KR101388680B1 - A binding molecules capable of neutralizing rabies virus - Google Patents

Abstract

The present invention relates to a binding molecule capable of neutralizing rabies virus, and more particularly, the binding molecule of the present invention is capable of neutralizing rabies viruses derived from individuals such as dogs, cattle, mongoose, bats, skunks and wolves. Because of this, it can be useful for treating patients infected with rabies virus from a wide range of individuals.

Description

Binding molecule capable of neutralizing rabies virus {A BINDING MOLECULES CAPABLE OF NEUTRALIZING RABIES VIRUS}

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

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

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

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

In the early stages, in addition to cold-like symptoms, you feel itching or fever at the bite. As 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 can also cause hypersensitivity to sunlight. Two to seven days after these symptoms are observed, the nerves and muscles of the whole body become paralyzed, leading to coma and dying from breathing problems.

Rabies can be treated and prevented by post-exposure prophylaxis via immediate topical wound protection and passive (anti-rabies immunoglobulin: hereinafter referred to as "anti-rabies antibody") and active (vaccine) immunization.

Currently developed anti-rabies antibodies include human derived rabies immunoglobulin (hereinafter referred to as "HRIG") and equine derived rabies immunoglobulin (hereinafter referred to as "ERIG"). HRIG's supply is difficult and prices are high. In addition, it is derived from human blood and has a high risk of infection, such as HIV, and is not high in efficacy because it is a polyclonal antibody. ERIG is derived from horses and has lower therapeutic efficiency than HRIG, which is why it is administered to patients at higher doses than HRIG. Although it is cheaper than HRIG, it is also not supplied smoothly, and anaphyaxis may be caused by antibodies derived from individuals different from humans. To overcome this drawback, the use of monoclonal antibodies that can neutralize rabies virus in post-exposure prophylaxis has been limited. Rabies-virus neutralizing murine single antibodies have been developed (Schumacher CL et al ., J. Clin . Invest . Vol. 84, p. 971-975, 1989), but short serum half-life, lack of ability to induce human effector function and human body Induced unwanted human anti-mouse antibody (HAMA) responses to murine antibodies are limited to direct administration to rabies-infected human patients.

Therefore, it is urgent to develop monoclonal antibodies capable of securing large-scale production and uniform quality by synthesizing through culturing because they are not derived from blood for the treatment of rabies.

It is an object of the present invention to provide a binding molecule that binds to rabies virus and has a neutralizing ability.

Another object of the present invention is to provide an immunoconjugate in which one or more tags are attached to the binding molecule.

It is another object of the present invention to provide a polynucleotide encoding said binding molecule.

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

Another object of the present invention is to provide a cell line transformed with said 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 of producing the 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 accomplish the above object, the present invention provides a binding molecule having a neutralizing ability by binding to a rabies virus.

The present invention also provides an immunoconjugate in which at least one tag is attached to the binding molecule.

The present invention also provides a polynucleotide encoding the binding molecule.

The present invention also provides an expression vector inserted with a polynucleotide encoding the binding molecule.

The present invention also provides a cell line wherein the expression vector is transformed into a host cell to produce the binding molecule of the present invention.

The present invention also provides a pharmaceutical composition further comprising the binding molecule and a pharmaceutically acceptable excipient.

The present invention also provides a composition for the treatment and prevention of rabies further comprising the binding molecule and a pharmaceutically acceptable excipient.

The present invention also provides a kit for diagnosing rabies comprising the binding molecule.

The present invention also provides a kit for the treatment and prevention of rabies comprising the binding molecule.

The present invention also provides a method for diagnosing rabies using the binding molecule.

The present invention also provides a method for treating and preventing rabies comprising administering to said subject an effective amount of said binding molecule.

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

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

Since the binding molecule capable of neutralizing the rabies virus of the present invention has a neutralizing ability against various rabies viruses, it is useful for the treatment and prevention of rabies in patients infected with rabies virus.

Figure 1 shows a chimeric antibody expression vector comprising the heavy and light chain genes of the present invention.
Figure 2 shows the results of in vivo animal experiments using the Chinese dog rabies virus (Rv342).

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

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

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

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

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

Hereinafter, the present invention will be described in detail.

The inventors of the Center for Disease Control (hereinafter referred to as "CDC") received a hybridoma cell that has been demonstrated to be capable of neutralizing a wide range of rabies viruses. The variable region sequence of was obtained. The chimeric antibody was then prepared by ligating the heavy and light chain variable regions to an IgG1 backbone. Since the chimeric antibody prepared as above in vivo and in In vitro experiments were carried out to test the neutralizing ability of various rabies viruses, it was confirmed that the monoclonal antibody of the present invention can be useful for treating patients infected with rabies virus derived from a wide range of individuals.

Accordingly, the present invention provides a binding molecule that binds to rabies virus and has a neutralizing ability.

In the present invention, the binding molecule is a variable having a CDR1 region comprising a polypeptide represented by SEQ ID NO: 27, a CDR2 region comprising a polypeptide represented by SEQ ID NO: 28 and a CDR3 region comprising a polypeptide represented by SEQ ID NO: 29 It is characterized by including an area.

Also in the present invention, the binding molecule has a CDR1 region comprising a polypeptide set forth in SEQ ID NO: 30, a CDR2 region comprising a polypeptide described in SEQ ID NO: 31 and a CDR3 region comprising a polypeptide described in SEQ ID NO: 32 And a variable region.

Also in the present invention, the binding molecule is a CDR1 region comprising a polypeptide as set out in SEQ ID NO: 27, a CDR2 region comprising a polypentide as set out in SEQ ID NO: 28 and a CDR3 region comprising a polypeptide as set out in SEQ ID NO: 29 A light chain variable region having a heavy chain variable region having a CDR1 region comprising a polypeptide represented by SEQ ID NO: 30, a CDR2 region comprising a polypeptide represented by SEQ ID NO: 31, and a CDR3 region comprising a polypeptide represented by SEQ ID NO: 32 It is characterized by including. The binding molecule is characterized in that the Fab fragment, Fv fragment, diabody (diabody), chimeric antibody, humanized antibody or human antibody.

In addition, in the present invention, the binding molecule is characterized in that it comprises a variable region comprising the polypeptide sequence set forth in SEQ ID NO: 35.

In the present invention, the binding molecule is characterized in that it comprises a variable region comprising a polypeptide sequence set forth in SEQ ID NO: 36.

Also in the present invention, the binding molecule is characterized in that it comprises a heavy chain variable region comprising a polypeptide sequence as set out in SEQ ID NO: 35 and a light chain variable region comprising a polypeptide sequence as set out in SEQ ID NO: 36. The binding molecule is characterized in that the Fab fragment, Fv fragment, diabody (diabody), chimeric antibody, humanized antibody or human antibody.

In the present invention, the binding molecule is characterized in that it comprises a heavy region comprising a variable region comprising a polypeptide sequence as set out in SEQ ID NO: 35 and a constant region comprising a polypeptide sequence as set out in SEQ ID NO: 37.

Also in the present invention, the binding molecule is characterized in that it comprises a light chain comprising a variable region comprising a polypeptide sequence as set out in SEQ ID NO: 36 and a constant region comprising a polypeptide sequence as set out in SEQ ID NO: 38.

Also in the present invention, the binding molecule is a heavy chain comprising a variable region comprising a polypeptide sequence as set out in SEQ ID NO: 35 and a constant region comprising a polypeptide sequence as set out in SEQ ID NO: 37 and a polypeptide sequence as set out in SEQ ID NO: 36 And a light chain comprising a constant region comprising a variable region comprising a and a polypeptide sequence set forth in SEQ ID NO: 38.

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

The binding molecule of the present invention is characterized in that the antibody.

In addition, the rabies virus is characterized in that it is derived from any one selected from the group consisting of dogs, cattle, mongoose, bats, skunks and wolves.

The present invention also provides an immunoconjugate in which at least one tag is attached to the binding molecule.

The present invention also provides a nucleic acid molecule encoding the binding molecule.

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

The present invention also provides an expression vector inserted with a nucleic acid molecule encoding the binding molecule.

As the expression vector, Celltrion's unique expression vector, MarEx vector (see patent application 10-2006-0020723), and pCDNA vectors commercially widely used, F, R1, RP1, Col, pBR322, ToL, Ti vectors; Cosmid; Phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμ, T-even, T2, T3, T7; It is preferable to use an expression vector selected from any one selected from the group consisting of plant viruses, but not limited thereto. All expression vectors known to those skilled in the art can be used in the present invention, and when selecting an expression vector, a target host may be selected. It depends on the nature of the cell. The introduction of the vector into the host cell may be performed by calcium phosphate transfection, viral infection, DEAE-dextran controlled transfection, lipofectamine transfection or electroporation, but is not limited thereto. An introduction method suitable for the expression vector and the host cell can be selected and used. Preferably, the vector contains one or more selection markers, but is not limited thereto, and may be selected depending on whether the product is produced using a vector that does not include the selection marker. The selection of the selection marker is selected by the host cell of interest, which uses methods already known to those skilled in the art and the present invention is not so limited.

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

The present invention also provides a cell line wherein the expression vector is transformed into a host cell to produce the binding molecule of the present invention.

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

The present invention also provides a pharmaceutical composition further comprising the binding molecule and a pharmaceutically acceptable excipient.

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

The present invention also provides a composition for the treatment and prevention of rabies further comprising the binding molecule and a pharmaceutically acceptable excipient.

The composition of the present invention may contain a pharmaceutically acceptable excipient in addition to the binding molecule having the rabies virus neutralizing ability of the present invention. Pharmaceutically acceptable excipients are well known to those skilled in the art.

In addition, the prophylactic and therapeutic composition of the present invention may include at least five different rabies therapeutics, and may also include several kinds of monoclonal antibodies, thereby exhibiting a synergistic effect on neutralizing activity.

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

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

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

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

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

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

In addition, the present invention provides a kit for diagnosing rabies comprising the binding molecule comprising the following steps: 1) a binding molecule having the rabies virus neutralizing ability of the present invention; And 2) a container.

In addition, the present invention provides a kit for treating and preventing rabies comprising the binding molecule comprising the following steps: 1) a binding molecule having the rabies virus neutralizing ability of the present invention; And 2) a container.

In addition, the present invention provides a method for diagnosing rabies using the binding molecule comprising the steps of: 1) contacting a sample of the subject with a binding molecule having a rabies virus neutralizing ability of the present invention; And 2) determining the rabies infection by analyzing the results of step 1).

The present invention also provides a method for treating and preventing rabies, comprising administering to the subject an effective amount of the binding molecule comprising the steps of: binding a rabbit virus neutralizing ability binding molecule In a therapeutically effective amount.

In addition, the present invention provides a method for producing a binding molecule having a neutralizing ability by binding to a rabies virus comprising the following steps: 1) culturing the cell line; And 2) recovering the expressed binding molecule.

The present invention also provides a method for detecting rabies virus, comprising the steps of: 1) contacting a sample of a subject with a binding molecule having the rabies virus neutralizing ability of the present invention; And 2) determining whether the binding molecule specifically binds to the sample of interest.

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

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

< Example >

Example  One: Hybridoma ( hybridoma Cell selection

Based on previous experiments and records on hybridoma cells stored in the Center for Disease Control (CDC), approximately 10 hybridoma cells were selected and cultured for each clone. Fab titers in the medium were tested for Rapid Fluorescent Focus Inhibition Test (RFFIT) (Smith, JS et al ., Geneva : World Health). Oragnization , pp . 181-191, 1996; And centers for Disease Control and Prevention, Morb . Mortal . Weekly Rep . 49 (RR-1), 1-21, 1999), and the results are shown in Table 1. As a result, a certain level (0.7 IU / ml) of the clones were selected and clones were named as follows: # 62-62-5, # 62-62-6, # 2-21-8, # 2 -21-14 and # 2-21-23.

Clone titer findings Sample ID Isotype Titer 62-111-1 IgG2a 0.03 62-105-06 Undetermined 0.03 62-62-5 IgG2a 0.7 62-80-6 IgG2b 0.7 2-21-20 IgG2a 0.1 62-28-5 IgG2a 0.03 62-114-6 IgG3 0.03 2-21-23 IgG2a 83 2-21-8 IgG2a 150 2-21-14 IgG2a 17.8 62-139-2 IgG1 0.03 2-21-17 Undetermined 0.03

Purified from hybridoma cells selected from these, monoclonal antibodies were obtained, and RFFIT was performed by titer-related experiments.

RFFIT Results Using Purified Clones Antibody name
(Antibody concentration) Titer IU / ml IU / mg 2-21-8
(3.5 mg / mL) 13500 158.82 45.4 62-80-6
(3.58 mg / mL) 60 0.71 0.2 2-21-23
(4.9 mg / mL) 7500 88.24 18.0 2-21-14
(1.9 mg / mL) 1900 22.35 11.7

The four clones of Table 2 were subjected to RFFIT for rabies virus stored in the US CDC. The US CDC has about 50 species of rabies viruses worldwide, and the list of viruses is shown in Table 3 below.

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

As a result, RFFIT results of the individual clones for the rabies virus described in Table 3 are listed in Table 4 below. The higher the value, the higher the neutralizing effect.

RFFIT Results Performed on US CDC No Rabies virus
Kinds SRIG 2-21-8
(IU / mg) 62-80-6
(IU / mg) 2-21-23
(IU / mg) 2-21-14
(IU / mg) One CVS-11 170 13500
(45.4) 60
(0.2) 7500
(18.0) 1900
(11.8) 2 Mongoose rsa 33 1300
(22.5) 45
(0.8) 1200
(14.8) 250
(8.0) 3 CASK 250 110
(0.3) 625
(1.4) 15625
(25.5) 2400
(10.1) 4 Tunissia dog 1100
42724
(22.2) 1800
(0.9) 42724
(15.9) 38206
(36.6) 5 Gabon dog 1000 10000
(5.7) 60
(0) 10000
(4.1) 1500
(1.6) 6 TXFX 250 24747
(56.6) 2700
(6.0) 113263
(184.9) 5700
(24.0) 7 THAI DOG 85 15625
(105.0) 125
(0.8) 9500
(45.6) 1100
(13.6) 8 Sonora dog 230 24747
(61.5) 480
(1.2) 34800
(61.8) 7500
(34.3) 9 002 Philippines 320 49326
(88.1) 75
(0.1) 40269
(51.4) 5100
(16.8) 10 DR MX 270 49326
(104.4) 16
(0) 6800
(10.3) 1200
(4.7) 11 DR Brazil 250 12000
(27.4) 440
(1.0) 12000
(19.6) 1400
(5.9) 12 Dog Phi 70 7000
57.1) 125
(1.0) 7000
(40.8) 1300
(19.5) 13 WA Bat 250 142857
(326.5) 440
(1.0) 151565
(247.5) 78124
(328.9) 14 3860ce Bat 250 17
(0) 3125
(7) <5
(0) 7
(0) 15 Dog arg 230 174692
(434.0) 1500
(3.6) 34800
(61.8) 6000
(27.5) 16 TX SK 250 174692
(399.3) <5
(0) 151565
(247.5) 8000
(33.7) 17 RAC 250 5400
(12.3) 14
(0) 5400
(8.8) 800
(3.4) 18 China 2005 1400 53887
(22.0) 8000
(3.2) 59744
(17.4) 42724
(32.1) 19 Rv342 China 290 9500
(18.1) 60
(0.1) 8000
(11.3) 10000
(36.3) 20 TX Coyote 250 38206
(87.3) 1600
(3.6) 40269
(65.7) 5100
(21.5) 21 rv61 170 230
(0.8) xxx 200
(0.5) xxx 22 AL Bat 170 170
(0.6) xxx 170
(0.4) xxx 23 LC NY 170 3125
(10.5) xxx 3125
(7.5) xxx 24 Bat ef 170 170
(0.6) xxx 170
(0.4) xxx 25 C1434 170 210
(0.7) xxx 230
(0.6) xxx 26 ABV (SM 4476) 145 125
(0.5) xxx 210
(0.6) xxx 27 Wu ABLV 320 390
(0.7) xxx 320
(0.4) xxx 28 AZ Bat 100 <5
(0) xxx 9
(0) xxx 29 VA 399 100 <5
(0) xxx <5
(0) xxx 30 TN 410 100 20432
(116.8) xxx <5
(0) xxx 31 TN 132 100 <5
(0) xxx <5
(0) xxx 32 SK 4384 230 70
(0.2) <5
(0) 1900
(3.4) xxx 33 AK FX 230 xxx 60
(0.1) 9500
(16.9) xxx 34 857r 180 250
(0.8) 1600
(5.0) 18086
(41.0) xxx 35 I-148 145 210
(0.8) 12
(0) 40269
(113.4) 3600
(26.1) 36 Mong PR 180 15625
(49.6) 320
(1.0) 174692
(396.1) xxx 37 Gray FX-AZ 180 170
(0.5) xxx 1500
(3.4) xxx 38 NC SK 180 1000
(3.2) xxx 13500
(30.6) xxx 39 323R 230 xxx xxx 8500
(15.1) xxx 40 RVHN 250 xxx > 11
(0) > 11
(0) xxx 41 MI 1625 250 xxx 133591
(298.5) 250
(0.4) xxx 42 I-151 145 210
(0.8) <5
(0) 38206
(107.5) 1600
(11.6) 43 TN 269 145 17
(0.1) <5
(0) 13
(0) <5
(0) 44 Sri lanka 145 95
(0.4) <5
(0) 75
(0.2) xxx 45 Myotis 145 54
(0.2) xxx 50
(0.1) xxx

(SRIG: Standard Rabies ImmunoGlobulin)

(xxx: do not perform)

Among them, 2-21-23 clone showed overall high efficacy and was delivered to Celltrion, and chimeric monoclonal antibody was prepared using the variable region of 2-21-23 clone and the constant region of human type antibody. .

Example  2: Hybridoma  cell( hybridoma cell Secreted by Chimeric  Antibody ( chimeric  Antibody) cDNA  secure

Example  2-1: Cell Culture

Hybridoma cells # 2-21-8, 2-21-14, 2-21-23, 62-80-6 were received from the US CDC and the cells were harvested from 5% fetal bovine serum (FBS; Sigma, 12003C). ) Was added to IMDM medium (Invitrogen 12440-053). During the culture period, mycoplasma contamination was examined using the Mycoplasma PCR ELISA kit (Roche, 11663925910), and it was confirmed that there was no mycoplasma in the culture.

Example  2-2: Hybridoma  Of mouse-type antibodies secreted by cells With heavy chains Light chain  Variable area DNA  secure

Total RNA was isolated from hybridoma # 2-21-23 in culture using RNeasy plus mini kit (Qiagen, 74134). Using the SMARTer PCR cDNA synthesis kit (Clontech, 634925) as a template for the isolated 1 ㎍ of total RNA, reverse transcription and 5 'and 3' fast cDNA terminal amplification polymerase chain reaction (5 ') were performed. and 3 'RACE PCR; Rapid Amplification of cDNA Ends Polymerase Chain Reaction at the same time to synthesize cDNA library having a specific sequence at the 5' and 3 'end. cDNA library synthesis reaction conditions are as follows. First, 1 μg of total RNA was mixed with 3 ′ SMART CDS Primer II A (5′-AAG CAG TGG TAT CAA CGC AGA GTA CT (30) N-1N-3 ′: SEQ ID NO: 1), followed by 72 ° C. The reaction was carried out for 3 minutes at 42 ° C. Thereafter, SMARTer II A Oligonucleotide (5'- AAG CAG TGG TAT CAA CGC AGA GTA-3 ': SEQ ID NO: 2) and reverse transcriptase were added and mixed, and reverse transcription reaction was performed at 42 ° C. for 60 minutes and at 70 ° C. for 10 minutes. By performing the above, first strand cDNA having a specific sequence at the 5 'end and 3' end was synthesized. First strand cDNA as a template, using a 5 'PCR Primer II A (5'-AAG CAG TGG TAT CAA CGC AGA GT -3 ': SEQ ID NO: 3) 1 minute heat denaturation at 95 ℃, 15 seconds at 95 ℃ , CDNA library having specific sequences at the 5 'and 3' ends was finally synthesized and amplified by polymerase chain reaction repeated 30 times at 65 ° C for 30 seconds and at 68 ° C for 30 seconds.

In order to amplify specific cDNA for the variable region of the mouse type antibody secreted by hybridoma cells in the synthesized cDNA library, polymerase chain reaction was performed using Advantage2 PCR Kit (Clontec, 639207). The sense primer used is a primer included in the SMARTer PCR cDNA synthesis kit used above. The 5 'PCR primer II A (5'- AAG CAG TGG TAT CAA CGC AGA) matches the specific sequence of the 5' end of the cDNA. GT-3 ': SEQ ID NO: 3). For the heavy chain, the antisense primer used a sequence (5'-GCC AGT GGA TAG AGC GAT G-3 ': SEQ ID NO: 4) targeting a sequence specific for the heavy chain constant region of IgG2a. Or 5′-GTG GGA AGA TGG AGA CAG TTG-3 ′ (SEQ ID NO: 5) and 5′-GGA CAG TCA GTT TGG-3 ′ (SEQ ID NO: 6) Was used. Using these primers, hybridoma cell # 2-21-23 is expressed through polymerase chain reaction repeated 30 times at 95 ° C for 30 seconds and at 68 ° C for 1 minute after thermal denaturation at 95 ° C for 1 minute. A cDNA fragment containing the entire variable region for the antibody was obtained. The cDNA fragments including the entire variable region of the heavy and light chains were cloned into TA vectors in the TOPO TA cloning kit (Invitrogen, K4500), and then sequenced. As a result of the sequencing analysis, in the case of the heavy chain, three IgG2a gamma chain variable region DNA sequences of one amino acid were obtained. In this case, it is considered that not one but three heavy chain variable regions are secured by mutation in the reverse transcriptase polymerase chain reaction (RT-PCR) process to secure cDNA fragments for the variable regions. In the case of the light chain, a variable region DNA sequence for one kappa chain was obtained.

Example  2-3: Chimeric  Antibody ( chimeric antibody ) Encrypted With heavy chains Light chain  cDNA production

Using overlapping PCR, a chimeric antibody was prepared by linking the variable region DNA sequence of the mouse type antibody chain to the constant region of a human type antibody. First, in order to proceed with the overlapping polymerase chain reaction, using the primers described in Table 5, thermal denaturation at 95 ° C. for 5 minutes, followed by 1 minute at 95 ° C., 1 minute at 57 ° C., and 1 minute at 72 ° C. Repeated times, polymerase chain reaction products for the mouse type light (kappa chain) and heavy (gamma chain) variable regions and polymerase chain reaction for the human type light (kappa chain) and heavy (gamma chain) constant regions. The product was obtained. Thereafter, the polymerase chain reaction product of the variable region and the constant region was used as a template, and PCR was performed under the same conditions as described above using HC F1 and HC R2 primers to secure a heavy chain connected to the variable region and the constant region. In the case of the light chain, polymerase chain reaction was carried out in the same manner using LC F1 and LC R2 primers to secure a light chain having a variable region and a constant region. Finally, each of the obtained heavy and light chains was cloned into a PCR2.1 TA cloning vector. After cloning, sequencing confirmed that no mutation occurred in the overlapping polymerase chain reaction.

Primer Information Primer
designation Explanation Base sequence HC F1 Sense primers for variable regions of mouse type heavy chains 5'- GCT AGC GCC ACC ATG AGA GTG CTG ATT CTT TTG TGC -3 '(SEQ ID NO: 7) HC R1 Antisense Primer for Variable Regions of Mouse Type Heavy Chains 5'- TGG TGG AGG CTG AGG AGA CGG TGA CTG AGG-3 '(SEQ ID NO: 8) HC F2 Sense Primer for the Constant Region of Human Type Heavy Chains 5'- CAC CGT CTC CTC AGC CTC CAC CAA GGG CCC -3 '(SEQ ID NO: 9) HC R2 Antisense Primer for the Constant Region of Human Type Heavy Chains 5'- GTT TAA ACG TGT CAT TTA CCC GGA GAC AGG GAG A -3 '(SEQ ID NO: 10) LC F1 Sense primers for variable regions of mouse type light chains 5'- GCT AGC GCC ACC ATG GAT TCA CAG GCC CAG GT -3 '(SEQ ID NO: 11) LC R1 Antisense Primers for Variable Regions of Mouse Type Light Chains 5'- GCA GCC ACA GTT CGT TTT ATT TCC AGC TTG GTC CCC -4 '(SEQ ID NO: 12) LC F2 Sense Primer for the Constant Region of Human Type Light Chains 5'- GGA AAT AAA ACG AAC TGT GGC TGC ACC ATC -3 '(SEQ ID NO: 13) LC R2 Antisense Primer for Constant Regions of Human Type Light Chains 5'- AGG GAT ATC GCG CGC TAA CAC TCT CCC CTG TTG AAG CTC -3 '(SEQ ID NO: 14)

Example  2-4: Chimeric  Antibody Expression Vector Construction

PCR2.1 TA cloning vectors containing the obtained heavy and light chains were treated with the restriction enzymes Nhe I and Pme I to obtain the heavy and light chain genes, and then the obtained heavy and light chain genes were respectively treated with the same restriction enzyme. It was inserted into pCT145 vector and pCT147 vector. The pCT145 and pCT147 vectors are Celltrion-specific vectors designed to clone the heavy and light chains of antibodies, respectively. Subsequently, in order to construct an expression vector including a heavy chain transcription unit (promoter-heavy chain gene-polya) and a light chain transcription unit (promoter-light chain gene-polya), a restriction enzyme Pac I was added to the pCT145 vector including the heavy chain gene. After treating Asc I and heavy chain transcription units, the same restriction enzyme was inserted into the pCT147 vector containing the light chain gene to insert the heavy chain transcription unit. Then, using restriction enzymes, vectors containing both heavy and light chain transcription units were selected and named pCT188 (see FIG. 1). The selected vector was extracted using Endofree plasmid maxi kit (QIAGEN, Germany, 12362), and finally the nucleotide sequence of the antibody was confirmed by sequencing using some of the extracted DNA. The confirmed sequences are shown in Table 6 below.

Mouse Type Monoclonal Antibody Sequence Information Sequence type order Heavy chain variable region CDR1
Polynucleotide AGTGATCATAGCTGGCAC (SEQ ID NO: 15) Heavy chain variable region CDR2
Polynucleotide TACATACACTACGATGGTAGCACTGACTACAACCCATCTCTCAAAAGT (SEQ ID NO: 16)
Heavy chain variable region CDR3
Polynucleotide GAAACGATCTACTATGGTAACCCCTATGGTATGGACTAC (SEQ ID NO: 17)
Light chain variable region CDR1
Polynucleotide AAATCCAGTCAGAGTCTGTTCAACAGTGGAACCCGAAAGAACTACTTGGCT (SEQ ID NO: 18)
Light chain variable region CDR2
Polynucleotide TGGGCATCCACTAGGGAATCT (SEQ ID NO: 19)
Light chain variable region CDR3
Polynucleotide AAGCAATCTTATAATCTGTACACG (SEQ ID NO: 20)
Heavy chain variable region # 1
Polynucleotide GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTACTCCGTCACCAGTGATCATAGCTGGCACTGGATCCGGCAGTTTCCAGAAAACAAACTGGAATGGATGGGCTACATACACTACGATGGTAGCACTGACTACAACCCATCTCTCAAAAGTCGAATCTTTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGAGAAACGATCTACTATGGTAACCCCTATGGTATGGACTACTGGGGTCAGGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 21) Heavy chain variable region # 6
Polynucleotide GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTACTCCGTCACCAGTGATCATAGCTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATACACTACGATGGTAGCACTGACTACAACCCATCTCTCAAAAGTCGAATCTTTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGAGAAACGATCTACTATGGTAACCCCTATGGTATGGACTACTGGGGTCAGGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 22) Heavy chain variable region # 7
Polynucleotide GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTACTCCGTCACCAGTGATCATAGCTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATACACTACGATGGTAGCACTGACTACAACCCATCTCTCAAAAGTCGAATCTTTGTCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGAGAAACGATCTACTATGGTAACCCCTATGGTATGGACTACTGGGGTCAGGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 23) Light chain variable region
Polynucleotide GACATTGTGATGTCACAGTCTCCGCCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGTTCAACAGTGGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCTCCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 24) Heavy chain variable region CDR1
Polypeptide SDHSWH (SEQ ID NO: 27)
Heavy chain variable region CDR2
Polypeptide YIHYDGSTDYNPSLKS (SEQ ID NO 28)
Heavy chain variable region CDR3
Polypeptide ETIYYGNPYGMDY (SEQ ID NO: 29)
Light chain variable region CDR1
Polypeptide KSSQSLFNSGTRKNYLA (SEQ ID NO: 30)
Light chain variable region CDR2
Polypeptide WASTRES (SEQ ID NO: 31)
Light chain variable region CDR3
Polypeptide KQSYNLYT (SEQ ID NO 32)
Heavy chain variable region # 1
Polypeptide DVQLQESGPDLVKPSQSLSLTCTVTGYSVTSDHSWHWIRQFPENKLEWMGYIHYDGSTDYNPSLKSRIFITRDTSKNQFFLQLNSVTTEDTATYYCARETIYYGNPYGMDYWGQGTSVTVSS (SEQ ID NO: 33) Heavy chain variable region # 6
Polypeptide DVQLQESGPDLVKPSQSLSLTCTVTGYSVTSDHSWHWIRQFPGNKLEWMGYIHYDGSTDYNPSLKSRIFITRDTSKNQFFLQLNSVTTEDTATYYCARETIYYGNPYGMDYWGQGTSVTVSS (SEQ ID NO: 34) Heavy chain variable region # 7
Polypeptide DVQLQESGPDLVKPSQSLSLTCTVTGYSVTSDHSWHWIRQFPGNKLEWMGYIHYDGSTDYNPSLKSRIFVTRDTSKNQFFLQLNSVTTEDTATYYCARETIYYGNPYGMDYWGQGTSVTVSS (SEQ ID NO: 35) Light chain variable region
Polypeptide DIVMSQSPPSLAVSAGEKVTMSCKSSQSLFNSGTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLSISSVQAEDLAVYYCKQSYNLYTFGGGTKLEIK (SEQ ID NO: 36)

Example  3: by transient transduction method Chimeric  Production of antibodies

FreeStyle ^TM Max (Invitrogen, 16447-100), a cationic polymer, was used for transient transduction in cells, and transduction was performed according to the manufacturer's instructions. The day before transduction, F2N cells grown on EX-CELL 293 Serum free media (Sigma, 14571C: hereinafter referred to as "EX-CELL 293 medium") were centrifuged and replaced with FreeStyle293 serum free media (Gibco, 12338). 50 ml (total 100 ml) were inoculated using two 250 ml shaker flasks at a concentration of 0.8 × 10 ⁶ cells per ml. On the day of transfection, 125 μg of pCT178 DNA containing the chimeric antibody gene and 125 μl of FreeStyle ^™ Max reagent were diluted in 2 ml volume using OptiPRO SFM II (Invitrogen, 12309) medium, and then mixed lightly. Immediately diluted FreeStyle ^™ Max reagent solution was mixed with a solution containing DNA, and then reacted at room temperature for 17 minutes. During the 17 min reaction at room temperature, the number of inoculated F2N cells to be used for transduction was measured, and the cell concentration was diluted to 1.0 × 10 ⁶ cells using the FreeStyle293 medium. After 17 minutes, transduction was performed by treating the F2N cells with a mixture solution of DNA and FreeStyle ^™ Max reagent. The day after transduction, the same amount of EX-CELL 293 medium was added to the transduced cells and cultured for 7 days to produce monoclonal antibodies.

Example  4: Chimeric  Identify efficacy of monoclonal antibodies

Example  4-1: in vitro  Experiment

Three chimeric forms of candidates were obtained from the monoclonal antibodies produced in Example 3, along with the original mouse antibody 2-21-23 prior to dilution to appropriate chimeric monoclonal antibodies by dilution to appropriate concentrations as shown in Table 7 below. Neutralizing capacity experiments were performed on representative rabies viruses, which were performed via RFFIT. The results are shown in Table 8.

Concentrations of 2-21-23 Mouse and Chimeric Monoclonal Antibodies Used in the Experiment Monoclonal antibody
(Antibody concentration) 1:10 Titer
(IU / mg) 1: 100 titer
(IU / mg) 1: 1000 tit
(IU / mg) Mouse antibody
2-21-23
(121 ug / mL) 1500
(198.3) 210
(27.8) 7
(0.9) Chimeric Antibody # 1
2-21-23
(116 ug / mL) 1400
(193.1) 95
(13.1) 11
(1.6) Chimeric Antibody # 6
2-21-23
(95 ug / mL) 1200
(202.1) 110
(18.5) 9
(1.5) Chimeric Antibody # 7
2-21-23
(186 ug / mL) 6800
(584.9) 270
(23.2) 25
(2.2)

Result of RFFIT of Monoclonal Antibody Converted to Chimeric Form Rabies virus types SRIG 2-21-23 mouse
Antibody
(121 ug / mL)
Titer (IU / mg) Chimeric Antibody # 1
2-21-23
(116 ug / mL)
Titer (IU / mg) Chimeric Antibody # 6
2-21-23
(95 ug / mL)
Titer (IU / mg) Chimeric Antibody # 7
2-21-23
(186 ug / mL)
Titer (IU / mg) China 2005 210 2200 (173.2) 540 (44.3) 230 (23.1) 5700 (291.9) Rv342
(China) 210 1600 (125.9) 320 (26.3) 200 (20.1) 8000 (409.6) Dog Thai 210 1300 (102.3) 320 (26.3) 170 (17.0) 4600 (235.5) Phi 002 180 2200 (202.0) 250 (23.9) 320 (37.4) 6000 (358.4) Phi dog 180 1500 (137.7) 9 (0.9) 45 (5.3) 2700 (161.3) Mong RSA 180 70 (6.4) 5 (0.5) 6 (0.7) 145 (8.7) Dog tune 230 1600 (115.0) 230 (17.2) 145 (13.3) 5700 (266.5) Dog gab 230 1500 (107.8) 70 (5.2) 145 (13.3) 1600 (74.8) DR MX
(Mexico) 230 1100 (79.1) 110 (8.2) 145 (13.3) 1500 (70.1) I-148
(India) 210 1900 (149.5) 290 (23.8) 125 (12.5) 7500 (384.0) TX SK 170 1600 (155.6) 75 (7.6) 60 (7.4) 4600 (291.0) SK 4384
(Texas) 170 440 (42.8) 42 (4.3) 25 (3.1) 1600 (101.2) RVHN
(Russia) 210 159750 (12573.8) 33489 (2749.5) 174692 (17513.0) 174692 (8944.8) Sri lanka 210 1100 (86.6) 9 (0.7) <5 (0) 2200 (112.6) Dog son 210 800 (63.0) 75 (6.2) 25 (2.5) 4600 (235.5)

Through this result, chimeric antibody # 7 2-21-23 showing high efficacy was selected.

Table 9 below shows the results of experiments compared to SRIG using viruses that were not made in Table 8 in earnest after the determination for chimeric antibody # 7 2-21-23.

Rabies virus types
SRIG
(actual titer) 2-21-23 chimeric antibody # 7 (9 ug / mL)
Titer
(IU / mg) Dog ARG 250 1300
(1155.6) 857r (27) 210 1400
(1481.5) AK Fox 210 1200
(1269.8) WA Bat 250 1300
(1155.6) rv61 170 1400
(1830.1) AL Bat 170 1200
(1568.6) Bat ef 170 1300
(1699.3) C1434 170 1400
(1830.1) Gray FX-AZ 180 340
(419.8) NC SK 180 230
(284.0) 323R 230 1400
(1352.7) I-151 145 1400
(2145.6) TN 269 145 60 (15/20 in well 1: 5)
(1.8) Myotis 145 1000
(1532.6)

Example  4-2: in vivo  Animal experiment

In order to investigate whether the 2-21-23 chimeric antibody # 7 selected in the above examples has a therapeutic effect against rabies virus in vivo, animal experiments were performed using a Syrian hamster as follows. . Chinese animal rabies virus Rv342 was used in this animal experiment.

The experimental group of animals was divided into four groups: 1. group injected with Rv342 virus only, 2. group injected with Rv342 virus and vaccine (Human diploid Imovax ^® Sanofi Pasteur), 3. Rv342 virus and 2-21-23 key Meric antibody # 7 was injected, 4. Rv342 virus, 2-21-23 chimeric antibody # 7 and vaccine (Human diploid Imovax â € ^“ Sanofi Pasteur). Rv342 virus was diluted 1/100 on the basis of MICLD50 / ml and injected 50 ul intramuscularly, and the vaccine was injected with 50 ul at least 2.5 IU of vaccine virus strain per ml (day 0, 3, 7, 14). Merric antibody # 7 was injected with 50 ul in 0.6 mg / mL. Vaccine and chimeric antibody # 7 were injected 24 hours after Rv342 virus injection, respectively.

Experimental results are shown in Table 10 and FIG. When the virus and 2-21-23 chimeric antibody # 7 were injected (group 3), all survived until the 45-day observation period, but only when the virus was injected (group 1) or the virus and vaccine were injected (group 2). All died. In addition, the virus, 2-21-23 chimeric antibody # 7, and vaccine injection (Group 4) showed a survival rate of 90% or more.

in In vivo animal test results Animal testing
group process Gun hamster
Number Survival hamster
Number Survival rate * One Rv342 virus 6 0 0% 2 Rv342 Virus + Vaccine 6 0 0% 3 Rv342 Virus + 2-21-23 Chimeric Antibody # 7 12 12 100% 4 Rv342 virus + 2-21-23 chimeric antibody # 7 + vaccine 12 11 91.7%

* Observation period: 45 days after virus infection

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

Attach an electronic file to a sequence list

Claims (25)

delete delete According to the Kabat method, a heavy chain having a CDR1 region comprising a polypeptide as set out in SEQ ID NO: 27, a CDR2 region comprising a polypeptide as set out in SEQ ID NO: 28 and a CDR3 region comprising a polypeptide as set out in SEQ ID NO: 29 A light chain variable region having a CDR1 region comprising a variable region and a polypeptide set forth in SEQ ID NO: 30, a CDR2 region comprising a polypeptide set forth in SEQ ID NO: 31 and a CDR3 region comprising a polypeptide set forth in SEQ ID NO: 32, A binding molecule that binds to rabies virus and has a neutralizing ability. delete delete A binding molecule having a neutralizing ability to bind to rabies virus, comprising a heavy chain variable region comprising a polypeptide sequence as set out in SEQ ID NO: 35 and a light chain variable region comprising a polypeptide sequence as set out in SEQ ID NO: 36. delete delete A variable region comprising a polypeptide region as set out in SEQ ID NO: 35 and a heavy chain comprising a constant region comprising a polypeptide sequence as set out in SEQ ID NO: 37 and a variable region comprising a polypeptide sequence as set out in SEQ ID NO: 36 and SEQ ID NO: 38 A binding molecule having a neutralizing ability to bind to rabies virus, comprising a light chain comprising a constant region comprising a polypeptide sequence as described. 10. The binding molecule of any one of claims 3, 6, or 9, wherein the binding molecule is an antibody. 7. The binding molecule of claim 3 or 6, wherein the binding molecule is a Fab fragment, Fv fragment, diabody, chimeric antibody, humanized antibody or human antibody. 10. The method of claim 3, 6 or 9, wherein the rabies virus is derived from any one selected from the group consisting of dogs, cattle, mongoose, bats, skunks and wolves. Binding molecule. An immunoconjugate in which at least one tag is additionally bound to the binding molecule of any one of claims 3, 6, or 9. 10. A nucleic acid molecule encoding the binding molecule of any one of claims 3, 6 or 9. 14. An expression vector into which the nucleic acid molecule of claim 14 is inserted. A cell line wherein the expression vector of claim 15 is transformed into a host cell to produce a binding molecule that binds to rabies virus and has a neutralizing ability. The method of claim 16, wherein the host cell is composed of CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK 293 cells and HEK293T cells Cell line, characterized in that any one selected from the group. delete A composition for treating and preventing rabies further comprising the binding molecule of any one of claims 3, 6, or 9 and a pharmaceutically acceptable excipient. 1) the binding molecule of any one of claims 3, 6 or 9; And
2) containers
Rabies diagnostic kit comprising a. 1) the binding molecule of any one of claims 3, 6 or 9; And
2) containers
Rabies treatment and prevention kit comprising a. 1) contacting a sample of a subject other than a human with a binding molecule of any one of claims 3, 6, or 9; And
2) determining whether the rabies infection by analyzing the results of step 1)
Rabies diagnostic method comprising a. A therapeutically effective amount of a binding molecule of any one of claims 3, 6, or 9 to a subject, except a human identified as infected with rabies.
Rabies treatment and prevention method comprising a. 1) culturing the cell line of claim 16; And
2) recovering the expressed binding molecule
A method of producing a binding molecule having a neutralizing ability by binding to a rabies virus comprising a. 1) contacting a sample of a subject other than a human with a binding molecule of any one of claims 3, 6, or 9; And
2) determining whether the binding molecule specifically binds to the target sample
Method for detecting a rabies virus comprising a.

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