KR20120135868A - FcεRI SPECIFIC HUMAN ANTIBODY AND COMPOSITION FOR TREATMENT OR DIAGNOSIS OF ALLERGY DISEASE COMPRISING THEREOF - Google Patents

Abstract

PURPOSE: A Fc epsilon RI-specific human antibody with high affinity to Fc region of IgE and a method for preparing the same are provided to effectively conjugation of IgE and Fc epsilon RI and to treat or diagnose allergic rhinitis, asthma, atopic dermatitis, and contact dermatitis. CONSTITUTION: A heavy chain variable region of a Fc epsilon RI-specific antibody contains CDR1, CDR2, and CDR3. The heave chain variable region has an amino acid of sequence number 2, 4, 6, 8, 10, 12, 14, or 16. A light chain variable region of the Fc epsilon RI-specific antibody contains CDR1, CDR2 and CDR3. The light chain variable region has an amino acid of sequence number 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. A pharmaceutical composition for treating allergic diseases contains the Fc epsilon-specific antibody, a fragment thereof, and one or more pharmaceutically acceptable carriers.

Description

FcεRI specific human antibody and composition for treatment or diagnosis of allergy disease comprising thereof

The present invention relates to a human antibody specific for FcεRI, a receptor having high affinity for Fc of IgE, and a method for obtaining the same, wherein the FcεRI specific antibody according to the present invention efficiently inhibits binding between IgE and FcεRI, By preventing the signaling caused by the IgE-mediated allergic rhinitis, asthma, atopic dermatitis, urticaria and contact dermatitis, such as allergic diseases can be utilized for the treatment or diagnosis.

Allergic reactions that cause hypersensitivity to certain foods or drugs include allergic diseases such as allergic rhinitis, asthma, atopic skin disease, urticaria and contact dermatitis, which have a form of an immediate response in the body. Immediate response in allergic reactions is mediated by immunoglobulin E (IgE). Most allergic reactions are caused by the binding of IgE to the surface of the allergic receptor, one of the receptors for the Fc portion (Fcε) of IgE is FcεRI.

IgE is a major factor involved in the immediate response to allergies and is involved in the final stages of the response. IgE is mainly synthesized in B lymphocytes, and the produced IgE binds to the FcεRI receptor with high affinity. FcεRI is a glycoprotein molecule and plays an important role in allergic reactions. In addition, FcεRI is expressed in mast cells, neutrophil cells, and coral cells, as well as in immune cells, dendritic cells (Allergy. 1995, 50, 193-199, Clin Exp Allergy. 1996, 26, 648- 655).

Human FcεRI has three different subunits: alpha chain, beta chain (signal amplification factor) and gamma chain (signal transfer factor). Tetramer FcεRI, composed of one alpha chain and one beta chain and two gamma chains, respectively, is mainly expressed on the cell membranes of tissue cells and basophils and plays an important role in type I allergic responses for cell activation. Only the alpha chain of FcεRI binds directly to IgE and the binding site for IgE spans the extracellular region of the alpha chain (Nature. 2000, 406, 259-266). Among them, FcεRI to which IgE binds has an α-subunit (FcεRIα), and FcεRIα has a size of about 60 kDa, and is composed of a hydrophobic domain existing in the cell membrane and a hydrophilic domain existing outside the cell membrane. IgE bound to FcεRI remains bound to the cell membrane for several weeks. Signal transduction mechanisms are activated by the binding of allergens and the binding of FcεRI to activate cells. This result in induces degranulation of cells. Pharmacological mediators, histamine (substances that respond late to hypersensitivity reactions) and serotonin, are released, causing type I allergic reactions. These substances, produced in mast cells and basophils, cause a variety of physiological allergic diseases. IgE specific for allergens continues to be expressed by B cells.

Allergic diseases are aggravated by the combination between IgE and FcεRI. FcεRI-expressing cells are known to increase in the blood of allergic patients, and the expression of FcεRI in the blood of allergic rhinitis, atopic, asthma and atopic dermatitis patients is increased. Although IgE is not expressed in basophils and mast cells, IgE and FcεRI bind in a very strong and safe form. This fact indicates that IgE is always around these cells.

Currently, allergen avoidance therapy, antiallergic drug use, and immunotherapy for certain allergens have been used for the treatment of allergic diseases (Clin. Immunol. 1997, 100 (2), 171-176, Allergy 1998, 53, 821-). 832, Allergy Clin. Immunol. 1998, 102, 631-636, Clin.Exp. Allergy. 1999, 29 (11), 1563-1571, Allergy Clin. Immunol. 2002, 110, 154-159). Among them, antihistamines and anti-inflammatory drugs of the corticosteroid system are widely used. Many allergens have been developed over the past few years, but only alleviate the symptoms, and there are no treatments that can cure the root cause of allergies. In addition, the efficacy of the drug is insufficient or has a number of disadvantages due to side effects. Therefore, the demand for a new concept of allergy treatment is increasing.

Traditional immunotherapy is a method of injecting allergens into allergic patients with increasing amounts over time. This method has been shown to be effective in the treatment of allergic rhinitis and in part to allergic asthma. This immunotherapy alters the T cell response from Th2 type to Th1 to alleviate the symptoms of allergic patients and induce allergen specific IgG responses. However, these immunotherapy also works to alleviate the symptoms of the patient, and has the disadvantage of not being a fundamental treatment.

In addition, as an antibody product targeting IgE, Omalizumab (Xolair) is used as a treatment for asthma patients. Omalizumap binds to IgE and inhibits the release of chemicals, a major cause of asthma and allergies. However, Omalizumab has side effects such as angioedema, anaphylactic reactions and other allergic conditions, and allergic bronchial spasms, and allergic granulomatous vasculitis and idiopathic severe thrombocytopenia have been reported through postmarketing results.

Therefore, the demand for the development of a therapeutic agent capable of effectively and fundamentally treating allergic diseases without side effects is continuously increasing.

 Allergy Clin. immunol. 2002, 110, 154-159

In order to solve the above problems, the present invention provides an antibody having the ability to specifically bind to FcεRI and a method for producing such an antibody, in order to provide a therapeutic agent that can effectively and fundamentally treat allergic diseases without side effects. For the purpose of

Still another object of the present invention is to provide a pharmaceutical composition for treating allergic diseases such as allergic rhinitis, asthma, atopic skin disease, urticaria and contact dermatitis, containing the FcεRI specific antibody.

Still another object of the present invention is to provide a method for suppressing allergic diseases such as allergic rhinitis, asthma, atopic skin disease, urticaria and contact dermatitis using the FcεRI specific antibody.

Another object of the present invention is to provide a therapeutic agent that can be used to diagnose or treat allergic diseases, particularly allergic rhinitis, asthma, atopic skin disease, urticaria and contact dermatitis, or to treat patients with the FcεRI specific antibody. A composition or kit for confirming efficacy in advance, and a method for diagnosing the disease using such composition or kit, or a method for confirming the efficacy of a therapeutic agent.

In order to achieve the above object, in the present invention, it is possible to specifically bind to FcεRI with high affinity (affinity), and is composed of human-derived sequences. Was prepared.

Specifically, in the present invention, using a phage display technique from a single-chain Fv (scFv) phage library to obtain a fully human antibody that binds to the FcεRI with high affinity and specificity. The preparation and phage display of the phage library can be carried out by manufacturing as known in the US Patent No. 7063943, US Patent No. 6172197 and the like.

The antibody according to the present invention obtained and provided by the above method comprises an FcεRI comprising CDR1, CDR2 and CDR3 included in any one amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16 CDR1, CDR2 included in any of the heavy chain variable regions of the specific antibody and in any amino acid sequence selected from SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40 And the light chain variable region of an FcεRI-specific antibody comprising CDR3.

In particular, the antibody according to the present invention

(a) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 2 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 18;

(b) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 4 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 20;

(c) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 6 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 22;

(d) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 6 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 24;

(e) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 8 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 26;

(f) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 10 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 28;

(g) a light chain variable region comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 12 and CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 28;

(h) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 8 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 30;

(i) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 14 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 32;

(j) a light chain variable region comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 16 and CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 34;

(k) a light chain variable region comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 8 and CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 36;

(l) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 16 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 38;

(m) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 12 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 40;

It is preferable that one of consisting of, in particular

(1) the heavy chain variable region of SEQ ID NO: 2 and the light chain variable region of SEQ ID NO: 18 (NPB 302);

(2) the heavy chain variable region of SEQ ID NO: 4 and the light chain variable region of SEQ ID NO: 20 (NPB 303);

(3) a heavy chain variable region of SEQ ID NO: 6 and a light chain variable region of SEQ ID NO: 22 (NPB 304);

(4) a heavy chain variable region of SEQ ID NO: 6 and a light chain variable region of SEQ ID NO: 24 (NPB 305);

(5) a heavy chain variable region of SEQ ID NO: 8 and a light chain variable region of SEQ ID NO: 26 (NPB 306);

(6) a heavy chain variable region of SEQ ID NO: 10 and a light chain variable region of SEQ ID NO: 28 (NPB 307);

(7) a heavy chain variable region of SEQ ID NO: 12 and a light chain variable region of SEQ ID NO: 28 (NPB 308);

(8) a heavy chain variable region of SEQ ID NO: 8 and a light chain variable region of SEQ ID NO: 30 (NPB 309);

(9) a heavy chain variable region of SEQ ID NO: 14 and a light chain variable region of SEQ ID NO: 32 (NPB 310);

(10) a heavy chain variable region of SEQ ID NO: 16 and a light chain variable region of SEQ ID NO: 34 (NPB 311);

(11) a heavy chain variable region of SEQ ID NO: 8 and a light chain variable region of SEQ ID NO: 36 (NPB 312);

(12) a heavy chain variable region of SEQ ID NO: 16 and a light chain variable region of SEQ ID NO: 38 (NPB 313);

(13) a heavy chain variable region of SEQ ID NO: 12 and a light chain variable region of SEQ ID NO: 40 (NPB 314);

It is more preferable that the configuration.

The fragment of the antibody according to the present invention as described above can be used for the same purpose, the fragment of the antibody in the present invention is a single chain antibody, diabodies, triabodies, tetrabodies, Fab fragments, F (ab) having a binding function to FcεRI ') ₂ fragments, Fd, scFv, domain antibodies, bispecific antibodies, minibodies, caps, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies, derivatives of antibody constant regions, Phosphorus receptors based on protein scaffolds, and the like, but are not limited thereto. As long as the binding function to FcεRI is maintained, fragments of the antibody according to the present invention may exhibit the same characteristics as the antibody according to the present invention. It is obvious to those skilled in the art.

In addition, as long as the characteristics of the antibody of the present invention are maintained, antibodies in which the mutation occurs in the variable region are included in the scope of the present invention. One such example is conservative substitution of amino acids in the variable region. Conservative substitutions mean substitutions with other amino acid residues that have properties similar to those of the original amino acid sequence. For example, lysine, arginine, and histidine have similar properties because they have a base side chain. Aspartic acid and glutamic acid It has similar characteristics in that it has a mountain side chain. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar characteristics in that they have a non-charged polar side chain, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine are nonpolar. Tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have side chains. Therefore, it will be apparent to those skilled in the art that amino acid substitutions within a group having similar characteristics as described above will not show any change in characteristics, so as long as the characteristics of the antibody according to the present invention are maintained, Antibodies that have undergone mutations due to conservative substitutions in are also included in the scope of the present invention.

The antibody or fragment thereof according to the present invention is also available in the form of a conjugate conjugated with another substance. Examples of the material that can be used by conjugation with the antibody or fragment thereof according to the present invention include, but are not limited to, an antibody for treating allergic diseases such as omalizumab, and a conventional allergic disease therapeutic agent such as an antihistamine or an anti-inflammatory agent. .

The present invention provides a pharmaceutical composition comprising the antibody or fragment thereof according to the present invention. The pharmaceutical composition may further include conventional pharmaceutically acceptable carriers and excipients. The pharmaceutical composition may be applied to allergic diseases mediated by IgE. Representative diseases include, but are not limited to, allergic rhinitis, asthma, atopic dermatitis, urticaria and contact dermatitis.

In addition, the present invention is to advance the efficacy of a therapeutic agent that can be used for the diagnosis or treatment of allergic diseases, in particular allergic rhinitis, asthma, atopic skin disease, urticaria and contact dermatitis, comprising the antibody or fragment thereof according to the present invention. And compositions or kits for identifying and methods of diagnosing the disease or confirming therapeutic efficacy using such compositions or kits.

The diagnosis of the disease or confirmation of the efficacy of the therapeutic agent may be performed by measuring the degree of inhibition of binding to IgE and FcεRI in the blood of a patient using a rapid method using color development, but is not limited thereto. 96-well plates and the like can also be used to quickly confirm the diagnostic or therapeutic efficacy of a large amount of samples.

The present invention also provides a polynucleotide sequence encoding the variable region of the antibody or fragment thereof according to the present invention. Such polynucleotide sequences are polynucleotides set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 39 May be selected from the sequence, and particularly preferably

(a) SEQ ID NO: 1 and SEQ ID NO: 17;

(b) SEQ ID NO: 3 and SEQ ID NO: 19;

(c) SEQ ID NO: 5 and SEQ ID NO: 21;

(d) SEQ ID NO: 5 and SEQ ID NO: 23;

(e) SEQ ID NO: 7 and SEQ ID NO: 25;

(f) SEQ ID NO: 9 and SEQ ID NO: 27;

(g) SEQ ID NO: 11 and SEQ ID NO: 27;

(h) SEQ ID NO: 7 and SEQ ID NO: 29;

(i) SEQ ID NO: 13 and SEQ ID NO: 31;

(j) SEQ ID NO: 15 and SEQ ID NO: 33;

(k) SEQ ID NO: 7 and SEQ ID NO: 35;

(l) SEQ ID NO: 15 and SEQ ID NO: 37;

(m) SEQ ID NO: 11 and SEQ ID NO: 39;

It can be selected from the combination according to.

The present invention provides a recombinant vector comprising the nucleotide sequence and a host cell comprising the same, and a method for producing an antibody that specifically binds to FcεRI according to the present invention using the recombinant vector or host cell. Particularly, it is preferable to prepare by expression and purification by genetic recombination method. Specifically, the variable regions encoding the antibodies specifically binding to FcεRI according to the present invention may be prepared separately or simultaneously by expression in one host cell. desirable.

As used herein, a "recombinant vector" refers to a gene construct that is an expression vector capable of expressing a protein of interest in a suitable host cell, and which contains essential regulatory elements operably linked to express the gene insert. In the present invention, "operably linked" means that the nucleic acid expression control sequence and the nucleic acid sequence encoding the protein of interest is functionally linked to perform a general function. Operative linkage with recombinant vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art. Can be easily used

Suitable expression vectors that may be used in the present invention may include signal sequences for membrane targeting or secretion in addition to expression control elements such as promoters, initiation codons, termination codons, polyadenylation signals, and enhancers. Initiation and termination codons are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein and must be functional in the subject and be in frame with the coding sequence when the gene construct is administered. Generic promoters can be either constitutive or inducible. Prokaryotic cells include, but are not limited to, the lac, tac, T3 and T7 promoters. Eukaryotic cells include monkey virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) such as long terminal repeat (LTR) promoter of HIV, Moloney virus, cytomegalovirus ), Epstein Barr virus (EBV), and Rhus saccomavirus (RSV) promoters, as well as promoters derived from? -Actin promoter, human heparobin, human muscle creatine, human metallothionein . The expression vector may comprise a selectable marker for selecting a host cell containing the vector. The selection marker is for selecting cells transformed with the vector, and markers conferring a selectable phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents or expression of surface proteins can be used. Since only cells expressing a selection marker survive in an environment treated with a selective agent, transformed cells can be selected. In addition, when the vector is a replicable expression vector, the vector may include a replication origin, which is a specific nucleic acid sequence from which replication is initiated. As the recombinant expression vector, various types of vectors such as plasmids, viruses, and cosmids can be used. The type of recombinant vector is not particularly limited as long as it functions to express a desired gene and to produce a desired protein in various host cells of prokaryotic and eukaryotic cells, but has a promoter with strong activity and strong expression, similar to natural state. Vectors that can produce large amounts of foreign protein in form are preferred.

Various expression host / vector combinations can be used to express the antibody or antibody fragment according to the invention. Suitable expression vectors for eukaryotic hosts may include, but are not limited to, expression control sequences derived from SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus and retrovirus. no. Expression vectors that can be used for bacterial hosts include bacterial plasmids obtained from Escherichia coli such as pET, pRSET, pBluescript, pGEX2T, pUC vector, col E1, pCR1, pBR322, pMB9 and derivatives thereof, , Phage DNA which can be exemplified by a wide variety of phage lambda derivatives such as lambda gt10 and lambda gt11, NM989, and other DNA phages such as M13 and filamentous single strand DNA phage. A useful expression vector for yeast cells is the 2 < 0 > C plasmid and its derivatives. The vector useful for insect cells is pVL941.

The recombinant vector is inserted into a host cell to form a transformant. Suitable host cells are Escherichia coli, Bacillus subtilis ( Bacillus). subtilis), genus Streptomyces (Streptomyces sp.), Pseudomonas species (Pseudomonas sp.), Proteus Mira Billy's (Proteus mirabilis ) or Staphylococcus sp. In addition, fungi such as Aspergillus sp., Fungi such as Pichia pastoris), Celebi jiae as Saccharomyces Mai Seth (Saccharomyces cerevisiae), Iasi survey car Rome Seth in (Schizosaccharomyces sp.) and Neuro Castello La Chrysler Corporation (Neurospora crassa) such as yeast and other lower eukaryotes, and from insects Eukaryotic cells such as cells of higher eukaryotes such as cells.

In addition, the host cell is preferably derived from plants, mammals, monkey kidney cells (COS7) cells, NSO cells, SP2 / 0, Chinese hamster ovary (CHO: Chinese hamster ovary) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells and HEK293 cells and the like are available, but are not limited to these. Particularly preferably CHO cells.

Transformation into a host cell in the present invention includes any method of introducing a nucleic acid into an organism, cell, tissue or organ and can be carried out by selecting a suitable standard technique according to the host cell as is known in the art. These methods include electroporation, plasma fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, agro bacterial mediated transformation, PEG, dextran sulfate, Lipofectamine and dry / inhibited mediated transformation methods and the like.

By culturing the transformant expressing the recombinant vector in a nutrient medium, it is possible to produce a large amount of FcεRI-specific antibody according to the present invention, the medium and culture conditions can be appropriately selected and used according to the host cell. Conditions such as temperature, pH of the medium and incubation time can be appropriately adjusted to be suitable for the growth of cells and the mass production of proteins during the culture. The recombinantly produced antibody or antibody fragment as described above can be recovered from the medium or cell digest, and can be isolated and purified by conventional biochemical separation techniques (Sambrook et al., Molecular Cloning: A laborarory Manual, 2nd). Ed., Cold Spring Harbor Laboratory Press (1989); Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182. Academic Press. Inc., San Diego, CA (1990)). These include electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunosorbent chromatography, size exclusion chromatography, etc.), isoelectric focusing, and various variations and complex methods thereof. These may be used but are not limited thereto, and in particular, it is preferable to separate and purify using Protein A.

Antibodies that specifically bind to FcεRI according to the present invention efficiently inhibit the binding between IgE and FcεRI at low concentrations, such as allergic rhinitis, asthma, atopic skin disease, urticaria and contact dermatitis mediated by IgE. It shows excellent therapeutic effect on various allergic diseases, and because it specifically binds and works only on FcεRI, there are almost no side effects and does not cause an immune response when administered in the body.

FcεRI specific antibodies according to the present invention may inhibit or treat IgE mediated allergic diseases by inhibiting binding between IgE and FcεRI. In particular, it can be usefully used for the treatment of allergic diseases such as allergic rhinitis, asthma, atopic skin disease, urticaria and contact dermatitis.

1 is a diagram showing the inhibitory effect of FcεRI and IgE on selected antibodies using a competitive ELISA technique.
2 shows the binding capacity of selected antibodies to human FcεRI.
Figure 3 shows the degranulation inhibitory effect of selected antibodies.
4 shows the PCA inhibitory effects of selected antibodies.
FIG. 5 shows changes in total cell number, macrophages, eosinophils and lymphocytes by selected antibodies in an OVA induced FcεRIα transgenic mouse asthma model.
Figure 6 shows the size exclusion chromatography results of the FcεRI specific antibody NPB311 according to the present invention.
Figure 7 confirms the structural stability of the FcεRI specific antibody NPB311 according to the present invention.
Figure 8 shows the degranulation inhibitory effect of the FcεRI specific antibody NPB311 according to the present invention.
9 is a view showing the effect of FcεRI specific antibody NPB311 according to the present invention on the secretion of beta-hexos aminidayes.
10 is a view showing the histamine secretion inhibitory effect of the FcεRI specific antibody NPB311 according to the present invention.
11 is a view showing the effect of FcεRI specific antibody NPB311 according to the present invention on the induction of histamine secretion.
12 is a diagram showing the hypersensitivity inhibitory effect of FcεRI specific antibody NPB311 according to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to embodiments and accompanying drawings. However, these are intended to explain the present invention in more detail, and the scope of the present invention is not limited by the following examples.

Example 1 Selection of FcεRI Specific Antibodies

ScFv DNA having randomly bound immunoglobulin heavy (H chain) and light (L chain) cDNAs was prepared, and a scFv phage library consisting of 10 ¹⁰ clones was prepared by introducing a phagemid vector. The phage library consists of two different libraries: a phage library made by synthesizing the CDR sequences of various human immunoglobulins in the form of a single chain scFv, and a phage library of scFv made of cDNA extracted from human peripheral blood. It includes.

The phage clones are then screened for display of scFv having the property of binding to human FcεRIα through a panning method, which is brought into contact with a soluble fragment of the α subunit (FcεRIα) of human FcεRI immobilized on a solid phase. , Obtained.

Specifically, FcεRI was coated on an Immuno tube at an concentration of 10 ug / ml at 4 ° C. for at least 12 hours. After blocking and negative selection, a phage library expressing scFv of human antibody was added to the tube and allowed to react for 1 hour, followed by 0.01% Tween-20. Washed 10 times with PBS (phosphate buffered saline) and phage eluted with 0.1M triethylamine. Phage ELISA (ELISA) was then performed to select phages with strong reactivity to FcεRI and to inhibit the interaction of FcεRI and IgE.

As a result,

(a) a heavy chain variable region of SEQ ID NO: 2 and a light chain variable region of SEQ ID NO: 18 (NPB 302);

(b) a heavy chain variable region of SEQ ID NO: 4 and a light chain variable region of SEQ ID NO: 20 (NPB 303);

(c) a heavy chain variable region of SEQ ID NO: 6 and a light chain variable region of SEQ ID NO: 22 (NPB 304);

(d) a heavy chain variable region of SEQ ID NO: 6 and a light chain variable region of SEQ ID NO: 24 (NPB 305);

(e) the heavy chain variable region of SEQ ID NO: 8 and the light chain variable region of SEQ ID NO: 26 (NPB 306);

(f) a heavy chain variable region of SEQ ID NO: 10 and a light chain variable region of SEQ ID NO: 28 (NPB 307);

(g) the heavy chain variable region of SEQ ID NO: 12 and the light chain variable region of SEQ ID NO: 28 (NPB 308);

(h) the heavy chain variable region of SEQ ID NO: 8 and the light chain variable region of SEQ ID NO: 30 (NPB 309);

(i) the heavy chain variable region of SEQ ID NO: 14 and the light chain variable region of SEQ ID NO: 32 (NPB 310);

(j) the heavy chain variable region of SEQ ID NO: 16 and the light chain variable region of SEQ ID NO: 34 (NPB 311);

(k) a heavy chain variable region of SEQ ID NO: 8 and a light chain variable region of SEQ ID NO: 36 (NPB 312);

(l) a heavy chain variable region of SEQ ID NO: 16 and a light chain variable region of SEQ ID NO: 38 (NPB 313);

(m) a heavy chain variable region of SEQ ID NO: 12 and a light chain variable region of SEQ ID NO: 40 (NPB 314);

All 13 scFvs were selected.

CDR sequences in each variable region selected are as described in Tables 1 and 2.

[ Example  2] competitive ELISA  Of antibodies selected through the method Fc ε RI and IgE  Confirmation of binding inhibitory effect

96-well plates are coated with 0.5 μg / ml FcεRI and then stopped with 2% skim milk. The FcεRI specific antibodies selected in Example 1 are diluted and added to each well and reacted at 37 ° C. for 1 hour. After washing the 96-well plate with PBS / Tween solution, 0.5 ug / ml of human IgE was added to each well and reacted at 37 ° C. for 1 hour, followed by washing with PBS / Twin again and then anti-IgE -HRP (1 ug / ml) is added to react for 1 hour at 37 ℃. Thereafter, 30 µl of the luminous reagent was added to measure light emission.

As a result, the selected antibodies showed a concentration-dependent effect of inhibiting the binding of FcεRI and IgE (see Fig. 1).

Example 3 Confirmation of FcεRI Binding Properties of Selected Antibodies Using Cell ELISA Method

Suspension of RBL-SX38 cells in 3% FBS (Feral Bovine Serum) and DMEM medium (Dulbecco's Modified Eagle's Medium) in 96-well plates and 37 ° C., 5% at 2 × 10 ⁴ cells / 100 μl in 96-well plates. 48 hours incubation in a CO ₂ incubator. After removing the culture medium, each cell was washed with PBS and fixed for 20 minutes by addition of 4% formaldehyde (formaldehyde). After washing with PBS and stopping with 2% skim milk, selected FcεRI specific antibodies were diluted and added to each well, reacted at 37 ° C. for 1 hour and then washed with PBS / twin. Thereafter, anti-rabbit-HRP 0.2 ug / ml was added and reacted at 37 ° C. for 1 hour, and luminescence was measured by adding 30 μl of a luminescent substrate solution.

As a result, it was confirmed that the selected antibodies NPB 302, NPB 303, NPB 305, NPB 307 and NPB 311 all have a binding ability to human FcεRI (see Fig. 2).

Example 4 Degranulation Inhibitory Effect of Selected Antibodies

RBL-SX38 cells were suspended in 3% FBS and DMEM medium in 96-well plates for beta hexosminidayes secretion, and then 37 ° C., 5% CO ₂ at 2 × 10 ⁴ cells / 100 ul in 96-plate. Incubated for 48 hours in the incubator.

After removal of the culture medium, each cell was washed once with PBS + a buffer (PBS, 0.4 mM MgCl ₂ , 1 mM CaCl ₂ , 5.6 mM glucose, 0.1% BSA) and then nitropheny-IgE (NP-; IgE) 0.1 μg / ml was dispensed and reacted at 37 ° C., 5% CO ₂ incubator for 30 minutes, and the selected FcεRI specific antibodies were diluted and treated in each well for 150 minutes in 37 ° C., 5% CO ₂ incubator. I was. Then, 30 μl of the supernatant containing beta hexoseminidayes reacted for 30 minutes in a 37 ° C., 5% CO ₂ incubator with NP-BSA (0.1 ug / ml), and intracellular beta hexose To obtain 30 μl of solution containing Aminidays, 60 μl of 0.1% Triton X-100 was lysed. Transfer each solution to a 96-well plate and add 1 mM of substrate buffer [4-p-nitrophenyl-N-acetyl-β-D-glucosaminide]. , Sodium citrate, 0.1 M, pH 4.5] and 30 μl of the solution were incubated at 37 ° C. for 1 hour to stop the reaction by adding a reaction stop solution (50 mM glycine, pH 10.7), and absorbance was measured at 405 nm.

As a result, it was confirmed that the amount of beta hexoseminidayes secretion was reduced, and thus the selected antibodies NPB 302, NPB 305 and NPB 311 inhibited the degranulation phenomenon (see Fig. 3).

Example 5 Confirmation of the Inhibition Effect of IgE and FcεRI of Selected Antibodies through PCA (Passive Cutaneous Anaphylaxis) Animal Experiment

Six-week-old females transformed to express human FcεRI were injected subcutaneously with 0.1 ug / 20 μl of NP-IgE in the left ear, while NP-IgE (0.1 ug / 20 μl) and NPB 302, NPB 311 (100 ug / 10 μl) was injected subcutaneously. After 24 hours, 1 mg of NP-BSA with 1% EVANS blue dye was intravenously injected into the tail. After 90 minutes, the ear was cut off, formaldehyde was added, the reaction was carried out at 55 ° C. for 24 hours, and the absorbance was measured at 620 nm.

As a result, as shown in Figure 5, it was confirmed that the treatment of the FcεRI-specific antibodies NPB 302 and NPB 311 selected in the present invention significantly inhibited PCA. Specifically, as a result of visual observation, hypersensitivity reaction was normally induced in the left ear and turned blue, but the right ear in which the hypersensitivity reaction was suppressed remained unchanged, and the absorbance was measured after separating the dye and separating the dye. Significant results were also obtained from the results. From these results, it was confirmed that NPB 302 and NPB 311, which are FcεRI specific antibodies selected in the present invention, inhibit the binding of IgE and FcεRI.

Example 6 Confirmation of the Effect of Selective Antibodies on Changes in Hypersensitivity Cell Number Using Asthma Model

100 ug of OVA (ovalbumin) and 2 mg of Alum were included in 200 μl solution. The mixed OVA-alum was continuously mixed at 37 ° C. for about 1 hour. The mixed OVA-alum was divided into 200 μl in a 1.5 ml tube and placed one by one in a 1 ml syringe. Subsequently, 6-week-old females transformed to express human FcεRI were intraperitoneally injected 200 μl over 0, 14 days and 2 times. On 28, 29 and 30 days, 30 μg of 1% OVA was inhaled through the nasal mucosa.

Selected antibodies NPB 302 and NPB 311 were injected at 27, 29 days at 3 mg / kg. After 24 hours the cell number in the bronchoalveolar lavage fluid was counted. The bronchial tubes of anesthetized mice were incised using zoletil, a tube was inserted, a 22-gauge needle was inserted into the trachea, and bronchoalveolar lavage was performed. . The collected bronchial alveolar lavage fluid was centrifuged at 4 ° C. and 5000 rpm for 5 minutes, and the supernatant was stored at −70 ° C. until the next experiment. Cells obtained by centrifugation were counted using a hemocytometer and plated on glass slides using cytospin 3 (Thermo, USA), and Diff-Quik ) Staining was performed. A total of 200 cells were counted to differentially calculate alveolar macrophages, eosinophils, lymphocytes and neutrophils.

As a result, it was observed that the total number of cells, macrophages, eosinophils and lymphocytes was increased in the bronchial alveolar lavage fluid of the asthma model transformed to express OVA-induced FcεRIα compared to the PBS group. However, when pretreatment with NPB 302 and NPB 311, it was confirmed that the total number of cells, macrophages, eosinophils and lymphocytes increased by OVA significantly decreased (see Fig. 5).

Example 7 Obtaining Pure Antibody Fragment NPB-311 (Fab)

After reacting B311 IgG with papain (pierce, Fab preparation kit) for 6 hours at 37 ° C, filter out Fc cut with protein A bead, collect FT and buffer with 50 mM phosphate buffer, 0.3 M sodium chloride buffer (pH 7.2). Change was performed. The B311 Fab was quantified and confirmed by SDS-PAGE gel running. Size exclusion chromatography was performed on Amersham AKTA Explorer using a 50 mM phosphage buffer and 150 mM NaCl buffer (pH 7.0) on a Hiprep 26/60 Sephacryl S-200H column. The flow rate was 1.5 ml / min, and a solution of about 160-180 ml was obtained by 4 ml using Frac-900 to obtain a fraction of a desired size (50 KD).

As a result of performing separation and purification of NPB-311 separated by Papain using a column, pure NPB-311 (Fab) was obtained (see FIG. 6).

[ Example  8] antibody fragment NPB -311 ( Fab ) stability

Dialysis membrane (Membra-Cel, Width 25mm, Diameter 16mm, MWCO: 3500) in 10 mM Ammonium carbonate buffer (pH 7.2) was dialyzed at 4 ° C. overnight. After storage at −80 ° C., Trep Tem. Mot. Pin31 (centrifuge for Vacuum concentrator) was used. : -88 ℃, Vacuum Pump: lyophilized at 9 Torr conditions.

10% SDS-PAGE was made using a 30% acrylamide mix and silver staining was performed after electrophoresis to confirm the band. After freeze-drying and confirming stability after refrigeration for two weeks, it was confirmed that there is no structural change (see FIG. 7).

[ Example  9] antibody fragments NPB -311 ( Fab )of Degranulation  Inhibitory effect

RBL-SX38 cells were suspended in 3% FBS (Feral Bovine Serum) and DMEM medium (Dulbecco's Modified Eagle's Medium) in 96-well plates for determination of beta hexosminidayes secretion, followed by 2 × 10 ⁴ in 96-plate. The cells / 100 ul were incubated for 48 hours in a 37 ℃, 5% CO ₂ incubator.

After removal of the culture medium, each cell was washed once with PBS + a buffer (PBS, 0.4 mM MgCl ₂ , 1 mM CaCl ₂ , 5.6 mM glucose, 0.1% BSA) and then nitropheny-IgE (NP-; IgE) 0.1 μg / ml was dispensed and reacted for 30 minutes in a 37 ° C., 5% CO ₂ incubator.

After the reaction, NPB-311 (Fab) was treated for each concentration and reacted for 150 minutes in 37 ℃, 5% CO ₂ incubator. After adding NP-BSA (0.1 ug / ml) for 30 minutes at 37 ℃, 5% CO ₂ incubator and 30 μl of the supernatant containing beta hexosminidayes secreted out of the cell 0.1% Triton X-100 Lysis with 60 μl yielded beta hexosminiminides in cells.

30 μl of the solution containing the beta hexosminiminides obtained above was transferred to a 96-well plate, and a substrate buffer [4-p-nitrophenyl-N-acetyl-β-D-glucosaminid (4-p-nitrophenyl -N-acetyl-β-D-glucosaminide) 1 mM, sodium citrate 0.1 M, pH 4.5] 30 µl was added and incubated at 37 ° C. for 1 hour, followed by addition of a reaction stop solution (50 mM glycine, pH 10.7). Was terminated and the absorbance was measured at 405 nm.

As a result, it was confirmed that the beta hexose miniday secretion was reduced through the treatment of NPB-311 (Fab), and as the concentration of NPB-311 (Fab) was increased, the beta hexose miniday secretion was further reduced. It occurred strongly and confirmed that the degranulation phenomenon was suppressed from the above result (refer FIG. 8).

[ Example  10] antibody fragments NPB -311 ( Fab ) Beta Hexos Aminidays  Effect on secretion

RBL-SX38 cells were suspended in 3% FBS (Feral Bovine Serum) and DMEM medium (Dulbecco's Modified Eagle's Medium) in 96-well plates for measuring beta hexosminidayes secretion, and then 2 × 10 ⁴ cells in 96-plate Incubated for 48 hours at 37 ℃, 5% CO ₂ incubator at / 100 ul.

After removing the culture medium, each cell was washed once with PBS + a buffer (PBS, 0.4 mM MgCl ₂ , 1 mM CaCl ₂ , 5.6 mM glucose, 0.1% BSA), and then 15 ug / ml NPB-311 (Fab) was added. The cells were added and reacted sequentially for 0, 2, 5, 10, 30, and 60 minutes, and NP-BSA (0.1 ug / ml) was added thereto for 30 minutes in a 37 ° C., 5% CO ₂ incubator.

In the above reaction, 30 μl of the supernatant containing beta hexosaminidase secreted out of the cell was lysed with 60 μl of 0.1% Triton X-100 to obtain beta hexoseminidayes in the cell.

Transfer 30 μl of the solution containing the beta hexoseminidayes to a 96-well plate, and transfer the substrate buffer [4-p-nitrophenyl-N-acetyl-β-D-glucosaminide (4-p-nitrophenyl-N-). 30 μl of acetyl-β-D-glucosaminide) 1 mM, sodium citrate 0.1 M, pH 4.5) was added thereto, and incubated at 37 ° C. for 1 hour, followed by addition of a reaction stop solution (50 mM glycine, pH 10.7) to terminate the reaction. The absorbance was measured at 405 nm.

As a result, it was confirmed that there is no change in the amount of beta hexose minidayes secretion, through which it was confirmed that there is no degranulation phenomenon of NPB-311 (Fab) alone (see Fig. 9).

[ Example  11] histamine ( histamine Secretion inhibitory effect

For measuring histamine secretion, first, RBL-SX38 cells were suspended in 10-well FBS and DMEM medium in 24-well plates, then 37 ° C., 5% CO ₂ at 2 × 10 ⁵ cells / 500 ul in 24-well plates. Incubated for 48 hours in the incubator.

After removing the culture medium, each cell was loaded with Tyrode's buffer (125 mM NaCl, 5 mM KCl, 0.4 mM MgCl ₂ , 1 mM CaCl ₂ , 5.6 mM glucose, 0.1% BSA, 10 mM Hepes, pH7. After washing twice with 2), 0.5 μg / ml of NP-IgE was dispensed and reacted in a culture medium at 37 ° C. in a 5% CO ₂ incubator for 24 hours. At this time, NPB-311 (Fab) was treated together. After 24 hours, the reaction was washed three times with T-load buffer, and then 0.1 ug / ml of NP-BSA was added thereto, followed by reaction for 30 minutes in a 37 ° C., 5% CO ₂ incubator.

Centrifuge 500 μl of the supernatant of the reaction to obtain a supernatant again, transfer 100 μl of the obtained supernatant to a 96-well plate, acetylate histamine, and then adjust the histamine content to the HTRF Kit (CISBIO International, France). Measured using.

As a result, as the concentration of NPB-311 (Fab) was increased, it was confirmed that the histamine secretion decreases more strongly, and from this result, it was confirmed that there is an effect of effectively suppressing the histamine secretion phenomenon (Fig. 10). Reference).

[ Example  12] antibody fragments NPB -311 ( Fab Only histamine ( histamine Secretion induction effect

In order to measure the amount of histamine secreted, RBL-SX38 cells were first suspended in 10-well FBS and DMEM medium in 24-well plates, followed by 37 ° C. and 5% CO ₂ incubator at 2 × 10 ⁵ cells / 500 ul in 24-well plates. Incubated for 48 hours.

After removal of the culture medium, each cell was loaded with Tyrode's buffer (125 mM NaCl, 5 mM KCl, 0.4 mM MgCl ₂ , 1 mM CaCl ₂ , 5.6 mM glucose, 0.1% BSA, 10 mM Hepes, pH7). .2) twice and then NPB-311 (Fab) 15 ug / ml was added to the cells and reacted sequentially for 0, 2, 5, 10, 30, 60 minutes and then NP-BSA (0.1 ug / ml ) Was added and reacted at 37 ° C. in a 5% CO ₂ incubator for 30 minutes.

The supernatant of the reaction was centrifuged to obtain a supernatant, 100 μl of the obtained supernatant was transferred to a 96-well plate and acetylated histamine, and then the histamine content was determined using an HTRF Kit (CISBIO International, France). Measured.

As a result, it was confirmed that there is no change in histamine, and through this, it was confirmed that there was no induction of histamine secretion phenomenon of NPB-311 (Fab) alone (see FIG. 11).

[ Example  13] surface Plasmon  resonance( Biacore ) NPB311 ( Fab )and sFc ε RI Affinity of

Using GE's BIAcore3000, the soluble sFcεRI was immobilized to 1100 RU at 10 mM sodium acetate (pH 5.0) under CM5 sensor chip, and the surface was activated using EDC / NHS, followed by 1 M ethanolamine ( ethanolamine) method. The control cells were fixed under the same conditions without soluble FcεRI. NPB311 (Fab) was diluted 1/2 to HBS-EP buffer with 100 to 0 nM 6point and flowed to the CM5 chip in the order of low to high concentration to measure kinetics and regenerated using 10 mM NaOH buffer. The affinity between NPB311 (Fab) and sFcεRI was measured by Biacore (SPR).

As a result, as can be seen in Table 3, KD = 4.07 × 10 ^-9 (M) was measured to determine that the affinity of NPB311 (Fab) and sFcεRI is good.

Example 14 Ca of Antibody Fragment NPB-311 (Fab) ²⁺  Secretion inhibitory effect

RBL-SX38 cells were added to 5 x 10E4 / 100 ul / well in black wall 96 wells, and after 37 ° C overnight incubation, NP-IgE to final 100 ng / ml or NPB-311 (Fab) to NP-IgE 500 , 100, 20, 4, 0.8, 0.16, 0 nM was added to 37 ℃, incubation for 2 hours. After washing once with PBS, FLIPR calcium 4 dye was added and 37 ° C., 1 hour incubation, NP-BSA was added, and the fluoresence of FLUO-4AM was measured 90 times at 2 second intervals.

When NP-IgE and NPB-311 (Fab) were simultaneously treated, as shown in Table 4, NPB-311 (Fab) was found to inhibit the release of calcium (calcium) in a concentration-dependent manner, From the above results, it was confirmed that the NPB-311 (Fab) antibody portion was coupled to FcεRIa to inhibit dimerization.

Example 15 Anaphylaxis Hypersensitivity of Antibody Fragment NPB-311 (Fab)

T / G mice, female 7-week-old mice were injected subcutaneously with 0.1 ug NP-IgE 20 ul in the left ear on Day 0, while simultaneously adding 0.1 ug NP-IgE 20 ul and 0.05 MPK NPB-311 (Fab) antibody mixture to the right. Subcutaneous injection into the ear. However, at this time, it was divided into two groups measured 30 minutes after the antibody injection and the next day measured.

After adding 1 mg / ml NP-BSA to 1% Evan's blue solution on Day 0 or Day 1, 100 ul was administered intravenously to observe the color change.

As a result, when subcutaneous injection of NPB-311 (Fab) was injected into human FcεRIa expressing transgenic mice, it was confirmed that anaphylaxis was suppressed. From the above results, NPB-311 (Fab) was bound to IgE-FcεRIa. It was confirmed that it interferes with (see Figure 12).

<110> NeoPharm Co., LTD. <120> FceRI specific human antibody and composition for treatment or          diagnosis of allergy disease comprising <160> 40 <170> Kopatentin 1.71 <210> 1 <211> 363 <212> DNA <213> Unknown <220> <223> HC1 <400> 1 gaggtgcagc tggtggagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaagg atcatcccta tccttggtat agcaaactac 180 gcacagaagt tccagggcag agtcacgatt accgcggaca aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagtgaac 300 ccattcggct attattacgc tatggacgtc tggggccaag ggaccacggt caccgtctct 360 agt 363 <210> 2 <211> 121 <212> PRT <213> Unknown <220> <223> HC1 <400> 2 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ser   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr              20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met          35 40 45 Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe      50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr  65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Val Asn Pro Phe Gly Tyr Tyr Tyr Ala Met Asp Val Trp Gly             100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 3 <211> 360 <212> DNA <213> Unknown <220> <223> HC2 <400> 3 gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagt ccggggagtc tctgaagatc 60 tcctgtaagg gttttggata cacgtttacc agaaactgga tcggatgggt gcgccagatg 120 cccgggaaag gcctggagtg gatgggtatc atctatcctg gggactctga taccagatac 180 agtccgtcct tccaaggcca ggtcaccatc tcagccgaca agtccatcag caccgcctac 240 ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc gagagatgtc 300 ggaaacggtg actacggttt ggacgtctgg ggccaaggga ccacggtcac cgtctctagt 360                                                                          360 <210> 4 <211> 120 <212> PRT <213> Unknown <220> <223> HC2 <400> 4 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Ser Gly Glu   1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Phe Gly Tyr Thr Phe Thr Arg Asn              20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met          35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe      50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr  65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys                  85 90 95 Ala Arg Asp Val Gly Asn Gly Asp Tyr Gly Leu Asp Val Trp Gly Gln             100 105 110 Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 5 <211> 378 <212> DNA <213> Unknown <220> <223> HC3 <400> 5 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggact cgcctttagc gattattata tgagctgggt ccgcctggct 120 ccagggaagg ggctggagtg ggtctcagtg atctctcctg gtagtggtag taaatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagatatgtt 300 ggtggtatga tgatgaagag ggatttttat tatgctatgg acgtctgggg ccagggtaca 360 ctggtcaccg tgagctca 378 <210> 6 <211> 126 <212> PRT <213> Unknown <220> <223> HC3 <400> 6 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Ala Phe Ser Asp Tyr              20 25 30 Tyr Met Ser Trp Val Arg Leu Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Val Ile Ser Pro Gly Ser Gly Ser Lys Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Tyr Val Gly Gly Met Met Met Lys Arg Asp Phe Tyr Tyr Ala             100 105 110 Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 125 <210> 7 <211> 363 <212> DNA <213> Unknown <220> <223> HC4 <400> 7 gaggtgcagt tggtacagtc tggagcagag gtgaaaaagc ccggggagtc tctgaagatc 60 tcttgtgagg gttctggata caggtttagc agctactgga tcgcctgggt gcgccagaag 120 cccggtaaag gcctggagtg gatggggatc atctatcctt atgactctga taccaaatat 180 ggcccgtcct tcggaggcca ggtcaccatc tcagccgaca agtccatcaa taccgcctat 240 ctggagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc gagactggct 300 ctgattcggg gaagtattat ttttgactat tggggcctgg gaaccctggt caccgtctct 360 agt 363 <210> 8 <211> 121 <212> PRT <213> Unknown <220> <223> HC4 <400> 8 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu   1 5 10 15 Ser Leu Lys Ile Ser Cys Glu Gly Ser Gly Tyr Arg Phe Ser Ser Tyr              20 25 30 Trp Ile Ala Trp Val Arg Gln Lys Pro Gly Lys Gly Leu Glu Trp Met          35 40 45 Gly Ile Ile Tyr Pro Tyr Asp Ser Asp Thr Lys Tyr Gly Pro Ser Phe      50 55 60 Gly Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Asn Thr Ala Tyr  65 70 75 80 Leu Glu Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys                  85 90 95 Ala Arg Leu Ala Leu Ile Arg Gly Ser Ile Ile Phe Asp Tyr Trp Gly             100 105 110 Leu Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 9 <211> 357 <212> DNA <213> Unknown <220> <223> HC5 <400> 9 gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagt ccggggagtc tctgaagatc 60 tcctgtaagg gttttggata caggtttacc agctactgga tcggctgggt gcgccagatg 120 cccgggaaag gcctggagtg gatggggatc atctatcctg gtgactctga taccagatac 180 agcccgtcct tccaaggcca ggtcaccatc tcagccgaca agtccatcag caccgcctac 240 ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc aaaagacaac 300 cccttcaccg gttcctttga tatctggggc ctagggacta tggtcaccgt ctctagt 357 <210> 10 <211> 119 <212> PRT <213> Unknown <220> <223> HC5 <400> 10 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Ser Gly Glu   1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Phe Gly Tyr Arg Phe Thr Ser Tyr              20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met          35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe      50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr  65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys                  85 90 95 Ala Lys Asp Asn Pro Phe Thr Gly Ser Phe Asp Ile Trp Gly Leu Gly             100 105 110 Thr Met Val Thr Val Ser Ser         115 <210> 11 <211> 357 <212> DNA <213> Unknown <220> <223> HC6 <400> 11 gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggagagtc tctgaagatc 60 tcctgtaagg gttctggata caccttcacc aaatactgga tcggctgggt gcgccagatg 120 cccggaaaag gcctggagtg gatggggatc atctatcctg gtgactctga tacaacatac 180 agcccgtcct tccaaggcca ggtcaccatc tcagccgaca agtccatcag caccgcctac 240 ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc aaaagacaac 300 cccttcaccg gttcctttga tatctggggc caagggacaa tggtcaccgt ctctagt 357 <210> 12 <211> 119 <212> PRT <213> Unknown <220> <223> HC6 <400> 12 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu   1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Lys Tyr              20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met          35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Thr Tyr Ser Pro Ser Phe      50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr  65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys                  85 90 95 Ala Lys Asp Asn Pro Phe Thr Gly Ser Phe Asp Ile Trp Gly Gln Gly             100 105 110 Thr Met Val Thr Val Ser Ser         115 <210> 13 <211> 360 <212> DNA <213> Unknown <220> <223> HC7 <400> 13 gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaagatc 60 tcctgtaagg gttctggata cagctttacc agctactgga tcggctgggt gcgccagatg 120 cccgggaaag gcctggagtg gatggggatc atctatcctg gtgactctga taccagatat 180 agcccgtcct tccaaggcca ggtcaccatc tcagccgaca agtccatgag ctccgcctac 240 ctgcagtgga acagcctgaa ggcctcggac accgccatgt attactgtgc gagagatgtc 300 ggaaacggtg actacggttt ggacgtctgg ggccaaggga ccacggtcac cgtctctagt 360                                                                          360 <210> 14 <211> 120 <212> PRT <213> Unknown <220> <223> HC7 <400> 14 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu   1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr              20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met          35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe      50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Met Ser Ser Ala Tyr  65 70 75 80 Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys                  85 90 95 Ala Arg Asp Val Gly Asn Gly Asp Tyr Gly Leu Asp Val Trp Gly Gln             100 105 110 Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 15 <211> 363 <212> DNA <213> Unknown <220> <223> HC8 <400> 15 gaggtgcagc tggtacagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaagg atcatcccta tccttggtat agcaaactac 180 gcacagaagt tccagggcag agtcacgatt accgcggaca aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagtgaac 300 ccattcggct attattacgc tatggacgtc tggggccaag ggaccacggt caccgtctct 360 agt 363 <210> 16 <211> 121 <212> PRT <213> Unknown <220> <223> HC8 <400> 16 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr              20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met          35 40 45 Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe      50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr  65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Val Asn Pro Phe Gly Tyr Tyr Tyr Ala Met Asp Val Trp Gly             100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 17 <211> 330 <212> DNA <213> Unknown <220> <223> LC1 <400> 17 cagtcggtgc tgactcagcc accctcagcg tctgggaccc cagggcagag ggtcaccatt 60 tcttgttctg ggagcacctc caacatcgga ggtaattctg tcaactggta ccagcaactc 120 ccaggagcgg cccccaaact gctcatctat gctaatattc agcggccctc aggggtccct 180 gaccgcttct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240 tctgaggatg aggctgatta ctactgtgct tcatgggatg acagccggaa tggttatgtc 300 ttcggaactg ggaccaaggt caccgtccta 330 <210> 18 <211> 110 <212> PRT <213> Unknown <220> <223> LC1 <400> 18 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Gly Asn              20 25 30 Ser Val Asn Trp Tyr Gln Gln Leu Pro Gly Ala Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Ala Asn Ile Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln  65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Asp Ser Arg                  85 90 95 Asn Gly Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu             100 105 110 <210> 19 <211> 333 <212> DNA <213> Unknown <220> <223> LC2 <400> 19 cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60 tcctgcactg gaaccagcag tgacattggt ggttatgact ttgtctcctg gtaccaacaa 120 cacccaggca aagcccccaa actcatcatt tatgacgtca ctaggcgacc ctcagagatt 180 tctaatcgct tctctgcctc taagtctggc aacacggcct ctctgaccat ctctgggctc 240 caggctgaag acgaggctga ttattactgc agttcatata caagcagcag caccccttat 300 gtcttcggaa ctgggaccaa ggtcaccgtc cta 333 <210> 20 <211> 111 <212> PRT <213> Unknown <220> <223> LC2 <400> 20 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln   1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Ile Gly Gly Tyr              20 25 30 Asp Phe Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu          35 40 45 Ile Ile Tyr Asp Val Thr Arg Arg Pro Ser Glu Ile Ser Asn Arg Phe      50 55 60 Ser Ala Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu  65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser                  85 90 95 Ser Thr Pro Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu             100 105 110 <210> 21 <211> 330 <212> DNA <213> Unknown <220> <223> LC3 <400> 21 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtagtg gctcttcatc taatattggc agtaatgctg tcaactggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat tatgatagtc atcggccaag cgaggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtggt acttgggatt ctagcctgag tggttatgtc 300 ttcggcggag gcaccaagct gacggtccta 330 <210> 22 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> LC3 <400> 22 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn              20 25 30 Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Tyr Asp Ser His Arg Pro Ser Glu Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg  65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu                  85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 23 <211> 330 <212> DNA <213> Unknown <220> <223> LC4 <400> 23 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccggacagag ggtcaccatc 60 tcttgtactg gctcttcatc taatattggc aataatgatg tctcctggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat tctaataatc agcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccggggatg aggctgatta ttactgtggt acttgggatt atagcctgag tggttatgtc 300 ttcggcggag gcaccaagct gacggtccta 330 <210> 24 <211> 110 <212> PRT <213> Unknown <220> <223> LC4 <400> 24 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn              20 25 30 Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg  65 70 75 80 Ser Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Tyr Ser Leu                  85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 25 <211> 333 <212> DNA <213> Unknown <220> <223> LC5 <400> 25 cagtccgtgc tgactcagcc gtcctcagtg tctggggccc cagggcagag ggtcaccatc 60 tcctgcactg ggagcagctc caatatcggg gcaggttatg atgtacattg gtatcaacat 120 cttccgggaa cagcccccaa acttctcatc tatggtatca acattcggcc ctcaggagtc 180 cccgaccgat tctctggctc caagtctgac acctcagcct ccctggccat cactgcactc 240 caggctgagg atgaggctga ttattactgc cagtcctatg acagcagcct gaggagtgtt 300 gtattcggcg gagggaccaa gctgaccgtc cta 333 <210> 26 <211> 111 <212> PRT <213> Unknown <220> <223> LC5 <400> 26 Gln Ser Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ala Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly              20 25 30 Tyr Asp Val His Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu          35 40 45 Leu Ile Tyr Gly Ile Asn Ile Arg Pro Ser Gly Val Pro Asp Arg Phe      50 55 60 Ser Gly Ser Lys Ser Asp Thr Ser Ala Ser Leu Ala Ile Thr Ala Leu  65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser                  85 90 95 Leu Arg Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 27 <211> 330 <212> DNA <213> Unknown <220> <223> LC6 <400> 27 cagtctgccc tgactcagcc tcgctcagtg tccgggtctc ctggacagtc agtcaccatc 60 tcctgcactg gaaccagcaa tgatgttggt ggttatacgt atgtgtcctg gtaccaacag 120 cacccaggca aagcccccaa actcatgatt tatgatgtca gtaagcggcc ctcaggggtc 180 cctgatcgct tctctggctc caagtctggc aacacggctt ccctgaccat ctctgggctg 240 caggctgagg atgaggctga ttattactgc agctcatatg caggcagtta cacttgggtg 300 ttcggcggag ggaccaagct gaccgtccta 330 <210> 28 <211> 110 <212> PRT <213> Unknown <220> <223> LC6 <400> 28 Gln Ser Ala Leu Thr Gln Pro Arg Ser Val Ser Gly Ser Pro Gly Gln   1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Asn Asp Val Gly Gly Tyr              20 25 30 Thr Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu          35 40 45 Met Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe      50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu  65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala Gly Ser                  85 90 95 Tyr Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 29 <211> 333 <212> DNA <213> Unknown <220> <223> LC7 <400> 29 cagtccgtgc tgactcagcc gtcctcagtg tctggggccc cagggcagag ggtcaccatt 60 tcctgcactg ggagcagctc caacatcggg gcagattatg atgtacactg gtaccagctg 120 gtttcaggag cagcccccaa actcctcatc tacggttaca ccaatcggcc ctcaggggtc 180 cctgaccgat tctctggctc caagtctggc acctcagcct ccctggtcat agctgggctc 240 caggctgagg atgaggctga ttattactgc cagtcctatg acagcagcct gagtgaagtg 300 gtattcggcg gagggaccaa gctgaccgtc cta 333 <210> 30 <211> 111 <212> PRT <213> Unknown <220> <223> LC7 <400> 30 Gln Ser Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ala Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Asp              20 25 30 Tyr Asp Val His Trp Tyr Gln Leu Val Ser Gly Ala Ala Pro Lys Leu          35 40 45 Leu Ile Tyr Gly Tyr Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe      50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Val Ile Ala Gly Leu  65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser                  85 90 95 Leu Ser Glu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 31 <211> 333 <212> DNA <213> Unknown <220> <223> LC8 <400> 31 cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60 tcctgcgtcg gaaccagcgg tgacattggt cgttttaatt atgtctcgtg gtaccaacaa 120 cacccaggca gggcccccaa actcatcatt tatgacgtca ctaggcgacc ctcagagatt 180 tctaatcgct tctctgcctc taagtctggc aacacggcct ctctgaccat ctctgggctc 240 caggctgaag acgaggctga ttattactgc agttcatata caagcagcag caccccttat 300 gtcttcggaa ctaggaccaa ggtcaccgtc cta 333 <210> 32 <211> 111 <212> PRT <213> Unknown <220> <223> LC8 <400> 32 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln   1 5 10 15 Ser Ile Thr Ile Ser Cys Val Gly Thr Ser Gly Asp Ile Gly Arg Phe              20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Arg Ala Pro Lys Leu          35 40 45 Ile Ile Tyr Asp Val Thr Arg Arg Pro Ser Glu Ile Ser Asn Arg Phe      50 55 60 Ser Ala Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu  65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser                  85 90 95 Ser Thr Pro Tyr Val Phe Gly Thr Arg Thr Lys Val Thr Val Leu             100 105 110 <210> 33 <211> 330 <212> DNA <213> Unknown <220> <223> LC9 <400> 33 caatctgtgc tgactcagcc cccctcagcg tctgggaccc ccgggcagac ggtctccatc 60 tcttgttctg gcagcagctc caacatcggc actaatattg taaattggta ccagcaactc 120 ccaggaacgg cccccaaact cctcatctat aggaatgatc agcggccctc aggggtccct 180 gaccgatttt ctggctccaa gtctggcacc tcaggttccc tggccatcag tgggctccag 240 tctgaggatg aggctgacta ttattgtgca gcatgggatg acagcctgaa tggtgggata 300 ttcggcggag ggaccaaact gaccgtccta 330 <210> 34 <211> 110 <212> PRT <213> Unknown <220> <223> LC9 <400> 34 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Thr Val Ser Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Asn              20 25 30 Ile Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Arg Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Gly Ser Leu Ala Ile Ser Gly Leu Gln  65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu                  85 90 95 Asn Gly Gly Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 35 <211> 330 <212> DNA <213> Unknown <220> <223> LC10 <400> 35 cagtctgccc tgactcagcc tcgctcagtg tccgggtctc ctggacagtc agtcaccatt 60 tcttgtactg gatcccgcgg tgacgttgga ggttatgact ttgtctcctg gtaccgtcaa 120 tacccaggcg aggcccccca actcattatt tatgatgtca gcgggaggcc atcaggagtc 180 cctgatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240 cagcctgaag atgaggctga ttattattgc tcttcatacg ccggcagtga gatttatgtc 300 ttcggaactg ggaccaaggt caccgtccta 330 <210> 36 <211> 110 <212> PRT <213> Unknown <220> <223> LC10 <400> 36 Gln Ser Ala Leu Thr Gln Pro Arg Ser Val Ser Gly Ser Pro Gly Gln   1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Ser Arg Gly Asp Val Gly Gly Tyr              20 25 30 Asp Phe Val Ser Trp Tyr Arg Gln Tyr Pro Gly Glu Ala Pro Gln Leu          35 40 45 Ile Ile Tyr Asp Val Ser Gly Arg Pro Ser Gly Val Pro Asp Arg Phe      50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu  65 70 75 80 Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala Gly Ser                  85 90 95 Glu Ile Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu             100 105 110 <210> 37 <211> 321 <212> DNA <213> Unknown <220> <223> LC11 <400> 37 gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtcggaga cagagtcacc 60 atcccttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccaatt tgcaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240 gaagattctg caacttacta ttgtcaacaa actgacagtt tcccgctcac tttcggcgga 300 ggaaccaagg tggaaatcaa a 321 <210> 38 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> LC11 <400> 38 Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Val Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Pro Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp              20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile          35 40 45 Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Thr Asp Ser Phe Pro Leu                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 39 <211> 330 <212> DNA <213> Unknown <220> <223> LC12 <400> 39 cagtctgccc tgactcagcc tgcctccatg tctgggtttc ctggacagtc gatcaccatc 60 tcctgcactg ggacccgcga tgatgttggg aattatgatc ttgtctcctg gtatcaacaa 120 cagccagcca aagcccccaa actcctcatt tatgaggaca ataagcggcc ctcaggggtc 180 tctagtcgct tctctggctc caagtctggc agcacggcct ccctgacaat ctctgggctc 240 cagactgagg acgaggctga ttattactgc tgctcatatg caggtgatta tacttatgtc 300 ttcggaactg ggaccaaggt caccgtccta 330 <210> 40 <211> 110 <212> PRT <213> Unknown <220> <223> LC12 <400> 40 Gln Ser Ala Leu Thr Gln Pro Ala Ser Met Ser Gly Phe Pro Gly Gln   1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Arg Asp Asp Val Gly Asn Tyr              20 25 30 Asp Leu Val Ser Trp Tyr Gln Gln Gln Pro Ala Lys Ala Pro Lys Leu          35 40 45 Leu Ile Tyr Glu Asp Asn Lys Arg Pro Ser Gly Val Ser Ser Arg Phe      50 55 60 Ser Gly Ser Lys Ser Gly Ser Thr Ala Ser Leu Thr Ile Ser Gly Leu  65 70 75 80 Gln Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala Gly Asp                  85 90 95 Tyr Thr Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu             100 105 110

Claims (21)

A heavy chain variable region of an FcεRI specific antibody comprising CDR1, CDR2 and CDR3 included in any one amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16. The heavy chain variable region of claim 1, wherein the heavy chain variable region of the FcεRI specific antibody has an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16. Light chain of an FcεRI-specific antibody comprising CDR1, CDR2 and CDR3 included in any of the amino acid sequences selected from SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40 Variable area. The light chain variable of FcεRI specific antibody according to claim 2, having an amino acid sequence selected from SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40. domain. A FcεRI specific antibody, or fragment thereof, comprising any one selected from among heavy chain variable regions according to claim 1 and any one selected from light chain variable regions according to claim 3. An FcεRI-specific antibody, or fragment of such an antibody, characterized by the combination of the following variable regions.
(a) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 2 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 18;
(b) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 4 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 20;
(c) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 6 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 22;
(d) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 6 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 24;
(e) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 8 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 26;
(f) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 10 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 28;
(g) a light chain variable region comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 12 and CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 28;
(h) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 8 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 30;
(i) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 14 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 32;
(j) a light chain variable region comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 16 and CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 34;
(k) a light chain variable region comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 8 and CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 36;
(l) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 16 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 38;
(m) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 12 and a light chain variable region comprising CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 40; An FcεRI-specific antibody, or fragment thereof, comprising the following heavy and light chain variable region sequences.
(a) a heavy chain variable region of SEQ ID NO: 2 and a light chain variable region of SEQ ID NO: 18 (NPB 302);
(b) a heavy chain variable region of SEQ ID NO: 4 and a light chain variable region of SEQ ID NO: 20 (NPB 303);
(c) a heavy chain variable region of SEQ ID NO: 6 and a light chain variable region of SEQ ID NO: 22 (NPB 304);
(d) a heavy chain variable region of SEQ ID NO: 6 and a light chain variable region of SEQ ID NO: 24 (NPB 305);
(e) the heavy chain variable region of SEQ ID NO: 8 and the light chain variable region of SEQ ID NO: 26 (NPB 306);
(f) the heavy chain variable region of SEQ ID NO: 10 and the light chain variable region of SEQ ID NO: 28 (NPB 307);
(g) the heavy chain variable region of SEQ ID NO: 12 and the light chain variable region of SEQ ID NO: 28 (NPB 308);
(h) the heavy chain variable region of SEQ ID NO: 8 and the light chain variable region of SEQ ID NO: 30 (NPB 309);
(i) the heavy chain variable region of SEQ ID NO: 14 and the light chain variable region of SEQ ID NO: 32 (NPB 310);
(j) the heavy chain variable region of SEQ ID NO: 16 and the light chain variable region of SEQ ID NO: 34 (NPB 311);
(k) a heavy chain variable region of SEQ ID NO: 8 and a light chain variable region of SEQ ID NO: 36 (NPB 312);
(l) a heavy chain variable region of SEQ ID NO: 16 and a light chain variable region of SEQ ID NO: 38 (NPB 313);
(m) a heavy chain variable region of SEQ ID NO: 12 and a light chain variable region of SEQ ID NO: 40 (NPB 314); 8. The antibody or fragment of claim 5, wherein the antibody or fragment of the antibody is a single chain antibody, diabody, triabbody, tetrabody, Fab fragment, F (ab ') ₂ having a binding function to FcεRI. Fragments, Fd, scFv, domain antibodies, bispecific antibodies, minibodies, caps, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies, derivatives of antibody constant regions, protein scaffolds FcεRI-specific antibodies, or fragments of such antibodies, characterized in that they are selected from phospholipids based on protein scaffolds. A pharmaceutical composition for the treatment of allergic diseases comprising an isolated antibody according to any one of claims 5 to 8, or a fragment of such an antibody and at least one pharmaceutically acceptable carrier. 10. The pharmaceutical composition of claim 9, wherein the allergic disease is at least one selected from allergic rhinitis, asthma, atopic skin disease, urticaria and contact dermatitis. The polynucleotide encoding the heavy chain light region or the light chain variable region of the FcεRI-specific antibody according to any one of claims 1 to 4. The method of claim 11, as set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39. Polynucleotide, characterized in that selected from the sequences. 9. A polynucleotide encoding a FccRi-specific antibody or fragment of such antibody according to any of claims 5-8. The polynucleotide encoding a FcεRI-specific antibody, or fragment of such antibody, according to claim 13, selected from a combination of the following polynucleotide sequences.
(a) SEQ ID NO: 1 and SEQ ID NO: 17;
(b) SEQ ID NO: 3 and SEQ ID NO: 19;
(c) SEQ ID NO: 5 and SEQ ID NO: 21;
(d) SEQ ID NO: 5 and SEQ ID NO: 23;
(e) SEQ ID NO: 7 and SEQ ID NO: 25;
(f) SEQ ID NO: 9 and SEQ ID NO: 27;
(g) SEQ ID NO: 11 and SEQ ID NO: 27;
(h) SEQ ID NO: 7 and SEQ ID NO: 29;
(i) SEQ ID NO: 13 and SEQ ID NO: 31;
(j) SEQ ID NO: 15 and SEQ ID NO: 33;
(k) SEQ ID NO: 7 and SEQ ID NO: 35;
(l) SEQ ID NO: 15 and SEQ ID NO: 37;
(m) SEQ ID NO: 11 and SEQ ID NO: 39; Recombinant expression vector comprising the polynucleotide sequence according to any one of claims 11-14. A host cell transformed with the recombinant expression vector of claim 15. The host cell of claim 16, wherein the host cell is selected from animal cells, plant cells, yeast, E. coli, and insect cells. The method of claim 16, wherein the transformed host cells are monkey kidney cells (COS7) cells, NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, young Baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells and 293 cells, Escherichia coli, Bacillus subtilis ( Bacillus subtilis), genus Streptomyces (Streptomyces sp.), Pseudomonas species (Pseudomonas sp.), Proteus Mira Billy's (Proteus such as Staphylococcus sp., Aspergillus sp., Pichia sp. pastoris , Saccharomyces cerevisiae ), Schizosaccharomyces sp. and Neurospora crassa . 20. A method of producing a FcεRI-specific antibody, fragment or variable region thereof, comprising culturing the vector or host cell according to any one of claims 15-18. A composition for diagnosing allergy diseases or for confirming the efficacy of an allergic disease treatment comprising the isolated antibody according to any one of claims 5 to 8, or a fragment of such antibody. 20. A kit for diagnosing an allergy disease or confirming the efficacy of an allergic disease treatment comprising a composition according to claim 20.

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