / or treatment of symptoms caused by a rabies-related virus, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier. .   The pharmaceutical composition according to claim 10, for preventing an onset of infection caused by rabies virus after exposure.   An amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4-12, which is a DNA encoding an anti-RV monoclonal antibody or antigen-binding fragment thereof capable of specifically binding to RV and neutralizing its biological activity And an isolated DNA selected from DNA that hybridizes with the DNA under highly stringent conditions.   The DNA according to claim 12, which encodes the amino acid sequences of SEQ ID NOs: 2 and 4.   The DNA according to claim 13, which encodes the amino acid sequences of SEQ ID NOs: 5 and 6.   A vector incorporating the DNA according to any one of claims 12 to 14.   A host cell into which the vector according to claim 15 has been introduced.   A method for producing the antibody or antigen-binding fragment according to any one of claims 1 to 9, comprising a step of culturing the host cell according to claim 16.