WO2015088256A1

WO2015088256A1 - Binding molecules capable of neutralizing rabies viruses

Info

Publication number: WO2015088256A1
Application number: PCT/KR2014/012171
Authority: WO
Inventors: 홍승서; 장신재; 김판겸; 김철민; 서지민; 안정선; 신지영; 맹기은; 김동조; 류수희; 박인창
Original assignee: (주)셀트리온
Priority date: 2013-12-12
Filing date: 2014-12-11
Publication date: 2015-06-18
Also published as: KR101739313B1; CN105814077A; KR20150068913A; CN105814077B

Abstract

The present invention relates to binding molecules capable of neutralizing rabies viruses. More specifically, the binding molecules of the present invention have an ability to neutralize rabies viruses derived from subjects, such as a dog, a cow, a mongoose, a bat, a skunk, a raccoon, a coyote, a fox, and a wolf, and thus can be favorably used to treat patients infected with rabies viruses derived from a broad range of subjects.

Description

Binding molecules that can neutralize rabies virus

The present invention relates to binding molecules capable of neutralizing rabies virus.

Rabies is a viral common infectious disease that primarily affects wildlife and pets, as well as mammals, including humans, causing acute brain disease. It is a fatal disease that occurs almost once in death, and is known to have the highest mortality rate with AIDS. The rabies is spread worldwide, with more than 10 million people receiving treatment after infection each year, with 40,000 to 70,000 deaths each year.

Rabies is transmitted from saliva and blood, usually from bites of dogs or cats infected with rabies. It can also be infected by most mammals, including skunks and bats.

Rabies virus shows actual onset symptoms after reaching the nerve tissue through the terminal nerve tissue of the body. Originally, the human brain has a blood brain barrier that blocks foreign substances, so viruses cannot penetrate, but the rabies virus passes through the blood barrier through the RVG (rabies virus glycoprotein) protein to the central nervous system. (central nervous system) Infects the brain.

In the early stages, in addition to cold-like symptoms, you feel itching or fever at the bite. As rabies progresses, anxiety, rabies (swallowing liquids such as water causes muscle cramps and severe pain, fearing water), fear of wind (wind makes the sense organs sensitive), excitement, paralysis , Neurological symptoms such as mental disorders. It can also cause hypersensitivity to sunlight. Two to seven days after these symptoms are observed, the nerves and muscles of the whole body become paralyzed, leading to coma and eventually dying from breathing problems.

Currently, there is a vaccine for rabies, but there is no treatment. Treatment after exposure to rabies includes post-treatment therapy (post-exposure prophylaxis). Post-treatment treatment consists of immediate topical wound protection and antibody administration for passive immunity (anti-rabies immunoglobulin: hereinafter referred to as "anti-rabies antibody") and vaccine administration for active immunity. Currently developed anti-rabies antibodies include human-derived rabies immunoglobulin (hereinafter referred to as "HRIG") and equine-derived rabies immunoglobulin (hereinafter referred to as "ERIG"). HRIG's supply is not smooth and expensive and it is a polyclonal antibody, so its efficacy per unit weight is not high. Also, since it is derived from human blood, there is a high risk of potential infection of human-derived diseases such as HIV. Although ERIG is cheaper than HRIG, it is also not well supplied and is much less effective than HRIG and is administered to patients at much higher doses. In addition, since the antibody is derived from a horse other than human, it may cause anaphyaxis. In order to overcome such a poor supply and disadvantages of polyclonal antibodies, the use of a monoclonal antibody capable of neutralizing the rabies virus has been proposed. In the 1980s, rabies-virus neutralizing murine single antibodies were developed (Schumacher CL et al ., J. Clin. Invest . Vol. 84, p. 971-975, 1989). Direct administration to human patients is limited due to the lack of immune response in the body and induction of human anti-mouse antibody (HAMA).

Therefore, for effective rabies treatment, it is not derived from blood and has high safety against potential infection, and it is possible to supply large-scale production by synthesis through culture, and it is composed only of antibodies with efficacy, ensuring uniform quality and high efficacy per unit amount. The development of human monoclonal antibodies is urgent.

The problem to be solved by the present invention is to provide a binding molecule having a neutralizing ability by binding to rabies virus.

In addition, another object of the present invention is to provide an immunoconjugate in which one or more tags are bonded to the binding molecule.

In addition, another object of the present invention is to provide a polynucleotide encoding the binding molecule.

In addition, another object of the present invention is to provide an expression vector inserted with a polynucleotide encoding the binding molecule.

In addition, another object of the present invention is to provide a cell line transformed with the expression vector.

In addition, another object of the present invention is to provide a composition comprising the binding molecule.

In addition, another object of the present invention is to provide a kit comprising the binding molecule.

In addition, another object of the present invention is to provide a method for diagnosing rabies using the binding molecule.

In addition, another object of the present invention is to provide a method for treating and preventing rabies using the binding molecule.

In addition, another object of the present invention is to provide a method of producing the binding molecule of the present invention by culturing the cell line.

In addition, another object of the present invention is to provide a method for detecting rabies virus using the binding molecule.

In order to solve the above problems, one embodiment of the present invention has a neutralizing activity when the rabies virus contains a wild type G protein, but the rabies virus is glycine (Gly) valine (Gal), glutamine (Glu) or 34 The inclusion of a G protein mutated to arginine (Arg) provides a binding molecule having no neutralizing activity. Here, the amino acid position is a numbered position except for a signal peptide of the G protein. Hereinafter, the numbering of amino acid positions of G protein is the same.

In one embodiment of the present invention, the binding molecule neutralizes the rabies escape virus in which glycine (Gly) at position 34 of the G protein is mutated to valine, glutamine (Glu), or arginine (Arg). It was confirmed that the epitope is at the antigenic site II of the G protein.

As one example, the binding molecule

According to the Kabat method,

a) a variable region comprising a CDR1 region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR3 region of SEQ ID NO: 3; or

b) a variable region comprising a CDR1 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5, and a CDR3 region of SEQ ID NO: 6

It may be a binding molecule comprising a.

As one example, the binding molecule

According to the Kabat method,

a) a variable region comprising a CDR1 region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR3 region of SEQ ID NO: 3; And

It may be a binding molecule comprising a.

As an example, the binding molecule may be a binding molecule including a variable region of SEQ ID NO: 25 and a variable region of SEQ ID NO: 26.

As one example, the binding molecule may be a binding molecule comprising a heavy chain of SEQ ID NO: 33 and a light chain of SEQ ID NO: 34.

In addition, another embodiment of the present invention has a neutralizing activity when the rabies virus includes a wild type G protein, but when the rabies virus includes a G protein in which serine (Ser) at position 331 is mutated to proline (Pro) Provided is a binding molecule having no neutralizing activity.

In one embodiment of the invention, the binding molecule is confirmed that the serine (Ser) at position 331 of the G protein does not neutralize the rabies escape virus (Escape virus) mutated to proline (Pro) epitope of the G protein It was confirmed that it is at antigenic site III.

As one example, the binding molecule

According to the Kabat method,

a) a variable region comprising a CDR1 region of SEQ ID NO: 7, a CDR2 region of SEQ ID NO: 8, and a CDR3 region of SEQ ID NO: 9; or

b) a variable region comprising a CDR1 region of SEQ ID NO: 10, a CDR2 region of SEQ ID NO: 11, and a CDR3 region of SEQ ID NO: 12

It may be a binding molecule comprising a.

As one example, the binding molecule

According to the Kabat method,

A variable region comprising a CDR1 region of SEQ ID NO: 7, a CDR2 region of SEQ ID NO: 8, and a CDR3 region of SEQ ID NO: 9; And

A variable region comprising a CDR1 region of SEQ ID NO: 10, a CDR2 region of SEQ ID NO: 11, and a CDR3 region of SEQ ID NO: 12

It may be a binding molecule comprising a.

As an example, the binding molecule may be a binding molecule including a variable region of SEQ ID NO: 27 and a variable region of SEQ ID NO: 28.

As an example, the binding molecule may be a binding molecule comprising a heavy chain of SEQ ID NO: 35 and a light chain of SEQ ID NO: 36.

In addition, another embodiment of the present invention has a neutralizing activity when the rabies virus includes a wild type G protein, the valine (Val) at position 210 is glutamic acid (Glu) or glutamic acid (Glu) at position 413 ) Contains a G protein mutated to aspartic acid (Asp) to provide a binding molecule having no neutralizing activity.

In one embodiment of the present invention, the binding molecule is a rabies escape virus in which valine (Val) at position 210 of G protein is mutated to glutamic acid (Glu) or glutamic acid at position 413 to aspartic acid (Asp). The failure to neutralize the escape virus confirmed that the epitope was in a new site of G protein, which has not been reported so far.

The G protein may be the G protein of CVS-11, but is not limited to this strain. More specifically, the wild type G protein may comprise the amino acid sequence of SEQ ID NO: 81.

As one example, the binding molecule

According to the Kabat method,

a) a variable region comprising a CDR1 region of SEQ ID NO: 13, a CDR2 region of SEQ ID NO: 14, and a CDR3 region of SEQ ID NO: 15;

b) a variable region comprising a CDR1 region of SEQ ID NO: 16, a CDR2 region of SEQ ID NO: 17, and a CDR3 region of SEQ ID NO: 18;

c) a variable region comprising a CDR1 region of SEQ ID NO: 19, a CDR2 region of SEQ ID NO: 20, and a CDR3 region of SEQ ID NO: 21; And

d) a variable region comprising a CDR1 region of SEQ ID NO: 22, a CDR2 region of SEQ ID NO: 23, and a CDR3 region of SEQ ID NO: 24

It may be a binding molecule, including any one variable region selected from the group consisting of.

As one example, the binding molecule

According to the Kabat method,

a) a variable region comprising a CDR1 region of SEQ ID NO: 13, a CDR2 region of SEQ ID NO: 14, and a CDR3 region of SEQ ID NO: 15; And

b) a variable region comprising a CDR1 region of SEQ ID NO: 16, a CDR2 region of SEQ ID NO: 17, and a CDR3 region of SEQ ID NO: 18

It may be a binding molecule comprising a.

As an example, the binding molecule may be a binding molecule including a variable region of SEQ ID NO: 29 and a variable region of SEQ ID NO: 30.

As an example, the binding molecule may be a binding molecule comprising a heavy chain of SEQ ID NO: 37 and a light chain of SEQ ID NO: 38.

As one example, the binding molecule

According to the Kabat method,

a) a variable region comprising a CDR1 region of SEQ ID NO: 19, a CDR2 region of SEQ ID NO: 20, and a CDR3 region of SEQ ID NO: 21; And

b) a variable region comprising a CDR1 region of SEQ ID NO: 22, a CDR2 region of SEQ ID NO: 23, and a CDR3 region of SEQ ID NO: 24

It may be a binding molecule comprising a.

As an example, the binding molecule may be a binding molecule including a variable region of SEQ ID NO: 31 and a variable region of SEQ ID NO: 32.

As an example, the binding molecule may be a binding molecule comprising a heavy chain of SEQ ID NO: 39 and a light chain of SEQ ID NO: 40.

In the present invention, the antigenic site, amino acid position, and epitope form of rabies virus G protein generally known are shown in Table 1.

Table 1

Antigenic site and amino acid position of rabies virus G protein

Antigenic Site	Amino acid	Epitope type
One	226-231	Linear
2	34-42, 198-200	Conformational
3	330-338	Conformational
4	251, 264	Linear

On the other hand, in the present invention, the CDRs of the variable regions were determined by conventional methods according to a system devised by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest (5 ^th ), National Institutes of Health, Bethesda) , MD. (1991)]. Although the CDRs used in the present invention were determined using the Kabat method, binding molecules including CDRs determined according to other methods such as the IMGT method, the Chothia method, and the AbM method are also included in the present invention.

In one embodiment of the invention, the binding molecule may be an antibody. In addition, the binding molecule may be, but is not limited to, Fab fragments, Fv fragments, diabodies, chimeric antibodies, humanized antibodies, or human antibodies. One embodiment of the present invention provides a fully human antibody that binds to rabies virus. As used herein, an antibody is used in its broadest sense and is specifically an intact monoclonal antibody, a polyclonal antibody, a multispecific antibody (eg, a bispecific antibody) formed from two or more intact antibodies, and a target. Antibody fragments that exhibit biological activity. Antibodies are proteins produced by the immune system that can recognize and bind specific antigens. In structural terms, the antibody typically has a Y-shaped protein consisting of four amino acid chains (two heavy chains and two light chains). Each antibody mainly has two regions, a variable region and a constant region. The variable region located at the distal portion of the arm of Y binds to and interacts with the target antigen. The variable region comprises a complementarity determining region (CDR) that recognizes and binds to a specific binding site on a particular antigen. The constant region located in the tail of Y is recognized and interacted with by the immune system. Target antigens generally have multiple binding sites called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, one antigen may have one or more corresponding antibodies.

In addition, the present invention includes functional variants of the antibody. Antibodies are considered functional variants of the antibodies of the invention if the variants can compete with the antibodies of the invention to specifically bind rabies virus or its G protein. Functional variants include, but are not limited to, derivatives having substantially similar primary structural sequences, including, for example, in vitro or in vivo modifications, chemicals and / or biochemicals. However, they are not found in the parental monoclonal antibodies of the invention. Such modifications include, for example, acetylation, acylation, covalent bonds of nucleotides or nucleotide derivatives, covalent bonds of lipids or lipid derivatives, crosslinking, disulfide bond formation, glycosylation, hydroxide, methylation, oxidation, PEGylation, proteolysis And phosphorylation and the like. Functional variants may optionally be antibodies comprising an amino acid sequence containing substitutions, insertions, deletions or combinations of one or more amino acids in comparison to the amino acid sequence of the parent antibody. Moreover, functional variants may include truncated forms of amino acid sequences at one or both of the amino terminus or carboxy terminus. The functional variant of the invention may have the same or different, higher or lower binding affinity compared to the parent antibody of the invention, but can still bind to rabies virus or its G protein. For example, the amino acid sequence of the variable region, including but not limited to, a framework, Hypervariable region, particularly the CDR 3 region, may be modified. Generally, the light or heavy chain region comprises three hypervariable regions, including three CDR regions, and a more conserved region, the framework region (FR). Hypervariable regions include amino acid residues from CDRs and amino acid residues from hypervariable loops. Functional variants within the scope of the present invention are about 50% -99%, about 60% -99%, about 80% -99%, about 90% -99%, about 95% -99%, Or about 97% -99% amino acid sequence identity. Gap or Bestfit known to those skilled in the art can be used in computer algorithms to optimally arrange the amino acid sequences to be compared and define similar or identical amino acid residues. The functional variant can be changed by or obtained by known general molecular biology methods including but not limited to PCR methods, mutagenesis using oligomeric nucleotides, and partial mutagenesis.

On the other hand, the rabies virus may be derived from any one selected from the group consisting of dogs, cattle, mongoose, bats, skunks, raccoons, coyotes, foxes and wolves, but is not limited thereto.

In addition, another embodiment of the present invention provides an immunoconjugate in which at least one tag is additionally bound to the binding molecule. For example, a drug may be further attached to the antibody according to the invention. That is, the antibody according to the present invention can be used in the form of an antibody-drug conjugate to which a drug is bound. The use of antibody-drug conjugates (ADCs), or immunoconjugates, for topical delivery of drugs enables targeted delivery of the drug moiety to infected cells. Unacceptable levels of toxicity can result. Increasing the selectivity of polyclonal and monoclonal antibodies (mAb) as well as drug-linking and drug-releasing properties can improve the maximal efficacy and minimal toxicity of ADC.

Conventional means of attaching the drug moiety to the antibody, ie, via covalent linkage, generally results in a heterogeneous molecular mixture in which the drug moiety is attached to numerous sites on the antibody. For example, cytotoxic drugs can typically be conjugated with an antibody, often through numerous lysine residues of the antibody, to produce a heterogeneous antibody-drug conjugate mixture. Depending on the reaction conditions, such heterogeneous mixtures typically have an antibody distribution between 0 and about 8 or more attached to the drug moiety. In addition, each subgroup of conjugates involving a particular integer ratio of drug moiety to antibody is a potentially heterogeneous mixture in which the drug moiety is attached to various sites on the antibody. Antibodies are large, complex, structurally diverse biomolecules, and often have many reactive functional groups. Reactivity with linker reagents and drug-linker intermediates depends on factors such as pH, concentration, salt concentration and cosolvent.

In addition, another embodiment of the present invention provides a nucleic acid molecule encoding the binding molecule.

Nucleic acid molecules of the invention include all nucleic acid molecules in which the amino acid sequence of an antibody provided herein is translated into a polynucleotide sequence, as known to those skilled in the art. Therefore, various polynucleotide sequences can be prepared by an open reading frame (ORF), all of which are also included in the nucleic acid molecules of the present invention.

In addition, another embodiment of the present invention provides an expression vector into which the nucleic acid molecule is inserted.

As the expression vector, Celltrion's unique expression vector MarEx vector (see Korean Patent Registration No. 10-1076602) and commercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, Ti vectors; Cosmid; Phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμ, T-even, T2, T3, T7; It is preferable to use an expression vector selected from any one selected from the group consisting of plant viruses, but not limited thereto. All expression vectors known to those skilled in the art can be used in the present invention, and when selecting an expression vector, a target host may be selected. It depends on the nature of the cell. The introduction of the vector into the host cell may be performed by calcium phosphate transfection, viral infection, DEAE-dextran controlled transfection, lipofectamine transfection or electroporation, but is not limited thereto. An introduction method suitable for the expression vector and the host cell can be selected and used. Preferably, the vector contains one or more selection markers, but is not limited thereto, and may be selected depending on whether the product is produced using a vector that does not include the selection marker. The selection of the selection marker is chosen by the host cell of interest, which uses methods already known to those skilled in the art and the present invention is not so limited.

In order to facilitate purification of the binding molecules of the present invention, a tag sequence may be inserted and fused to an expression vector. The tag may include, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag. Any tag that facilitates purification known to those skilled in the art may be used in the present invention.

In addition, another embodiment of the present invention provides a cell line wherein the expression vector is transformed into a host cell, thereby binding to rabies virus to produce a binding molecule having a neutralizing ability.

In the present invention, the cell line may include, but is not limited to, cells of mammalian, plant, insect, fungal or cellular origin. The mammalian cells include any one selected from the group consisting of CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK 293 cells and HEK293T cells. It is preferable to use one as a host cell, but is not limited thereto, and all cells usable as mammalian host cells known to those skilled in the art are available.

In addition, another embodiment of the present invention provides a pharmaceutical composition for treating and preventing rabies further comprising the binding molecule and a pharmaceutically acceptable excipient.

In addition, another embodiment of the present invention provides a rabies diagnostic composition further comprising the binding molecule and a pharmaceutically acceptable excipient.

Compositions of the present invention may include pharmaceutically acceptable excipients in addition to binding molecules having the ability to neutralize rabies virus. Pharmaceutically acceptable excipients are well known to those skilled in the art.

In addition, the prophylactic and therapeutic compositions of the present invention may include at least one other rabies therapeutic agent, and may also include several types of monoclonal antibodies, thereby exhibiting a synergistic effect on neutralizing activity.

In addition, the prophylactic and therapeutic composition of the present invention may further include at least one other therapeutic or diagnostic agent. Such therapeutic agents include, but are not limited to, anti-viral agents. Such agents can be antibodies, small molecules, organic or inorganic compounds, enzymes, polynucleotide sequences, anti-viral peptides, and the like.

The prophylactic and therapeutic compositions of the present invention are sterile and stable under the conditions of manufacture and storage, and may be in powder form for reconstitution in a suitable pharmaceutically acceptable excipient upon or prior to delivery. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and lyophilization, which produce further desired components from the powder of the active ingredient and its presterilized-filtered solution. The compositions of the present invention may be in solution and may be added and / or mixed before or at the time of delivery of the appropriate pharmaceutically acceptable excipient to provide a unit dosage injectable form. Preferably the pharmaceutically acceptable excipients used in the present invention are suitable for drug concentration, can maintain a suitable flowability, and can be delayed if necessary.

Selection of the optimal route of administration of the prophylactic and therapeutic compositions of the present invention involves several factors including the relationship of the physico-chemical properties of the active molecules in the composition, the urgency of the clinical situation and the plasma concentration of the active molecule to the desired therapeutic effect Affected by For example, monoclonal antibodies of the invention can be prepared with carriers that prevent their rapid release, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used in the present invention. Monoclonal antibodies can also be coated with or administered with a substance or compound that prevented the inactivation of the antibody. For example, monoclonal antibodies can be administered with a suitable carrier—liposomes or diluents.

Methods of administering the prophylactic and therapeutic compositions of the invention can be divided orally and parenterally, and the preferred route of administration is intravenous but not limited thereto.

The oral forms include tablets, troches, medicinal drops, aqueous or oily suspensions, powders or dispersible granules, emulsions, rigid capsules, soft gelatin capsules, syrups or elixirs, pills, dragees, solutions, gels or It may be formulated as a slurry. These formulations include, but are not limited to, pharmaceutical excipients containing inert diluents, granulating or disintegrating agents, binders, brightening agents, preservatives, colorants, flavoring or sweetening agents, vegetable or mineral oils, wetting agents and thickeners.

The parenteral form may be in the form of an aqueous or non-aqueous isotonic sterile non-toxic injection or infusion solution or suspension. The solution or suspension may be a drug such as 1,3-butanediol, Ringus's solution, Hanks' solution, isotonic sodium chloride solution, oils, fatty acids, local anesthetics, preservatives, buffers, viscosity or solubility that are nontoxic to the receptor at the dosage and concentration applied. Agents, water soluble antioxidants, oil soluble antioxidants and metal chelating agents.

It is preferable that the binding molecule of the present invention used in the diagnostic composition is detectably labeled. Various methods available for labeling biomolecules are well known to those skilled in the art and are contemplated within the scope of the present invention. Examples of marker types that can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds and bioluminescent compounds. Commonly used markers include phosphors (eg, fluresin, rhodamine, Texas red, etc.), enzymes (eg, horseradish peroxidase, β-galactosidase, alkaline phosphatase), radioisotopes (eg, 32P or 125I), biotin, digoxigenin, colloidal metals, chemiluminescent or bioluminescent compounds (eg dioxetane, luminol or acridinium). Labeling methods such as covalent binding of enzymes or biotinyl groups, iodide methods, phosphorylation methods, biotinylation methods and the like are well known in the art. Detection methods include, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzyme reactions, and the like. Commonly used detection assays include radioisotopes or non-radioisotope methods. These include Western blotting, overlay-assay, Radioimmuno Assay (RIA) and Immunity Radioimmunometric Assay (IRMA), Enzyme Immuno Assay (EIA), Enzyme Linked Immuno Sorbent Assay (ELISA), Fluorescent Immuno Assay (CIA) and Chemiluminoluminescent Immune Assay).

In addition, another embodiment of the present invention

a) said binding molecule; And

b) containers

It provides a rabies diagnostic kit comprising a.

In addition, another embodiment of the present invention

a) said binding molecule; And b) a container

It provides a kit for the treatment and prevention of rabies comprising.

In addition, another embodiment of the present invention

a) contacting the sample with the binding molecule; And

b) determining whether rabies infection is analyzed by analyzing the result of step a)

It provides a rabies diagnostic method comprising a.

In addition, another embodiment of the present invention

a) contacting the sample with the binding molecule; And

b) detecting the reaction of the binding molecule with the sample of interest

It provides a method for providing information for the diagnosis of rabies comprising.

In addition, another embodiment of the present invention provides a method for treating and preventing rabies, comprising administering to a subject a therapeutically effective amount of said binding molecule. For example, immunity to rabies virus can be conferred within 1, 2, 3 or several days by administering the human monoclonal antibody of the present invention when traveling to a rabies risk zone.

In addition, another embodiment of the present invention

a) culturing the cell line; And

b) recovering the expressed binding molecule

Provides a method of producing a binding molecule having a neutralizing ability by binding to a rabies virus comprising a.

In addition, another embodiment of the present invention

a) contacting the sample with the binding molecule; And

b) determining whether the binding molecule specifically binds to the sample of interest

It provides a method for detecting a rabies virus comprising a.

In the rabies virus detection method of the present invention, the subject sample includes, but is not limited to, blood, serum, saliva, sputum, saliva, sweat, tissue or other biological material from a (potentially) infected subject. Sample preparation is possible by the following method. The (potential) subject of infection may be a human subject, but may also be animals suspected of being a carrier of rabies virus. The subject sample may first be manipulated to make it more suitable for the detection method. Preferably, the binding molecule or immunoconjugate of the invention is contacted with the subject sample under conditions that allow the formation of an immunological complex between the binding molecule and the rabies virus or its antigenic component present in the subject sample. Formation of immunological complexes indicative of the presence of rabies virus in the subject sample is detected and measured by appropriate means. Such methods include, but are not limited to, immunoassays such as radioimmunoassay (RIA), ELISA, immunofluorescence, immunohistochemistry, FACS, BIACORE, Western blot analysis.

In addition, another embodiment of the present invention

According to the Kabat method,

a) a variable region comprising a CDR1 region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR3 region of SEQ ID NO: 3, and a CDR1 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5, and a CDR3 of SEQ ID NO: 6 Binding molecules comprising a variable region comprising a region;

b) a variable region comprising a CDR1 region of SEQ ID NO: 7, a CDR2 region of SEQ ID NO: 8, and a CDR3 region of SEQ ID NO: 9, and a CDR1 region of SEQ ID NO: 10, a CDR2 region of SEQ ID NO: 11, and a CDR3 of SEQ ID NO: 12 Binding molecules comprising a variable region comprising a region;

c) a variable region comprising a CDR1 region of SEQ ID NO: 13, a CDR2 region of SEQ ID NO: 14, and a CDR3 region of SEQ ID NO: 15, and a CDR1 region of SEQ ID NO: 16, a CDR2 region of SEQ ID NO: 17, and a CDR3 of SEQ ID NO: 18 Binding molecules comprising a variable region comprising a region; And

d) a variable region comprising a CDR1 region of SEQ ID NO: 19, a CDR2 region of SEQ ID NO: 20, and a CDR3 region of SEQ ID NO: 21, and a CDR1 region of SEQ ID NO: 22, a CDR2 region of SEQ ID NO: 23, and a CDR3 of SEQ ID NO: 24 Binding molecules comprising variable regions comprising regions

Provided are compositions (hereinafter "cocktail compositions") comprising two or more binding molecules selected from the group consisting of:

In one embodiment, the cocktail composition is

According to the Kabat method,

a) a variable region comprising a CDR1 region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR3 region of SEQ ID NO: 3, and a CDR1 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5, and a CDR3 of SEQ ID NO: 6 Binding molecules comprising a variable region comprising a region; And

b) a variable region comprising a CDR1 region of SEQ ID NO: 7, a CDR2 region of SEQ ID NO: 8, and a CDR3 region of SEQ ID NO: 9, and a CDR1 region of SEQ ID NO: 10, a CDR2 region of SEQ ID NO: 11, and a CDR3 of SEQ ID NO: 12 Binding molecules comprising a variable region comprising a region.

In another embodiment, the cocktail composition is

According to the Kabat method,

a) a variable region comprising a CDR1 region of SEQ ID NO: 7, a CDR2 region of SEQ ID NO: 8, and a CDR3 region of SEQ ID NO: 9, and a CDR1 region of SEQ ID NO: 10, a CDR2 region of SEQ ID NO: 11, and a CDR3 of SEQ ID NO: 12 Binding molecules comprising a variable region comprising a region; And

b) a variable region comprising a CDR1 region of SEQ ID NO: 19, a CDR2 region of SEQ ID NO: 20, and a CDR3 region of SEQ ID NO: 21, and a CDR1 region of SEQ ID NO: 22, a CDR2 region of SEQ ID NO: 23, and a CDR3 of SEQ ID NO: 24 Binding molecules comprising a variable region comprising a region.

Hereinafter, the words used in the present invention are defined as follows.

As used herein, the term "binding molecule" refers to an intact immunoglobulin, or an immunoglobulin that binds to an antigen, including monoclonal antibodies, such as chimeric, humanized or human monoclonal antibodies, eg For example, it refers to a variable domain comprising an immunoglobulin fragment that competes with an intact immunoglobulin for binding to a rabies virus or a G protein (Glycoprotein) or fragment thereof outside the virus. Regardless of the structure, the antigen-binding fragment binds to the same antigen recognized by intact immunoglobulins. An antigen-binding fragment comprises two or more contiguous amino acid residues of the binding molecule, five or more contiguous amino acid residues, ten or more contiguous amino acid residues, and 15 or more contiguous amino acid residues. At least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 35 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, 70 or more contiguous amino acid residues, 80 or more contiguous amino acid residues, 90 or more contiguous amino acid residues, 100 or more contiguous amino acid residues, 125 or more contiguous amino acid residues, 150 or more contiguous amino acid residues, 175 or more contiguous amino acid residues, 200 At least 2 consecutive amino acid residues, or at least 250 consecutive amino acid residues It may comprise a peptide or polypeptide comprising the amino acid sequence.

Antigen-binding fragments are especially Fab, F (ab '), F (ab') ₂ , Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single- Chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, polypeptides containing one or more fragments of immunoglobulin sufficient to bind a particular antigen to the polypeptide, and the like It includes. The fragments may be produced synthetically or by enzymatic or chemical digestion of complete immunoglobulins or may be produced genetically by recombinant DNA techniques. Production methods are well known in the art.

As used herein, the term "pharmaceutically acceptable excipient" refers to an inert material that is combined into an active molecule, such as a drug, agent, or binding molecule, to produce an acceptable or convenient dosage form. Pharmaceutically acceptable excipients are nontoxic or are excipients that are acceptable to the recipient for their intended use, at least in the doses and concentrations in which the toxicity is used, and with other components of the formulation including drugs, preparations or binding powders. It is compatible.

As used herein, the term "therapeutically useful amount" refers to the amount of the binding molecule of the invention effective for the prophylaxis or treatment of or before or after exposure to the rabies virus.

The inventors of the present invention inoculated rabies vaccine to 15 healthy adult subjects and collected blood to separate PBMCs, and selected candidate antibodies through phage display and B cell sorting using the isolated PBMCs. After measuring the basic titers of the selected candidate antibodies, the antibodies showing a certain level of neutralization ability are representative of each continent and animal that are prevalent in the world at the Center for Disease Control (CDC). In vivo and in vitro experiments were performed on four antibodies that have been shown to neutralize rabies virus, and neutralization ability tests were performed on a variety of rabies viruses. It has been found that it can be usefully used to treat patients infected with rabies virus.

Binding molecules capable of neutralizing the rabies virus of the present invention have been found to possess neutralizing ability against various rabies viruses, and thus are useful for the treatment and prevention of rabies.

1 depicts human antibody expression vectors comprising the heavy and light chain genes of the present invention.

Figure 2 shows the ELISA results using the G-protein of rabies virus.

Figure 3 is a graph showing the survival rate of the mouse against the SV2 virus in animal experiments.

Hereinafter, the present invention will be described in detail with reference to Examples. The following examples are intended to illustrate the present invention in detail, but the present invention is not limited thereto.

실시예Example

실시예 1: 광견병 바이러스에 특이적인 인간 항체 클론의 선별Example 1: Screening for Human Antibody Clones Specific to Rabies Virus

실시예 1-1: 광견병 백신 예방접종자의 혈액으로부터 PBMC 분리Example 1-1 Isolation of PBMCs from Blood of Rabies Vaccine Vaccines

Volunteers in this example consisted of healthy adults vaccinated against the rabies vaccine, which was approved by the Institutional Review Board (IRB). Volunteers confirmed negative responses to other infectious viruses, ie VDRL and HBsAg, and negative responses to anti-HCV and anti-HIV antibodies. A total of the volunteers received rabies vaccination once a year, and those who had not been vaccinated previously received 3 shots. At this time, the vaccinated rabies vaccine was Verorab® (Sanofi Pasteur). Two weeks after the last inoculation, approximately 50 ml of whole blood was obtained and the peripheral blood mononuclear cells (PBMC) were isolated using the Ficoll-Paque PLUS (GE Healthcare) method.

The separated PBMCs were washed twice with phosphate buffer solution and then adjusted to a concentration of 1x10 ⁷ cells / ml with freezing medium (RPMI (Gibco, cat No: A10491-01): FBS: DMSO = 5: 4: 1). (Liquid Nitrogen Tank) was stored.

실시예 1-2: 항체 디스플레이된 파지 라이브러리(phage library) 제작Example 1-2 Preparation of Antibody Displayed Phage Library

Total RNA was extracted from the PBMC isolated in Example 1-1 using TriReagent (Molecular Research Center), and cDNA was synthesized using the SuperScriptTM First-Strand cDNA synthesis system (Invitrogen, USA).

For the construction of antibody libraries from synthesized cDNAs, see the literature ( Barbas C. et. Al . Phage display a laboratory manual. 2001. CSHL Press). In brief, the variable region of the light and heavy chains of the antibody was amplified by PCR (polymerase chain reaction) using high fidelity Taq polymerase (Roche) and degenerative primer set from the synthesized cDNA. Amplified DNA fragments were isolated by gel extraction kit (Qiagen) after 1% agarose gel electrophoresis. Using the isolated variable region fragment as a template, the variable heavy chain region and the light chain variable region were linked to each other and amplified by scFv-type genes. Separated. The amplified scFv gene was digested with SfiI (Roche) restriction enzyme for 12 hours and then isolated using 1% agarose gel electrophoresis and gel extraction kit (Qiagen). Phagemid vector was also cleaved with SfiI restriction enzyme, isolated, mixed with the scFv gene, T4 DNA ligase (Roche), and reacted at 16 ° C. for 12 hours. The reaction solution was mixed with ER2738 competent cell (Lucigen) and transformed by electroporation. The transformed ER2738 was added to VCSM13 helper phage (Agilent Technologies) for 12 hours after shaking culture to prepare a phage library.

실시예 1-3: 파지 라이브러리를 이용한 항체의 선별Example 1-3 Screening of Antibodies Using Phage Library

See Laboratory techniques in rabies (4 ^th edition, World Health Organization) for isolation of rabies virus G protein to be used to screen antibody fragments that bind to rabies virus G protein from phage libraries.

Briefly, rabies virus was incubated in BHK-21 or Vero cells for 3 or 4 days, after which culture medium was obtained, and the virus was concentrated using an ultracentrifuge. The concentrated virus isolated the G protein located on the surface of the virus using octyl beta glucopytanoside chemicals. The isolated protein body was quantitatively and qualitatively analyzed by ELISA.

The phage library culture prepared in Example 1-2 was centrifuged to remove host cells, 4% PEG and 0.5 M NaCl were added and centrifuged at 9000 rpm for 15 minutes to settle the phage and remove the supernatant. The precipitated phages were diluted in 1% BSA / TBS and placed in an antigen-binding ELISA plate and reacted at room temperature for 2 hours. After removing the reaction solution, the ELISA plate was washed with PBS containing 0.05% tween20, 60 μl of 0.1 M glycine-HCl (pH 2.2) was added to remove the antigen-binding phage, and 2 M Tris (pH 9.1) was used. Neutralized. The detached phage was infected with ER2738, incubated with VCSM13 helper phage, and used for the next panning. Candidate antibody segments were selected after two or three panning.

실시예 1-4: 파지 효소 면역분석(phage enzyme immunoassay)Example 1-4 phage enzyme immunoassay

After two pannings, a portion of the infected ER2738 was plated onto LB plates before adding helper phage to obtain colonies. The colonies formed in the culture medium were shaken and cultured. When the OD ₆₀₀ was 0.7 or more, VCSM13 helper phage was added and shaken at 37 ° C for at least 12 hours. The culture medium was centrifuged to remove host cells and a supernatant containing phage was prepared.

For phage enzyme immunoassay, G-protein was adsorbed on 96-well microtiter plates and 150 μl of 3% BSA / PBS was added and reacted at 37 ° C. for 1 hour. The prepared phage supernatant was diluted 1: 1 with 6% BSA / PBS and placed in each well and left at 37 ° C. for 2 hours. Each well was washed three times with PBS containing 0.05% Tween 20. Then, anti-M13 antibody labeled with horseradish peroxidase was added and allowed to stand at 37 ° C for 1 hour. Each well was washed three times with PBS containing 0.05% Tween 20, followed by adding ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid] -diammonium salt) and measuring the absorbance at 405 nm. Antibodies were selected.

실시예 1-5: FACS를 이용한 항체의 선별Example 1-5 Screening Antibodies Using FACS

After approval from the Institutional Review Board (IRB), 50 ml of whole blood was collected from healthy individuals who received the rabies vaccine. Whole blood collection was done within 7 days after vaccination and PBMC was isolated from whole blood using Ficoll-Plaque PLUS (GE Helthcare) method. The isolated PBMCs were stored under liquid nitrogen until the B cells were separated by FACS method.

In order to increase the antibody expression of thawed PBMC, 5.5 days of incubation was performed in a cell culture medium containing four cytokines (IL-4, IL-6, IL-21, CD40L) at a temperature of 37 ° C. and 5% CO ₂ . Flow cytometry was performed to isolate B cells expressing antibodies specific to rabies virus in cultured PBMCs. Flow cytometry was performed by labeling PBMCs with fluorescently labeled antibodies or antigens in the presence of an FcγR blocker, and then separating only the labeled cells using a flow cytometer (FACS aria II, BD biosciences) and placing them in 96-well PCR plates (applied biosystems). . Antibodies or antigens used for labeling are as follows.

TABLE 2

Antigens and Antibodies Used on Labels for Antibody Selection Using FACS

antigen	Antibodies	dyeing
FITC-labeled rabies virus G protein	APC-efluor 780 labeled anti human CD3 antibody (Ebioscience), PerCP-Cy5.5 labeled anti human CD19 antibody (Ebioscience), PE-Cy7 labeled anti human CD27 antibody (Ebioscience)	PI fluorescent staining

FITC-labeled rabies virus G protein was constructed to obtain B cells expressing rabies virus specific antibodies. The above-described purified rabies virus G protein was experimented manually using a FITC antibody labeling kit (Pierce) manufactured by Pierce, to obtain a FITC-labeled rabies virus G protein.

실시예 1-6: 단일 세포에서의 cDNA 합성 및 항체 유전자의 증폭Example 1-6 cDNA Synthesis in Single Cells and Amplification of Antibody Genes

CDNA was synthesized using a superscript III first strand synthesis system kit manufactured by Invitrogen in single cells separated from each well of a 96 well plate. Synthesis was performed according to the manual provided in the kit. The process of obtaining antibody genes from the synthesized cDNA was performed by modifying the method described in Thomas Tiller et al (Journal of Immunological Methods, 2008). In brief, the amplification of the heavy and light chain genes was carried out by PCR through the first PCR process, and the amplification of the heavy and the light chain genes was performed again using the PCR product obtained through the first PCR through the nested PCR. . After agarose electrophoresis of the amplified heavy and light chain genes, the bands of the heavy and light chain genes were identified, incised with a blade, the agarose fragment containing DNA was transferred to a 1.5ml tube, and the DNA was purified from agarose using a PCR purification kit (Qiagen). The bay was dissolved and purified. The purified and heavy and kappa light chains were cloned into a PCDNA3.1 vector containing a portion of the antibody after treatment with Not 1 and Age1 restriction enzymes. Lambda light chains were cloned into a modified PCDNA 3.1 vector containing a portion of the antibody after treatment with Age1, Xho1 restriction enzymes as well.

실시예 1-7: ELISA를 이용한 후보 항체의 선별Example 1-7 Screening for Candidate Antibodies Using ELISA

The genes of the candidate antibodies selected in Examples 1-4, 1-5, and 1-6 were expressed in animal cell expression vectors, followed by ELISA using the antibodies secreted into the culture medium. G-protein of rabies virus was attached to the ELISA plate and expressed antibody was added. After washing off the unbound antibody, candidate antibodies bound to the antigen were selected using an anti-human antibody conjugated with horseradish peroxidase.

As a result of ELISA using G-protein of rabies virus, candidates showing antigen-specific binding could be selected as shown in FIG. 2.

실시예 1-8: 바이러스 중화능을 이용한 후보 항체의 선별Example 1-8: Selection of Candidate Antibodies Using Virus Neutralization Ability

In Example 1-7, about 80 candidate antibodies showing some high binding properties were tested for basic viral activity using CVS-11.

Over 50 antibodies with a constant (1000 IU / mg) titer were selected. The antibodies thus selected were subjected to RFFIT for rabies virus worldwide, which is being stored by the Center for Disease Control (CDC). The US CDC has about 50 species of rabies viruses worldwide, and the list of viruses is shown in Table 3 below. Among the primary screenings, neutralization test was performed with about 6 viruses and the results are shown in Table 4. Y is an antibody with a neutralizing ability, N represents an antibody without neutralizing ability, and M represents an antibody with some neutralizing ability.

TABLE 3

US CDC-owned rabies virus list

No.	Rabies virus types	origin
One	CVS-11
2	Mongoose rsa	Mongoose, South Africa
3	CASK	Skunk, California, USA
4	Dog tune	Dog, Tunisia
5	dog gab	Dog, Gabon
6	TXFX	Gray fox, TX
7	Dog thai	Dog, Thailand
8	Dong son	Dog, Mexico
9	Phi 002	Human / dog, Philippines
10	DR MX	Bat, Mexico
11	DR Brazil	Bat, Brazil
12	phi dog	Human / dog, Philippines
13	WA Bat	Bat, Washington, USA
14	3860 Bat	Bat, California, USA
15	Dog arg	Dog, Argentina
16	TX SK	Skunk, Texas, USA
17	RAC	Raccoon, Georgia, USA
18	China 2005	Dog, China
19	Rv342 China	Cow / dog, China
20	TX Coyote	Coyote, Texas, USA
21	rv61	Human (ex dog), United Kingdom (ex India)
22	AL Bat	Bat, California, USA
23	LC NY	Bat, New York, USA
24	Bat ef	Bat, Pennsylvania, USA
25	C1434	Bat, Alabama, USA
26	ABV (SM 4476)	Australia Bat Virus
27	Wu ABLV	Australia Bat Lyssa Virus
28	AZ Bat	Bat, Arizona
29	VA 399	Bat, Virginia, USA
30	TN 410	Bat, Tennessee, USA
31	TN 132	Bat, Tennessee, USA
32	SK 4384	Skunk, Texas, USA
33	AK FX	Arctic Fox, Alaska, USA
34	857r	Raccoon dog, Russia, Far East
35	I-148	Dog, India
36	Mongoose PR	Mongoose, Puerto-Rico
37	Gray FX-AZ	Gray Fox, Arizona, USA
38	NC SK	Skunk, Wisconsin, USA
39	323R	Dog / Coyote, Texas, USA
40	RVHN	Human (ex wolf), Russia, Arctic
41	MI1625	Bat, Tennessee, USA
42	I-151	Dog, India
43	TN-269	Bat, Tennessee, USA
44	Sri lanka	Cow, Sri Lanka
45	Myotis	Bat, Washington, USA

Table 4

RFFIT results performed on US CDC (primary screen, 6 viruses, 50 antibodies)

Virus	Gabon Dog	China dog	CASK	TN 410 Bat	I-148 Dog	3860 CA Bat
mAb No.	Gabon Dog	China dog	CASK	TN 410 Bat	I-148 Dog	3860 CA Bat
One	Y	Y	Y	M	Y	Y
2	Y	Y	Y	N	Y	M
3	Y	Y	Y	Y	Y	Y
4	Y	Y	Y	Y	Y	Y
5	Y	Y	Y	Y	Y	Y
6	Y	Y	Y	Y	Y	Y
7	Y	Y	Y	Y	Y	M
8	Y	Y	Y	N	N	N
9	Y	Y	Y	N	Y	M
10	Y	Y	Y	Y	Y	Y
11	M	Y	Y	N	Y	Y
12	Y	M	Y	-	-	-
13	Y	Y	Y	-	-	-
14	Y	Y	Y	Y	Y	Y
15	Y	Y	Y	M	Y	Y
16	Y	Y	Y	N	Y	Y
17	Y	Y	N	N	Y	Y
18	Y	Y	N	N	Y	Y
19	Y	Y	N	N	Y	Y
20	Y	Y	Y	Y	Y	N
21	-	-	Y	Y	Y	Y
22	-	-	Y	N	Y	N
23	-	-	Y	N	Y	N
24	-	-	Y	N	Y	N
25	-	-	Y	N	Y	M
26	-	-	Y	Y	Y	Y
27	-	-	Y	M	Y	Y
28	-	-	Y	N	Y	Y
29	-	-	Y	N	Y	M
30	-	-	Y	N	Y	M
31	-	-	Y	N	Y	N
32	-	-	Y	N	Y	N
33	-	-	Y	N	Y	Y
34	-	-	Y	N	Y	Y
35	-	-	Y	M	Y	M
36	-	-	Y	N	Y	Y
37	-	-	Y	N	Y	M
38	-	-	M	N	Y	N
39	-	-	-	N	Y	M
40	-	-	-	N	Y	N
41	-	-	-	Y	Y	Y
42	-	-	-	Y	Y	Y
43	-	-	-	N	M	M
44	-	-	-	N	Y	Y
45	-	-	-	N	Y	Y
46	-	-	-	N	Y	M
47	-	-	-	N	Y	Y
48	-	-	-	N	Y	Y
49	-	-	-	Y	Y	Y
50	-	-	-	N	Y	M

Based on these results, 15 antibodies were selected, and the antibodies were tested for neutralization ability against 50 viruses (Table 3), and the results are shown in Table 5 below.

Table 5

Neutralization test of 15 antibodies

No.

Virus

Antibodies

One

2

3

4

5

6

7

8

9

10

11

12

13

14

15

One

Dog Tun Dog, Tunisia

o

2

TXFX Gray fox, TX

o

3

Dog son Dog, Mexico

o

4

DR MX TN / MX COW) Bat, Mexico

o

5

Dog Arg Dog, Argentina

o

x

6

TX SK Skunk, Texas, USA

o

7

rv61 Human (ex dog), UK (ex India)

o

x

o

x

o

8

857r Raccoon dog, Russia, Far East

o

-

9

Phi 002 (231) Human / dog, Philippines

o

-

10

WA Bat Bat, Washington, USA

o

-

11

LC NY Bat, New York, USA

x

o

x

12

Bat Ef Bat, Pennsyl-vania, USA

x

o

x

o

x

o

x

o

13

RAC Raccoon, Georgia, USA

x

o

x

o

x

o

14

TX Coyote Coyote, Texas, USA

o

x

o

x

15

Mongoose PR Mongoose, Puerto-Rico

o

x

16

I-151 Dog, India

o

x

o

17

Gabon dog Dog, Gabon

o

x

o

x

o

18

Sri Lanka Cow, Sri Lanka

o

19

Gray AZ fox Gray Fox, Arizona, USA

o

x

o

x

20

NC SK Skunk, Wisconsin, USA

o

21

China dog 2005 Dog, China

o

22

Dog thai Dog, Thailand

o

x

23

phi dog Human / dog, Philippines

o

x

o

24

Rv342 China Cow / dog, China

o

25

TN-269 Bat, Tennessee USA

x

o

x

o

x

o

x

26

Wu ABLV Australian bat lyssa, Genotype 7

o

27

ABV (SM 4476) Australian bat lyssa, Genotype 7

o

28

DR Brazil Bat, Brazil

x

o

x

o

x

o

x

29

AK FX Arctic Fox, Alaska, USA

o

30

323R Dog / Coyote, Texas, USA

o

x

31

TX SK 4384 Skunk, Texas, USA

o

32

ERA

o

33

I-148 Dog, India

o

x

o

x

o

x

o

34

CASK Skunk, California, USA

o

x

35

AZ Bat Bat, Arizona

o

x

o

x

o

x

36

3860 CA Bat Bat, California, USA

o

x

o

x

o

x

37

TN410 Bat, Tennessee, USA

x

o

x

o

x

38

AL Bat Bat, California, USA

o

x

o

39

EBLV 1 A09-3484 Genotype 5

o

x

o

40

EBLV 2 A03-4659 Genotype 6

x

o

x

o

x

o

x

o

41

Duvenhag eGenotype 4

o

x

o

x

o

x

42

TN 132 Bat, Tennessee, USA

x

o

x

o

x

43

Mongoose RSA Mongoose, South Africa

o

44

Myotis Bat, Washington, USA

o

45

C1434 Bat, Alabama, USA

o

x

o

46

VA 399 Bat, Virginia, USA

x

o

x

o

x

o

x

47

MI1625 Bat, Tennessee, USA

o

x

o

x

Of these, four clones of Tables 3, 5, 10, and 11 (hereinafter referred to as “This Antibody 1”, “This Antibody 2”, “This Antibody 3”) ”,“ This is Antibody 4 ”). In Table 6 below, conformational epitope means a binding site having a tertiary protein structure.

Table 6

Neutralizing Ability and Epitope of Four Finally Selected Human Antibodies

ID (No.)	Neutralization capacity (IU / mg)	Epitope
This is Antibody 1 (No. 3 in Table 5)	7353	3 (Conformational)
This is Antibody 2 (No. 5 in Table 5)	7941	New site
This is Antibody 3 (No. 10 in Table 5)	5294	New site
This is Antibody 4 (No. 11 in Table 5)	2059	2 (Conformational)

실시예 2: 인간 항체 발현 벡터 제작Example 2: Construction of Human Antibody Expression Vectors

PCR2.1 TA cloning vectors containing the obtained heavy and light chains were treated with the restriction enzymes Nhe I and Pme I to obtain the heavy and light chain genes, and then the obtained heavy and light chain genes were respectively treated with the same restriction enzyme. pCT145 and pCT146 vectors were inserted. The pCT145 and pCT146 vectors are Celltrion-specific vectors designed to clone the heavy and light chains of antibodies, respectively. Subsequently, in order to construct an expression vector including a heavy chain transcription unit (promoter-heavy chain gene-polya) and a light chain transcription unit (promoter-light chain gene-polya), a restriction enzyme Pac I was added to the pCT145 vector including the heavy chain gene. After treating Asc I and heavy chain transcription units, the same restriction enzyme was inserted into the pCT146 vector containing the light chain gene to insert the heavy chain transcription unit. Then, using a restriction enzyme, a vector containing both a heavy chain and a light chain transcription unit was selected and named pCT188 (see FIG. 1, Korean Patent Registration No. 10-1076602, Patent Holder: Celltrion Co., Ltd.). The selected vector was extracted using Endofree plasmid maxi kit (QIAGEN, Germany, 12362), and finally the nucleotide sequence of the antibody was confirmed by sequencing using some of the extracted DNA.

실시예 3: 일시적 형질도입 방법에 의한 인간 항체의 생산Example 3: Production of Human Antibodies by Transient Transduction Method

FreeStyle ^TM Max (Invitrogen, 16447-100), a cationic polymer, was used for transient transduction in cells, and transduction was performed according to the manufacturer's instructions. The day before transduction, centrifugation of F2N cells (refer to Korean Patent No. 10-1005967, patent holder: Celltrion) grown on EX-CELL 293 Serum free media (Sigma, 14571C: hereinafter referred to as "EX-CELL 293 medium"). After separation, the medium was replaced with FreeStyle293 serum free media (Gibco, 12338), and 50 ml (100 ml total) were inoculated using two 250 ml shaker flasks at a concentration of 0.8 × 10 ⁶ cells per ml. On the day of transfection, 125 μg of the pCT178 DNA containing the antibody gene and 125 μl of the FreeStyle ^™ Max reagent were diluted in 2 ml volume using OptiPRO SFM II (Invitrogen, 12309) medium, and the mixture was gently mixed. Immediately diluted FreeStyle ^™ Max reagent solution was mixed with a solution containing DNA, and then reacted at room temperature for 17 minutes. During the 17 min reaction at room temperature, the number of inoculated F2N cells to be used for transduction was measured, and the cell concentration was diluted to 1.0 × 10 ⁶ cells using the FreeStyle293 medium. After 17 minutes, transduction was performed by treating the F2N cells with a mixture solution of DNA and FreeStyle ^™ Max reagent. The day after transduction, the same amount of EX-CELL 293 medium was added to the transduced cells and cultured for 7 days to produce monoclonal antibodies.

실시예 4: 최종 선별된 인간 단일클론 항체의 중화효능 확인Example 4 Confirmation of Neutralizing Effect of Final Selected Human Monoclonal Antibodies

Four final screened antibodies were tested for in vitro neutralization of approximately 50 rabies viruses worldwide, which were performed via RFFIT. The results are shown in Table 7.

TABLE 7

Results of RFFIT of Human Monoclonal Antibodies

No.	virus	ID	This thing antibody 1	This is Antibody 2	This is Antibody 3	This is Antibody 4
No.	virus	Working conc. (Ug / ml)	0.20
One	Mongoose RSAMongoose, South Africa	Titer	320	280	1000	270
		IU / mL	9.1	8.0	28.6	7.7
		IU / mg	9143	8000	28571	7714
2	Dog TunDog, Tunisia	Titer	270	230	280	230
		IU / mL	2.7	2.3	2.8	2.3
		IU / mg	2700	2300	2800	2300
3	Dog thaiDog, Thailand	Titer	1300	1100	1300	320
		IU / mL	9.3	7.9	9.3	2.3
		IU / mg	9286	7857	9286	2286
4	Dog sonDog, Mexico	Titer	1100	390	1000	340
		IU / mL	8.8	3.1	8.0	2.7
		IU / mg	8800	3120	8000	2720
5	Phi 002 (231) Human / dog, Philippines	Titer	250	125	250	250
		IU / mL	2.5	1.3	2.5	2.5
		IU / mg	2500	1250	2500	2500
6	DR MXBat, Mexico	Titer	180	110	230	250
		IU / mL	1.7	1.0	2.2	2.4
		IU / mg	1714	1048	2190	2381
7	DR BrazilBat, Brazil	Titer	45	50	50	56
		IU / mL	1.8	2.0	2.0	2.2
		IU / mg	1800	2000	2000	2240
8	WA BatBat, Washington, USA	Titer	250	180	270	270
		IU / mL	2.0	1.4	2.2	2.2
		IU / mg	2000	1440	2160	2160
9	rv61Human (ex dog), UK (ex India)	Titer	280	230	270	200
		IU / mL	2.4	2.0	2.3	1.7
		IU / mg	2435	2000	2348	1739
10	AL BatBat, California, USA	Titer	1100	1000	540	1100
		IU / mL	7.6	6.9	3.7	7.6
		IU / mg	7586	6897	3724	7586
11	AZ BatBat, Arizona	Titer	1300	800	1100	125
		IU / mL	11.3	7.0	9.6	1.1
		IU / mg	11304	6957	9565	1087
12	phi dogHuman / dog, Philippines	Titer	280	250	230	75
		IU / mL	1.04	0.93	0.85	0.28
		IU / mg	5185	4630	4259	1389
13	Rv342 ChinaCow / dog, China	Titer	1300	900	1000	280
		IU / mL	2.4	1.6	1.8	0.5
		IU / mg	11818	8182	9091	2545
14	TX SK 4384Skunk, Texas, USA	Titer	250	145	170	75
		IU / mL	0.71	0.41	0.49	0.21
		IU / mg	3571	2071	2429	1071
15	AK FXArctic Fox, Alaska, USA Failed	Titer	270	110	75	60
		IU / mL	2.00	0.81	0.56	0.44
		IU / mg	10000	4074	2778	2222
16	Gray FX-AZ (AZ fox) Gray Fox, Arizona, USA	Titer	170	60	125	16
		IU / mL	5.67	2.00	4.17	0.53
		IU / mg	28333	10000	20833	2667
17	323RDog / Coyote, Texas, USA	Titer	625	280	280	60
		IU / mL	3.47	1.56	1.56	0.33
		IU / mg	17361	7778	7778	1667
18	RVHNHuman (ex wolf), Russia, Arctic	Titer	42	40	42	54
		IU / mL	0.31	0.30	0.31	0.40
		IU / mg	1556	1481	1556	2000
19	TN-269Bat, Tennessee, USA	Titer	390	320	360	145
		IU / mL	3.12	2.56	2.88	1.16
		IU / mg	15600	12800	14400	5800
20	China dog 2005 Dog, China	Titer	13	9	13	12
		IU / mL	0.46	0.32	0.46	0.43
		IU / mg	2321	1607	2321	2143
21	TN410Bat, Tennessee, USA	Titer	200	125	75	19
		IU / mL	5.33	3.33	2.00	0.51
		IU / mg	26667	16667	10000	2533
22	Dog ArgDog, Argentina	Titer	170	95	210	145
		IU / mL	0.43	0.24	0.53	0.36
		IU / mg	2125	1188	2625	1813
23	TX SK 4380Skunk, Texas, USA	Titer	70	56	70	50
		IU / mL	0.56	0.45	0.56	0.40
		IU / mg	2800	2240	2800	2000
24	RACRaccoon, Georgia, USA	Titer	145	75	70	95
		IU / mL	1.07	0.56	0.52	0.70
		IU / mg	5370	2778	2593	3519
25	TX CoyoteCoyote, Texas, USA	Titer	170	56	60	56
		IU / mL	1.00	0.33	0.35	0.33
		IU / mg	5000	1647	1765	1647
26	Mongoose PRMongoose, Puerto-Rico	Titer	56	54	125	54
		IU / mL	0.40	0.39	0.89	0.39
		IU / mg	2000	1929	4464	1929
27	I-151Dog, India	Titer	320	170	480	125
		IU / mL	1.88	1.00	2.82	0.74
		IU / mg	9412	5000	14118	3676
28	Sri LankaCow, Sri Lanka	Titer	50	40	65	54
		IU / mL	0.40	0.32	0.52	0.43
		IU / mg	2000	1600	2600	2160
29	Wu ABLVAustralian bat lyssa, Genotype 7	Titer	440	540	270	250
		IU / mL	0.68	0.83	0.42	0.38
		IU / mg	3385	4154	2077	1923
30	ABV (SM 4476) Australian bat lyssa, Genotype 7	Titer	210	85	170	54
		IU / mL	1.56	0.63	1.26	0.40
		IU / mg	7778	3148	6296	2000
31	3860 CA BatBat, California, USA	Titer	210	56	75	56
		IU / mL	1.50	0.40	0.54	0.40
		IU / mg	7500	2000	2679	2000
32	Gabon dogDog, Gabon	Titer	170	145	180	65
		IU / mL	1.06	0.91	1.13	0.41
		IU / mg	5313	4531	5625	2031
33	CVS-11	Titer	250	270	180	70
		IU / mL	1.47	1.59	1.06	0.41
		IU / mg	7353	7941	5294	2059
34	EBLV 1 A09-3484Genotype 5	Titer	540	230	180	56
		IU / mL	5.40	2.30	1.80	0.56
		IU / mg	27000	11500	9000	2800
35	EBLV 2 A03-4659Genotype 6	Titer	280	200	270	54
		IU / mL	3.11	2.22	3.00	0.60
		IU / mg	15556	11111	15000	3000
36	DuvenhageGenotype 4	Titer	180	60	95	13
		IU / mL	7.20	2.40	3.80	0.44
		IU / mg	36000	12000	19000	2200
37	EBLV 1 A09-3485Genotype 5	Titer	180	75	95	50
		IU / mL	6.67	2.78	3.52	1.85
		IU / mg	33333	13889	17593	9259
38	EBLV 2 A09-3483 Genotype 6	Titer	125	70	125	50
		IU / mL	5.56	3.11	5.56	2.22
		IU / mg	27778	15556	27778	11111
39	CASKSkunk, California, USA	Titer	54	54	56	50
		IU / mL	0.43	0.43	0.45	0.40
		IU / mg	2160	2160	2240	2000
40	Bat EfBat, Pennsylvania, USA	Titer	50	42	50	50
		IU / mL	1.05	0.88	1.05	1.05
		IU / mg	5263	4421	5263	5263
41	C1434Bat, Alabama, USA	Titer	36	14	40	19
		IU / mL	0.96	0.37	1.07	0.51
		IU / mg	4800	1867	5333	2533
42	VA 399Bat, Virginia, USA	Titer	50	50	42	10
		IU / mL	2.78	2.78	2.33	0.56
		IU / mg	13889	13889	11667	2778
43	TN 132Bat, Tennessee, USA	Titer	280	180	170	56
		IU / mL	10.00	6.43	6.07	2.00
		IU / mg	50000	31243	30357	10000
44	TXFX Gray fox, TX	Titer	250	125	110	56
		IU / mL	1.85	0.93	0.81	0.41
		IU / mg	9259	4630	4074	2074
45	I-148Dog, India	Titer	10	11	10	13
		IU / mL	0.40	0.44	0.40	0.52
		IU / mg	2000	2200	2000	2600
46	LC NYBat, New York, USA	Titer	45	42	50	11
		IU / mL	2.14	2.00	2.38	0.52
		IU / mg	10714	10000	11905	2619
47	857rRaccoon dog, Russia, Far East	Titer	54	56	56	56
		IU / mL	2.70	2.80	2.80	2.80
		IU / mg	2700	2800	2800	2800
48	NC SKSkunk, Wisconsin, USA	Titer	270	250	280	280
		IU / mL	2.16	2.00	2.24	2.24
		IU / mg	2160	2000	2240	2240
49	MI1625Bat, Tennessee, USA	Titer	70	45	50	11
		IU / mL	2.50	1.61	1.79	0.39
		IU / mg	12500	8036	8929	1964
50	MyotisBat, Washington, USA	Titer	280	280	270	230
		IU / mL	2.00	2.00	1.93	1.64
		IU / mg	10000	10000	9643	8214
51	ERA	Titer	250	60	145	50
		IU / mL	1.79	0.43	1.04	0.36
		IU / mg	8929	2143	5179	1786

실시예 5: 최종 선별된 인간 단일클론항체의 항원결합부위(antigenic site) 판별Example 5 Antigen Site Determination of Final Selected Human Monoclonal Antibodies

In order to know the antigen-binding site (antigenic site) of the selected antibody through the above experiment was conducted to find the escape mutant virus (escape mutant virus).

having infectivity of 10 ⁶ per ml gives reacted for CVS-11 virus a 96 well cell was subsequently diluted 1.5-fold in a culture plate at 0.5 ~ 1 IU / ml of antibodies and 37 ℃ 1 hour. After 1 hour of reaction, 2x10 ⁵ cells / ml of BHK cells were added and cultured for 3 days. After 3 days the virus was obtained, cells were fixed and stained for expression of Rabies Nucleocapsid protein for CVS-11 virus infection (Jenobiotech, 9061). In the second passage, the same experiment is repeated, increasing the amount of antibody to the virus obtained in the first passage. The passage information progressed for each antibody is shown in Table 8. In the first or second passages, virus 4-5 clones, which were found to be infected or increased incidence, even though the virus obtained from CVS-11 was found to be uninfected or in small amounts was gradually increased. After amplification, RNA was isolated using a QIAamp Viral RNA Mini kit (QIAGEN, 52904).

Table 8

Escape Mutation Virus Experiment

Antibodies	Passage 1	Passage 2	Passage 3	Amplification
This is Antibody 4	1 IU / ml	10 IU / ml	-	5 clones
This thing antibody 1	1 IU / ml	5 IU / ml	10 IU / ml	4 clones
This is Antibody 3	0.5 IU / ml	2 IU / ml	7 IU / ml	5 clones

RNA was used as a template to synthesize cDNA using SuperScript III First-strand Synthesis system for RT-PCR (Invitrogen, 18080-051), followed by amplification with Takara ExTaq (Takara, RR001A), followed by sequencing. Proceeded. As a result of sequencing, the epitope and mutated nucleotide and amino acid information of each antibody are shown in Table 9.

In the case of antibody 2, it was confirmed that it was unable to neutralize Mutant virus which has a mutation in amino acid 210 obtained through antibody 3, and thus it was found that the antigenic site is a new site like antibody 3.

Table 9

Antibody-binding site confirmation result

Antibodies	Epitope	Nucleotide Variation	Amino acid mutations	Amino acid position *
This is Antibody 4	II	GGA-> GTA	Gly (G)-> Val (V)	34
		GGA-> GAA	Gly (G)-> Glu (E)	34
		GGA-> AGA	Gly (G)-> Arg (R)	34
This thing antibody 1	III	TCA-> CCA	Ser (S)-> Pro (P)	331
This is Antibody 3	New site	GTG-> GAG	Val (V)-> Glu (E)	210
This is Antibody 3	New site	GAG-> GAT	Glu (E)-> Asp (D	413

* The amino acid position is the numbered position except for the signal peptide (Signal peptide) of the rabies virus G protein

실시예 6: CR4098 항체로 생성된 탈출바이러스에 대한 중화능력 확인Example 6: Confirmation of neutralizing ability against escape virus produced by CR4098 antibody

실시예 6-1. CR4098 항체에 대한 탈출바이러스 생성Example 6-1. Escape Virus Generation Against CR4098 Antibodies

DNA sequences for CR4098 antibodies (Crucell) were obtained from patents (see US7579446) and the National Center for Biotechnology Information (NCBI) database, and were cloned into pCT146 expression vectors (see Example 2) synthesized in the engineering (oil globules at Daejeon). Delivered to Celltrion. The sequence of the synthesized part was confirmed by restriction digestion analysis and DNA sequencing. CR4098 antibodies were produced in a short run fashion in the F2N78 cell line as in Example 3.

Then, an experiment was performed to generate an escape mutant virus for the antibody using CR4098.

having infectivity of 10 ⁶ per ml gives reacted for CVS-11 virus a 96 well cells for 1 hour at then successively 1.5-fold dilution of the culture plates 10 ~ 40 IU / ml of antibodies CR4098 and 37 ℃. After 1 hour reaction, add 2x10 ⁵ cells / ml of BHK cells and incubate for 3 days. After 3 days the virus was obtained, cells were fixed and stained for expression of Rabies Nucleocapsid protein for CVS-11 virus infection (Jenobiotech, 9061). In the second passage, the same experiment is repeated with the amount of CR4098 antibody added to the virus from the first passage. In the first or second passages, viruses 4-5 clones that were found to have developed or increased incidence, even though the virus obtained from CVS-11 was found to be uninfected or in small amounts, were gradually increased in CR4098 antibodies. After amplification, RNA was isolated using a QIAamp Viral RNA Mini kit (QIAGEN, 52904).

RNA was used as a template to synthesize cDNA using SuperScript III First-strand Synthesis system for RT-PCR (Invitrogen, 18080-051), followed by amplification with Takara ExTaq (Takara, RR001A), followed by sequencing. Proceeded. Sequencing results showed that the altered sequence of escaped mutant virus for the CR4098 antibody was in N336K.

실시예 6-2. CR4098 항체로 생성된 탈출바이러스에 대한 중화능력 실험Example 6-2. Neutralization test against escape virus produced by CR4098 antibody

With respect to escape virus (N336K) produced by the CR4098 antibody, it was confirmed whether the antibody of the present invention has a neutralizing ability.

Neutralizing ability test results, all antibodies of the present invention showed a neutralizing effect on the CR4098 antibody escape virus (N336K), it was confirmed that only CR4098 antibody has no neutralizing ability.

Table 10

Neutralization test results for escape virus produced by CR4098 antibody

Virus	Antibodies	IU / mg
CR4098 mutant (N336K)	CR4098	All infection
	This is Antibody 4	1352
	This thing antibody 1	3081
	This is Antibody 3	2336

실시예 7: 이 건 항체의 인도 분리 광견병 바이러스에 대한 중화 능력 시험Example 7 Test of Neutralization Ability of the Antibodies Against Indian Isolated Rabies Virus

7-1. in-vitro 실험 7-1. in-vitro experiment

This is the wild type virus of Table 11 in India using the antibodies 1, 3, 4, Mix 1 (this antibody 1 + this antibody 4) and Mix 2 (this antibody 1 + this antibody 3). The neutralization competence test (RFFIT) was performed. Experimental progress was performed at the National Institute Mental Health and Science (NIMHANS). Each antibody was tested in Neuro 2 a cells at a concentration of about 1 ug and 100 FFD ₅₀ of each virus below. RFFIT results showed neutralizing capacity for each virus in all five antibodies.

Table 11

Outbreak of Rabies Virus in India

Virus abbreviation	A virus isolated animal	Region where virus was isolated
SV1	Dog	Kerala, India
SV2	Dog	Kerala, India
SV3	Human	Karnataka, India
SV4	Human	Karnataka, India
SV5	Dog	Chennai, Tamilnadu, India
SV6	Dog	Chennai, Tamilnadu, India

7-2. in-vivo 실험7-2. in-vivo experiment

In vitro experiments were performed in mice following in vitro experiments, and as shown in Table 12, all antibodies in each animal test group showed high neutralizing ability. Ten mice were used in each animal experimental group, and 100 LD50 (in 0.1 mL) was used for the virus. Three hours after virus inoculation (muscle injection), each antibody (about 1 ug) was inoculated in the same place (muscle injection), after which the survival rate was observed up to 30 days. Figure 3 is a graph showing the survival rate of the mouse for the SV2 virus (SV1 ~ SV6) of the total six viruses in animal experiments.

Survival rates corresponding to each of the SV1 to SV6 viruses in this animal experiment are shown in Table 12.

Table 12

Neutralizing ability test for rabies virus in India

	SV1	SV2	SV3	SV4	SV5	SV6
This thing antibody 1	80	90	90	80	90	90
This is Antibody 3	90	90	100	90	90	100
This is Antibody 4	100	100	100	100	100	100
This is Antibody 1 + This is Antibody 4	100	100	100	100	100	100
This is Antibody 1 + This is Antibody 3	100	100	100	100	100	100
Control	0	0	0	0	0	0

While the present invention has been described with reference to exemplary embodiments, it is understood that those skilled in the art can make various changes without departing from the scope of the present invention, and that elements can be replaced by equivalents. You will know. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out the invention, but that the invention will include all embodiments falling within the scope of the appended claims.

Claims

Rabies has neutralizing activity when it contains wild type G protein, but when rabies virus contains G protein mutated to glycine (Vly), glutamine (Glu), or arginine (Arg) at position 34 Binding molecules that do not have neutralizing activity.
A binding molecule that has neutralizing activity when the rabies virus contains a wild type G protein, but has no neutralizing activity when the rabies virus contains a G protein in which serine at position 331 is mutated to proline (Pro).
Rabies virus has neutralizing activity when it contains wild type G protein, but rabies virus is mutated by valine at position 210 to glutamic acid (Glu) or at position 413 to glutamic acid (Glu) as aspartic acid (Asp) A binding molecule that does not have neutralizing activity when it contains a G protein.
The method according to any one of claims 1 to 3,

The G protein is a binding molecule, characterized in that the G protein of CVS-11.
The binding molecule of claim 4, wherein the wild type G protein comprises the amino acid sequence of SEQ ID NO: 81. 6.
The method of claim 1,

The binding molecule

According to the Kabat method,

a) a variable region comprising a CDR1 region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR3 region of SEQ ID NO: 3; And

b) a variable region comprising a CDR1 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5, and a CDR3 region of SEQ ID NO: 6

A binding molecule comprising a.
The method of claim 2,

The binding molecule

According to the Kabat method,

a) a variable region comprising a CDR1 region of SEQ ID NO: 7, a CDR2 region of SEQ ID NO: 8, and a CDR3 region of SEQ ID NO: 9; And

b) a variable region comprising a CDR1 region of SEQ ID NO: 10, a CDR2 region of SEQ ID NO: 11, and a CDR3 region of SEQ ID NO: 12

A binding molecule comprising a.
The method of claim 3,

The binding molecule

According to the Kabat method,

a) a variable region comprising a CDR1 region of SEQ ID NO: 13, a CDR2 region of SEQ ID NO: 14, and a CDR3 region of SEQ ID NO: 15; And

b) a variable region comprising a CDR1 region of SEQ ID NO: 16, a CDR2 region of SEQ ID NO: 17, and a CDR3 region of SEQ ID NO: 18

A binding molecule comprising a.
The method of claim 3,

The binding molecule

According to the Kabat method,

a) a variable region comprising a CDR1 region of SEQ ID NO: 19, a CDR2 region of SEQ ID NO: 20, and a CDR3 region of SEQ ID NO: 21; And

b) a variable region comprising a CDR1 region of SEQ ID NO: 22, a CDR2 region of SEQ ID NO: 23, and a CDR3 region of SEQ ID NO: 24

A binding molecule comprising a.
The method of claim 6,

The binding molecule is a binding molecule, characterized in that it comprises a variable region of SEQ ID NO: 25 and a variable region of SEQ ID NO: 26.
The method of claim 7, wherein

The binding molecule is a binding molecule, characterized in that it comprises a variable region of SEQ ID NO: 27 and a variable region of SEQ ID NO: 28.
The method of claim 8,

The binding molecule is a binding molecule, characterized in that it comprises a variable region of SEQ ID NO: 29 and a variable region of SEQ ID NO: 30.
The method of claim 9,

The binding molecule is a binding molecule, characterized in that it comprises a variable region of SEQ ID NO: 31 and a variable region of SEQ ID NO: 32.
The method of claim 10,

The binding molecule comprises a heavy chain of SEQ ID NO: 33 and a light chain of SEQ ID NO: 34.
The method of claim 11,

The binding molecule comprises a heavy chain of SEQ ID NO: 35 and a light chain of SEQ ID NO: 36.
The method of claim 12,

The binding molecule comprises a heavy chain of SEQ ID NO: 37 and a light chain of SEQ ID NO: 38.
The method of claim 13,

The binding molecule is a binding molecule, characterized in that it comprises a heavy chain of SEQ ID NO: 39 and a light chain of SEQ ID NO: 40.
The method according to any one of claims 1 to 17,

The binding molecule is characterized in that the binding molecule.
The method according to any one of claims 1 to 17,

The binding molecule is a binding molecule, characterized in that the Fab fragment, Fv fragment, diabody (diabody), chimeric antibody, humanized antibody or human antibody.
The method according to any one of claims 1 to 17,

The rabies virus is a binding molecule, characterized in that derived from any one selected from the group consisting of dogs, cows, mongoose, bats, skunks, raccoons, coyotes, foxes and wolves.
An immunoconjugate in which at least one tag is additionally bound to a binding molecule of any one of claims 1 to 17.
A nucleic acid molecule encoding the binding molecule of any one of claims 1 to 17.
An expression vector in which the nucleic acid molecule of claim 22 is inserted.
A cell line wherein the expression vector of claim 23 is transformed into a host cell to produce a binding molecule that binds to rabies virus and has a neutralizing ability.
The method of claim 24,

The host cell is any one selected from the group consisting of CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK 293 cells and HEK293T cells. Cell line, characterized in that.
A pharmaceutical composition for treating and preventing rabies further comprising the binding molecule of any one of claims 1 to 17 and a pharmaceutically acceptable excipient.
a) the binding molecule of any one of claims 1-17; And

b) containers

Rabies diagnostic kit comprising a.
a) the binding molecule of any one of claims 1-17; And

b) containers

Rabies treatment and prevention kit comprising a.
a) contacting a subject sample with a binding molecule of any one of claims 1 to 17; And

b) determining whether rabies infection is analyzed by analyzing the result of step a)

Rabies diagnostic method comprising a.
a) contacting a subject sample with a binding molecule of any one of claims 1 to 17; And

b) detecting the reaction of the binding molecule with the sample of interest

How to provide information for diagnosing rabies comprising.
18. A method of administering to a subject a therapeutically effective amount of the binding molecule of any one of claims 1 to 17.

Rabies treatment and prevention method comprising a.
a) culturing the cell line of claim 24; And

b) recovering the expressed binding molecule

A method of producing a binding molecule having a neutralizing ability by binding to a rabies virus comprising a.
a) contacting a subject sample with a binding molecule of any one of claims 1 to 17; And

b) determining whether the binding molecule specifically binds to the sample of interest

Method for detecting a rabies virus comprising a.