KR101739313B1 - A binding molecules capable of neutralizing rabies virus - Google Patents

Abstract

The present invention relates to a binding molecule capable of neutralizing rabies virus. More specifically, the binding molecule of the present invention is useful as a vaccine against rabies viruses derived from individuals such as dogs, cows, mongooses, bats, skunks, raccoons, coyotes, foxes and wolves , It can be used to treat patients infected with rabies viruses derived from a wide range of individuals.

Description

A BINDING MOLECULES CAPABLE OF NEUTRALIZING RABIES VIRUS "

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

Rabies is a common infectious disease of viral pathogens and affects not only wildlife and pets but also affects mammals, including humans, and causes acute brain diseases. It is a fatal disease that occurs almost once when it occurs, and it is known as the disease with the highest mortality rate with AIDS. These rabies are spread all over the world, with more than 10 million people being treated every year, with between 40,000 and 70,000 deaths each year.

Rabies are infected with saliva and blood. They usually become sick when they are infested with dogs or cats infected with rabies. It can also be infected by most mammals, including skunks and bats.

The rabies virus, after reaching the cranial nerves through the terminal nerve tissue of the body, shows actual onset symptoms. Originally, the blood brain barrier exists in the human brain to block external substances, so that viruses can not penetrate. However, rabies virus passes through the blood barrier through RVG (rabies virus glycoprotein) protein, (central nervous system) infects the brain.

In addition to symptoms similar to a cold in the early days, itching or fever in the bite area. As the rabies progresses, anxiety, aphthae (swelling of water and other fluids cause spasm of the muscles, severe pain, fear of water), fear of wind (wind makes the senses more sensitive), excitement, paralysis , And psychiatric disorders. It also causes hypersensitivity to sunlight. Two to seven days after these symptoms are observed, the nerves or muscles of the whole body are paralyzed and fall into a coma, resulting in respiratory failure.

Currently, vaccinations against rabies are available but no treatment is available. Treatment after exposure to rabies is post-cure treatment (post-exposure prophylaxis). The post-cure treatment consists of immediate local wound protection and antibody administration for passive immunization (anti-rabies antibody), and vaccination for active immunization. Currently developed anti-rabies antibodies include human derived rabies immunoglobulin (HRIG) and equine derived rabies immunoglobulin (ERIG). In the case of HRIG, the supply is not smooth and expensive, and polyclonal antibody is not effective per unit weight. Also, since it is derived from human blood, there is a high risk of potential infection of human-caused diseases such as HIV. Although ERIG is less expensive than HRIG, it is also less effective than HRIG, and its therapeutic efficiency is lower than that of HRIG. In addition, it can cause anaphylaxis because it is an antibody derived from horse, which is different from human. To overcome these drawbacks of poor supply and polyclonal antibodies, the use of monoclonal antibodies that neutralize rabies viruses has been proposed. In the 1980s, rabies-virus neutralizing murine monoclonal antibodies were developed (Schumacher CL et al ., J. Clin . Invest . Vol. 84, p. 971-975, 1989), the short half-life in the human body, the absence of the immune response by the antibody, and the induction of human anti-mouse antibody (HAMA) .

Therefore, for the effective rabies treatment, it is not derived from the blood and has a high safety against potential infection, and it is possible to produce and supply on a large scale through synthesis through cultivation. It is composed of only antibodies having efficacy, The development of human monoclonal antibodies is urgent.

A problem to be solved by the present invention is to provide a binding molecule capable of binding to rabies virus and having neutralizing ability.

Another problem to be solved by the present invention is to provide an immunoconjugate having one or more tags attached to the binding molecule.

Another object of the present invention is to provide a polynucleotide encoding the binding molecule.

Another object of the present invention is to provide an expression vector having a polynucleotide encoding the binding molecule inserted therein.

Another object of the present invention is to provide a cell line transformed with the above-mentioned expression vector.

Another object of the present invention is to provide a composition comprising the binding molecule.

Another object of the present invention is to provide a kit comprising the binding molecule.

Another object of the present invention is to provide a method for diagnosing rabies using the binding molecule.

Another object of the present invention is to provide a method for treating and preventing rabies using the binding molecule.

Another object of the present invention is to provide a method for producing a binding molecule of the present invention by culturing the cell line.

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-mentioned problems, one embodiment of the present invention is a method for preventing or treating a rabies virus, which comprises neutralizing activity when a rabies virus contains a wild type G protein, wherein the rabies of the rabies virus is valine, valine, glutamine Provides a binding molecule that does not have neutralizing activity when it contains a G protein that has been mutated to arginine (Arg). Here, the amino acid position is a numbered position excluding the signal peptide of the G protein. Hereinafter, the numbering of amino acid positions of the G protein is the same.

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

As an example, the binding molecule

According to the Kabat method,

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

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

, ≪ / RTI >

As an example, the binding molecule

According to the Kabat method,

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

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

, ≪ / RTI >

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

In another embodiment of the present invention, the rabies virus has a neutralizing activity when it contains a wild-type G protein, but when the rabies virus contains a G protein whose serine at position 331 is mutated to proline (Pro) Thereby providing a binding molecule having no neutralizing activity.

In one embodiment of the present invention, the binding molecule does not neutralize the escape virus, which is a serine (Ser) at position 331 of G protein, mutated into proline (Pro) antigenic site III.

As an example, the binding molecule

According to the Kabat method,

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

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

, ≪ / RTI >

As an example, the binding molecule

According to the Kabat method,

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

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

, ≪ / RTI >

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

In another embodiment of the present invention, the rabies virus has a neutralizing activity when it contains a wild-type G protein, but the rabies virus at the 210 position is substituted with glutamic acid (Glu) or the glutamic acid at position 413 ) Contains a G protein that is mutated to aspartic acid (Asp), it provides a binding molecule that does not have neutralizing activity.

In one embodiment of the invention, the binding molecule is selected from the group consisting of Val (Val) at position 210 of G protein is replaced with glutamic acid (Glu), or glutamate (Glu) at position 413 is mutated into aspartic acid (Escape virus), confirming that the epitope is in a new site of a previously unreported G protein (New site).

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

As an example, the binding molecule

According to the Kabat method,

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

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

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

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

And a variable region selected from the group consisting of:

As an example, the binding molecule

According to the Kabat method,

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

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

, ≪ / RTI >

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

As an example, the binding molecule

According to the Kabat method,

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

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

, ≪ / RTI >

As an example, the binding molecule may be a binding molecule comprising 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, which are generally known so far, are shown in 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

Meanwhile, in the present invention, the CDRs of the variable regions have been determined in a conventional manner according to a system designed by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest (5 ^th ), National Institutes of Health, Bethesda , ≪ / RTI > MD. (1991)). Although the CDR crystal used in the present invention uses the Kabat method, a binding molecule comprising a CDR determined according to other methods such as the IMGT method, Chothia method, AbM method and the like is 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 a Fab fragment, an Fv fragment, a diabody, a chimeric antibody, a humanized antibody, or a human antibody, but is not limited thereto. An embodiment of the present invention provides a fully human antibody that binds to rabies virus. As used herein, antibodies are used in their broadest sense and specifically include multispecific antibodies (e. G., Bispecific antibodies) formed from intact monoclonal antibodies, polyclonal antibodies, two or more intact antibodies, Lt; RTI ID = 0.0 > biologically < / RTI > Antibodies are proteins produced by the immune system that can recognize and bind specific antigens. In its structural aspect, the antibody typically has a Y-shaped protein consisting of four amino acid chains (two heavy chains and two light chains). Each antibody has two regions, mainly a variable region and a constant region. The variable region located at the distal end of the arm of Y binds 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 specific antigen. The constant region in the tail of Y is recognized and interacted by the immune system. Target antigens typically have multiple binding sites called epitopes, which are recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen can have one or more corresponding antibodies.

The present invention also 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 for specific binding to rabies virus or its G protein. Functional variants are not necessarily to be the primary structural sequence comprises a substantially similar derivatives, but limited to, for example, ex vivo (in vitro ) or in vivo ( in vivo variants, chemicals, and / or biochemicals, which are not found in the parental monoclonal antibodies of the invention. Such modifications include, for example, acetylation, acylation, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, crosslinking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, And phosphorylation. A functional variant may optionally be an antibody comprising an amino acid sequence that contains substitutions, insertions, deletions, or combinations of one or more amino acids compared to the amino acid sequence of the parent antibody. Moreover, functional variants may comprise truncated forms of the amino acid sequence at one or both of the amino terminus or the carboxy terminus. The functional variants of the present invention may have the same, different, higher or lower binding affinity as the parent antibody of the invention, but still bind to the rabies virus or its G protein. In one example, the amino acid sequence of the variable region, including but not limited to the framework structure, the hypervariable region, particularly the CDR3 region, can be modified. Generally, the light or heavy chain region comprises three hypervariable regions and more conserved regions, i.e., the framework regions (FR), which comprise three CDR regions. The hypervariable region comprises an amino acid residue from the CDR and an amino acid residue from the hypervariable loop. Functional variants falling within the scope of the present invention are those that comprise about 50% to about 99%, about 60% to about 99%, about 80% to about 99%, about 90% to about 99% Or about 97% to 99% amino acid sequence homology. Gap or Bestfit known to those skilled in the art of computer algorithms may be used to optimally align the amino acid sequences to be compared and to define similar or identical amino acid residues. Functional variants can be obtained by, but not limited to, altering the parent antibody or a portion thereof by known general molecular biology methods, including PCR methods, mutagenesis using oligonucleotides and partial mutagenesis, or by organic synthesis.

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

Yet another embodiment of the present invention provides an immunoconjugate wherein one or more tags are additionally attached to the binding molecule. For example, a drug may be further attached to an antibody according to the present invention. That is, the antibody according to the present invention can be used in the form of an antibody-drug conjugate conjugated with a drug. When an antibody-drug conjugate (ADC), that is, an immunoconjugate, is used for local delivery of a drug, the drug moiety can be targeted to the infected cells. If the drug is administered without conjugation, This can lead to unacceptable levels of toxicity. By increasing the selectivity of polyclonal and monoclonal antibodies (mAbs) as well as drug-linking and drug-releasing properties, the maximum efficacy and minimal toxicity of the ADC can be improved.

Conventional means of attaching a drug moiety to an antibody, i. E., Through a covalent bond, generally result in a heterogeneous molecular mixture in which the drug moiety is attached to a number of sites on the antibody. For example, a cytotoxic drug can typically be conjugated to an antibody, often through numerous lysine residues of the antibody, to produce a heterogeneous antibody-drug conjugate mixture. Depending on the reaction conditions, such a heterogeneous mixture typically has an antibody distribution of 0 to about 8 or more attached to the drug moiety. In addition, each aliquot of the conjugate with a specific integer ratio 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 and structurally diverse biomolecules, often with many reactive functional groups. Reactivity with linker reagents and drug-linker intermediates is dependent on such factors as pH, concentration, salt concentration and cosolvent.

Yet another embodiment of the present invention provides a nucleic acid molecule encoding said binding molecule.

The nucleic acid molecule of the present invention includes all of the nucleic acid molecules translated into a polynucleotide sequence as known to those skilled in the art by the amino acid sequence of the antibody provided herein. Therefore, various polynucleotide sequences by ORF (open reading frame) can be produced, and these are all included in the nucleic acid molecule of the present invention.

Another embodiment of the present invention provides an expression vector into which the nucleic acid molecule is inserted.

As the above expression vector, MarEx vector (refer to Korean Patent No. 10-1076602) and commercially widely used pCDNA vector, F, R1, RP1, Col, pBR322, ToL, Ti vector which is an expression vector unique to Celltrion; Cosmids; Lambda, lambdoid, M13, Mu, p1 P22, Qμ, T-even, T2, T3, T7 and the like; Plant virus, but it is not limited thereto. Any expression vector known to the person skilled in the art as an expression vector can be used in the present invention. In selecting an expression vector, a target host It depends on the nature of the cell. May be performed by calcium phosphate transfection, viral infection, DEAE-dextran controlled transfection, lipofectamine transfection, or electroporation methods, but not limited thereto, by those skilled in the art An expression vector and a suitable introduction method for the host cell can be selected and used. Preferably, the vector contains one or more selectable markers, but is not limited thereto, and a vector that does not include a selectable marker may also be used to select the product according to whether the product is produced or not. Selection of the selection marker is selected by the desired host cell, which uses the method already known to those skilled in the art, so the present invention is not limited thereto.

In order to facilitate the purification of the binding molecules of the present invention, the tag sequence can be inserted into the expression vector and fused. Such tags include, but are not limited to, hexa-histidine tags, hemagglutinin tags, myc tags, or flag tags, all of which are known to those skilled in the art and that facilitate purification.

In another embodiment of the present invention, there is provided a cell line transforming the expression vector into a host cell to produce a binding molecule having a neutralizing ability by binding to rabies virus.

In the present invention, the cell line can include, but is not limited to, cells of mammalian, plant, insect, fungal or cellular origin. The mammalian cells may be selected from the group consisting of CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, HT1080 cells, A549 cells, HEK 293 cells and HEK293T cells Although it is preferable to use one as a host cell, it is not limited thereto, and cells usable as a mammalian host cell known to a person skilled in the art are all available.

Another embodiment of the present invention provides a pharmaceutical composition for treating and preventing rabies, which further comprises the binding molecule and a pharmaceutically acceptable excipient.

Another embodiment of the present invention provides a rabies diagnostic composition further comprising the binding molecule and a pharmaceutically acceptable excipient.

The composition of the present invention may contain 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 composition for the prevention and treatment of the present invention may include at least one other rabies therapeutic agent, and may also include various kinds of monoclonal antibodies, thereby exhibiting a synergistic effect on the neutralizing activity.

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

The compositions for the prophylaxis and treatment of the present invention may be sterile and stable under the conditions of manufacture and storage and may be in powder form for reconstitution into suitable pharmaceutically acceptable excipients at the time of delivery or prior to delivery. In the case of bactericidal powders for the preparation of bactericidal injection solutions, the preferred method of preparation is vacuum drying and lyophilization to produce additional desired components from powders of active ingredients and their presterilized-filtered solutions. The compositions of the present invention may be in a solution form and suitable pharmaceutically acceptable excipients may be added and / or mixed prior to or during delivery to provide a unit dose scanning form. Preferably, the pharmaceutically acceptable excipients used in the present invention are suitable for the drug concentration, maintain proper flowability, and may delay the absorption if desired.

Optimal pathway selection for administration of the compositions for the prevention and treatment of the present invention depends on several factors, including the physico-chemical properties of the active molecules in the composition, the urgency of the clinical situation, and the relationship of the plasma concentration of the active molecule to the desired therapeutic effect Lt; / RTI > For example, the monoclonal antibodies of the invention may be formulated with a carrier that blocks 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 may be used in the present invention. Monoclonal antibodies may also be coated or co-administered with a substance or compound that inhibits the inactivation of the antibody. For example, monoclonal antibodies can be administered with suitable carrier-liposomes or diluents.

The method of administering the composition for prevention and treatment of the present invention can be divided into oral and parenteral routes, and the preferred route of administration is intravenous, but not limited thereto.

The oral form can be in the form of tablets, troches, medicinal drops, aqueous or oily suspensions, powders or dispersible granules, emulsions, rigid capsules, soft gelatine capsules, syrups or elixirs, tablets, dragees, Can be formulated as a slurry agent. These formulations include, but are not limited to, pharmaceutical excipients containing inert diluents, granulating or disintegrating agents, binders, polishes, preservatives, colorants, flavoring or sweetening agents, vegetable or mineral oils, wetting agents and thickening agents.

The parenteral form may be in the form of an aqueous or non-aqueous isotonic sterile non-toxic injection or infusion solution or suspension. Solutions or suspensions may be formulated with pharmaceutically acceptable additives such as agents, oils, fatty acids, topical anesthetics, preservatives, buffers, viscosity or solubility enhancement such as 1,3-butanediol, Ringer's solution, Hanks solution, isotonic sodium chloride solution, Soluble antioxidants, oil-soluble antioxidants, and metal chelating agents.

Preferably, the binding molecules of the invention used in the diagnostic composition are detectably labeled. Various methods that can be used to label biomolecules are well known to those skilled in the art and are contemplated within the scope of the present invention. Examples of marking classes that can be used in the present invention include enzymes, radioactive isotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds and bioluminescent compounds. Commonly used markers include fluorescent substances (e.g., fluorescein, rhodamine, Texas red, etc.), enzymes (such as horseradish peroxidase,? -Galactosidase, alkaline phosphatase), radioactive isotopes 32P or 125I), biotin, digoxigenin, colloidal metals, chemiluminescent or bioluminescent compounds (such as dioxetane, luminol or acridinium). Methods of labeling enzymes or biotinyl groups such as covalent bonding, iodination, phosphorylation, biotinylation, 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. Detection assays commonly used include radioisotope or non-radioisotope methods. Among them, they include Western blotting, overlay-analysis, RIA (Radioimmunoassay) and IRMA (Immune Radiometric Immunoassay), EIA (Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno Sorbent Assay), FIA (Fluorescent Immuno Assay) Assay).

In another embodiment of the present invention,

a) the binding molecule; And

b) container

The present invention also provides a rabies diagnostic kit.

In another embodiment of the present invention,

a) the binding molecule; And b)

The present invention provides a kit for treating and preventing rabies.

In another embodiment of the present invention,

a) contacting the binding partner with the target sample; And

b) analyzing the result of step a) to determine whether it is rabies-infected

The present invention also provides a method for diagnosing rabies.

In another embodiment of the present invention,

a) contacting the binding partner with the target sample; And

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

A method for providing information for rabies diagnosis.

Another embodiment of the present invention provides a method for treating and preventing rabies, comprising administering to the subject a therapeutically effective amount of the binding molecule. For example, administration of the human monoclonal antibody of the invention when traveling in a rabies-critical area may confer immunity against rabies virus within one, two, three or several days.

In another embodiment of the present invention,

a) culturing the cell line; And

b) recovering the expressed binding molecule

Which is capable of binding to rabies viruses containing a neutralizing ability.

In another embodiment of the present invention,

a) contacting the binding partner with the target sample; And

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

The present invention provides a method for detecting rabies virus.

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 (potentially) Sample preparation is possible. The (potentially) infected subject may be a human subject, but may also be suspected animals as carriers of rabies virus. The target sample can be manipulated first to make it more suitable for the detection method. Preferably, the binding molecules or immunogenic adjuvants of the invention are contacted with the subject sample under conditions that permit the formation of an immunological complex between the binding molecule and the rabies virus or its antigenic component present in the sample of interest. The formation of immunological complexes, which indicate the presence of rabies viruses in the sample of interest, 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 another embodiment of the present invention,

According to the Kabat method,

a) a variable region comprising the CDR1 region of SEQ ID NO: 1, the CDR2 region of SEQ ID NO: 2 and the CDR3 region of SEQ ID NO: 3, and the CDR1 region of SEQ ID NO: 4, the CDR2 region of SEQ ID NO: 5, A binding molecule comprising a variable region comprising a region;

b) a variable region comprising the CDR1 region of SEQ ID NO: 7, the CDR2 region of SEQ ID NO: 8, and the CDR3 region of SEQ ID NO: 9 and the CDR1 region of SEQ ID NO: 10, the CDR2 region of SEQ ID NO: 11, A binding molecule comprising a variable region comprising a region;

c) a variable region comprising the CDR1 region of SEQ ID NO: 13, the CDR2 region of SEQ ID NO: 14 and the CDR3 region of SEQ ID NO: 15 and the CDR1 region of SEQ ID NO: 16, the CDR2 region of SEQ ID NO: A binding molecule comprising a variable region comprising a region; And

d) a variable region comprising the CDR1 region of SEQ ID NO: 19, the CDR2 region of SEQ ID NO: 20 and the CDR3 region of SEQ ID NO: 21 and the CDR1 region of SEQ ID NO: 22, the CDR2 region of SEQ ID NO: 23, A binding molecule comprising a variable region comprising a region

(Hereinafter referred to as "cocktail composition").

In one embodiment, the cocktail composition comprises

According to the Kabat method,

a) a variable region comprising the CDR1 region of SEQ ID NO: 1, the CDR2 region of SEQ ID NO: 2 and the CDR3 region of SEQ ID NO: 3, and the CDR1 region of SEQ ID NO: 4, the CDR2 region of SEQ ID NO: 5, A binding molecule comprising a variable region comprising a region; And

b) a variable region comprising the CDR1 region of SEQ ID NO: 7, the CDR2 region of SEQ ID NO: 8, and the CDR3 region of SEQ ID NO: 9 and the CDR1 region of SEQ ID NO: 10, the CDR2 region of SEQ ID NO: 11, And a variable region comprising an < RTI ID = 0.0 > region. ≪ / RTI >

In another embodiment, the cocktail composition comprises

According to the Kabat method,

a) a variable region comprising the CDR1 region of SEQ ID NO: 7, the CDR2 region of SEQ ID NO: 8, and the CDR3 region of SEQ ID NO: 9 and the CDR1 region of SEQ ID NO: 10, the CDR2 region of SEQ ID NO: 11, A binding molecule comprising a variable region comprising a region; And

b) a variable region comprising the CDR1 region of SEQ ID NO: 19, the CDR2 region of SEQ ID NO: 20, and the CDR3 region of SEQ ID NO: 21 and the CDR1 region of SEQ ID NO: 22, the CDR2 region of SEQ ID NO: 23, And a variable region comprising an < RTI ID = 0.0 > region. ≪ / RTI >

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

The term "binding molecule" as used herein includes intact immunoglobulins comprising monoclonal antibodies, such as chimeric, humanized or human monoclonal antibodies, or immunoglobulin binding to an antigen, e. 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) outside thereof or a fragment thereof. Regardless of structure, the antigen-binding fragments bind to the same antigen recognized by the intact immunoglobulin. The antigen-binding fragment may comprise at least two contiguous amino acid residues of a binding molecule, at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, At least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 30 consecutive amino acid residues, at least 35 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, At least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, More than two consecutive amino acid residues, or more than 250 consecutive amino acid residues It may comprise a peptide or polypeptide comprising the amino acid sequence.

The antigen-binding fragments may in particular be selected from the group consisting of Fab, F (ab ') ₂ , Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single- Such as a polypeptide containing one or more fragments of an immunoglobulin sufficient to bind to a particular antigen to a polypeptide, such as a monoclonal antibody, a monoclonal antibody, a diabody, a triabody, a tetrabody, . The fragment may be produced synthetically or by enzymatic or chemical degradation of the complete immunoglobulin, or may be genetically engineered by recombinant DNA technology. Methods of generation are well known in the art.

As used herein, the term " pharmaceutically acceptable excipient "means an inert material combined with an active molecule, such as a drug, agent, or binding molecule to produce an acceptable or convenient dosage form. Pharmaceutically acceptable excipients are non-toxic or at least those excipients which are acceptable for their intended use in the recipient at the dosages and concentrations for which they are used and include other components of the formulation including drugs, Can be compatible.

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

The inventors of the present invention collected 15 rabbit vaccine in healthy adult subjects, collected the PBMCs, isolated PBMCs, and screened candidate antibodies by phage display and B cell sorting. The primary antibody titers of the selected candidate antibodies were measured, and antibodies exhibiting neutralizing ability above a certain level were collected from the Center for Disease Control (hereinafter referred to as "CDC"), representative of each continent and animal In vivo and in vitro experiments were carried out on four antibodies that were proved to be capable of neutralizing rabies virus, and the neutralization ability test was carried out on various rabies viruses. Thus, monoclonal antibodies of the present invention were obtained from a wide variety of individuals It has been confirmed that it can be useful for treating patients infected with rabies virus.

Since the binding molecule capable of neutralizing the rabies virus of the present invention has been confirmed to have neutralizing ability against various rabies viruses, it is useful for treating and preventing rabies.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows a human antibody expression vector comprising the heavy and light chain genes of the present invention.
Figure 2 shows ELISA results using rabbit virus G-protein.
3 is a graph showing the survival rate of the mouse against the SV2 virus in an animal experiment

Hereinafter, the present invention will be described in detail with reference to examples. The following examples are intended to illustrate the present invention without limiting it thereto.

Example

Example  1: Selection of human antibody clones specific for rabies virus

Example  1-1: From the blood of a rabies vaccine vaccine PBMC  detach

In this example, volunteers consisted of healthy adults who were vaccinated against rabies vaccines, which were approved by the IRB. Applicants have confirmed negative responses to other contagious viruses, VDRL and HBsAg, and negative responses to anti-HCV and anti-HIV antibodies. A total of three vaccinations were conducted for those who had been vaccinated with rabies one year before, and those who were not previously vaccinated. At this time, the vaccinated rabies vaccine was Verorab® (Sanofi Pasteur). Two weeks after the last inoculation, about 50 ml of whole blood was obtained and PBMC (peripheral blood mononuclear cells) were isolated using the Ficoll-Paque PLUS (GE Healthcare) method.

The separated PBMCs were washed twice with phosphate buffered saline and incubated at a concentration of 1 x ¹⁰ cells / ml in a freezing medium (RPMI (Gibco, cat No: A10491-01): FBS: DMSO = 5: 4: (Liquid Nitrogen Tank).

Example  1-2: Antibody-displayed phage library ( phage library Production

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

Production of the antibody library from the synthesized cDNA was reference to the Prior Art (Barbas C. et. Al. Phage display a laboratory manual. 2001. CSHL Press). Briefly, high frequency fidelity Taq polymerase (Roche) and degenerative primer sets were used to amplify the variable regions of the light and heavy chains of the antibody by polymerase chain reaction (PCR). The amplified DNA fragments were separated using gel extraction kit (Qiagen) after 1% agarose gel electrophoresis. Using the separated variable region fragment as the template, the overlapping PCR method was used to construct the scFv-type gene by linking the variable region of the antibody heavy chain to the light chain. The gene was amplified using 1% agarose gel electrophoresis and gel extraction kit (Qiagen) Respectively. The amplified scFv gene was digested with SfiI (Roche) restriction enzyme for 12 hours and then separated using 1% agarose gel electrophoresis and gel extraction kit (Qiagen) method. The phagemid vector was cleaved with SfiI restriction enzyme, and then the scFv gene was mixed with T4 DNA ligase (Roche), followed by reaction at 16 ° C for 12 hours. The reaction mixture was mixed with ER2738 competent cell (Lucigen) and transformed by electroporation method. Transfected ER2738 was cultured in shaking, and VCSM13 helper phage (Agilent Technologies) was added and cultured for 12 hours to prepare phage library.

Example  1-3: Screening of Antibodies Using Phage Library

The isolation of rabies virus G protein to be used for screening antibody fragments binding to rabies virus G protein from phage libraries was made in laboratory techniques in rabies (4 ^th edition, World Health Organization).

Briefly, rabies virus was cultured in BHK-21 or Vero cells for three to four days, and then the culture medium was obtained and the virus was concentrated using an ultracentrifuge. The enriched virus was isolated from the G protein on the virus surface using octyl beta glucopytanoside chemistry. The separated protein bodies were quantitatively and qualitatively analyzed by ELISA.

The phage library culture prepared in Example 1-2 was centrifuged to remove the host cells, and 4% PEG and 0.5 M NaCl were added thereto, followed by centrifugation at 9000 rpm for 15 minutes to precipitate the phage and remove the supernatant. The precipitated phage were diluted in 1% BSA / TBS and placed in an ELISA plate coupled with the antigen and reacted at room temperature for 2 hours. After removing the reaction solution, the ELISA plate was washed with PBS containing 0.05% tween 20, and then 60 μl of 0.1 M glycine-HCl (pH 2.2) was added to remove the phage bound to the antigen. The phage was removed using 2 M Tris Neutralized. The removed phage were infected with ER2738 and then cultured in a VCSM13 helper phage to be used for next panning. After 2 or 3 panning, candidate antibody fragments were selected.

Example  1-4: Phage Enzyme Immunoassay ( phage enzyme immunoassay )

A portion of ER2738 infected after two rounds of panning was plated on LB plates to obtain a colony before helper phage was added. The formed colonies were shake cultured. When OD ₆₀₀ reached 0.7 or more, VCSM13 helper phage was added and cultured at 37 占 폚 for 12 hours or more with shaking. The culture was centrifuged to remove the host cells and a supernatant containing the phage was prepared.

For phage enzyme immunoassay, G-protein was adsorbed on a 96-well microtiter plate 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, placed in each well, and allowed to stand at 37 DEG C for 2 hours. Each well was washed three times with PBS containing 0.05% Tween 20, and incubated with anti-M13 antibody labeled with horseradish peroxidase for 1 hour at 37 ° C. Each well was washed three times with PBS containing 0.05% Tween 20. ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid] -diammonium salt) was added and the absorbance at 405 nm was measured. Antibodies were selected.

Example  1-5: FACS Screening of antibodies using

After receiving approval from the Clinical Review Board (IRB), 50 ml of whole blood was collected from a healthy human who received the rabies vaccine. The whole blood collection period was within 7 days after vaccination and PBMC were isolated from whole blood using Ficoll-Plaque PLUS (GE Helthcare) method. Isolated PBMCs were stored under liquid nitrogen until FACS method isolation of B cells.

The cells were incubated at 37 ° C and 5% CO ₂ for 5.5 days in cell culture medium containing four cytokines (IL-4, IL-6, IL-21, CD40L) to enhance antibody expression of thawed PBMCs. Flow cytometry was performed to isolate B cells expressing antibodies specific for rabies virus in cultured PBMCs. In flow cytometry, PBMCs were labeled with fluorescently labeled antibodies or antigens in the presence of FcγR blockers, and then only the labeled cells were isolated using a flow cytometer (FACS aria II, BD biosciences) and placed in a 96-well PCR plate (applied biosystems) . Antibodies or antigens used on the label are as follows.

Antigens and antibodies used for labeling for antibody selection using FACS antigen Antibody 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 - specific antibodies. The above-described purified rabies virus G protein was subjected to manual FITC antibody labeling kit (Pierce) manufactured by Pierce to obtain FITC-labeled rabies virus G protein.

Example  1-6: Single cell cDNA  Synthesis and amplification of antibody genes

CDNA was synthesized by using the superscript III first strand synthesis system kit manufactured by Invitrogen in a single cell of each well of 96 well plate. The synthetic method was carried out according to the manual provided in the kit. The procedure for obtaining the antibody gene from the synthesized cDNA was carried out by modifying the method described in Thomas Tiller et al (Journal of Immunological Methods, 2008). Briefly, the heavy and light chain genes were amplified by PCR through the first PCR procedure, and the second and third PCR amplifications were performed by nested PCR using the PCR product obtained by the first PCR as the template and the heavy and light chain genes were amplified again . The amplified heavy and light chain genes were subjected to agarose gel electrophoresis, and then the heavy and light chain gene bands were identified. The agarose sections containing the DNA were transferred to a 1.5 ml tube, And then purified. The purified DNA was labeled with Not 1, Age 1 After restriction enzyme treatment, the vector was cloned into a PCDNA3.1 vector containing a part of the antibody. Lambda light chain was cloned in the 3.1 PCDNA vector strain containing the portion of the antibody then similarly treated with Age1, Xho1 restriction enzymes.

Example  1-7: ELISA Screening for candidate antibodies using

The genes of the candidate antibodies selected in Examples 1-4, 1-5, and 1-6 were expressed in an animal cell expression vector and ELISA was performed using antibodies secreted from the culture. The rabies virus G-protein was attached to the ELISA plate and the expressed antibody was added. The unbound antibody was washed and the anti-human antibody conjugated with horseradish peroxidase was used to select a candidate antibody bound to the antigen.

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

Example  1-8: Virus Neutralizing ability  Selection of candidate antibodies used

In Example 1-7, about 80 candidate antibodies exhibiting some high binding characteristics were subjected to a basic virus activity lowering experiment using CVS-11.

We selected more than 50 antibodies with more than double activity (1000 IU / mg). The antibody thus selected was subjected to RFFIT on the worldwide rabies virus stored in the Center for Disease Control (CDC). The US CDC has about 50 rabies viruses all over the world, and the list of viruses is shown in Table 3 below. In the first screening, six viruses were tested for neutralization ability. The results are shown in Table 4. Y is an antibody showing neutralizing ability, N is an antibody having no neutralizing ability, and M is an antibody showing some neutralizing ability.

List of CDC rabies virus in the United States No . Rabies virus type  origin One CVS-11 2 Mongoose RSA Mongoose, South Africa 3 CASK Skunk, California, USA 4 Dog Tun 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

RFFIT results from the 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 . 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 screened. These antibodies were tested for neutralizing ability against 50 viruses (Table 3). The results are shown in Table 5 below.

15 neutralizing ability test No. Virus Antibody One 2 3 4 5 6 7 8 9 10 11 12 13 14 15 One Dog Tun Dog, Tunisia o o o o o o o o o o o o o o o 2 TXFX Gray fox, TX o o o o o o o o o o o o o o o 3 Dog last Dog, Mexico o o o o o o o o o o o o o o o 4 DR MX TN / MX COW) Bat, Mexico o o o o o o o o o o o o o o o 5 Dog Arg Dog, Argentina o o o o o o o o o o o o x x 6 TX SK Skunk, Texas, USA o o o o o o o o o o o o o o o 7 rv61 Human (ex dog), UK (ex India) o o o o o x o o o o o x o o o 8 857r Raccoon dog, Russia, Far East o o o o o o o o o o o o o o - 9 Phi 002 (231) Human / dog, Philippines o o o o o o o o o o o o o o - 10 WA Bat Bat, Washington, USA o o o o o o o o o o o o o o - 11 LC NY Bat, New York, USA x x x o x x x x x x x x x x x 12 Bat Ef Bat, Pennsyl-vania, USA x o o x o o o o o x o o o x o 13 RAC Raccoon, Georgia, USA x x o o o o o x o o x o o o o 14 TX Coyote Coyote, Texas, USA o o o o o o o o o o x o o o x 15 Mongoose PR Mongoose, Puerto-Rico o o o o o o o o o o o o o x x 16 I-151 Dog, India o o o x o o o o o o o o o o o 17 Gabon dog Dog, Gabon o o o o o o x o o o x o o o o 18 Sri Lanka Cow, Sri Lanka o o o o o o o o o o o o o o o 19 Gray AZ fox Gray Fox, Arizona, USA o o o o o o o o o x x o o o x 20 NC SK Skunk, Wisconsin, USA o o o o o o o o o o o o o o o 21 China dog 2005 Dog, China o o o o o o o o o o o o o o o 22 Dog thai Dog, Thailand o o o o o o o o o o o o o o x 23 phi dog Human / dog, Philippines o o o o o o o o o o o o o x o 24 Rv342 China Cow / dog, China o o o o o o o o o o o o o o o 25 TN-269 Bat, Tennessee USA x x o o o x x o x o x x x x x 26 Wu ABLV Australian bat lyssa, Genotype 7 o o o o o o o o o o o o o o o 27 ABV (SM 4476) Australian bat lyssa, Genotype 7 o o o o o o o o o o o o o o o 28 DR Brazil Bat, Brazil x o o x o o o o x o o o o o x 29 AK FX Arctic Fox, Alaska, USA o o o o o o o o o o o o o o o 30 323R Dog / Coyote, Texas, USA o o o o o o o o o o o o o x x 31 TX SK 4384 Skunk, Texas, USA o o o o o o o o o o o o o o o 32 ERA o o o o o o o o o o o o o o o 33 I-148 Dog, India o o o x o o x o o o o o o x o 34 CASK Skunk, California, USA o o o o o o o o o o o o o x x 35 AZ Bat Bat, Arizona o o o o o x o x o o o o x x x 36 3860 CA Bat Bat, California, USA o o o o o x x o o o o o x o x 37 TN410 Bat, Tennessee, USA x x o o o x x o o o x x x x x 38 AL Bat Bat, California, USA o o o o o o o o o o x o o o o 39 EBLV 1 A09-3484 Genotype 5 o o o o o x x o o o o o o o o 40 EBLV 2 A03-4659 Genotype 6 x o o o o x x o o o x o x o o 41 Duvenhag eGenotype 4 o o o o o x x o o o o x x o x 42 TN 132 Bat, Tennessee, USA x x x o o x x o x x x x x x x 43 Mongoose RSA Mongoose, South Africa o o o o o o o o o o o o o o o 44 Myotis Bat, Washington, USA o o o o o o o o o o o o o o o 45 C1434 Bat, Alabama, USA o o o o x o o o o o o o o o o 46 VA 399 Bat, Virginia, USA x x x o o x x o x o x x x x x 47 MI1625 Bat, Tennessee, USA o o o o o x x o o o x x x x x

In consideration of the antigen binding site and the basic titer, 4 clones of 3, 5, 10 and 11 of Table 5 (hereinafter referred to as " Tissue Antibody 1 "," Tissue Antibody 2 ","&Quot;," this cancer antibody 4 ") was selected. The conformational epitope in Table 6 below refers to a binding site having a tertiary protein structure.

The final neutralizing ability of four selected human antibodies and the epitope ID  ( No .) Neutralization ability IU / mg ) Epitope This dry-cell antibody 1 (No. 3 in Table 5) 7353 3 (Conformational) This dry-type antibody 2 (No. 5 in Table 5) 7941 New Site This canine antibody 3 (10 in Table 5) 5294 This canine antibody 4 (11 in Table 5) 2059 2 (Conformational)

Example  2: Production of human antibody expression vector

By treatment with restriction enzymes Nhe I and Pme I to PCR2.1 TA cloning vector containing the heavy and light chains to secure the after securing the heavy chain gene and light chain gene, processes the obtained heavy chain gene and light chain gene into the same restriction enzymes, respectively, pCT145 vector and pCT146 vector. The pCT145 and pCT146 vectors are Celltrion-specific vectors designed to clone the heavy and light chains of the antibody, respectively. Subsequently, in order to prepare an expression vector containing both a heavy chain transcription unit (promoter-heavy chain gene-polyAE) and a light chain transcription unit (promoter-light chain gene-polyAE), pCT145 vector containing the heavy chain gene was ligated with restriction enzyme Pac I And Asc I to obtain a heavy chain transcription unit. Then, a heavy chain transcription unit was inserted into the pCT146 vector containing the light chain gene by treating with the same restriction enzyme. Thereafter, the vector containing the heavy chain transcription unit and the light chain transcription unit at the same time was selected using restriction enzymes and named pCT188 (see FIG. 1, Korean Patent Registration No. 10-1076602, patentee: Celltrion, Inc.). The selected vector was extracted using the Endofree plasmid maxi kit (QIAGEN, Germany, 12362). Finally, the base sequence of the antibody was confirmed by sequencing using some of the extracted DNA.

Example  3: Production of human antibody by transient transduction method

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

Example  4: Determination of the neutralization efficacy of the final screened human monoclonal antibody

The final selection of four antibodies was carried out using RFFIT, in vitro neutralization capacity experiments, with about 50 rabies viruses worldwide. The results are shown in Table 7.

Results of RFFIT of human monoclonal antibodies No . virus ID This
Antibody 1 This
Antibody 2 This
Antibody 3 This
Antibody 4 Working conc. (Ug / ml) 0.20 One Mongoose RSA
Mongoose, 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 Tun
Dog, Tunisia Titer 270 230 280 230 IU / mL 2.7 2.3 2.8 2.3 IU / mg 2700 2300 2800 2300 3 Dog thai
Dog, Thailand Titer 1300 1100 1300 320 IU / mL 9.3 7.9 9.3 2.3 IU / mg 9286 7857 9286 2286 4 Dog is last
Dog, 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 MX
Bat, Mexico Titer 180 110 230 250 IU / mL 1.7 1.0 2.2 2.4 IU / mg 1714 1048 2190 2381 7 DR Brazil
Bat, Brazil Titer 45 50 50 56 IU / mL 1.8 2.0 2.0 2.2 IU / mg 1800 2000 2000 2240 8 WA Bat
Bat, Washington, USA Titer 250 180 270 270 IU / mL 2.0 1.4 2.2 2.2 IU / mg 2000 1440 2160 2160 9 rv61
Human (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 Bat
Bat, 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 Bat
Bat, Arizona Titer 1300 800 1100 125 IU / mL 11.3 7.0 9.6 1.1 IU / mg 11304 6957 9565 1087 12 phi dog
Human / 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 China
Cow / 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 4384
Skunk, 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 FX
Arctic 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 323R
Dog / 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 RVHN
Human (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-269
Bat, 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 TN410
Bat, 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 Arg
Dog, 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 4380
Skunk, Texas, USA Titer 70 56 70 50 IU / mL 0.56 0.45 0.56 0.40 IU / mg 2800 2240 2800 2000 24 RAC
Raccoon, 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 Coyote
Coyote, 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 PR
Mongoose, 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-151
Dog, India Titer 320 170 480 125 IU / mL 1.88 1.00 2.82 0.74 IU / mg 9412 5000 14118 3676 28 Sri Lanka
Cow, 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 ABLV
Australian 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 3860ce Bat
Bat, 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 dog
Dog, 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 A09-3484
Genotype 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 A03-4659
Genotype 6 Titer 280 200 270 54 IU / mL 3.11 2.22 3.00 0.60 IU / mg 15556 11111 15000 3000 36 Duvenhage
Genotype 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 A09-3485
Genotype 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 CASK
Skunk, 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 Ef
Bat, Pennsylvania, USA Titer 50 42 50 50 IU / mL 1.05 0.88 1.05 1.05 IU / mg 5263 4421 5263 5263 41 C1434
Bat, 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 399
Bat, 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 132
Bat, 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-148
Dog, India Titer 10 11 10 13 IU / mL 0.40 0.44 0.40 0.52 IU / mg 2000 2200 2000 2600 46 LC NY
Bat, 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 857r
Raccoon 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 SK
Skunk, Wisconsin, USA Titer 270 250 280 280 IU / mL 2.16 2.00 2.24 2.24 IU / mg 2160 2000 2240 2240 49 MI1625
Bat, Tennessee, USA Titer 70 45 50 11 IU / mL 2.50 1.61 1.79 0.39 IU / mg 12500 8036 8929 1964 50 Myotis
Bat, 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

Example  5: Antigen binding site of the finally selected human monoclonal antibody ( antigenic site ) Discrimination

Experiments were conducted to find escape mutant viruses in order to know the antigenic sites of the selected antibodies.

CVS-11 viruses with an infectivity of 10 ⁶ / ml are diluted 1.5-fold in succession to 96-well cell culture plates and reacted with 0.5-1 IU / ml antibody at 37 ° C for 1 hour. After 1 hour of reaction, 2 x 10 ⁵ cells / ml of BHK cells were added and cultured for 3 days. After 3 days, the virus was obtained, and the cells were fixed and stained for expression of Rabies Nucleocapsid protein (Jenobiotech, 9061). In the second passage, the same experiment as above is repeated by increasing the amount of antibody to the virus from the first passage. The information on the progress of each antibody is shown in Table 8. In the first or second passage, viruses 4-5 clones that have been identified as having an infection or increased infection even though the virus has been found to be infectious or infected with a small amount of CVS- After amplification, the RNA was isolated using QIAamp Viral RNA Mini kit (QIAGEN, 52904).

Escape mutation virus experiment Antibody Passage  One Passage  2 Passage  3 Amplification This key antibody 4 1 IU / ml 10 IU / ml - 5 clones Tissue Antibody 1 1 IU / ml 5 IU / ml 10 IU / ml 4 clones This gun antibody 3 0.5 IU / ml 2 IU / ml 7 IU / ml 5 clones

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

In the case of this mutant 2, it was confirmed that the mutant virus mutated at amino acid 210 obtained through this mutant 3 could not be neutralized. As a result, the antigenic site was found to be a new site like this mutant antibody 3.

Antibody Antigen binding site confirmation result Antibody Epitope Nucleotide variation Amino acid mutation Amino acid position * This key antibody 4 II GGA-> GTA Gly (G) - > Val (V) 34 GGA-> GAA Gly (G) - > Glu (E) 34 GGA-> AGA Gly (G) - > Arg (R) 34 Tissue Antibody 1 III TCA-> CCA Ser (S) - > Pro (P) 331 This gun antibody 3 New site GTG-> GAG Val (V) - > Glu (E) 210 GAG-> GAT Glu (E) -> Asp (D 413

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

Example  6: CR4098  Identification of neutralizing ability against escape viruses produced by antibodies

Example  6-1. CR4098  Generation of escape viruses against antibodies

The DNA sequence for the CR4098 antibody (Crucell) was obtained from the patent (see US7579446) and NCBI (National Center for Biotechnology Information) database and synthesized in Ennomixs (Yuseong-Gu, Daejeon) and cloned into pCT146 expression vector Celltrion. The sequence of the synthesized part was confirmed by restriction enzyme cleavage analysis and DNA sequencing. The CR4098 antibody was produced in the F2N78 cell line as in Example 3 in a short-term production mode.

Then, CR4098 was used to generate an escape mutant virus for the antibody.

CVS-11 viruses with an infectivity of 10 ⁶ per ml are serially diluted 1.5-fold in 96-well cell culture plates and reacted with 10-40 IU / ml of CR4098 antibody at 37 ° C for 1 hour. After 1 hour of reaction, 2 x 10 ⁵ cells / ml of BHK cells are added and cultured for 3 days. After 3 days, the virus was obtained and the cells were fixed and stained for expression of Rabies Nucleocapsid protein (CVS-11 virus infection) (Jenobiotech, 9061). In the second passage, the same experiment as above is repeated by increasing the amount of CR4098 antibody in the virus from the first passage. In the first or second passage, viruses 4-5 clones were found to have infections or increased levels of infection even though the viruses obtained from CVS-11 were found to be infected or infected in small quantities, gradually increasing the antibody to CR4098 After amplification, RNA was isolated using QIAamp Viral RNA Mini kit (QIAGEN, 52904).

CDNA was synthesized using SuperScript III first strand synthesis system for RT-PCR (Invitrogen, 18080-051) with RNA as a template, followed by amplification with Takara ExTaq (Takara, RR001A), followed by sequencing . Sequencing Results The altered sequence of the escape mutant virus for the CR4098 antibody was seen at N336K.

Example  6-2. CR4098  Neutralization ability test for escape virus produced by antibody

For the escape virus (N336K) generated by the CR4098 antibody, it was confirmed whether the antibody of the present invention was capable of neutralizing.

As a result of the neutralization ability test, it was confirmed that all antibodies of the present invention showed neutralizing effect against CR4098 antibody-expressing virus (N336K), and only CR4098 antibody had no neutralizing ability.

Neutralization ability test results for escape viruses produced by CR4098 antibody Virus Antibody IU / mg CR4098 mutant
(N336K) CR4098 All infection This key antibody 4 1352 Tissue Antibody 1 3081 This gun antibody 3 2336

Example  7: Detection of neutralizing ability against rabies virus isolated from the goat antibody

7-1. in - vitro  Experiment

The wild-type virus of Table 11, which has been infected in India using this testicular antibody 1, 3, 4, Mix 1 (this key antibody 1 + key antibody 4) and Mix 2 (this key antibody 1 + key antibody 3) (RFFIT). The experiment was conducted by the National Institute of Mental Health and Science (NIMHANS). Each antibody was tested at Neuro 2 a cell with a concentration of about 1 μg and 100 FFD ₅₀ of each virus below. The RFFIT results showed neutralizing ability for each virus in all five antibodies.

Rabies virus in India Abbreviation of virus Virus-isolated animal Region where the 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  Experiment

Following the in vitro experiments above, in vivo experiments were carried out in mice. As shown in Table 12, all antibodies in the animal test groups showed high neutralizing ability. For each animal test group, 10 mice were used, and 100 LD50 (0.1 mL) was used for the virus used. Three hours after the virus inoculation (intramuscular injection), each antibody (about 1 ug) was inoculated in the same place (intramuscular injection), and survival rate was observed up to 30 days. FIG. 3 is a graph showing the survival rate of the mouse against the SV2 virus among the six viruses (SV1 to SV6) in animal experiments.

The survival rates of the SV1 to SV6 viruses in this animal experiment are shown in Table 12.

Separation of this Tumor Antibody by Animal Experiments Neutralization test for rabies virus SV1 SV2 SV3 SV4 SV5 SV6 Tissue Antibody 1 80 90 90 80 90 90 This gun antibody 3 90 90 100 90 90 100 This key antibody 4 100 100 100 100 100 100 This tumor antibody 1 +
This key antibody 4 100 100 100 100 100 100 This tumor antibody 1 +
This gun 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 will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. You will know. Accordingly, it is not intended that the invention be limited to the particular embodiments disclosed as the best mode contemplated for carrying out the invention but that the invention be construed as including all the embodiments falling within the scope of the appended claims.

<110> CELLTRION INC. <120> A BINDING MOLECULES CAPABLE OF NEUTRALIZING RABIES VIRUS <130> CPD2014033KR <160> 81 <170> Kopatentin 2.0 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 11B6 heavy chain CDR1 <400> 1 Arg Arg Arg Asp Tyr Trp Gly   1 5 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> 11B6 heavy chain CDR2 <400> 2 Ser Phe Phe His Arg Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Glu Ser   1 5 10 15 <210> 3 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 11B6 heavy chain CDR3 <400> 3 His Pro Ser Thr Pro Phe Ala Glu Tyr Leu Leu Leu Pro Asp Ala Phe   1 5 10 15 Asp Ser         <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 11B6 light chain CDR1 <400> 4 Arg Ala Ser Gln Ser Val Arg Ser Ser Leu Ala   1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 11B6 light chain CDR2 <400> 5 Gly Ala Ser Thr Arg Ala Thr   1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 11B6 light chain CDR3 <400> 6 Gln Gln Tyr Ser Asp Trp Pro Leu Thr   1 5 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> NP-19-9 heavy chain CDR1 <400> 7 Ser Tyr Asp Ile Ser   1 5 <210> 8 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> NP-19-9 heavy chain CDR2 <400> 8 Trp Ile Ser Thr Tyr Lys Gly Asn Thr Asn Phe Ala Gln Lys Phe Gln   1 5 10 15 Asp     <210> 9 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> NP-19-9 heavy chain CDR3 <400> 9 Ala Lys Ser His Pro Phe Tyr Asp Phe Trp Ser Ala Tyr Tyr Val Pro   1 5 10 15 Gly Ala Phe Asp Ile              20 <210> 10 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> NP-19-9 light chain CDR1 <400> 10 Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn   1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> NP-19-9 light chain CDR2 <400> 11 Asp Ala Ser Asn Leu Glu Thr   1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> NP-19-9 light chain CDR3 <400> 12 Gln Gln Tyr Asp Asn Leu Pro Leu Thr   1 5 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> NP-19-11 heavy chain CDR1 <400> 13 Ser Tyr Asp Val Ser   1 5 <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> NP-19-11 heavy chain CDR2 <400> 14 Trp Ile Ser Ala Tyr Arg Gly Asn Thr Lys Tyr Ala Gln Lys Phe Gln   1 5 10 15 Gly     <210> 15 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> NP-19-11 heavy chain CDR3 <400> 15 Ala Lys Ser His Pro Tyr Tyr Asp Phe Trp Ser Gly Tyr Tyr Val Pro   1 5 10 15 Gly Ala Phe Asp Ile              20 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> NP-19-11 light chain CDR1 <400> 16 Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Thr   1 5 10 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> NP-19-11 light chain CDR2 <400> 17 Asp Ala Ser Asn Leu Glu Thr   1 5 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> NP-19-11 light chain CDR3 <400> 18 Gln Gln Tyr Asp Asn Leu Pro Pro Thr   1 5 <210> 19 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> NP-19-35 heavy chain CDR1 <400> 19 Ser Tyr Asp Val Ser   1 5 <210> 20 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> NP-19-35 heavy chain CDR2 <400> 20 Trp Ile Ser Ala Tyr Arg Gly Asn Thr Lys Tyr Ala Gln Lys Phe Gln   1 5 10 15 Gly     <210> 21 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> NP-19-35 heavy chain CDR3 <400> 21 Ala Lys Ser His Pro Tyr Tyr Asp Phe Trp Ser Gly Tyr Tyr Val Pro   1 5 10 15 Gly Ala Phe Asp Ile              20 <210> 22 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> NP-19-35 light chain CDR1 <400> 22 Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn   1 5 10 <210> 23 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> NP-19-35 light chain CDR2 <400> 23 Asp Ala Ser Asn Leu Glu Thr   1 5 <210> 24 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> NP-19-35 light chain CDR3 <400> 24 Gln Gln Tyr Asp Asn Leu Pro Pro Thr   1 5 <210> 25 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> 11B6 heavy chain variable region <400> 25 Glu Val Gln Leu Leu Gln Glu Ser Gly Pro Gly Leu Ala Lys Pro Ser   1 5 10 15 Glu Thr Leu Ser Leu Ile Cys Thr Val Ser Gly Gly Ser Ile Ser Arg              20 25 30 Arg Arg Asp Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Arg Gly Leu          35 40 45 Glu Trp Ile Gly Ser Phe Phe His Arg Gly Ser Thr Tyr Tyr Asn Pro      50 55 60 Ser Leu Glu Ser Arg Val Ser Ile Ser Val Asp Pro Ser Lys Asn Gln  65 70 75 80 Phe Ser Leu His Leu Ser Ser Val Thr Val Ala Asp Thr Ala Val Tyr                  85 90 95 Tyr Cys Ala Arg His Pro Ser Thr Pro Phe Ala Glu Tyr Leu Leu Leu             100 105 110 Pro Asp Ala Phe Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 125 Ser     <210> 26 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 11B6 light chain variable region <400> 26 Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly   1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Ser Ser              20 25 30 Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile          35 40 45 Ser Gly Ala Ser Thr Arg Ala Thr Asp Ile Pro Ala Arg Leu Ser Gly      50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Val Ser Ser Leu Gln Ser  65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Asp Trp Pro Leu                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 27 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> NP-19-9 heavy chain variable region <400> 27 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Asp Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met          35 40 45 Gly Trp Ile Ser Thr Tyr Lys Gly Asn Thr Asn Phe Ala Gln Lys Phe      50 55 60 Gln Asp Arg Val Thr Leu Thr Thr Asp Thr Ser Thr Thr Thr Ala Tyr  65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Ala Lys Ser His Pro Phe Tyr Asp Phe Trp Ser Ala Tyr Tyr             100 105 110 Val Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Val Val Thr Val         115 120 125 Ser Ser     130 <210> 28 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> NP-19-9 light chain variable region <400> 28 Glu Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr              20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile          35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Leu                  85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys             100 105 <210> 29 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> NP-19-11 heavy chain variable region <400> 29 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Asp Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met          35 40 45 Gly Trp Ile Ser Ala Tyr Arg Gly Asn Thr Lys Tyr Ala Gln Lys Phe      50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr  65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Ala Lys Ser His Pro Tyr Tyr Asp Phe Trp Ser Gly Tyr Tyr             100 105 110 Val Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val         115 120 125 Ser Ser     130 <210> 30 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> NP-19-11 light chain variable region <400> 30 Glu Leu Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr              20 25 30 Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile          35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Pro                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 31 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> NP-19-35 heavy chain variable region <400> 31 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Asp Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met          35 40 45 Gly Trp Ile Ser Ala Tyr Arg Gly Asn Thr Lys Tyr Ala Gln Lys Phe      50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr  65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Ala Lys Ser His Pro Tyr Tyr Asp Phe Trp Ser Gly Tyr Tyr             100 105 110 Val Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val         115 120 125 Ser Ser     130 <210> 32 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> NP-19-35 light chain variable region <400> 32 Glu Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr              20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile          35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Pro                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 33 <211> 459 <212> PRT <213> Artificial Sequence <220> <223> 11B6 heavy chain <400> 33 Glu Val Gln Leu Leu Gln Glu Ser Gly Pro Gly Leu Ala Lys Pro Ser   1 5 10 15 Glu Thr Leu Ser Leu Ile Cys Thr Val Ser Gly Gly Ser Ile Ser Arg              20 25 30 Arg Arg Asp Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Arg Gly Leu          35 40 45 Glu Trp Ile Gly Ser Phe Phe His Arg Gly Ser Thr Tyr Tyr Asn Pro      50 55 60 Ser Leu Glu Ser Arg Val Ser Ile Ser Val Asp Pro Ser Lys Asn Gln  65 70 75 80 Phe Ser Leu His Leu Ser Ser Val Thr Val Ala Asp Thr Ala Val Tyr                  85 90 95 Tyr Cys Ala Arg His Pro Ser Thr Pro Phe Ala Glu Tyr Leu Leu Leu             100 105 110 Pro Asp Ala Phe Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 125 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser     130 135 140 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 145 150 155 160 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr                 165 170 175 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr             180 185 190 Ser Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln         195 200 205 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp     210 215 220 Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 225 230 235 240 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro                 245 250 255 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr             260 265 270 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn         275 280 285 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg     290 295 300 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 305 310 315 320 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser                 325 330 335 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys             340 345 350 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp         355 360 365 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe     370 375 380 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 385 390 395 400 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe                 405 410 415 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly             420 425 430 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr         435 440 445 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys     450 455 <210> 34 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> 11B6 light chain <400> 34 Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly   1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Ser Ser              20 25 30 Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile          35 40 45 Ser Gly Ala Ser Thr Arg Ala Thr Asp Ile Pro Ala Arg Leu Ser Gly      50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Val Ser Ser Leu Gln Ser  65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Asp Trp Pro Leu                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 35 <211> 460 <212> PRT <213> Artificial Sequence <220> <223> NP-19-9 heavy chain <400> 35 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Asp Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met          35 40 45 Gly Trp Ile Ser Thr Tyr Lys Gly Asn Thr Asn Phe Ala Gln Lys Phe      50 55 60 Gln Asp Arg Val Thr Leu Thr Thr Asp Thr Ser Thr Thr Thr Ala Tyr  65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Ala Lys Ser His Pro Phe Tyr Asp Phe Trp Ser Ala Tyr Tyr             100 105 110 Val Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Val Val Thr Val         115 120 125 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser     130 135 140 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 145 150 155 160 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu                 165 170 175 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu             180 185 190 Tyr Ser Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr         195 200 205 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val     210 215 220 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 225 230 235 240 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe                 245 250 255 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val             260 265 270 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe         275 280 285 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro     290 295 300 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 305 310 315 320 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val                 325 330 335 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala             340 345 350 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg         355 360 365 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly     370 375 380 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 385 390 395 400 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser                 405 410 415 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln             420 425 430 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His         435 440 445 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys     450 455 460 <210> 36 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> NP-19-9 light chain <400> 36 Glu Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr              20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile          35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Leu                  85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 37 <211> 460 <212> PRT <213> Artificial Sequence <220> <223> NP-19-11 heavy chain <400> 37 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Asp Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met          35 40 45 Gly Trp Ile Ser Ala Tyr Arg Gly Asn Thr Lys Tyr Ala Gln Lys Phe      50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr  65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Ala Lys Ser His Pro Tyr Tyr Asp Phe Trp Ser Gly Tyr Tyr             100 105 110 Val Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val         115 120 125 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser     130 135 140 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 145 150 155 160 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu                 165 170 175 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu             180 185 190 Tyr Ser Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr         195 200 205 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val     210 215 220 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 225 230 235 240 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe                 245 250 255 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val             260 265 270 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe         275 280 285 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro     290 295 300 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 305 310 315 320 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val                 325 330 335 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala             340 345 350 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg         355 360 365 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly     370 375 380 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 385 390 395 400 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser                 405 410 415 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln             420 425 430 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His         435 440 445 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys     450 455 460 <210> 38 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> NP-19-11 light chain <400> 38 Glu Leu Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr              20 25 30 Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile          35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Pro                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 39 <211> 460 <212> PRT <213> Artificial Sequence <220> <223> NP-19-35 heavy chain <400> 39 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Asp Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met          35 40 45 Gly Trp Ile Ser Ala Tyr Arg Gly Asn Thr Lys Tyr Ala Gln Lys Phe      50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr  65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Ala Lys Ser His Pro Tyr Tyr Asp Phe Trp Ser Gly Tyr Tyr             100 105 110 Val Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val         115 120 125 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser     130 135 140 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 145 150 155 160 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu                 165 170 175 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu             180 185 190 Tyr Ser Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr         195 200 205 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val     210 215 220 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 225 230 235 240 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe                 245 250 255 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val             260 265 270 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe         275 280 285 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro     290 295 300 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 305 310 315 320 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val                 325 330 335 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala             340 345 350 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg         355 360 365 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly     370 375 380 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 385 390 395 400 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser                 405 410 415 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln             420 425 430 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His         435 440 445 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys     450 455 460 <210> 40 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> NP-19-35 light chain <400> 40 Glu Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr              20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile          35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Pro                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 41 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 11B6 heavy chain CDR1 <400> 41 cggcggagag actactgggg c 21 <210> 42 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> 11B6 heavy chain CDR2 <400> 42 agcttcttcc accggggcag cacctactac aaccccagcc tggaaagc 48 <210> 43 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> 11B6 heavy chain CDR3 <400> 43 caccccagca cccccttcgc cgagtacctg ctgctgcccg acgccttcga tagc 54 <210> 44 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> 11B6 light chain CDR1 <400> 44 agagccagcc agagcgtgcg gagcagcctg gcc 33 <210> 45 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 11B6 light chain CDR2 <400> 45 ggcgccagca ccagagccac c 21 <210> 46 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 11B6 light chain CDR3 <400> 46 cagcagtaca gcgactggcc cctgacc 27 <210> 47 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> NP-19-9 heavy chain CDR1 <400> 47 agctatgata tcagc 15 <210> 48 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> NP-19-9 heavy chain CDR2 <400> 48 tggatcagca cttacaaggg taacacaaac tttgcacaaa agttccagga c 51 <210> 49 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> NP-19-9 heavy chain CDR3 <400> 49 gccaaatccc acccgtttta cgatttttgg agtgcttact atgtccccgg tgcttttgat 60 atc 63 <210> 50 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> NP-19-9 light chain CDR1 <400> 50 caggcgagtc aggacattag caactattta aat 33 <210> 51 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NP-19-9 light chain CDR2 <400> 51 gatgcatcca atttggaaac a 21 <210> 52 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NP-19-9 light chain CDR3 <400> 52 caacagtatg ataatctccc ccttact 27 <210> 53 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> NP-19-11 heavy chain CDR1 <400> 53 agctatgatg tcagc 15 <210> 54 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> NP-19-11 heavy chain CDR2 <400> 54 tggatcagcg cttacagggg taacacaaag tatgcacaga agttccaggg c 51 <210> 55 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> NP-19-11 heavy chain CDR3 <400> 55 gccaaatccc acccgtatta tgatttttgg agtgggtact atgtccccgg ggcttttgat 60 atc 63 <210> 56 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> NP-19-11 light chain CDR1 <400> 56 caggcgagtc aggacattag caactattta act 33 <210> 57 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NP-19-11 light chain CDR2 <400> 57 gatgcatcca atttggaaac a 21 <210> 58 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NP-19-11 light chain CDR3 <400> 58 caacagtatg ataatctccc tccaact 27 <210> 59 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> NP-19-35 heavy chain CDR1 <400> 59 agctatgatg tcagc 15 <210> 60 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> NP-19-35 heavy chain CDR2 <400> 60 tggatcagcg cttacagggg taacacaaag tatgcacaga agttccaggg c 51 <210> 61 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> NP-19-35 heavy chain CDR3 <400> 61 gccaaatccc acccgtatta tgatttttgg agtgggtact atgtccccgg tgcttttgat 60 atc 63 <210> 62 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> NP-19-35 light chain CDR1 <400> 62 caggcgagtc aggacattag caactattta aat 33 <210> 63 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NP-19-35 light chain CDR2 <400> 63 gatgcatcca atttggaaac a 21 <210> 64 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NP-19-35 light chain CDR3 <400> 64 caacagtatg ataatctccc tccaact 27 <210> 65 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> 11B6 heavy chain variable region <400> 65 gaagtgcagc tgctgcagga aagcggccct ggcctggcca agcccagcga gacactgagc 60 ctgatctgca ccgtgtccgg cggcagcatc agccggcgga gagactactg gggctggatc 120 agacagcccc ctggcagagg cctggaatgg atcggcagct tcttccaccg gggcagcacc 180 tactacaacc ccagcctgga aagccgggtg tccatcagcg tggaccccag caagaaccag 240 ttcagcctgc acctgagcag cgtgaccgtg gccgacaccg ccgtgtacta ctgcgccaga 300 caccccagca cccccttcgc cgagtacctg ctgctgcccg acgccttcga tagctggggc 360 cagggcaccc tggtgacagt gtccagc 387 <210> 66 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> 11B6 light chain variable region <400> 66 gacatcgtga tgacccagag ccccgccacc ctgagcgtgt ccccaggcga gagagccacc 60 ctgtcctgca gagccagcca gagcgtgcgg agcagcctgg cctggtatca gcagaggcca 120 ggccaggccc ccagactgct gatcagcggc gccagcacca gagccaccga catccctgcc 180 cctgcagagc 240 gaggacttcg ccgtgtacta ctgccagcag tacagcgact ggcccctgac cttcggcgga 300 ggcaccaagg tggaaatcaa g 321 <210> 67 <211> 390 <212> DNA <213> Artificial Sequence <220> <223> NP-19-9 heavy chain variable region <400> 67 gaggtgcagc tggtggagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta cacctttacc agctatgata tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcagcactt acaagggtaa cacaaacttt 180 gcacaaaagt tccaggacag agtcaccctt accacagaca catccacgac cacagcctac 240 atggagctga ggagcctgac atctgacgac acggccgtgt attactgtgc gagagccaaa 300 tcccacccgt tttacgattt ttggagtgct tactatgtcc ccggtgcttt tgatatctgg 360 ggccaaggga cagtggtcac cgtctcttca 390 <210> 68 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> NP-19-9 light chain variable region <400> 68 gagctcgtga tgacgcagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc aggcgagtca ggacattagc aactatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctacgat gcatccaatt tggaaacagg ggtcccatca 180 aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 240 gaagatattg caacatatta ctgtcaacag tatgataatc tcccccttac tttcggccct 300 gggaccaaag tggatatcaa a 321 <210> 69 <211> 390 <212> DNA <213> Artificial Sequence <220> <223> NP-19-11 heavy chain variable region <400> 69 gaggtgcagc tggtggagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta cacctttacc agctatgatg tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcagcgctt acaggggtaa cacaaagtat 180 gcacagaagt tccagggcag agtcaccatg accacagaca catccacgag cacagcctac 240 atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc gagagccaaa 300 tcccacccgt attatgattt ttggagtggg tactatgtcc ccggggcttt tgatatctgg 360 ggccaaggga caatggtcac cgtctcttca 390 <210> 70 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> NP-19-11 light chain variable region <400> 70 gagctccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc aggcgagtca ggacattagc aactatttaa cttggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctacgat gcatccaatt tggaaacagg ggtcccatca 180 aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 240 gaagatattg caacatatta ctgtcaacag tatgataatc tccctccaac tttcggcgga 300 gggaccaagg tggaaatcaa a 321 <210> 71 <211> 390 <212> DNA <213> Artificial Sequence <220> <223> NP-19-35 heavy chain variable region <400> 71 gaggtgcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta cacctttacc agctatgatg tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcagcgctt acaggggtaa cacaaagtat 180 gcacagaagt tccagggcag agtcaccatg accacagaca catccacgag cacagcctac 240 atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc gagagccaaa 300 tcccacccgt attatgattt ttggagtggg tactatgtcc ccggtgcttt tgatatctgg 360 ggccaaggga caatggtcac cgtctcttca 390 <210> 72 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> NP-19-35 light chain variable region <400> 72 gagctcgtga tgactcagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc aggcgagtca ggacattagc aactatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctacgat gcatccaatt tggaaacagg ggtcccatca 180 aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 240 gaagatattg caacatatta ctgtcaacag tatgataatc tccctccaac tttcggcgga 300 gggaccaagc tggagatcaa a 321 <210> 73 <211> 1380 <212> DNA <213> Artificial Sequence <220> <223> 11B6 heavy chain <400> 73 gaagtgcagc tgctgcagga aagcggccct ggcctggcca agcccagcga gacactgagc 60 ctgatctgca ccgtgtccgg cggcagcatc agccggcgga gagactactg gggctggatc 120 agacagcccc ctggcagagg cctggaatgg atcggcagct tcttccaccg gggcagcacc 180 tactacaacc ccagcctgga aagccgggtg tccatcagcg tggaccccag caagaaccag 240 ttcagcctgc acctgagcag cgtgaccgtg gccgacaccg ccgtgtacta ctgcgccaga 300 caccccagca cccccttcgc cgagtacctg ctgctgcccg acgccttcga tagctggggc 360 cagggcaccc tggtgacagt gtccagcgcc agcaccaagg gccccagcgt gttccctctg 420 gcccccagca gcaagagcac atctggcgga acagccgccc tgggctgcct ggtgaaagac 480 tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac 540 accttcccgg ctgtcctaca gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg 600 ccctccagca gcttgggcac ccagacctac atctgcaacg tgaatcacaa gcccagcaac 660 accaaggtgg acaagaaagt tgagcccaaa tcttgtgaca aaactcacac atgcccaccg 720 tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 780 gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 840 gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 900 acaaagccgc gggaggagca gtacaacagc acgtaccggg tggtcagcgt cctcaccgtc 960 ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 1020 ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 1080 tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 1140 gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1200 aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1260 aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1320 catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaatga 1380                                                                         1380 <210> 74 <211> 645 <212> DNA <213> Artificial Sequence <220> <223> 11B6 light chain <400> 74 gacatcgtga tgacccagag ccccgccacc ctgagcgtgt ccccaggcga gagagccacc 60 ctgtcctgca gagccagcca gagcgtgcgg agcagcctgg cctggtatca gcagaggcca 120 ggccaggccc ccagactgct gatcagcggc gccagcacca gagccaccga catccctgcc 180 cctgcagagc 240 gaggacttcg ccgtgtacta ctgccagcag tacagcgact ggcccctgac cttcggcgga 300 ggcaccaagg tggaaatcaa gcggaccgtg gccgctccca gcgtgttcat cttcccaccc 360 agcgacgagc agctgaagtc cggcacagcc agcgtggtgt gcctgctgaa caacttctac 420 ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caactcccag 480 gaaagcgtga ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540 ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600 ctgtccagcc ccgtgaccaa gagcttcaac cggggcgagt gctga 645 <210> 75 <211> 1383 <212> DNA <213> Artificial Sequence <220> <223> NP-19-9 heavy chain <400> 75 gaggtgcagc tggtggagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta cacctttacc agctatgata tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcagcactt acaagggtaa cacaaacttt 180 gcacaaaagt tccaggacag agtcaccctt accacagaca catccacgac cacagcctac 240 atggagctga ggagcctgac atctgacgac acggccgtgt attactgtgc gagagccaaa 300 tcccacccgt tttacgattt ttggagtgct tactatgtcc ccggtgcttt tgatatctgg 360 ggccaaggga cagtggtcac cgtctcttca gcttccacca agggcccatc ggtcttcccc 420 ctggcaccct cctccaagag cacctctggg ggcacagcag ccctgggctg cctggtcaag 480 gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgccctgac cagcggcgtg 540 cacaccttcc cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc 600 gtgccctcca gcagcttggg cacccagacc tacatctgca acgtgaatca caagcccagc 660 aacaccaagg tggacaagaa agttgagccc aaatcttgtg acaaaactca cacatgccca 720 ccgtgcccag cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc 780 aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 840 cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 900 aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 960 gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc 1020 ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag 1080 gtgtacaccc tgcccccatc ccgggaggag atgaccaaga accaggtcag cctgacctgc 1140 ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg 1200 gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac 1260 agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 1320 atgcatgagg ctctgcacaa ccactacaca cagaagagcc tctccctgtc tccgggtaaa 1380 tga 1383 <210> 76 <211> 645 <212> DNA <213> Artificial Sequence <220> <223> NP-19-9 light chain <400> 76 gagctcgtga tgacgcagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc aggcgagtca ggacattagc aactatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctacgat gcatccaatt tggaaacagg ggtcccatca 180 aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 240 gaagatattg caacatatta ctgtcaacag tatgataatc tcccccttac tttcggccct 300 gggaccaaag tggatatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645 <210> 77 <211> 1383 <212> DNA <213> Artificial Sequence <220> <223> NP-19-11 heavy chain <400> 77 gaggtgcagc tggtggagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta cacctttacc agctatgatg tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcagcgctt acaggggtaa cacaaagtat 180 gcacagaagt tccagggcag agtcaccatg accacagaca catccacgag cacagcctac 240 atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc gagagccaaa 300 tcccacccgt attatgattt ttggagtggg tactatgtcc ccggggcttt tgatatctgg 360 ggccaaggga caatggtcac cgtctcttca gcttccacca agggcccatc ggtcttcccc 420 ctggcaccct cctccaagag cacctctggg ggcacagcag ccctgggctg cctggtcaag 480 gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgccctgac cagcggcgtg 540 cacaccttcc cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc 600 gtgccctcca gcagcttggg cacccagacc tacatctgca acgtgaatca caagcccagc 660 aacaccaagg tggacaagaa agttgagccc aaatcttgtg acaaaactca cacatgccca 720 ccgtgcccag cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc 780 aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 840 cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 900 aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 960 gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc 1020 ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag 1080 gtgtacaccc tgcccccatc ccgggaggag atgaccaaga accaggtcag cctgacctgc 1140 ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg 1200 gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac 1260 agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 1320 atgcatgagg ctctgcacaa ccactacaca cagaagagcc tctccctgtc tccgggtaaa 1380 tga 1383 <210> 78 <211> 645 <212> DNA <213> Artificial Sequence <220> <223> NP-19-11 light chain <400> 78 gagctccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc aggcgagtca ggacattagc aactatttaa cttggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctacgat gcatccaatt tggaaacagg ggtcccatca 180 aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 240 gaagatattg caacatatta ctgtcaacag tatgataatc tccctccaac tttcggcgga 300 gggaccaagg tggaaatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645 <210> 79 <211> 1383 <212> DNA <213> Artificial Sequence <220> <223> NP-19-35 heavy chain <400> 79 gaggtgcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta cacctttacc agctatgatg tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcagcgctt acaggggtaa cacaaagtat 180 gcacagaagt tccagggcag agtcaccatg accacagaca catccacgag cacagcctac 240 atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc gagagccaaa 300 tcccacccgt attatgattt ttggagtggg tactatgtcc ccggtgcttt tgatatctgg 360 ggccaaggga caatggtcac cgtctcttca gcctccacca agggcccatc ggtcttcccc 420 ctggcaccct cctccaagag cacctctggg ggcacagcag ccctgggctg cctggtcaag 480 gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgccctgac cagcggcgtg 540 cacaccttcc cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc 600 gtgccctcca gcagcttggg cacccagacc tacatctgca acgtgaatca caagcccagc 660 aacaccaagg tggacaagaa agttgagccc aaatcttgtg acaaaactca cacatgccca 720 ccgtgcccag cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc 780 aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 840 cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 900 aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 960 gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc 1020 ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag 1080 gtgtacaccc tgcccccatc ccgggaggag atgaccaaga accaggtcag cctgacctgc 1140 ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg 1200 gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac 1260 agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 1320 atgcatgagg ctctgcacaa ccactacaca cagaagagcc tctccctgtc tccgggtaaa 1380 tga 1383 <210> 80 <211> 645 <212> DNA <213> Artificial Sequence <220> <223> NP-19-35 light chain <400> 80 gagctcgtga tgactcagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc aggcgagtca ggacattagc aactatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctacgat gcatccaatt tggaaacagg ggtcccatca 180 aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 240 gaagatattg caacatatta ctgtcaacag tatgataatc tccctccaac tttcggcgga 300 gggaccaagc tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645 <210> 81 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> CVS-11 strain G protein <400> 81 Met Val Pro Gln Val Leu Leu Phe Val Pro Leu Leu Gly Phe Ser Leu   1 5 10 15 Cys Phe Gly Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro              20 25 30 Trp Ser Pro Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val          35 40 45 Val Glu Asp Glu Gly Cys Thr Asn Leu Ser Glu Phe Ser Tyr Met Glu      50 55 60 Leu Lys Val Gly Tyr Ile Ser Ala Ile Lys Val Asn Gly Phe Thr Cys  65 70 75 80 Thr Gly Val Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr                  85 90 95 Val Thr Thr Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala             100 105 110 Cys Arg Ala Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu         115 120 125 Glu Ser Leu His Asn Pro Tyr Pro Asp Tyr His Trp Leu Arg Thr Val     130 135 140 Arg Thr Thr Lys Glu Ser Leu Ile Ile Ile Ser Pro Ser Val Thr Asp 145 150 155 160 Leu Asp Pro Tyr Asp Lys Ser Leu His Ser Arg Val Phe Pro Gly Gly                 165 170 175 Lys Cys Ser Gly Ile Thr Val Ser Ser Thr Tyr Cys Ser Thr Asn His             180 185 190 Asp Tyr Thr Ile Trp Met Pro Glu Asn Pro Arg Pro Arg Thr Pro Cys         195 200 205 Asp Ile Phe Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Asn Lys     210 215 220 Thr Cys Gly Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly 225 230 235 240 Ala Cys Arg Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp                 245 250 255 Gly Thr Trp Val Ala Met Gln Thr Ser Asp Glu Thr Lys Trp Cys Pro             260 265 270 Pro Asp Gln Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu         275 280 285 His Leu Val Val Glu Glu Leu Val Lys Lys Arg Glu Glu Cys Leu Asp     290 295 300 Ala Leu Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu 305 310 315 320 Ser His Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile                 325 330 335 Phe Asn Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg             340 345 350 Thr Trp Asn Glu Ile Ile Pro Ser Lys Gly Cys Leu Lys Val Gly Gly         355 360 365 Arg Cys His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu     370 375 380 Gly Pro Asp Gly His Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu 385 390 395 400 Gln Gln His Met Glu Leu Leu Lys Ser Ser Val Ile Pro Leu Met His                 405 410 415 Pro Leu Ala Asp Pro Ser Thr Val Phe Lys Glu Gly Asp Glu Ala Glu             420 425 430 Asp Phe Val Glu Val His Leu Pro Asp Val Tyr Lys Gln Ile Ser Gly         435 440 445 Val Asp Leu Gly Leu Pro Asn Trp Gly Lys Tyr Val Leu Met Thr Ala     450 455 460 Gly Ala Met Ile Gly Leu Val Leu Ile Phe Ser Leu Met Thr Trp Cys 465 470 475 480 Arg Arg Ala Asn Arg Pro Glu Ser Lys Gln Arg Ser Phe Gly Gly Thr                 485 490 495 Gly Arg Asn Val Ser Val Thr Ser Gln Ser Gly Lys Val Ile Pro Ser             500 505 510 Trp Glu Ser Tyr Lys Ser Gly Gly Glu Ile Arg Leu         515 520

Claims (33)

A binding molecule having a neutralizing activity against rabies virus, which is any one of binding molecules selected from the group consisting of binding molecules of i) to iv)
According to the Kabat method
i) a) a heavy chain variable region comprising the CDR1 region of SEQ ID NO: 1, the CDR2 region of SEQ ID NO: 2, and the CDR3 region of SEQ ID NO: 3; And b) a binding molecule comprising a light chain variable region comprising the CDR1 region of SEQ ID NO: 4, the CDR2 region of SEQ ID NO: 5, and the CDR3 region of SEQ ID NO: 6;
ii) a) a heavy chain variable region comprising the CDR1 region of SEQ ID NO: 7, the CDR2 region of SEQ ID NO: 8, and the CDR3 region of SEQ ID NO: 9; And b) a binding molecule comprising a light chain variable region comprising the CDR1 region of SEQ ID NO: 10, the CDR2 region of SEQ ID NO: 11, and the CDR3 region of SEQ ID NO: 12;
iii) a) a heavy chain variable region comprising the CDR1 region of SEQ ID NO: 13, the CDR2 region of SEQ ID NO: 14, and the CDR3 region of SEQ ID NO: 15; And b) a binding molecule comprising a light chain variable region comprising the CDR1 region of SEQ ID NO: 16, the CDR2 region of SEQ ID NO: 17, and the CDR3 region of SEQ ID NO: 18; And
iv) a) a heavy chain variable region comprising the CDR1 region of SEQ ID NO: 19, the CDR2 region of SEQ ID NO: 20, and the CDR3 region of SEQ ID NO: 21; And b) a light chain variable region comprising the CDR1 region of SEQ ID NO: 22, the CDR2 region of SEQ ID NO: 23, and the CDR3 region of SEQ ID NO:
The method according to claim 1,
The binding molecule is characterized in that asparagine (Asn) at position 336 (numbering except positions 1 to 19) of SEQ ID NO: 81 has a neutralizing activity against rabies virus including G protein mutated to lysine (Lys) .
delete delete delete delete delete delete delete The method according to claim 1,
Wherein the binding molecule comprises a variable region of SEQ ID NO: 25 and a variable region of SEQ ID NO: 26.
The method according to claim 1,
Wherein the binding molecule comprises a variable region of SEQ ID NO: 27 and a variable region of SEQ ID NO: 28.
The method according to claim 1,
Wherein the binding molecule comprises a variable region of SEQ ID NO: 29 and a variable region of SEQ ID NO: 30.
The method according to claim 1,
Wherein the binding molecule comprises a variable region of SEQ ID NO: 31 and a variable region of SEQ ID NO: 32.
11. The method of claim 10,
Wherein the binding molecule comprises the heavy chain of SEQ ID NO: 33 and the light chain of SEQ ID NO: 34.
12. The method of claim 11,
Wherein the binding molecule comprises the heavy chain of SEQ ID NO: 35 and the light chain of SEQ ID NO: 36.
13. The method of claim 12,
Wherein the binding molecule comprises the heavy chain of SEQ ID NO: 37 and the light chain of SEQ ID NO: 38.
14. The method of claim 13,
Wherein the binding molecule comprises a heavy chain of SEQ ID NO: 39 and a light chain of SEQ ID NO: 40.
18. The method according to any one of claims 1, 2, and 10 to 17,
Wherein the binding molecule is an antibody.
18. The method according to any one of claims 1, 2, and 10 to 17,
Wherein the binding molecule is a Fab fragment, an Fv fragment, a diabody, a chimeric antibody, a humanized antibody or a human antibody.
18. The method according to any one of claims 1, 2, and 10 to 17,
Wherein said rabies virus is derived from any one member selected from the group consisting of dogs, cows, mongooses, bats, skunks, raccoons, coyotes, foxes and wolves.
An immunogenic composition comprising a binding molecule according to any one of claims 1, 2 and 10 to 17 in addition to one or more tags.
17. A nucleic acid molecule encoding a binding molecule of any one of claims 1, 2, and 17 to 17.
22. An expression vector into which the nucleic acid molecule of claim 22 is inserted.
A cell line transforming a host cell with the expression vector of claim 23 to produce a binding molecule capable of binding to a rabies virus and having a neutralizing ability.
25. The method of claim 24,
The host cell may be any one selected from the group consisting of CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, HT1080 cells, A549 cells, HEK 293 cells and HEK293T cells Lt; / RTI &gt;
A pharmaceutical composition for the treatment and prevention of rabies, which additionally comprises the binding molecule of any one of claims 1, 2, and 10 to 17 and a pharmaceutically acceptable excipient.
a) a binding molecule according to any one of claims 1, 2, and 10 to 17; And
b) container
A rabies diagnostic kit.
a) a binding molecule according to any one of claims 1, 2, and 10 to 17; And
b) container
And a kit for treating and preventing rabies.
delete a) contacting a binding molecule of any one of claims 1, 2, and 10 to 17 with a sample of interest; And
b) detecting the reaction of the binding molecule with the sample of interest
/ RTI &gt; for rabies &lt; RTI ID = 0.0 &gt; diagnosis. &Lt; / RTI &gt;
Administering to a subject other than human a therapeutically effective amount of a binding molecule of any one of claims 1, 2, and 10 to 17
/ RTI &gt; A method of treating and preventing rabies, comprising the steps of:
a) culturing the cell line of claim 24; And
b) recovering the expressed binding molecule
To a rabies virus containing the neutralizing ability.
a) contacting a binding molecule of any one of claims 1, 2, and 10 to 17 with a sample of interest; And
b) measuring whether the binding molecule specifically binds to the sample of interest
Gt; a &lt; / RTI &gt; rabies virus.

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