KR102642178B1 - Mutant anti-rabies antibody with improved serum half-lives and neutralizing rabies virus - Google Patents

Abstract

The present invention relates to a rabies virus neutralizing antibody whose blood half-life and rabies virus neutralizing efficacy is increased by mutation, and a method for producing the same, and more specifically to a heavy chain (HC) represented by SEQ ID NO: 1 and a rabies virus neutralizing antibody represented by SEQ ID NO: 2. It is about a rabies virus neutralizing antibody comprising a light chain (LC), wherein the 462nd amino acid of the heavy chain is substituted with lysine (K) or the 463rd amino acid is substituted with phenylalanine (F).
The present invention includes a heavy chain (HC) represented by SEQ ID NO: 1 and a light chain (LC) represented by SEQ ID NO: 2, wherein the 462nd amino acid of the heavy chain is substituted with lysine (K) or the 463rd amino acid is substituted with phenylalanine (F). The rabies virus neutralizing antibody, which is characterized in that it has a greatly increased half-life in the blood compared to commercial antibodies and existing plant-derived antibodies, and also has an excellent rabies virus neutralizing effect, can be usefully used in the prevention and treatment of rabies disease.

Description

Mutant anti-rabies antibody with improved serum half-lives and neutralizing rabies virus}

The present invention relates to a rabies virus neutralizing antibody whose blood half-life and rabies virus neutralizing efficacy are increased by mutation, and more specifically, to a heavy chain (HC) represented by SEQ ID NO: 1 and a light chain (LC) represented by SEQ ID NO: 2. It relates to a rabies virus neutralizing antibody, wherein the 462nd amino acid of the heavy chain represented by SEQ ID NO: 1 is substituted with lysine (K) and the 463rd amino acid is substituted with phenylalanine (F).

Rabies is a viral zoonotic disease that mainly affects wild animals and pets, as well as mammals, including humans, causing acute brain disease. It is a fatal disease that almost causes death once it occurs, and is known to be the disease with the highest mortality rate along with AIDS. This rabies disease is widespread worldwide, with more than 10 million people receiving treatment each year after infection, and between 40,000 and 70,000 deaths each year.

Rabies is transmitted through saliva and blood, and is mainly caused by being bitten by a dog or cat infected with rabies. It can also be infected by most mammals, including skunks and bats.

The rabies virus reaches the brain nerve tissue through the body's terminal nerve tissue and then shows actual symptoms. Originally, the human brain has a blood brain barrier that blocks external substances, so viruses cannot penetrate, but the rabies virus passes through the blood barrier through the RVG (rabies virus glycoprotein) protein and enters the central nervous system. (central nervous system) Infects the brain.

In the beginning, in addition to cold-like symptoms, you may feel itching or heat at the site of the bite. As rabies progresses, anxiety, phobias (when swallowing liquids such as water causes muscle spasms and severe pain, causing fear of water), fear of wind (wind irritates sense organs), excitement, and paralysis. , neurological abnormalities such as mental abnormalities appear. It can also cause hypersensitivity to sunlight. 2-7 days after these symptoms are observed, nerves and muscles throughout the body become paralyzed, the patient falls into a coma, and eventually dies from respiratory failure.

Currently, there are vaccinations for rabies, but there is no cure. Treatment after exposure to rabies includes post-bite therapy (post-exposure prophylaxis). Post-bite treatment consists of immediate local wound protection, antibody administration for passive immunity (anti-rabies immunoglobulin: hereinafter referred to as “anti-rabies antibody”), and vaccine administration for active immunity. Currently developed anti-rabies antibodies include human derived rabies immunoglobulin (hereinafter referred to as “HRIG”) and equine derived rabies immunoglobulin (hereinafter referred to as “ERIG”). In the case of HRIG, supply is not smooth, it is expensive, and since it is a polyclonal antibody, the efficacy per unit weight is not high. Additionally, because it is derived from human blood, there is a high risk of potential infection from human-derived diseases such as HIV. Although ERIG is cheaper than HRIG, it is also not in smooth supply, and its treatment efficiency is lower than that of HRIG, so it is administered to patients at much higher doses. In addition, because the antibodies are derived from horses, a different animal from humans, they may cause anaphyaxis. In order to overcome the poor supply and shortcomings of polyclonal antibodies, the use of a monoclonal antibody that can neutralize the rabies virus was proposed. A rabies-virus neutralizing murine monoclonal antibody was developed in the 1980s (Schumacher CL et al., J. Clin. Invest. Vol. 84, p. 971-975, 1989), but due to its short half-life in the human body and its Direct administration to human patients is limited due to disadvantages such as the absence of an immune response in the body and the induction of human anti-murine antibodies (HAMA).

The neonatal Fc receptor (FcRn) produced in vivo binds to the Fc portion of the antibody and plays a role in controlling the homeostasis of the antibody (IgG) in the body. The higher the binding force between FcRn and antibody (IgG), the longer it can stay in the body by preventing decomposition of the antibody in vivo. At this time, the efficiency of the antibody can be increased by mutating the Fc portion of the antibody (IgG) to increase its binding force with FcRn.

Therefore, for effective treatment of rabies, there is an urgent need to develop antibodies that have increased binding affinity to FcRn, extend half-life in the blood, improve neutralization efficacy, and can be mass-produced.

Accordingly, the present inventors produced an antibody with mutated amino acids in the heavy chain in order to improve the half-life in the blood of a rabies treatment drug and improve its neutralizing effect, and completed the present invention by confirming that it was effectively expressed in plants.

Therefore, the object of the present invention includes a heavy chain (HC) represented by SEQ ID NO: 1 and a light chain (LC) represented by SEQ ID NO: 2, wherein the 462nd amino acid of the heavy chain represented by SEQ ID NO: 1 is lysine (K) and 463. To provide a rabies virus neutralizing antibody characterized in that the first amino acid is substituted with phenylalanine (F).

Another object of the present invention is to provide a polynucleotide encoding the rabies virus neutralizing antibody.

Another object of the present invention is to provide a vector containing the above polynucleotide.

Another object of the present invention is to provide plants or tissues transformed with the vector.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating rabies comprising the antibody or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to achieve the above object, the present invention includes a heavy chain (HC) represented by SEQ ID NO: 1 and a light chain (LC) represented by SEQ ID NO: 2, and the 462nd amino acid of the heavy chain represented by SEQ ID NO: 1 is Provided is a rabies virus neutralizing antibody characterized in that lysine (K) and the 463rd amino acid are substituted with phenylalanine (F).

Additionally, a polynucleotide encoding the rabies virus neutralizing antibody is provided.

Additionally, a vector containing the polynucleotide is provided.

Additionally, plants or tissues transformed with the vector are provided.

Additionally, a pharmaceutical composition for preventing or treating rabies comprising the antibody or a pharmaceutically acceptable salt thereof as an active ingredient is provided.

Hereinafter, the present invention will be described in detail.

The present invention includes a heavy chain (HC) represented by SEQ ID NO: 1 and a light chain (LC) represented by SEQ ID NO: 2, wherein the 462nd amino acid of the heavy chain represented by SEQ ID NO: 1 is lysine (K) and the 463rd amino acid is Provided is a rabies virus neutralizing antibody characterized by substitution with phenylalanine (F).

The present inventors modified the heavy chain base sequence of the existing human SO57 antibody to optimize it for plant codons and confirmed that it is effectively expressed in crops such as tobacco, and are disclosing it herein for the first time.

In the present invention, the term 'antibody' is used confusingly with 'immunoglobulin (hereinafter referred to as "Ig")', and is a general term for proteins that selectively act on antigens and participate in biological immunity. Antibodies are made up of two pairs: light and heavy chains. The light and heavy chains of these antibodies are polypeptides composed of multiple domains. In a whole antibody, each heavy chain includes a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region includes heavy chain constant domains CH1, CH2 and CH3 (antibody classes IgA, IgD and IgG) and optionally heavy chain constant domain CH4 (antibody classes IgE and IgM). Each light chain includes a light chain variable domain (VL) and a light chain constant domain (CL). The variable domains VH and VL can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed within more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. (Janeway, CA , JR., et al. (2001). Immunobiology., ^5th ed., Garland Publishing; and Woof, J., Burton, D., Nat Rev Innunol 4 (2004) 89-99). The two pairs of heavy and light chains (HC/LC) can specifically bind to the same antigen. Accordingly, the entire antibody is a bivalent, monospecific antibody.

The antibody of the present invention includes a heavy chain (HC) represented by SEQ ID NO: 1 and a light chain (LC) represented by SEQ ID NO: 2, wherein the 462nd amino acid of the heavy chain represented by SEQ ID NO: 1 is lysine (K) and the 463rd amino acid. This is a rabies virus neutralizing antibody characterized by substitution with phenylalanine (F).

In one embodiment of the present invention, the rabies virus neutralizing antibody SO57, which is characterized in that the 462nd amino acid of the heavy chain is substituted with lysine (K) and the 463rd amino acid is substituted with phenylalanine (F), has different binding affinity to FcRn and rabies virus neutralizing ability. It was confirmed that the efficacy was increased the most compared to antibodies substituted with .

Additionally, in the present invention, the antibody may further include an endoplasmic reticlum signal peptide (same meaning as endoplasmic reticulum targeting sequence). The endoplasmic reticulum signal peptide (ER signal sequence) refers to an amino acid sequence that allows a protein to be recognized by a signal recognition particle on the endoplasmic reticulum and allows the protein to translocate into the ER lumen. In the present invention, the type and amino acid sequence of the endoplasmic reticulum signal peptide are not limited as long as it is a plant endoplasmic reticulum signal peptide known to those skilled in the art. For example, literature such as US 20130295065 and WO 2009158716 can be referred to. In the present invention, the endoplasmic reticulum signal peptide is preferably KDEL (SEQ ID NO: 14), HDEL (SEQ ID NO: 15), SEKDEL (SEQ ID NO: 16), KHEDL (SEQ ID NO: 17), KEEL (SEQ ID NO: 18), SEHEDL (SEQ ID NO: 19) ) It may be any one polypeptide selected from the group consisting of, and most preferably it may be a polypeptide represented by KDEL.

The insertion site of the endoplasmic reticulum signal peptide is not limited as long as it does not affect the antigen recognition and binding ability of the antibody, but may preferably be the end of the antibody heavy chain protein peptide sequence, and more preferably the C-terminus of the antibody heavy chain protein peptide sequence. It may be a part.

By inserting the nucleotide encoding the KDEL into a specific gene (in the present invention, an antibody expression gene), KDEL is exposed at the end of the amino acid sequence of the final product. This induces the produced protein to exist in the endoplasmic reticulum within the transformed cell rather than being secreted outside the plant cell. The protein produced in the host cell into which the specific gene is introduced is stored in the endoplasmic reticulum by the KDEL sequence and undergoes post-translatonal modification that can occur in the plant. This plays an important role in solving problems caused by increased expression of antibody proteins in cells and differences in sugar structures between different species, which play an important role in the immune response between different species. In antibodies produced through the endoplasmic reticulum (ER) storage process using the KDEL sequence, the high-mannose glycan-type structure (also called high-mannose glycan-type) triggers an immune response through the mannose receptor in dendritic cells or macrophages. (Zhe Lu, et al., Expression of GA733-Fc Fusion Protein as a Vaccine Candidate for Colorectal Cancer in Transgenic Plants, Journal of Biomedicine and Biotechnology Volume 2012, Article ID 364240, 11 pages, doi: 10.1155/2012/364240).

The antibody of the present invention may be expressed in eukaryotes such as E. coli, yeast, and mammals, but is preferably expressed in plants.

The plant may preferably be a seed plant, which is a plant that reproduces by making and spreading seeds, and includes both gymnosperms and angiosperms, and angiosperms include both dicotyledonous plants and monocotyledonous plants. Seed plants are largely composed of roots, stems, and leaves as nutritional organs, and flowers as reproductive organs, and there are seeds, which are plants in an undeveloped embryonic state. The plant in the present invention may be a whole plant with a complete structure or a part of a plant consisting of plant organs, tissues, such as roots, stems, leaves, flowers, and seeds, or a plurality of plant cells. Parts of a plant may be connected to the whole plant or may be separate.

Additionally, the types of cells or tissues of transformed plants according to the present invention are not limited as long as they are derived from plants. Plant tissues include, for example, epidermal tissue composed of epidermal cells that make up the surface of the plant, stomata tissue composed of two guard cells in the epidermis, hairs formed from epidermal cells, root hairs formed from epidermal cells of the root, and stem. Parenchyma tissue including the part of the root excluding the vascular bundle and mesophyll tissue (hedge tissue, spongy tissue) that performs photosynthesis, olfactory tissue enlarged in the parenchyma tissue of the stem or petiole, sclerenchymal tissue with enlarged cell walls, involved in the movement of water within the plant. There are xylem or tracheids, xylem parenchyma, xylem containing xylem fibers, phloem parenchyma responsible for transporting organic substances, phloem tissue containing phloem fibers, semicells, etc.

Additionally, plants in the present invention include cultures of plant cells or tissues. The 'culture' refers to the ability of plant cells or tissues to form the shape of the entire plant or to regenerate the plant body, thereby converting parts of the plant, such as cells, tissues, organs, embryos, and seeds, into nutrients. It refers to the product cultured in the added medium. The cell/tissue culture products of the plant may include, for example, embryo culture, slice culture, callus culture, suspension culture, drug culture (pot culture), and protoplast culture, but are not limited thereto. Protoplast refers to the cell contents obtained by removing the plant cell wall and isolating it.

The plant of the present invention can be used without limitation as long as it is a plant capable of mass producing protein, but more specifically, tobacco, Arabidopsis, corn, rice, soybean, canola, alfalfa, sunflower, alfalfa, sorghum, wheat, cotton, peanut, It may be selected from the group consisting of tomatoes, potatoes, lettuce and peppers, and is preferably tobacco. The tobacco in the present invention is a plant of the Nicotiana genus and is not particularly limited in type as long as it can overexpress proteins. The present invention can be carried out by selecting an appropriate variety according to the purpose of transformation method and mass production of protein. You can. For example , Nicotiana benthamiana L. or Nicotiana tabacum cv. Varieties such as xanthi are available.

Additionally, the present invention provides a polynucleotide encoding the rabies virus neutralizing antibody.

In the present invention, ‘polynucleotide’ means ‘nucleic acid’ and refers to deoxyribonucleotide (DNA) or ribonucleotide (RNA) in single- or double-stranded form. Unless otherwise limited, known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides are also included. 'mRNA' is an RNA that transfers the genetic information of the base sequence of a specific gene to ribosomes that form polypeptides during the protein synthesis process.

The polynucleotide of the present invention may be a polynucleotide encoding the rabies virus neutralizing antibody of the present invention, and preferably includes a heavy chain (HC) represented by SEQ ID NO: 1 and a light chain (LC) represented by SEQ ID NO: 2, It may be a polynucleotide encoding a rabies virus neutralizing antibody, wherein the 462nd amino acid of the heavy chain represented by SEQ ID NO: 1 is substituted with lysine (K) and the 463rd amino acid is substituted with phenylalanine (F).

Additionally, the present invention provides a vector containing the above polynucleotide.

In the present invention, a 'vector' is a recombinant expression vector capable of expressing a desired protein or nucleic acid (RNA) in a suitable host cell, and is an essential control operably linked to enable expression of the polynucleotide (gene) insert. refers to a genetic construct containing an element. 'Operably linked' means that a nucleic acid expression control sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked to perform a general function, and the gene is linked by the expression control sequence. It means connected so that it can be manifested. The ‘expression control sequence’ refers to a DNA sequence that regulates the expression of an operably linked polynucleotide sequence in a specific host cell. Such regulatory sequences include a promoter to effect transcription, optional operator sequences to regulate transcription, sequences encoding suitable mRNA ribosome binding sites, sequences regulating termination of transcription and translation, initiation codons, stop codons, and polyadenylation. Includes signals and enhancers.

The vector of the present invention includes a heavy chain (HC) represented by SEQ ID NO: 1 and a light chain (LC) represented by SEQ ID NO: 2, and the 462nd amino acid of the heavy chain represented by SEQ ID NO: 1 is lysine (K) and the 463rd amino acid. It is characterized by expressing a rabies virus neutralizing antibody characterized in that the amino acid is substituted with phenylalanine (F).

The heavy chain included in the vector of the present invention may have the amino acid sequence represented by SEQ ID NO: 3, and the light chain may have the amino acid sequence represented by SEQ ID NO: 2.

For the expression of antibodies, the light and heavy chains are included in the vector. The promoters for the expression of the light and heavy chains may be the same or different promoters, and this may be appropriate depending on the host cell in which the antibody is expressed, the level of expression, etc. It can be adjusted.

For convenience of classification, in the present invention, the promoter for the light chain is called the first promoter, and the promoter for the heavy chain is called the second promoter.

For the purpose of the present invention, the expression control sequences and other elements essential for gene expression are preferably derived from the host plant or optimized for expression in the plant. For example, the promoter of a plant gene or a promoter of a gene that uses a plant as a host or can be expressed in a plant is operably linked to the polynucleotide of the antibody with a mutated heavy chain portion represented by SEQ ID NO: 1 according to the present invention and used in a recombinant expression vector. Can be inserted. Any plant-derived promoter can be selected and used as long as it is commonly used in the art, for example, ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu), beta- Conglycinin promoter, phaseolin promoter, ADH (alcohol dehydrogenase) promoter, heat shock promoter, ADF (actin depolymerization factor) promoter, tissue-specific promoter, etc. can be used without limitation. In addition, the octopine polymerase (synthase) promoter, nopaline polymerase promoter, mannopine polymerase promoter, and the 35S and 19S promoters of cauliflower mosaic virus (CaMV), which are derived from viruses, can be used. In addition to the promoter, it may additionally include additional expression control sequences such as an enhancer that can increase transcription efficiency. The promoter is a constitutive promoter that continuously expresses a gene in all plant cells, or expresses a gene only in a specific plant tissue/organ or during a specific plant development period, or is activated by a specific stimulus such as light or hormones or the environment. It may be an inducible promoter having. The first or second promoter used to express the antibody of the present invention is preferably the CaMV 35S promoter (Ca2p) or the potato protease inhibitor II promoter (Pin2P), and more preferably the first promoter. may be the promoter of potato protease repressor II (Pin2P) and the second promoter may be the CaMV 35S promoter (Ca2p).

For the expression of antibodies, light and heavy chains are included in the vector. The termination sequences of the light and heavy chains may be the same or different from each other, and may be appropriately adjusted depending on the host cell to be expressed, expression level, etc. .

For convenience of classification, in the present invention, the termination sequence for the light chain is called the first termination sequence, and the termination sequence for the heavy chain is called the second termination sequence.

In addition, the poly A signal sequence for polyadenylation of the 3'-end of mRNA suitable for the present invention is derived from the nopaline polymerase or octopine polymerase gene of Agrobacterium tumefaciens . , the 3' terminal portion of the protease inhibitor I or II gene of tomato or potato, the CaMV 35S terminator, and the OCS terminator (octopine synthase terminator) sequence. Preferably, the first or second termination sequence of the present invention may be a terminator sequence (T2) derived from the 3' terminal portion of the potato protease inhibitor I and II genes or the nopaline polymerase gene. More preferably, the first termination sequence is the 3' end of the protease inhibitor II gene of potato (Terminator of Pin2 gene from potato, T1), and the second termination sequence is a termination sequence derived from the nopaline polymerase gene ( It may be Terminator of nopaline synthase (NOS) gene, T2).

The recombinant expression vector of the present invention is a vector commonly used in the cloning field, especially in the field of plant-based protein expression, and its type is not particularly limited as long as it can be appropriately selected by a person skilled in the art. A polynucleotide encoding the antibody of the present invention is operably linked to a promoter suitable for plant expression, and elements suitable for gene expression in plants, such as 5' untranslated region sequence, RNA A recombinant expression vector containing the polynucleotide of the present invention can be prepared by using a transcription termination sequence and a polyadenylation signal sequence using genetic recombination techniques known in the art. In addition, the recombinant expression vector according to the present invention may contain a selection marker and/or a replication origin for selecting host cells when constructing the recombinant expression vector, and the expression vector may also be used to express antibodies and, if necessary, It includes a signal sequence for targeting or secretion to the cell membrane or other organelles to the extent that it does not impair activity, and can be manufactured in various ways depending on the purpose, including the sequence of a reporter or marker gene. .

The signal sequence for targeting or secretion to the organelle may preferably be an endoplasmic reticlum signal peptide (same meaning as endoplasmic reticulum signal peptide). The endoplasmic reticulum signal peptide (ER signal sequence) refers to an amino acid sequence that allows a protein to be recognized by a signal recognition particle on the endoplasmic reticulum and allows the protein to translocate into the ER lumen. In the present invention, the type and amino acid sequence of the endoplasmic reticulum signal peptide are not limited as long as it is a plant endoplasmic reticulum signal peptide known to those skilled in the art. For example, literature such as US 20130295065 and WO 2009158716 can be referred to. In the present invention, the endoplasmic reticulum signal peptide is preferably KDEL (SEQ ID NO: 14), HDEL (SEQ ID NO: 15), SEKDEL (SEQ ID NO: 16), KHEDL (SEQ ID NO: 17), KEEL (SEQ ID NO: 18), SEHEDL (SEQ ID NO: 19) ) It may be any one polypeptide selected from the group consisting of, and most preferably it may be a polypeptide represented by KDEL.

The insertion site of the endoplasmic reticulum signal peptide is not limited as long as it does not affect the antigen recognition and binding ability of the antibody, but may preferably be the end of the antibody heavy chain protein peptide sequence, and more preferably the C-terminus of the antibody heavy chain protein peptide sequence. It may be a part.

The recombinant expression vector for expression in plants for implementing the present invention is preferably an Agrobacterium binary vector. The 'binary vector' refers to the separation of the tumor inducible plasmid (Ti plasmid), a tumor-inducing gene, into two plasmids in Agrobacterium-mediated transformation, that is, the recombinant gene is transferred to the plant's genome. It refers to a vector that is divided into a plasmid with the LB (left border) and RB (right border) sequences required to move the recombinant gene, and a plasmid that encodes the protein needed to move the recombinant gene. Preferably, the pBI121 binary vector can be used. The Agrobacterium used in the present invention can be any type commonly used for plant transformation.

In one embodiment of the present invention, a vector (SEQ ID NO: 21) having the cleavage map shown in FIG. 1 was prepared and plants were transformed.

Additionally, the present invention provides plants or tissues transformed with the above recombinant expression vector.

The present invention also provides plants transformed with a recombinant expression vector. The transformed plant expresses an antibody containing the heavy chain variant portion of the present invention, may be a cell, tissue, or culture of the plant, and includes part or the entire plant.

The types of plants are the same as described above.

The method for transforming plant cells or tissues can be any plant transformation method known in the art without limitation. A person skilled in the art can select and perform a known transformation method appropriate for a specific plant, taking into account the characteristics of the plant selected as the host. Plant transformation methods include, for example, a method of fusing liposomes containing a recombinant expression vector with plant protoplasts, a method of injecting the recombinant expression vector into plant protoplasts using PEG, and direct incorporation of the recombinant expression vector into plant cells. Injection method, fine particle bombardment method, gene gun, electroporation method, transformation method using virus, vacuum infiltration method, floral meristem dipping method, etc. can be used. , Preferably, a transformation method using Agrobacterium can be used.

The 'transformation method using Agrobacterium' is a method of transferring foreign genes to plant cells using Agrobacterium, a gram-negative soil bacterium that causes tumors in the roots and stems of plants. T-DNA (transfer DNA) of the tumor-inducing plasmid (Ti plasmid) found in Agrobacterium such as Agrobacterium tumefaciens and Agrobacterium rhizogenes . This is a method that utilizes the phenomenon of insertion into the plant genome. In transformation using Agrobacterium, a binary plasmid (or binary vector) containing the foreign gene to be introduced into the plant (exogenous DNA) and T-DNA (LB and RB sequences located on both edges of the foreign gene) and T- It is common to use a binary system consisting of two plasmids: a helper plasmid that allows DNA to be inserted into the plant genome; The transformation method using Agrobacterium can be used on various plant tissues such as leaves, stems, and roots, and young tissues tend to be easily transformed.

Using the transformation method using Agrobacterium in the present invention, antibodies can be expressed transiently or stably expressed. For transient expression, a part of the plant, for example, a leaf of the plant, is infected and transformed with Agrobacterium containing a recombinant expression vector, and after a period of time for sufficient expression of the protein of interest, the infected part of the plant is obtained. can do. For stable expression, plant cells or tissues can be cultured, infected with Agrobacterium, transformed, then further cultured to select appropriate transformants, and after re-differentiation, transformed plants can be cultured with a complete structure. . By obtaining and germinating seeds from the transformed plants, transformed plants can be stably obtained in the next generation.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating rabies disease comprising the rabies virus neutralizing antibody as an active ingredient.

The antibody according to the present invention has excellent rabies virus neutralizing efficacy and can remain in the body for a long time due to its increased half-life in the blood, so it can be usefully used as a pharmaceutical composition for preventing or treating rabies disease.

The composition according to the present invention can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration. Can be administered, preferably intravascularly.

Additionally, the composition according to the present invention can be formulated in various ways depending on the route of administration by methods known in the art along with a pharmaceutically acceptable carrier. 'Pharmaceutically acceptable' refers to a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient and does not usually cause gastrointestinal upset, allergic reactions such as dizziness, or similar reactions when administered to humans. . The carrier includes all types of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes.

When the composition of the present invention is administered parenterally, the composition of the present invention may be formulated with a suitable parenteral carrier in the form of injections, transdermal administration, and nasal inhalation according to methods known in the art. The above injections must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. For injections, examples of suitable carriers include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), mixtures thereof, and/or vegetable oils. You can. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanol amine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. etc. can be used. In order to protect the injection from microbial contamination, it may additionally contain various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. Additionally, in most cases, the injection may additionally contain an isotonic agent such as sugar or sodium chloride.

In the case of transdermal administration, forms such as ointments, creams, lotions, gels, external solutions, paste preparations, linear preparations, and airol preparations are included. In the above, ‘transdermal administration’ means that the composition of the present invention is topically administered to the skin and an effective amount of the active ingredient contained in the composition is delivered into the skin. For example, the composition of the present invention can be prepared in an injectable formulation and administered by lightly pricking the skin with a 30-gauge thin injection needle or applying it directly to the skin. These formulations are described in a generally known text in pharmaceutical chemistry, Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania.

For inhalation administration, the compositions according to the invention may be administered from a pressurized pack or nebulizer using a suitable propellant, such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be conveniently delivered in the form of an aerosol spray. For pressurized aerosols, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in inhalers or insufflators can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

As for other pharmaceutically acceptable carriers, those described in the following literature may be referred to (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The composition according to the invention may also contain one or more buffers (e.g. saline or PBS), carbohydrates (e.g. glucose, mannose, sucrose or dextran), antioxidants, bacteriostatic agents, chelating agents (e.g. For example, EDTA or glutathione), adjuvants (e.g. aluminum hydroxide), suspending agents, thickening agents and/or preservatives.

Additionally, the composition of the present invention can be formulated in various ways using methods known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal. Additionally, the composition of the present invention can be administered in combination with known compounds that are effective in preventing or treating rabies.

In one embodiment of the present invention, it was confirmed that the antibody of the present invention was well expressed in tobacco plants, and it was confirmed that mass production and purification of the antibody of the present invention was possible.

In another embodiment of the present invention, it was confirmed that the antibody of the present invention had superior FcRn affinity than the wild SO57 antibody and the MST antibody and MN antibody in which other parts of the heavy chain amino acid sequence were substituted, and the antibody according to the present invention had the best half-life in the blood. I confirmed that it was long.

In another embodiment of the present invention, it was confirmed that the antibody of the present invention has a superior rabies virus neutralizing effect than the wild SO57 antibody, the MST antibody with other parts of the heavy chain amino acid sequence substituted, and the MN antibody, and can be usefully used in the treatment of rabies disease. was confirmed.

Therefore, the present invention includes a heavy chain (HC) represented by SEQ ID NO: 1 and a light chain (LC) represented by SEQ ID NO: 2, wherein the 462nd amino acid of the heavy chain represented by SEQ ID NO: 1 is lysine (K) and the 463rd amino acid. A rabies virus neutralizing antibody characterized by substitution with phenylalanine (F) and a polynucleotide encoding the same, a vector containing the same, a plant cell or tissue transformed with the recombinant expression vector, and the rabies virus neutralizing antibody as an active ingredient. It provides a pharmaceutical composition comprising:

The rabies virus neutralizing antibody of the present invention has a greatly increased half-life in the blood compared to commercial antibodies and existing plant-derived antibodies, and also has an excellent rabies virus neutralizing effect, so it can be usefully used in the prevention and treatment of rabies disease.

Figure 1 shows a cleavage map of the transformation vectors used in the present invention.
Figure 2 shows the results of transforming tobacco and confirming the expression of rabies antibody protein by Western blot.
Figure 3 shows mass production of transgenic tobacco plants under optimized conditions (left), and commercial antibodies (mAb ^H , This is the result of confirming the purification of the 2nd international standard for anti-rabies innunoglobulin, Human NIBSC, RAI, NIBSC) and three antibodies (MST, MN, HN).
Figure 4 shows the results of analyzing the binding affinity between three antibodies (MST, MN, HN) and human FcRn (hFcRn) using Enzyme-Linked ImmunoSorbent Assay (ELISA).
Figure 5 shows the results of Surface Plasmon Resonance (SPR) analysis of the binding affinity between three antibodies (MST, MN, HN) and human FcRn (hFcRn) using an XPR36 surface instrument (bio-rad).
Figure 6 shows the results of an activity analysis against the rabies virus, CVS11, using the Rapid Fluorescent Focus Inhibition Test (RFFIT) of a rabies antibody treatment derived from tobacco plants.
Figure 7 shows the results of analyzing the N-glycans structures obtained from WT and three antibody (MST, MN, HN) variants using MALDI-TOF mass spectrum analysis. (■: GlcNAc, ○: mannose, △: xlylose )

Hereinafter, the present invention will be described in detail.

However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

<Example 1>

Preparation of binary vectors for expressing SO57 in plant-based systems

The heavy chain of the SO57 antibody (GenBank accession no. PCR was performed on AY172957). Accordingly, the sequences of antibodies with mutated MST (M281Y/S283T/T285E), MN (M457L/N463S), and HN (H462K/N463F) positions were prepared.

PCR was performed 30 times under the following conditions: 94°C for 20 seconds, 53°C for 20 seconds, and 72°C for 90 seconds.

primer sequence antibody primer sequence MST 5'-CACCACGCATGTGACCTCAGGCTCCCGGGTGATATAGAGGGTGTCCTTGGGTTTTGG-3'
(SEQ ID NO: 6) 5'-CCAAAACCCAAGGACACCCTCTATATCACCCGGGAGCCTGAGGTCACATGCGTGGTG-3' (SEQ ID NO: 7) M.N. 5'-CTGCGTGTAGTGGCTGTGCAGAGCCTCATGC-3' (SEQ ID NO: 8) 5'-GCATGAGGCTCTGCACAGCCACTACACGCAG-3' (SEQ ID NO: 9) 5'-TTCTCATGCTCCGTGTTGCATGAGGCTCTGC-3' (SEQ ID NO: 10) 5'-GCAGAGCCTCATGCAACACGGAGCATGAGAA-3' (SEQ ID NO: 11) H.N. 5'-GCTCTTCTGCGTGTA GTGGAACTTCAGAGCTCATGCATCACG-3'
(SEQ ID NO: 12) 5'-CGTGATGCATGAGGCTCTGAA GTTCCACTACACGCAGAAGAGC-3'
(SEQ ID NO: 13)

Primers containing NcoI and , was introduced using primers containing PstI and BamHI. HCK combined with the mutated heavy chain and KDEL was placed together with the AMV translation enhancer, and the light chain (SEQ ID NO: 2, GenBank accession no. AY172960) was placed under the control of Pin2P (potato Pin2 gene promoter).

Three cleavage maps of the pBI121 binary vector into which the heavy and light chains of the SO57 antibody with mutated heavy chain amino acid sequences were introduced are shown in Figure 1 .

<Example 2>

Transient expression of SO57 antibody in tobacco cells

The three types of modified antibody genes prepared in <Example 1> were introduced into tobacco plant cells using Agrobacterium (LBA4404) to prepare transgenic plants expressing different antibodies.

The expression of each modified rabies antibody protein was confirmed through Western blot. The modified rabies antibody was confirmed by detecting rabbit anti-human Fcγ and goat anti-human F(ab')₂ conjugated with horseradish peroxidase (HRP) as secondary antibodies.

As shown in Figure 2 , as a result of confirming the bands in the heavy chain (50 kDa) and light chain (25 kDa), it was confirmed that the antibody of the present invention was well expressed in tobacco plants.

<Example 3>

Purification of modified SO57 antibody from tobacco

Tobacco plants transformed with three types of antibodies were mass-produced under optimized conditions (see left of Figure 3), and three types of rabies were identified through protein purification using a Protein A affinity column using a down streaming process. The antibody therapeutic agent was purified. Approximately 200-250 g of tobacco plant leaves for each gene were ground and purified, and commercially available antibodies (mAb ^H , The purification results of the 2nd international standard for anti-rabies innunoglobulin, Human NIBSC, RAI, NIBSC), existing antibodies, and three antibodies (MST, MN, HN) were confirmed (right of Figure 3).

As shown in Figure 3 , all three types of antibodies were well purified without significant differences from existing antibodies. These results confirm that no problems were found that may reduce production efficiency due to mutation of the sugar structure when the protein is expressed in plants, and confirm that the antibody according to the present invention is well produced in tobacco plants.

<Example 4>

Confirmation of affinity of modified antibody with FcRn (ELISA)

The binding affinity of the three antibodies (MST, MN, HN) and human FcRn (hFcRn) was analyzed by competitive ELISA at pH 6.0 and pH 7.4, respectively. 100 ㎕ each of the existing antibody (WT) and the three antibodies (MST, MN, HN) were incubated overnight at 4°C and attached to a 96 well plate, followed by hFcRn treatment at 37°C for 1 hour and 30 minutes, followed by secondary detection of hFcRn. After treating each antibody (6-His Antibody, Bethyl) at 100 ㎕ RT for 2 hours, absorbance was measured (Friguet B. et al., J. Immunol. Meth., 77; 305-319, 1985) and compared. did.

As shown in Figure 4 , at pH 6.0, the affinity for MST, MN, and HN increased by about 12, 16, and 18 times, respectively, compared to WT, and at pH 7.4, it increased by about 2, 15, and 17 times, respectively. Through this, it was confirmed that the affinity of the HN antibody with FcRn was significantly superior to that of existing antibodies and MST and MN antibodies, and accordingly, it was confirmed that the antibody's decomposition was inhibited in vivo and could stay longer than the comparative antibodies.

<Example 5>

Confirmation of affinity of modified antibody with FcRn (SPR analysis)

The binding affinity of three antibodies (MST, MN, HN) and human FcRn (hFcRn) was measured using surface plasmon resonance (SPR) using an XPR36 surface instrument (bio-rad) at pH 6.0 and pH 7.4, respectively. was analyzed. After attaching hFcRn to the biochip, WT and three antibodies (MST, MN, HN) were flowed, and the affinity was measured and compared.

As shown in Figure 5 , the SPR results also showed the same tendency as the hFcRn and ELISA results. At pH 6.0, WT, MST, MN, and HN showed 100, 140, 180, and 195 RU, respectively, and at pH 7.4, they showed 20, 20, 70, and 100 RU, respectively. Through this, it was confirmed that the FcRn affinity of HN was significantly superior to that of existing antibodies and MST and MN antibodies, and accordingly, it was confirmed that the antibody degradation was inhibited in vivo and could stay longer than the comparative antibodies.

<Example 6>

Confirmation of rabies virus neutralizing efficacy of modified antibody

Analysis of the activity of the modified antibody against rabies virus CVS11 was performed using a neutralization in vitro assay (RFFIT). Each antibody was diluted at 1:2.5, 1:12.5, 1:62.5, and 1:312.5 to a CVS of 32 to 100 FFD50, dispensed onto a slide, then added with mouse neuroblastoma (N2a) cells and incubated at 35°C for 20 hours. I order it. After fixation with acetone, it was stained using the indirect immunofluorescent antibody method, reacted at 37°C for 30 minutes, and then observed under a fluorescence microscope (x200).

As a result, as shown in Figure 6, all three types of antibodies were changed compared to commercially available antibodies (the 2nd international standard for anti-rabies innunoglobulin, Human NIBSC, RAI, NIBSC) and antibodies before the amino acid sequence change. It showed higher neutralization efficacy. Through the above experimental results, it was confirmed that the rabies virus neutralizing efficacy of HN was the most excellent compared to existing antibodies and MST and MN antibodies, and that the efficacy varied depending on the position of the amino acid substitution.

As a result of combining the results of the above examples, the antibody of the present invention is well expressed and purified in plants and can be mass-produced, has the best FcRn affinity and can stay in the body the longest, and has a different comparative antibody neutralizing effect against rabies virus. It was confirmed that it has superior technical characteristics than others, and it was confirmed that it can be usefully used as a treatment for rabies.

<Example 7>

N-glycan analysis of modified antibodies (MST, MN, HN) using MALDI-TOF

After permethylation treatment, the N-glycans structures obtained from WT and three antibody variants (MST, MN, HN) were analyzed using MALDI-TOF mass spectral analysis.

As shown in Figure 7, the gray square represents GlcNAc, the white circle represents mannose, and the white triangle represents xlylose. When looking at the sugar structures of WT and the three antibodies (MST, MN, HN), they showed an oligomannose (OM) type structure. In the case of MST, a sugar structure containing β(1,2)-xylose was observed. However, the structure is evaluated to be very small. And, it was confirmed that most sugar structures were composed of antibodies with 7 mannoses. Based on the results of this study, it appears that even if there is a modification of the amino acid in the Fc portion, the modification of the sugar structure is almost insignificant.

Therefore, in addition to the experimental results of Example 3, the antibody of the present invention more clearly confirmed that no problems were found that may reduce production efficiency due to mutation of the sugar structure when the protein is expressed in plants, according to the present invention. This result supports that antibodies can be well produced in plants without modification of the sugar structure.

The rabies virus neutralizing antibody comprising a heavy chain (HC) represented by SEQ ID NO: 1 and a light chain (LC) represented by SEQ ID NO: 2 of the present invention, wherein the heavy chain region has the mutation H462K or N463F, is a commercially available antibody. And, the half-life in the blood is greatly increased compared to existing plant-derived antibodies, and the rabies virus neutralizing effect is also excellent, so it can be usefully used in the prevention and treatment of rabies disease, so it has industrial applicability.

<110> CHUNG ANG University industry Academic Cooperation Foundation <120> Mutant anti-rabies antibody with improved serum half-lives and neutralizing rabies virus <130> NP17-0053P <150> KR 10-2017-0100556 <151> 2017-08- 08 <160> 21 <170> KoPatentIn 3.0 <210> 1 <211> 480 <212> PRT <213> Artificial Sequence <220> <223> Human SO57 wild type HC <400> 1 Met Asp Trp Thr Trp Arg Phe Leu Phe Val Val Ala Ala Ala Thr Gly 1 5 10 15 Val Gln Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe 35 40 45 Asn Arg Tyr Thr Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala 65 70 75 80 Gln Arg Phe Gln Gly Arg Leu Thr Ile Thr Ala Asp Glu Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Phe Cys Ala Arg Glu Asn Leu Asp Asn Ser Gly Thr Tyr Tyr Tyr 115 120 125 Phe Ser Gly Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val 130 135 140 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 145 150 155 160 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 165 170 175 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 180 185 190 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 195 200 205 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 210 215 220 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 225 230 235 240 Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 245 250 255 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 260 265 270 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 275 280 285 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 290 295 300 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 305 310 315 320 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 325 330 335 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 340 345 350 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 355 360 365 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 370 375 380 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 385 390 395 400 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 405 410 415 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 420 425 430 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 435 440 445 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 450 455 460 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Lys Asp Glu Leu 465 470 475 480 <210> 2 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> Human SO57 wild type LC <400> 2 Met Ser Val Pro Thr Met Ala Trp Ala Leu Leu Leu Leu Ser Leu Leu 1 5 10 15 Thr Gln Gly Thr Gly Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Arg 20 25 30 Ser Val Ser Gly Ser Pro Gly Gln Ser Val Thr Ile Ser Cys Thr Gly 35 40 45 Thr Ser Ser Asp Ile Gly Gly Tyr Asn Phe Val Ser Trp Tyr Gln Gln 50 55 60 His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp Ala Thr Lys Arg 65 70 75 80 Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr 85 90 95 Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr 100 105 110 Tyr Cys Cys Ser Tyr Ala Gly Asp Tyr Thr Pro Gly Val Val Phe Gly 115 120 125 Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser 130 135 140 Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala 145 150 155 160 Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val 165 170 175 Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr 180 185 190 Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu 195 200 205 Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln 210 215 220 Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu 225 230 235 240 Cys Ser <210> 3 <211> 480 <212> PRT <213> Artificial Sequence <220> <223> Human SO57 : HN HC <400> 3 Met Asp Trp Thr Trp Arg Phe Leu Phe Val Val Ala Ala Ala Thr Gly 1 5 10 15 Val Gln Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe 35 40 45 Asn Arg Tyr Thr Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala 65 70 75 80 Gln Arg Phe Gln Gly Arg Leu Thr Ile Thr Ala Asp Glu Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Phe Cys Ala Arg Glu Asn Leu Asp Asn Ser Gly Thr Tyr Tyr Tyr 115 120 125 Phe Ser Gly Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val 130 135 140 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 145 150 155 160 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 165 170 175 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 180 185 190 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 195 200 205 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 210 215 220 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 225 230 235 240 Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 245 250 255 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 260 265 270 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 275 280 285 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 290 295 300 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 305 310 315 320 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 325 330 335 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 340 345 350 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 355 360 365 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 370 375 380 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 385 390 395 400 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 405 410 415 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 420 425 430 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 435 440 445 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu Lys Phe His 450 455 460 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Lys Asp Glu Leu 465 470 475 480 <210> 4 <211> 480 <212> PRT <213> Artificial Sequence <220> <223> Human SO57 : MST HC <400> 4 Met Asp Trp Thr Trp Arg Phe Leu Phe Val Val Ala Ala Ala Thr Gly 1 5 10 15 Val Gln Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe 35 40 45 Asn Arg Tyr Thr Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala 65 70 75 80 Gln Arg Phe Gln Gly Arg Leu Thr Ile Thr Ala Asp Glu Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Phe Cys Ala Arg Glu Asn Leu Asp Asn Ser Gly Thr Tyr Tyr Tyr 115 120 125 Phe Ser Gly Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val 130 135 140 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 145 150 155 160 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 165 170 175 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 180 185 190 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 195 200 205 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 210 215 220 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 225 230 235 240 Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 245 250 255 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 260 265 270 Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val 275 280 285 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 290 295 300 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 305 310 315 320 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 325 330 335 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 340 345 350 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 355 360 365 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 370 375 380 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 385 390 395 400 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 405 410 415 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 420 425 430 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 435 440 445 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 450 455 460 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Lys Asp Glu Leu 465 470 475 480 <210> 5 <211> 480 <212> PRT <213> Artificial Sequence <220> <223> Human SO57 : MN HC <400> 5 Met Asp Trp Thr Trp Arg Phe Leu Phe Val Val Ala Ala Ala Thr Gly 1 5 10 15 Val Gln Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe 35 40 45 Asn Arg Tyr Thr Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala 65 70 75 80 Gln Arg Phe Gln Gly Arg Leu Thr Ile Thr Ala Asp Glu Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Phe Cys Ala Arg Glu Asn Leu Asp Asn Ser Gly Thr Tyr Tyr Tyr 115 120 125 Phe Ser Gly Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val 130 135 140 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 145 150 155 160 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 165 170 175 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 180 185 190 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 195 200 205 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 210 215 220 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 225 230 235 240 Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 245 250 255 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 260 265 270 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 275 280 285 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 290 295 300 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 305 310 315 320 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 325 330 335 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 340 345 350 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 355 360 365 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 370 375 380 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 385 390 395 400 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 405 410 415 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 420 425 430 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 435 440 445 Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His 450 455 460 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Lys Asp Glu Leu 465 470 475 480 <210> 6 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> MST primer 1 <400> 6 caccacgcat gtgacctcag gctcccgggt gatatagagg gtgtccttgg gttttgg 57 <210> 7 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> MST primer 2 <400> 7 ccaaaaccca aggacaccct ctatatcacc cgggagcctg aggtcacatg cgt ggtg 57 <210> 8 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> MN primer 1 <400> 8 ctgcgtgtag tggctgtgca gagcctcatg c 31 <210> 9 <211> 31 <212> DNA <213> Artificial Sequence <220> <223 > MN primer 2 <400> 9 gcatgaggct ctgcacagcc actacacgca g 31 <210> 10 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> MN primer 3 <400> 10 ttctcatgct ccgtgttgca tgaggctctg c 31 <210> 11 <211> 31 <212 > DNA <213> Artificial Sequence <220> <223> MN primer 4 <400> 11 gcagagcctc atgcaacacg gagcatgaga a 31 <210> 12 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> HN primer 1 <400> 12 gctcttctgc gtgtagtgga acttcagagc ctcatgcatc acg 43 <210> 13 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> HN primer 2 <400> 13 cgtgatgcat gaggctctga agttccacta cacgcagaag a gc 43 <210> 14 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> KDEL <400> 14 Lys Asp Glu Leu 1 <210> 15 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> HDEL <400> 15 His Asp Glu Leu 1 <210> 16 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> SEKDEL <400> 16 Ser Glu Lys Asp Glu Leu 1 5 < 210> 17 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> KHEDL <400> 17 Lys His Glu Asp Leu 1 5 <210> 18 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> KEEL <400> 18 Lys Glu Glu Leu 1 <210> 19 <211 > 6 <212> PRT <213> Artificial Sequence <220> <223> SEHEDL <400> 19 Ser Glu His Glu Asp Leu 1 5 <210> 20 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> KDEL <400> 20 aaggacgaac tt 12 <210> 21 <211> 16454 <212> DNA <213> Artificial Sequence <220> <223> HN vector <400> 21 tgagcgtcgc aaaggcgctc ggtcttgcct tgctcgtcgg tgatgtactt c accagctcc 60 gcgaagtcgc tcttcttgat ggagcgcatg gggacgtgct tggcaatcac gcgcacccc 120 cggccgtttt agcggctaaa aaagtcatgg ctctgccctc gggcggacca cgcccatcat 180 gaccttgcca agctcgtcct gcttctcttc gatcttcgcc agcagggcga ggatcgtggc 240 atcaccgaac cgcgcc gtgc gcgggtcgtc ggtgagccag agtttcagca ggccgcccag 300 gcggcccagg tcgccattga tgcgggccag ctcgcggacg tgctcatagt ccacgacgcc 360 cgtgattttg tagccctggc cgacggccag caggtaggcc gacaggctca tgccggccgc 420 c gccgccttt tcctcaatcg ctcttcgttc gtctggaagg cagtacacct tgataggtgg 480 gctgcccttc ctggttggct tggtttcatc agccatccgc ttgccctcat ctgttacgcc 540 ggcggtagcc ggccagcctc gcagagcagg attcccgttg agcaccgcca ggtgcgaata 600 agggacagtg aagaaggaac acccgctcgc gggtgggcct acttcaccta tcctgcccgg 660 ctgacgccgt tggatacacc aaggaaagt c tacacgaacc ctttggcaaa atcctgtata 720 tcgtgcgaaa aaggatggat ataccgaaaa aatcgctata atgaccccga agcagggtta 780 tgcagcggaa aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 840 gcagggtcgg aacaggagag cgc acgaggg agcttccagg gggaaacgcc tggtatcttt 900 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 960 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1020 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1080 ttaccgcctt tgagtgagct gataccgctc g ccgcagccg aacgaccgag cgcagcgagt 1140 cagtgagcga ggaagcggaa gagcgccaga aggccgccag agaggccgag cgcggccgtg 1200 aggcttggac gctagggcag ggcatgaaaa agcccgtagc gggctgctac gggcgtctga 1260 cgcggtggaa agggggaggg g atgttgtct acatggctct gctgtagtga gtgggttgcg 1320 ctccggcagc ggtcctgatc aatcgtcacc ctttctcggt ccttcaacgt tcctgacaac 1380 gagcctcctt ttcgccaatc catcgacaat caccgcgagt ccctgctcga acgctgcgtc 1440 cggaccggct tcgtcgaagg cgtctatcgc ggcccgcaac agcggcgaga gcggagcctg 1500 ttcaacggtg ccgccgcgct cgccggcatc gctg tcgccg gcctgctcct caagcacggc 1560 cccaacagtg aagtagctga ttgtcatcag cgcattgacg gcgtccccgg ccgaaaaacc 1620 cgcctcgcag aggaagcgaa gctgcgcgtc ggccgtttcc atctgcggtg cgcccggtcg 1680 cgtgccgg ca tggatgcgcg cgccatcgcg gtaggcgagc agcgcctgcc tgaagctgcg 1740 ggcattcccg atcagaaatg agcgccagtc gtcgtcggct ctcggcaccg aatgcgtatg 1800 attctccgcc agcatggctt cggccagtgc gtcgagcagc gcccgcttgt tcctgaagtg 1860 ccagtaaagc gccggctgct gaacccccaa ccgttccgcc agtttgcgtg tcgtcagacc 1920 gtctacgccg acctcgttca acaggtccag ggcggcacgg atcactgtat tcggctgcaa 1980 ctttgtcatg cttgacactt tatcactgat aaacataata tgtccaccaa cttatcagtg 2040 ataaagaatc cgcgcgttca atcggaccag cggaggctgg tccggaggcc agacgtgaaa 2100 cccaacatac ccctgatcgt aattctgagc act gtcgcgc tcgacgctgt cggcatcggc 2160 ctgattatgc cggtgctgcc gggcctcctg cgcgatctgg ttcactcgaa cgacgtcacc 2220 gcccactatg gcattctgct ggcgctgtat gcgttggtgc aatttgcctg cgcacctgtg 2280 ctgggcgcgc tgtcggatcg tttcgggcgg cggccaatct tgctcgtctc gctggccggc 2340 gccagatctg gggaaccctg tggttggcat gcacatacaa atggacgaac gg ataaacct 2400 tttcacgccc ttttaaatat ccgattattc taataaacgc tcttttctct taggtttacc 2460 cgccaatata tcctgtcaaa cactgatagt ttaaactgaa ggcgggaaac gacaatctga 2520 tcatgagcgg agaattaagg gagtcacgtt atgacccccg ccgatgacg c gggacaagcc 2580 gttttacgtt tggaactgac agaaccgcaa cgttgaagga gccactcagc cgcgggtttc 2640 tggagtttaa tgagctaagc acatacgtca gaaaccatta ttgcgcgttc aaaagtcgcc 2700 taaggtcact atcagctagc aaatatttct tgtcaaaaat gctccactga cgttccataa 2760 attcccctcg gtatccaatt agagtctcat attcactctc aatccaaata atctgcaccg 2820 gatctggatc gt ttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt 2880 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg 2940 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg 30 00 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg 3060 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg 3120 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca 3180 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc 3240 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc 3300 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca 3360 aggcgcgcat gcccgacggc gatgatctcg tcgtgaccca tggcgatgcc tgcttgccga 3420 atatcat ggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg 3480 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg 3540 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg 3600 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga 3660 ccaagcgacg cccaacctg c catcacgaga tttcgattcc accgccgcct tctatgaaag 3720 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct 3780 catgctggag ttcttcgccc acgggatctc tgcggaacag gcggtcgaag gtgccgatat 3840 catt acgaca gcaacggccg acaagcacaa cgccacgatc ctgagcgaca atatgatcgg 3900 gcccggcgtc cacatcaacg gcgtcggcgg cgactgccca ggcaagaccg agatgcaccg 3960 cgatatcttg ctgcgttcgg atattttcgt ggagttcccg ccacagaccc ggatgatccc 4020 cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc 4080 gatgattatc atataatttc tgttga atta cgttaagcat gtaataatta acatgtaatg 4140 catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata 4200 cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc 4260 tatgttacta gatcgggcct cctgtcaat g ctggcggcgg ctctggtggt ggttctggtg 4320 gcggctctga gggtggtggc tctgagggtg gcggttctga gggtggcggc tctgagggag 4380 gcggttccgg tggtggctct ggttccggtg attttgatta tgaaaagatg gcaaacgcta 4440 ataagggggc tatgaccgaa aatgccgatg aaaacgcgct acagtctgac gctaaaggca 4500 aacttgattc tgtcgctact gattacggtg ctgctatcga tggtt tcatt ggtgacgttt 4560 ccggccttgc taatggtaat ggtgctactg gtgattttgc tggctctaat tcccaaatgg 4620 ctcaagtcgg tgacggtgat aattcacctt taatgaataa tttccgtcaa tatttacctt 4680 ccctccctca atcggttgaa tgtcgcc ctt ttgtctttgg cccaatacgc aaaccgcctc 4740 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag 4800 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt 4860 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca 4920 caggaaacag ctatgaccat gattacgcca agctttataa aaagctaaat aagatagagg 4980 g tattttcgt aaacaaataa ctaattcta gaaagtattc aatgcttatt agttataata 5040 caacaaacca aacaagcaat aaaaactaat ccccaacata acttatccca gcataactta 5100 tcccatcata acttattcca tcataacttg ttttcaaacc aaatgacccc taaagctata 5160 tatgcaaaag gaaaaaggg c taaaaatacc cctagactat ttgaaaagat ttaaatatac 5220 ccctcattca tttaatttggt taaaaatatc cctcaatcta tcaatccttt ttaaaattat 5280 tgttattctt attttttaat taggtgacat cttcctattg gataaaaaaa aaaaatttct 5340 acctatcaaa tctttctcga cccaattcaa agaacttgtt cattaattat tatttttcca 5400 gcaaaatga a aatttttagt taggatgata aaatttatcc aaatttctac caccacccaa 5460 aactaaatct tccaattcag aaaaaatcat tcattacaac tcttggattt gtttgtatga 5520 tgtgttagaa gcagaaatgg gtttaaaagg tgaattaaat ggtattttta actaaagaaa 5580 tgaatgcggg atatatttaa gttttttcag ataattcatg aatattttta tcctctttcc 5640 gtgtgcaata atagagttcc aacttaatta tcacgtggac ttataagaaa ccgatgctgg 5700 atgataatta tttaaaaaaa caagcaagtc ggggtcagta cagcttgctg aataggagaa 5760 attaaagata gcaaccagga aaagttaaga gcttttggct cctccgtcca attataaccg 5820 tccactatat atatttgaga ctcgtattga gaaacaataa ataataagaa tgatattact 5880 atattacctt ttgaatatat taaatttaat gctttgaaaa atatatttga taatgaataa 5940 tatctaatag cagcaagaat aaaatagaca caaacaagta aaattactca ttgatctttc 6000 aaattagaca agtattatcg gacatctact tttagtatag taaacaagta a agatcggat 6060 aaagagagta ataaagaaga agcaagcgta agtaccttgc caaaataatt aactaacaag 6120 cacatctttt ttttttatca aatattaata aaaataattt atattaatat gaagaaaaaa 6180 aaggttttag tttgctatct ttttgatcac tcgtttgcta taaataggtg gaggaggaca 6240 gacactcttc accccaaaat taaaagaaaa a gaggcaggg ctgcagatga gtgtccccac 6300 catggcctgg gctctgctcc tcctcagcct cctcactcag ggcacaggat cctgggctca 6360 gtctgccctg actcagcctc gctcagtgtc cgggtctcct ggacagtcag tcaccatctc 6420 ctgcactgga accagcagtg atattggtgg ttataacttt gtctcctggt accaacaaca 6480 cccaggcaaa gcccccaaac tcatgattta tgatgccact aagcggccct caggggtccc 6540 tgatcgcttc tctggctcca agtctggcaa cacggcctcc ctgaccatct ctgggctcca 6600 ggctgaggat gaggctgatt attactgctg ctcatatgca ggcgactaca ccccgggcgt 6660 ggttttcggc ggagggacca agctgacc gt cctaggtcag cccaaggctg ccccctcggt 6720 cactctgttc ccgccctcct ctgaggagct tcaagccaac aaggccacac tggtgtgtct 6780 cataagtgac ttctacccgg gagccgtgac agtggcctgg aaggcagata gcagccccgt 6840 caaggcggga gtgg agacca ccacaccctc caaacaaaagc aacaacaagt acgcggccag 6900 cagctacctg agcctgacgc ctgagcagtg gaagtcccac agaagctaca gctgccaggt 6960 cacgcatgaa gggagcaccg tggagaagac agtggcccct acagaatgtt catagggatc 7020 cgcatgccac cctgcaatgt gaccctagac ttgtccatct tctggattgg ccaacttaat 7080 taatgtatga aataaaagga tgcacacata gtgacatgct aatcactata atgtg ggcat 7140 caaagttgtg tgttatgtgt aattactaat tatctgaata agagaaagag atcatccata 7200 tttcttatcc taaatgaatg tcacgtgtct ttataattct ttgatgaacc agatgcattt 7260 tattaaccaa ttccatatac atataaatgg catgcaagct tgcatgcctg caggtccga t 7320 tgagactttt caacaaaggg taatatccgg aaacctcctc ggattccatt gcccagctat 7380 ctgtcacttt attgtgaaga tagtggaaaa ggaaggtggc tcctacaaat gccatcattg 7440 cgataaagga aaggccatcg ttgaagatgc ctctgccgac agtggtccca aagatggacc 7500 cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtct t caaagcaagt 7560 ggattgatgt gatggtccga ttgagacttt tcaacaaagg gtaatatccg gaacctcct 7620 cggattccat tgcccagcta tctgtcactt tattgtgaag atagtggaaa aggaaggtgg 7680 ctcctacaaa tgccatcatt gcgataaagg aaaggccatc gtt gaagatg cctctgccga 7740 cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc 7800 aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa gggatgacgc 7860 acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat ttcatttgga 7920 gaggagatct ttttatttt aattttcttt caaatacttc caccatggac tggacctgga 7980 ggttcctctt tgtggtggca gcagctacag gtgtccagtc ccaggtgcag ctggtgcagt 8040 ctggggctga ggtgaagaag cctgggtcct cggtgaaggt ctcctgcaag gcttctggag 8100 gcaccttcaa caggtatact gtcaactggg tgcgacaggc ccct ggacaa gggcttgagt 8160 ggatgggagg catcatccct atctttggta cagcaaacta cgcacagagg ttccagggca 8220 gactcaccat taccgcggac gaatccacga gcacagccta catggagctg agcagcctga 8280 gatctgatga cacggccgtg tatttctgtg cgagagagaa tctcgataat tcggggactt 8340 attattattt ctcaggctgg ttcgacccct ggggccaggg aaccctggtc accgtctcct 8400 cagcc tccac caagggccca tcggtcttcc ccctggcacc ctcctccaag agcacctctg 8460 ggggcacagc ggccctgggc tgcctggtca aggactactt ccccgaaccg gtgacggtgt 8520 cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc ctacag tcct 8580 caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg ggcacccaga 8640 cctacatctg caacgtgaat cacaagccca gcaacaccaa ggtggacaag agagttgagc 8700 ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa ctcctggggg 8760 gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc tcccggaccc 8820 ctgaggtc ac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc aagttcaact 8880 ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag gagcagtaca 8940 acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg ctgaatggca 9000 aggagtacaa gtgcaaggtc tccaaacaaag ccctcccagc ccccatcgag aaaaccatct 9060 ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca tcccgggagg 9120 agatgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat cccagcgaca 9180 tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc acgcctcccg 9240 tgctggactc cgacggctcc ttcttcctct atagcaagct caccgtggac aagagcaggt 9300 ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgaag ttccactaca 9360 cgcagaagag cctctccctg tccccgggta aaaaggacga actttgatct agacggatcc 9420 ccgatcgttc aa acatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg 9480 cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat 9540 gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat 9600 acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat 9660 ctatgttact agatcgggaa ttcactggcc gtcgtt ttac aacgtcgtga ctgggaaaac 9720 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat 9780 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgcccgc 9840 tcctttcgct ttcttcc ctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 9900 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaaa 9960 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 10020 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 10080 caaccctatc tcgggctatt cttttgattt ata agggatt ttgccgattt cggaaccacc 10140 atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct 10200 cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc 10260 accccagtac attaaaaacg tccgcaat gt gttattaagt tgtctaagcg tcaatttgtt 10320 tacaccacaa tatatcctgc caccagccag ccaacagctc cccgaccggc agctcggcac 10380 aaaatcacca ctcgatacag gcagcccatc agtccgggac ggcgtcagcg ggagagccgt 10440 tgtaaggcgg cagactttgc tcatgttacc gatgctattc ggaagaacgg caactaagct 10500 gccgggtttg aaacacggat gatctcgcgg agggtagcat gttg attgta acgatgacag 10560 agcgttgctg cctgtgatca aatatcatct ccctcgcaga gatccgaatt atcagccttc 10620 ttattcattt ctcgcttaac cgtgacaggc tgtcgatctt gagaactatg ccgacataat 10680 aggaaatcgc tggataaagc cgctgaggaa gctgagtggc gctatttctt tagaagtgaa 10740 cgttgacgat atcaactccc ctatccattg ctcaccgaat ggtacaggtc ggggacccga 10800 agttccgact gtcggcctga tgcatccccg gctgatcgac cccagatctg gggctgagaa 10860 agcccagtaa ggaaacaact gtaggttcga gtcgcgagat cccccggaac caaaggaagt 10920 aggttaaacc cgctccgatc aggccgagcc acgccaggcc gagaacattg gttcc tgtag 10980 gcatcgggat tggcggatca aacactaaag ctactggaac gagcagaagt cctccggccg 11040 ccagttgcca ggcggtaaag gtgagcagag gcacgggagg ttgccacttg cgggtcagca 11100 cggttccgaa cgccatggaa accgcccccg ccaggcccgc t gcgacgccg acaggatcta 11160 gcgctgcgtt tggtgtcaac accaacagcg ccacgcccgc agttccgcaa atagccccca 11220 ggaccgccat caatcgtatc gggctaccta gcagagcggc agagatgaac acgaccatca 11280 gcggctgcac agcgcctacc gtcgccgcga ccccgcccgg caggcggtag accgaaataa 11340 acaacaagct ccagaatagc gaaatattaa gtgcgccgag gatgaagatg cgcatccacc 1 1400 agattcccgt tggaatctgt cggacgatca tcacgagcaa taaacccgcc ggcaacgccc 11460 gcagcagcat accggcgacc cctcggcctc gctgttcggg ctccacgaaa acgccggaca 11520 gatgcgcctt gtgagcgtcc ttggggccgt cctcctgt tt gaagaccgac agcccaatga 11580 tctcgccgtc gatgtaggcg ccgaatgcca cggcatctcg caaccgttca gcgaacgcct 11640 ccatgggctt tttctcctcg tgctcgtaaa cggacccgaa catctctgga gctttcttca 11700 gggccgacaa tcggatctcg cggaaatcct gcacgtcggc cgctccaagc cgtcgaatct 11760 gagccttaat cacaattgtc aattttaatc ctctgtttat cggcagttcg tagagcgcgc 11820 cgt gcgtccc gagcgatact gagcgaagca agtgcgtcga gcagtgcccg cttgttcctg 11880 aaatgccagt aaagcgctgg ctgctgaacc cccagccgga actgacccca caaggcccta 11940 gcgtttgcaa tgcaccaggt catcattgac ccaggcgtgt tccaccaggc cgctgcctcg 12000 caactcttcg caggcttcgc cgacctgctc gcgccacttc ttcacgcggg tggaatccga 12060 tccgcacatg aggcggaagg tttccagctt gagcgggtac ggctcccggt gcgagctgaa 12120 atagtcgaac atccgtcggg ccgtcggcga cagcttgcgg tacttctccc atatgaattt 12180 cgtgtagtgg tcgccagcaa acagcacgac gatttcctcg tcgatcagga cctggcaacg 12240 ggacgtt ttc ttgccacggt ccaggacgcg gaagcggtgc agcagcgaca ccgattccag 12300 gtgcccaacg cggtcggacg tgaagcccat cgccgtcgcc tgtaggcgcg acaggcattc 12360 ctcggccttc gtgtaatacc ggccattgat cgaccagccc aggtcctggc a aagctcgta 12420 gaacgtgaag gtgatcggct cgccgatagg ggtgcgcttc gcgtactcca acacctgctg 12480 ccacaccagt tcgtcatcgt cggcccgcag ctcgacgccg gtgtaggtga tcttcacgtc 12540 cttgttgacg tggaaaaatga ccttgttttg cagcgcctcg cgcgggattt tcttgttgcg 12600 cgtggtgaac agggcagagc gggccgtgtc gtttggcatc gctcgcatcg tgtccggcca 12660 cggcgcaata tcgaacaagg aaagctgcat ttccttgatc tgctgcttcg tgtgtttcag 12720 caacgcggcc tgcttggcct cgctgacctg ttttgccagg tcctcgccgg cggtttttcg 12780 cttcttggtc gtcatagttc ctcgcgtgtc g atggtcatc gacttcgcca aacctgccgc 12840 ctcctgttcg agacgacgcg aacgctccac ggcggccgat ggcgcgggca gggcaggggg 12900 agccagttgc acgctgtcgc gctcgatctt ggccgtagct tgctggacca tcgagccgac 12960 ggactggaag gtttcgcggg gcgcacgcat gacggtgcgg cttgcgatgg tttcggcatc 13020 ctcggcggaa aaccccgcgt cgatcagttc ttgcctgtat gccttccggt caaacgtccg 13080 attcattcac cctcct tgcg ggattgcccc gactcacgcc ggggcaatgt gcccttattc 13140 ctgatttgac ccgcctggtg ccttggtgtc cagataatcc accttatcgg caatgaagtc 13200 ggtcccgtag accgtctggc cgtccttctc gtacttggta ttccgaatct tgccctgcac 13 260 gaataccagc gaccccttgc ccaaatactt gccgtgggcc tcggcctgag agccaaaaca 13320 cttgatgcgg aagaagtcgg tgcgctcctg cttgtcgccg gcatcgttgc gccacatcta 13380 ggtactaaaa caattcatcc agtaaaatat aatattttat tttctcccaa tcaggcttga 13440 tccccagtaa gtcaaaaaat agctcgacat actgttcttc cccgatatcc tcc ctgatcg 13500 accggacgca gaaggcaatg tcataccact tgtccgccct gccgcttctc ccaagatcaa 13560 taaagccact tactttgcca tctttcacaa agatgttgct gtctcccagg tcgccgtggg 13620 aaaagacaag ttcctcttcg ggcttttccg tcttta aaaa atcatacagc tcgcgcggat 13680 ctttaaatgg agtgtcttct tcccagtttt cgcaatccac atcggccaga tcgttattca 13740 gtaagtaatc caattcggct aagcggctgt ctaagctatt cgtataggga caatccgata 13800 tgtcgatgga gtgaaagagc ctgatgcact ccgcatacag ctcgataatc ttttcagggc 13860 tttgttcatc ttcatactct tccgagcaaa ggacgccatc ggcctcactc atgagcagat 1 3920 tgctccagcc atcatgccgt tcaaagtgca ggacctttgg aacaggcagc tttccttcca 13980 gccatagcat catgtccttt tcccgttcca catcataggt ggtcccttta taccggctgt 14040 ccgtcatttt taaatagg ttttcatttt ctccccaccag cttatatacc ttagcaggag 14100 acattccttc cgtatctttt acgcagcggt atttttcgat cagttttttc aattccggtg 14160 atattctcat tttagccatt tattatttcc ttcctctttt ctacagtatt taaagatacc 14220 ccaagaagct aattataaca agacgaactc caattcactg ttccttgcat tctaaaacct 14280 taaataccag aaaacagctt tttcaaagtt gttttcaaag ttggcgtata acatagtatc 14340 gacggagccg att ttgaaac cacaattatg ggtgatgctg ccaacttact gatttagtgt 14400 atgatggtgt ttttgaggtg ctccagtggc ttctgtgtct atcagctgtc cctcctgttc 14460 agctactgac ggggtggtgc gtaacggcaa aagcaccgcc ggacatcagc gctatctctg 14520 ctctcactgc cgtaaaacat ggcaactgca gttcacttac accgcttctc aacccggtac 14580 gcaccagaaa atcattgata tggccatgaa tggcgttgga tgccgggcaa cagcccgcat 14640 tatgggcgtt ggcctcaaca cgattttacg tcacttaaaa aactcaggcc gcagtcggta 14700 acctcgcgca tacagccggg cagtgacgtc atcgtctgcg cggaaatgga cgaacagtgg 14760 ggctat gtcg gggctaaatc gcgccagcgc tggctgtttt acgcgtatga cagtctccgg 14820 aagacggttg ttgcgcacgt attcggtgaa cgcactatgg cgacgctggg gcgtcttatg 14880 agcctgctgt caccctttga cgtggtgata tggatgacgg atgg ctggcc gctgtatgaa 14940 tcccgcctga agggaaagct gcacgtaatc agcaagcgat atacgcagcg aattgagcgg 15000 cataacctga atctgaggca gcacctggca cggctgggac ggaagtcgct gtcgttctca 15060 aaatcggtgg agctgcatga caaagtcatc gggcattatc tgaacataaa acactatcaa 15120 taagttggag tcattaccca attatgatag aatttacaag ctataaggtt attgtcctgg 15180 gtttcaagca ttagtccatg caagttttta t gctttgccc attctataga tatattgata 15240 agcgcgctgc ctatgccttg ccccctgaaa tccttacata cggcgatatc ttctatataa 15300 aagatatatt atcttatcag tattgtcaat atattcaagg caatctgcct cctcatcctc 15360 ttcatcctct tcgtcttggt ag ctttttaa atatggcgct tcatagagta attctgtaaa 15420 ggtccaattc tcgttttcat acctcggtat aatcttacct atcacctcaa atggttcgct 15480 gggtttatcg cacccccgaa cacgagcacg gcacccgcga ccactatgcc aagaatgccc 15540 aaggtaaaaa ttgccggccc cgccatgaag tccgtgaatg ccccgacggc cgaagtgaag 15600 ggcaggccgc cacccaggcc gccgccctca ctg cccggca cctggtcgct gaatgtcgat 15660 gccagcacct gcggcacgtc aatgcttccg ggcgtcgcgc tcgggctgat cgcccatccc 15720 gttactgccc cgatcccggc aatggcaagg actgccagcg ctgccatttt tggggtgagg 15780 ccgttc gcgg ccgaggggcg cagcccctgg ggggatggga ggcccgcgtt agcgggccgg 15840 gagggttcga gaaggggggg cacccccctt cggcgtgcgc ggtcacgcgc acagggcgca 15900 gccctggtta aaaacaaggt ttataaatat tggtttaaaa gcaggttaaa agacaggtta 15960 gcggtggccg aaaaacgggc ggaaaccctt gcaaatgctg gattttctgc ctgtggacag 16020 cccctcaaat gtcaataggt gcgcccctca tctgtcagca ctctgcc cct caagtgtcaa 16080 ggatcgcgcc cctcatctgt cagtagtcgc gcccctcaag tgtcaatacc gcagggcact 16140 tatccccagg cttgtccaca tcatctgtgg gaaactcgcg taaaatcagg cgttttcgcc 16200 gatttgcgag gctggccagc tccacg tcgc cggccgaaat cgagcctgcc cctcatctgt 16260 caacgccgcg ccgggtgagt cggcccctca agtgtcaacg tccgcccctc atctgtcagt 16320 gagggccaag ttttccgcga ggtatccaca acgccggcgg ccgcggtgtc tcgcacacgg 16380 cttcgacggc gtttctggcg cgtttgcagg gccatagacg gccgccagcc cagcggcgag 16440ggcaaccagc ccgg 16454

Claims (10)

It includes a heavy chain (HC) represented by SEQ ID NO: 1 and a light chain (LC) represented by SEQ ID NO: 2, wherein the 462nd amino acid of the heavy chain represented by SEQ ID NO: 1 is lysine (K) and the 463rd amino acid is phenylalanine (F ) A rabies virus neutralizing antibody characterized by substitution.
The rabies virus neutralizing antibody according to claim 1, wherein the antibody is expressed in plants.
The method of claim 1, wherein the antibody contains an endoplasmic reticulum selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19 at the terminal portion of the antibody heavy chain protein peptide sequence. An antibody further comprising a signal peptide.
A polynucleotide encoding the rabies virus neutralizing antibody of claim 1.
A vector containing the polynucleotide of claim 4.
The method of claim 5, wherein the vector comprises: a first promoter, a polynucleotide encoding the light chain represented by SEQ ID NO: 2 operably linked thereto, and a first terminator; and
A vector comprising a second promoter, a polynucleotide encoding a heavy chain represented by SEQ ID NO: 3 operably linked thereto, a polynucleotide encoding an endoplasmic reticulum signal peptide, and a second terminator,
A vector characterized in that the endoplasmic reticulum signal peptide is represented by one selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.
delete The vector according to claim 5, wherein the vector is an HN vector having a cleavage map indicated as HN among the cleavage maps shown in FIG. 1.
Plant cells or plant tissues transformed with the vector of claim 5.
A pharmaceutical composition for preventing or treating rabies comprising the antibody of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.