KR20130071961A - Fcrn specific human antibody and composition for treatment of autoimmune diseases - Google Patents

Abstract

The present invention relates to a human antibody specific for FcRn, a receptor having high affinity for IgG, a method for preparing the same, a composition for treating autoimmune diseases comprising such an antibody, and a method for treating and diagnosing autoimmune diseases using the same. The FcRn specific antibody according to the present invention is used for the treatment of auto-immune disease by non-competitively binding to FcRn with IgG and reducing the amount of serum pathogenic auto-antibody in the blood. And it can be used for diagnosis.

Description

FcRn specific human antibody and composition for treating autoimmune diseases, including the same

The present invention relates to human antibodies specific for FcRn (abbreviation for neonatal Fc receptor, also called fetal Fc-receptor, FcRP, FcRB.Brambell receptor), a method for producing the same, comprising such antibodies The present invention relates to a composition for treating autoimmune diseases and a method for treating and diagnosing autoimmune diseases using the same, wherein the FcRn specific antibody according to the present invention is non-competitively bound to FcRn with IgG and the like. By reducing the amount of auto-antibody can be used for the treatment and diagnosis of auto-immune disease (auto-immune disease).

Antibodies are immune proteins that bind to specific antigens. In most animals, including humans and mice, heavy and light chain polypeptides are paired. Each chain consists of two distinct regions, called variable and constant domains, where the variable regions of the heavy and light chains exhibit significant sequence diversity between antibodies and specific binding to the target antigen. The constant region is responsible for binding to a number of natural proteins that have low sequence diversity between antibodies and induce significant biochemical reactions. In humans, there are five isotype antibodies, including IgA (including IgA1 and IgA2 subclasses), IgD, IgE, IgG (subdivisions of IgG1, IgG2, IgG3 and IgG4), and IgM. These isotypes are mainly classified according to the characteristics of the constant region.

IgG antibodies, which are most abundant in the blood, protect organisms against invading pathogens, promote recruitment of immune system-related substances, and mediate inflammatory responses in tissues, are composed of two heavy and two light chains. It is a tetrameric protein. IgG heavy chains at the N-terminus in the order of VH-CH1-CH2-CH3, representing heavy chain variable domain (VH), heavy chain constant domain 1 (CH1), heavy chain constant domain 2 (CH2) and heavy chain constant domain 3 (CH3), respectively. It consists of four immunoglobulin domains linked to the C terminus.

The IgG light chain also consists of two immunoglobulin domains linked from the N-terminus to the C-terminus in the order of VL-CL, representing the light chain variable domain (VL) and the light chain constant domain (CL), respectively.

Under normal conditions, the half-life of most IgGs except the IgG3 isotype in serum is about 22-23 days in humans, which is relatively long compared to the serum half-life of other plasma proteins. The long half-life of IgG in the blood is strongly bound to neonatal Fc receptor (FcRn), a kind of Fc gamma receptor, in the endosomes at pH 6.0. The gradual lysosome pathway can be avoided, and circulating back to the cell membrane, IgG rapidly dissociates from FcRn in the bloodstream at a weak basic pH (-7.4). By this receptor-mediated recycling mechanism, FcRn is known to effectively block the degradation of IgG in lysosomes, extending the half-life of IgG (Roopenian et al. J. Immunol. 170: 3528, 2003). .

FcRn has also been shown to mediate the uptake of IgG antibodies from breast milk in the rat neonatal gut and to promote their transport into the circulatory system. FcRn has also been isolated from the human placenta, which is known to mediate maternal IgG uptake and transport into the fetal circulation. In adults, FcRn is expressed in epithelial tissues of the lungs, intestines, kidneys, and many tissues, including the surface of the nose, vagina, and biliary tree.

FcRn is typically a non-covalent heterodimer that resides in endosomes of endothelial cells and epithelial cells. FcRn is a membrane bound receptor with three heavy chain alpha domains (α1, α2 and α3) and one water soluble light chain β2-microglobulin (β2m) domain. Structurally, it belongs to the population of major histocompatibility complex class 1 molecules with β2m as a common light chain. The αFcRn chain comprises three heavy chain domains, α2, α3 and α3, a β2m light chain domain, an extracellular domain with one oligosaccharide, and a single-pass transmembrane. ) And a relatively short cytoplasmic tail and have a molecular weight of about 46 kD (Burmeister et al. Nature 372: 366, 1994.).

In these mice, when engineered to "knockout" the genes encoding β2m and FcRn heavy chains in mice to express their role in FcRn for IgG homeostasis, these proteins were not expressed. Serum half-life and concentrations of IgG have been drastically reduced (Junghans et al, Proc. Natl. Acad. Sci. 93: 5512, 1996), suggesting an FcRn dependent mechanism for IgG homeostasis. In addition, although anti-human FcRn antibodies can be produced in these FcRn knockout mice, and these antibodies have been proposed to prevent binding of IgG to FcRn, such antibodies have not yet been produced or tested (WO 02/43658 A). .

The possibility that inhibition of IgG binding to FcRn can reduce the IgG serum half-life by reducing IgG recycling to treat autoimmune diseases caused by autoantibodies has been demonstrated in a mouse model of autoimmune dermal bullous disease (Li et al. J. Clin. Invest. 115: 3440, 2005). Therefore, agents that block or antagonize the binding of FcRn to IgG can be used in methods for treating or preventing autoimmune diseases and inflammatory diseases mediated by IgG.

Autoimmune diseases and alloimmune diseases are diseases mediated by pathogenic antibodies, such as immune neutropenia, guillain-barré syndrome, epilepsy, autoimmune cerebral encephalitis, Isaac syndrome, nevus syndrome, vulgaris, vesicles, vesicles, Acquired epidermal bleeding, gestational pseudo bullous, mucosal membrane bullous, antiphospholipid syndrome, autoimmune anemia, autoimmune grave disease, goodpasture syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus and idiopathic thrombocytopenia Idiopathic thrombocytopenic purpura (hereinafter referred to as 'ITP') is a representative disease. In the case of ITP, peripheral platelet destruction progresses due to the generation of autoantibodies that bind to specific platelet membrane glycoproteins. Antiplatelet antibodies opsoniaztion platelets and lead to rapid platelet destruction by reticulum (eg macrophages).

In general, attempts have been made to increase platelet concentration by suppressing the immune system in order to treat ITP. ITP is more prevalent in women than in men, and more frequently in children than in adults. The prevalence is 1 in 10,000 people. Chronic ITP is one of the major blood diseases in both adults and children. In addition, in the United States and around the world, it causes a large amount of admission fee and treatment costs. There are 20,000 new cases in the United States each year, and the costs associated with the treatment and special treatment of ITP are enormous. Most ITP pediatric patients have a very low number of platelets, resulting in sudden bleeding (a typical symptom: contusion, small red spots on the skin, nosebleeds, and gum bleeding). Children sometimes recover without treatment, but many doctors recommend careful observation, palliative care, and treatment by gamma globulin intravenous injection.

Extensive IgG injection (IVIG) is applied to various autoimmune diseases to treat autoimmune diseases (Arnson autoimmunity 42: 553 (2009)). The IVIG effect is explained by various mechanisms, but one of them is also explained by the mechanism of increasing clearance of pathogenic antibodies by competition with endogenous IgG against FcRn. Intravenous administration of large amounts of human immunoglobulin (IgG) (IVIG) increases the number of platelets in children suffering from immune ITP and has been shown to be beneficial in the treatment of multiple other autoimmune diseases. Many studies have identified mechanisms for the effectiveness of IVIG in the treatment of autoimmune diseases. In relation to ITP, early studies have shown that the effect of IVIG is primarily due to blocking Fc receptors involved in phagocytosis of antibody-opsonized platelets. Subsequent studies have shown that Fc-deficient IVIG results in increased platelet counts in some ITP patients, and recently the effect of IVIG stimulates FcγRIIb expression on macrophage cells that leads to inhibition of platelet phagocytosis. There are also reports that it is due to.

Nevertheless, such IVIG treatments have great side effects and are quite expensive to administer. In addition, other therapies used in the treatment of autoimmune / homogenous diseases other than IVIG include polyclonal anti-D immunoglobulins, corticosteroids, and immunosuppressive agents including chemotherapeutic agents, cytokine plasma, and the like. Surgical treatments such as separation and exchange, in vitro antibody adsorption (eg, using a Prosorba column) or spleen extraction. However, like IVIG, these therapies are limited in their use due to insufficient efficacy and high cost. In addition, competitive inhibition of the hypothetical mechanism of IVIG inhibition of FcRn binding results in a substantial increase in clearance of pathogenic antibodies due to low affinity for FcRn at the physiological pH of IgG (ie, pH 7.2-7.4). Significantly large amounts of IVIG are required to obtain VE, with typical clinical doses of IVIG being about 2 g / kg).

The use of inhibitors with the concept of treating autoimmune diseases by competitively inhibiting binding of IgG to FcRn is one of the promising therapies, but for now due to the high affinity for FcRn of endogenous IgG and the concentration of endogenous IgG in the blood It is known that a significant amount of inhibitor is required and therefore has the same limitations as current IVIG therapies.

Therefore, it has a high affinity for FcRn, it is possible to remove the pathogenic antibody in a small amount, there is an urgent need for the development of human antibodies that can reduce the immunogenicity.

 Burmeister et al. Nature 372: 366, 1994.  Roopenian et al. J. Immunol. 170: 3528, 2003.  Li et al. J. Clin. Invest. 115: 3440, 2005.

In order to solve the above problems, the present invention provides an antibody capable of specifically binding to FcRn and a method for producing such antibody, in order to effectively provide a therapeutic agent capable of effectively and essentially treating autoimmune diseases including ITP. The purpose is to provide. FcRn-specific antibodies according to the present invention are specific to FcRn and bind pH independantly (pH independent) to prevent non-competitive binding of antibody Fc to FcRn, thereby causing a bioimmune disease. Autoimmune diseases can be treated by reducing internal autoantibodies.

Still another object of the present invention is ITP containing the FcRn specific antibody, immune neutropenia, guillain-barré syndrome, epilepsy, autoimmune cerebral encephalitis, Isaac syndrome, nevus syndrome, vulgaris, vesicles, bleb , Autologous epidermal bleeding, gestational pseudo bullous, mucosal membrane bullous, antiphospholipid syndrome, autoimmune anemia, autoimmune Grave disease, Goodpasture syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis and lupus It is to provide a pharmaceutical composition for the treatment of immune diseases.

Another object of the present invention is ITP using the FcRn specific antibody, immune neutropenia, Guillain-Barré syndrome, epilepsy, autoimmune cerebral encephalitis, Isaac syndrome, nevus syndrome, vulgaris spear, deciduous scab, vesicular scab, Acquired epidermal bullous, gestational pseudo bullous, mucosal membrane bullous, antiphospholipid syndrome, autoimmune anemia, autoimmune grave disease, goodpasture syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis and lupus It provides a method for preventing or treating a disease and a method for diagnosing.

In order to achieve the above object, in the present invention, it has a high affinity to FcRn (specific) and can bind independantly to pH, and is composed of human-derived sequences, and almost no induction of immune response when administered in vivo. Fully human antibodies were prepared.

The antibodies provided herein are polyclonal or monoclonal antibodies with specificity for FcRn, preferably in the form of monoclonal antibodies against human FcRn, in particular human monoclonal antibodies, FcRn In combination with, it acts as a non-competitive inhibitor of IgG. Binding of the pathogenic antibody to FcRn is inhibited by FcRn binding with the antibody according to the present invention, thereby promoting clearance, ie elimination, of the pathogenic antibody from the body of the individual, thereby reducing the half-life.

As used herein, the term “pathogenic antibody” refers to an antibody causing a pathological condition or disease. Such antibodies include antiplatelet antibodies, antiacetylcholine antibodies, antinucleic acid antibodies, anti-phospholipid antibodies, anti Collagen antibodies (anti-collagen antibody), anti-ganglioside antibody (anti-ganglioside antibody), anti-desmoglein antibody (anti-desmoglein antibody) and the like, but are not limited thereto.

Antibodies according to the present invention have the advantage of enabling non-competitive inhibition of the binding of pathogenic antibodies to FcRn at physiological pH, pH 7.0-7.4. US 2002 / 0138863A et al. Describe that an antibody against FcRn must bind to FcRn at the same site important for IgG binding to Fc so that binding of IgG to FcRn is inhibited when the antibody binds to FcRn. FcRn binds pH-dependently to its ligand, ie IgG, and exhibits substantially no affinity for IgG at physiological pH other than acidic. Thus, anti-FcRn antibodies that specifically bind FcRn at physiological pH act as non-competitive inhibitors of binding of IgG to FcRn, in which case the binding of anti-FcRn antibodies to FcRn is not affected by the presence of IgG. Thus, antibodies according to the invention which bind FcRn non-competitively with IgG by pH-independence mediate FcRn mediation of IgG at much lower concentrations than competing inhibitors, i.e., existing antibodies that competitively bind FcRn with IgG. It has the advantage of being able to treat diseases caused by signaling. In addition, in the process of intracellular migration in the state of binding to FcRn, the anti-FcRn antibody according to the present invention maintains binding to FcRn with a higher affinity than IgG in the blood, so endosome is an acidic pH environment where IgG and FcRn can bind. It is possible to inhibit the binding of IgG and FcRn in the interior, and has the effect of promoting the clearance of IgG.

In summary, unlike the antibodies disclosed in the prior art, the antibody according to the present invention has an affinity for FcRn even at physiological pH, that is, an environment in which IgG does not bind to FcRn, that is, pH 7.0 to 7.4, and has affinity for FcRn than IgG in blood at pH 6.0. It has a higher affinity for and acts as a non-competitive inhibitor.

In the present invention, a phage display technique from a single-chain Fv (scFv) phage library was used to obtain a fully human antibody that binds to FcRn with high affinity and specificity. Preparation of phage libraries and phage display can be carried out by manufacturing as known in US 7,063,943B, US 6,172,197B and the like.

The antibody according to the present invention obtained and provided by the above method is an FcRn 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 Have a heavy chain variable region of a specific antibody or a heavy chain variable region having 90%, preferably at least 95% sequence homology with such heavy chain variable region,

A light chain variable region, or such a light chain variable region, of an FcRn specific antibody comprising CDR1, CDR2 and CDR3 included in any one amino acid sequence selected from SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30 and 32 It has a light chain variable region having 90%, preferably at least 95% sequence homology.

In addition, the antibody according to the present invention is any one of the heavy chain variable region of the FcRn specific antibody having an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16, or such heavy chain variable FcRn specific antibody having a heavy chain variable region having 90%, preferably at least 95%, homology with the region and any amino acid sequence selected from SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, and 32 Any one of the light chain variable regions of or a light chain variable region having 90%, preferably 95% or more sequence homology with such a light chain variable region.

In particular, the antibody according to the present invention,

(a) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the amino acid sequence of SEQ ID NO: 2, or a heavy chain variable region having at least 90%, preferably at least 95% sequence homology with the amino acid of SEQ ID NO: 18 A light chain variable region comprising CDR1, CDR2 and CDR3 included in the sequence, or a light chain variable region having at least 90%, preferably at least 95% homology thereof;

(b) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the amino acid sequence of SEQ ID NO: 4, or a heavy chain variable region having at least 90%, preferably at least 95% sequence homology with the amino acid of SEQ ID NO: 20 A light chain variable region comprising CDR1, CDR2 and CDR3 included in the sequence, or a light chain variable region having at least 90%, preferably at least 95% homology thereof;

(c) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the amino acid sequence of SEQ ID NO: 6, or a heavy chain variable region having at least 90%, preferably at least 95% sequence homology with the amino acid of SEQ ID NO: 22 A light chain variable region comprising CDR1, CDR2 and CDR3 included in the sequence, or a light chain variable region having at least 90%, preferably at least 95% homology thereof;

(d) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the amino acid sequence of SEQ ID NO: 8, or a heavy chain variable region having at least 90%, preferably at least 95% sequence homology with the amino acid of SEQ ID NO: 24 A light chain variable region comprising CDR1, CDR2 and CDR3 included in the sequence, or a light chain variable region having at least 90%, preferably at least 95% homology thereof;

(e) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the amino acid sequence of SEQ ID NO: 10, or a heavy chain variable region having at least 90%, preferably at least 95% sequence homology with the amino acid of SEQ ID NO: 26 A light chain variable region comprising CDR1, CDR2 and CDR3 included in the sequence, or a light chain variable region having at least 90%, preferably at least 95% homology thereof;

(f) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the amino acid sequence of SEQ ID NO: 12, or a heavy chain variable region having at least 90%, preferably at least 95% sequence homology with the amino acid of SEQ ID NO: 28 A light chain variable region comprising CDR1, CDR2 and CDR3 included in the sequence, or a light chain variable region having at least 90%, preferably at least 95% homology thereof;

(g) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the amino acid sequence of SEQ ID NO: 14, or a heavy chain variable region having at least 90%, preferably at least 95% sequence homology with the amino acid of SEQ ID NO: 30 A light chain variable region comprising CDR1, CDR2 and CDR3 included in the sequence, or a light chain variable region having at least 90%, preferably at least 95% homology thereof; And

(h) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the amino acid sequence of SEQ ID NO: 16, or a heavy chain variable region having at least 90%, preferably at least 95% sequence homology with the amino acid of SEQ ID NO: 32 A light chain variable region comprising CDR1, CDR2 and CDR3 included in the sequence, or a light chain variable region having at least 90%, preferably at least 95% homology thereof;

It has a heavy and light chain variable region selected from.

More preferably, the antibody according to the present invention,

(a) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2, or a heavy chain variable region having 90% or more, preferably 95% or more sequence homology therewith, and a light chain variable region having the amino acid sequence of SEQ ID NO: 18, or Light chain variable region having at least 90%, preferably at least 95% sequence homology;

(b) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4, or a heavy chain variable region having at least 90%, preferably at least 95% sequence homology with a light chain variable region having the amino acid sequence of SEQ ID NO: 20, or Light chain variable region having at least 90%, preferably at least 95% sequence homology;

(c) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 6, or a heavy chain variable region having 90% or more, preferably 95% or more sequence homology therewith, and a light chain variable region having the amino acid sequence of SEQ ID NO: 22, or Light chain variable region having at least 90%, preferably at least 95% sequence homology;

(d) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8, or a heavy chain variable region having 90% or more, preferably 95% or more sequence homology therewith, and a light chain variable region having the amino acid sequence of SEQ ID NO: 24, or Light chain variable region having at least 90%, preferably at least 95% sequence homology;

(e) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10, or a heavy chain variable region having 90% or more, preferably 95% or more sequence homology therewith, and a light chain variable region having the amino acid sequence of SEQ ID NO: 26, or Light chain variable region having at least 90%, preferably at least 95% sequence homology;

(f) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 12, or a heavy chain variable region having 90% or more, preferably 95% or more sequence homology therewith, and a light chain variable region having the amino acid sequence of SEQ ID NO: 28, or Light chain variable region having at least 90%, preferably at least 95% sequence homology;

(g) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14, or a heavy chain variable region having 90% or more, preferably 95% or more sequence homology therewith, and a light chain variable region having the amino acid sequence of SEQ ID NO: 30, or Light chain variable region having at least 90%, preferably at least 95% sequence homology; And

(h) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16, or a heavy chain variable region having sequence identity of at least 90%, preferably at least 95%, and a light chain variable region having the amino acid sequence of SEQ ID NO: 32, or Light chain variable region having at least 90%, preferably at least 95% sequence homology;

It has a heavy and light chain variable region selected from.

Such an antibody according to the present invention also includes fragments of the antibody. Fragments of antibodies in the present invention include single chain antibodies, diabodies, triabodies, tetrabodies, Fab fragments, F (ab ') ₂ fragments, Fd, scFv, domain antibodies, bispecific antibodies, which have a binding function to FcRn, Minibodies, scaps, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies, derivatives of antibody constant regions, phosphorus based on protein scaffolds, and the like. It is obvious to one skilled in the art that, as long as the binding function to FcRn is maintained, fragments of antibodies of any form according to the invention will exhibit the same properties as antibodies according to the invention.

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. An example is an antibody in which a conservative substitution of amino acids in a variable region occurs. 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. Substances that can be used by conjugation with the antibody or fragment thereof according to the present invention generally include therapeutic agents used in the treatment of autoimmune diseases and substances capable of inhibiting FcRn activity, or blood, serum, lymph, or other tissues. It is physically connected with a moiety that enhances its stabilization and / or maintenance in the circulation. For example, an FcRn-binding antibody can be linked to a polymer, such as a non-antigenic polymer such as polyalkylenepolyalkylene oxide or polyethylene oxide. Suitable polymers will vary substantially by weight. Polymers having a molecular number average weight in the range of about 200 to about 35,000 (or about 1,000 to about 15,000, and 2,000 to about 12,500) can be used. For example, FcRn-binding antibodies can be conjugated to water soluble polymers such as hydrophilic polyvinyl polymers such as polyvinyl alcohol and polyvinylpyrrolidone. An unlimited list of such polymers includes, but is not limited to, homopolymers, copolymers, and block copolymers of polyalkylene oxides such as polyethylene glycol (PEG) or polypropylene glycol, polyoxyethylenated polyols, and the like. It doesn't happen. As a clue, the water solubility of these block copolymers should be maintained.

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 pharmaceutically acceptable carrier should be compatible with the active ingredients of the antibody and the like according to the present invention, and may include saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol and one of these components or Two or more components may be used in combination, and other conventional additives such as antioxidants, buffers, bacteriostatic agents, etc. may be added as necessary. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions and the like. It may also be formulated and provided in various forms, such as powders, tablets, capsules, solutions, injections, ointments, syrups, and the like, in unit-dose or multi-dose containers, such as sealed ampoules and bottles. May be provided.

The pharmaceutical composition may be applied to all autoimmune diseases mediated by IgG and FcRn. Representative diseases include immune neutropenia, guillain-barré syndrome, epilepsy, autoimmune cerebral encephalitis, Isaac syndrome, birthmark syndrome, vulgaris ulcer, Deciduous blisters, bullous blisters, acquired epidermal blisters, gestational blisters, mucosal membrane blisters, antiphospholipid syndrome, autoimmune anemia, autoimmune grave disease, goodpasture syndrome, Myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus and idiopathic thrombocytopenic purpura (ITP) and the like, but are not limited thereto.

The present invention provides a method for alleviating an autoimmune or allogeneic state comprising administering an antibody according to the invention or a fragment of such an antibody to a subject in need thereof, while also providing certain anti-FcRn therapies.

The method or anti-FcRn therapy for alleviating an autoimmune or alloimmune condition according to the invention can be achieved by administering to a subject a pharmaceutical composition according to the invention, wherein the pharmaceutical composition according to the invention is intended for oral or parenteral administration. Specifically, for example, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal, sublingual or topical administration are possible, but are not limited thereto. It is not. The dosage of the composition according to the present invention may vary in range depending on the weight, age, sex, health condition, diet, time of administration, method, excretion rate and severity of the disease of the patient, and is easily available to those skilled in the art. It can be determined as a single or repeated dose. In general, 1 to 200 mg / kg, more preferably 1 to 40 mg / kg, may be administered to a patient with autoimmune or alloimmune symptoms, and such a dosing regimen is preferred that the serum endogenous IgG concentration is 75% of the pretreatment value. It can be designed to reduce it to less than%, and an intermittent or chronic (continuous) dosing policy can be applied, taking into account the patient's condition.

The present invention provides a diagnostic composition comprising the antibody or fragment thereof according to the present invention and a diagnostic method using the same. In other words, the antibody or fragment thereof which binds to FcRn according to the present invention and the fragment according to the present invention has diagnostic utility in vitro and in vivo.

In one aspect, the invention provides a method for detecting the presence of FcRn in vitro or in vivo.

In vitro detection methods include, for example, (1) contacting a sample with an FcRn-binding antibody; (2) detecting the formation of a complex between the FcRn-binding antibody and the sample; And / or (3) contacting a reference sample (eg, a control sample) with the antibody; (4) determining the extent of formation of the complex between the antibody and the sample as compared to in the reference sample. Changes in the formation of complexes in a sample or individual, such as a statistically significant change compared to a control sample or individual, may mean the presence of FcRn in the sample.

In vivo detection methods include the steps of (1) administering an FcRn-binding antibody to a subject; (2) detecting the formation of a complex between the FcRn-binding antibody and the subject. Detection can include determining the location or time point of complex formation. FcRn-binding antibodies can be labeled directly or indirectly with a detectable substance that facilitates detection of bound or unbound antibodies. Suitable detectable materials include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Complex formation between FcRn-binding antibodies and FcRn can be detected by measuring or visualizing antibodies bound to or not bound to FcRn. Traditional detection assays such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry can be used. In addition to labeling of FcRn-binding antibodies, the presence of FcRn can be analyzed in samples by competition immunoassay using criteria labeled with a detectable substance and unlabeled FcRn-binding antibodies. In one example of such an assay, the amount of labeled reference bound to a biological sample, labeled criteria, and FcRn-binding antibody, and bound to an unlabeled antibody is determined. The amount of FcRn in the sample is inversely proportional to the amount of labeled reference bound to the FcRn-binding antibody.

Antibodies or fragments thereof according to the invention for use for diagnostic purposes can be labeled with fluorophores or chromophores. Since antibodies and other proteins absorb light with wavelengths up to approximately 310 nm, the fluorescent moiety should be selected to have substantial absorption at wavelengths above 310 nm, preferably above 400 nm. Various suitable fluorescent materials and chromophores can be labeled with a group of fluorophores. One group of fluorophores is the xanthene dye, which includes fluorescein and rhodamine. Another group of fluorescent compounds is naphthylamine. Once labeled with a fluorophore or chromophore, the antibody can be used to detect the presence or localization of FcRn in the sample using, for example, a fluorescence speculum (eg, confocal or deconvolution).

The presence or localization of FcRn using the antibody or fragment thereof according to the present invention can be detected by various methods as follows.

Histological Analysis: Antibodies or fragments thereof according to the invention can be synthesized with a label (eg, a tablet or epitope tag) or detectably labeled by conjugating a label or label-binding group, for example. The antibody is then contacted with a fixed cut of tissue placed on a tissue sample, eg, a microscope slide. After incubation for binding, tissue samples are washed to remove unbound antibodies. The tissue sample is then analyzed using, e.g., a speculum, to confirm that the antibody is bound to the tissue sample. Naturally, the antibody may not be labeled at the time of binding. After binding and washing, the antibody is labeled to allow detection.

Protein Arrays : The antibodies or fragments thereof according to the invention can be used immobilized on protein arrays. Protein arrays can be used, for example, as diagnostic tools for selecting medical samples (eg, isolated cells, blood, serum, biopsies, etc.). Naturally, the protein array may also include other ligands that bind to FcRn or other target molecules, for example. Polypeptides for arrays can be sprinkled at high speed using commercially available robotic devices (eg, from Genetic MicroSystems or BioRobotics). The array substrate can be, for example, nitrocellulose, plastic, glass, for example surface-modified glass. The array may also comprise a porous matrix, such as acrylamide, agarose, or other polymer.

FACS (Fluorescence Activated Cell Sorting): Antibodies or fragments thereof according to the present invention can be used to label cells, eg, cells in a sample (eg, a patient sample). Antibodies may also be attached to fluorescent compounds. The cells can then be sorted using a fluorescence activated cell sorter (eg, Becton Dickinson). As the cells pass through the sorter, the laser beam excites the fluorescent compound, the detector counts the cells passing through and detects the fluorescence to determine whether the fluorescent compound is attached to the cell. The amount of label bound to each cell can be quantified and analyzed to characterize the sample.

The presence of FcRn or FcRn-expressing tissues in vivo according to the present invention can be confirmed through an in vivo image detection method. The method comprises the steps of: (1) administering an anti-FcRn antibody conjugated to a detectable marker to an individual (eg, an autoimmune disease patient); (ii) exposing the subject to a means for detecting a detectable marker for an FcRn-expressing tissue or cell. For example, the subject is imaged by, for example, NMR or other tomography means. Examples of labels useful for diagnostic imaging include radioactive labels, fluorescent labels, positron emitting isotopes, chemiluminescent materials, luminases, and the like. Radiolabeled antibodies can also be used for in vitro diagnostic tests. The specific activity of the isotope-labeled antibody depends on the half-life, isotopic purity of the radiolabel, and how the label is incorporated into the antibody.

The invention also relates to antibodies that bind FcRn, and to diagnostic uses, such as in vitro, for example, in biopsies or cells from a sample, eg, an autoimmune disease patient, or in vivo, For example, there is provided a kit comprising instructions for the use of an FcRn-binding antibody or antigen-binding fragment thereof for detecting FcRn by imaging an individual. The kit may further comprise at least one additional reagent, such as a label or additional diagnostic agent. In the case of in vivo use, the antibody may be formulated as a pharmaceutical composition.

The present invention also provides a polynucleotide sequence encoding the variable region of the antibody or fragment thereof according to the present invention.

The polynucleotide sequence encoding the heavy chain variable region of the antibody or fragment thereof according to the present invention is any one selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 and 15, or 90% thereof, preferably 95% or more sequence homology,

The polynucleotide sequence encoding the light chain variable region has any one selected from 17, 19, 21, 23, 25, 27, 29 and 31, or a sequence having 90%, preferably 95% or more sequence homology thereto. .

The polynucleotide sequence encoding the antibody or fragment thereof according to the invention

A polynucleotide sequence encoding any one selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 and 15, or a heavy chain variable region having 90%, preferably 95% or more sequence homology thereto;

Consisting of a polynucleotide sequence encoding any of 17, 19, 21, 23, 25, 27, 29 and 31, or a light chain variable region having 90%, preferably 95% or more sequence homology therewith,

 Particularly preferably

(a) a sequence having sequence identity of SEQ ID NO: 1 or greater than or equal to 90%, preferably greater than or equal to 95%, and a sequence sequence having greater than or equal to SEQ ID NO: 17 or greater than or equal to 90%, preferably greater than or equal to 95% sequence identity;

(b) a sequence having SEQ ID NO: 3 or at least 90%, preferably at least 95%, homology with SEQ ID NO: 3 and a sequence having SEQ ID NO: 19 or at least 90%, preferably at least 95% homology with it;

(c) a sequence having sequence identity of SEQ ID NO: 5 or at least 90%, preferably at least 95%, and SEQ ID NO: 21 or at least 90%, preferably at least 95% of this;

(d) a sequence having SEQ ID NO: 7 or at least 90%, preferably at least 95% sequence homology with SEQ ID NO: 7 and a sequence having SEQ ID NO: 23 or at least 90%, preferably at least 95% homology with it;

(e) SEQ ID NO: 9 or a sequence having at least 90%, preferably at least 95% sequence homology with SEQ ID NO: 9 or a sequence having at least 90%, preferably at least 95% sequence homology with SEQ ID NO.

(f) a sequence having SEQ ID NO: 11 or at least 90%, preferably at least 95% sequence homology with SEQ ID NO: 11 or a sequence having SEQ ID NO: 27 or at least 90%, preferably at least 95% sequence homology thereto;

(g) a sequence having SEQ ID NO: 13 or at least 90%, preferably at least 95% sequence homology with SEQ ID NO: 13 or at least 90%, preferably at least 95% sequence homology with SEQ ID NO. And

(h) a sequence having sequence identity of SEQ ID NO: 15 or at least 90%, preferably at least 95%, and SEQ ID NO: 31 or at least 90%, preferably at least 95% of this;

≪ / RTI >

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 FcRn according to the present invention using the recombinant vector or host cell. In particular, it is preferable to prepare by expression and purification by genetic recombination method. Specifically, the variable regions encoding the antibodies specifically binding to FcRn 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" refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein 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), for example the long terminal repeat (LTR) promoter of HIV, moronivirus, cytomegalovirus (CMV) ), Epstein bar virus (EBV), Loose sacoma virus (RSV) promoters, as well as promoters derived from β-actin promoter, human heroglobin, human muscle creatine, human metallothionein, but are not limited thereto. . The expression vector may comprise a selectable marker for selecting a host cell containing the vector. The selection marker is for selecting cells transfected with the vector, and markers may be used that confer a selectable phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents or expression of surface proteins. Since only cells expressing a selection marker survive in an environment treated with a selective agent, the transfected 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 recombinant vector is not particularly limited as long as it expresses a desired gene in various host cells of prokaryotic and eukaryotic cells and produces a desired protein. However, it has a promoter showing strong activity and a promoter similar to a natural state A vector capable of producing a large amount of an exogenous protein in a form is preferable.

Combinations of various expression hosts / vectors 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. . Expression vectors that can be used in bacterial hosts include broader hosts such as bacterial plasmids derived from Escherichia coli , such as pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9 and derivatives thereof, RP4. Plasmids with ranges, phage DNA that can be exemplified by a wide variety of phage lambda derivatives such as λgt10 and λgt11, NM989, and other DNA phages such as M13 and filamentary single-stranded DNA phages. Useful expression vectors for yeast cells are 2 ° C. plasmids and derivatives thereof. A useful vector for insect cells is pVL941.

The recombinant vector is inserted into a host cell to form a transfectant, and suitable host cells are Escherichia coli, Bacillus subtilis ( Bacillus subtilis). subtilis ), Streptomyces sp.), Pseudomonas sp.), Proteus mirabilis or Staphylococcus prokaryotic cells such as sp. Also, the genus Aspergillus fungi such as sp.), blood Chiapas pastoris (Pichia pastoris , Saccharomyces cerevisiae ), genus Schizosaccharomyces sp.) and Neurospora eukaryotic cells such as yeast such as crassa , other lower eukaryotic cells, and cells of higher eukaryotes such as cells from insects.

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.

In the present invention, “transfection” or “transformation” into a host cell includes any method of introducing a nucleic acid into an organism, cell, tissue or organ and selects appropriate standard techniques according to the host cell as known in the art. These methods include electroshock electroporation, plasma fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, agro bacteria mediated transfection, PEG, dextran sulfate, lipofectamine, and dry / inhibitory mediated transfection methods, and the like.

By culturing the transfectant expressing the recombinant vector in a nutrient medium, a large amount of the FcRn-specific antibody according to the present invention can be produced, and 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.

Human antibodies specific for FcRn, a receptor having high affinity for IgG provided in the present invention, have high affinity and specificity, have almost no problems due to immunogenicity, and are non-competitive to IgG for FcRn. It can be used very effectively for the treatment and diagnosis of autoimmune diseases because it has the property of combining and reducing the amount of pathogenic auto-antibody in the blood.

1 is a diagram showing the result of screening an antibody variable domain that binds only to αFcRn but not to β2m in human FcRn, and shows a phage prepared from a human antibody cDNA library with αFcRn (heavy chain), β2m, and α-Myc. This bound 96-well plate was treated by ELISA to phage phages that bound to αFcRn (heavy chain) but not β2m at pH6.0 and pH7.4. Indicates.
FIG. 2 is a diagram showing the binding ability of the hit antibody to the cell surface human FcRn (hFcRn). FIG. 2 shows eight selected antibodies that bind to human FcRn on the cell surface. Treatment with HEK293 cells overexpressing human FcRn confirmed the antibody binding to cell surface FcRn at pH 6.0 and pH 7.4. The binding of the antibody to FcRn was achieved by treating the cells with cells at each pH and then using Fluorescent activated cell sorter (FACS) using Alexa488 labeled anti-human goat antibody. Confirmed through the analysis, the results of the cell surface binding capacity of the selected antibody was expressed as a relative value based on the MFI of human IgG1 (hIgG1).
FIG. 3 shows the results of observing at the cellular level whether selected antibodies that bind to cell surface human FcRn can inhibit human IgG binding to human FcRn. FIG. 3 shows that binding to HEK293 cells overexpressing human FcRn. Eight selected screened antibodies and unlabeled human IgG1 were treated with 250 nM, 500 nM and 1000 nM to confirm the inhibitory ability by decreasing the cell surface binding ability of Alexa488 labeled human IgG1.
4 is a diagram showing the results confirmed through cross-reactivity of mice, rats and monkeys whether the antibody that binds human FcRn also binds to other species of FcRn. FITC-labeled anti-mouse, anti-rat, anti-treated with IgG antibodies of different species to cell lines NIH3T3L1, Rat-2, Cos7 cells overexpressing mouse, rat, monkey FcRn, and recognized antibodies of each species The results of analysis of antibody fluorescence values of each species bound to the cell surface by treatment with human goat antibodies by FACS. The intensity of fluorescence was expressed in relative proportions of MFI values of three selected antibodies, anti-mouse, anti-rat FcRn antibody and cynomolgus IgG antibody based on the MFI of human IgG1.
FIG. 5 shows catabolic activity of hIgG1 by HL161_1A and HL161_11G antibodies selected from Tg276 (hFcRn + / +, hβ2m + / +, mFcRn − / −, mβ2m − / −), transgenic mice expressing human FcRn. (Cabolism) shows the results of confirming the effect, 5 mg / kg Biotinylated human IgG1 intraperitoneal injection (IP injection) to Tg276 mice (24, 48, 72, 96 hours after injection) The result obtained by intraperitoneal injection of 10 mg / kg of human IgG1, HL161_1A, HL161_11H or PBS. Sampling proceeded 6, 12, 24, 48, 72, 96, 120, 168 hours after the administration of Biotin-IgG. Blood was collected before administration of the drug at 24, 48, 72, 96 hours by Biotin-IgG by ELISA. The residual amount of was confirmed. As a result, the remaining amount of the 6-hour blood sample was 100%, and the remaining amount of each time period was expressed as a relative ratio.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for explaining the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. will be.

Example 1 Preparation of Soluble FcRn Expression Vector

FcRn is a heterodimeric protein in which transmembrane anchored α-heavy chains (αFcRn) and β chains (β2m) are non-covalently linked. Therefore, the expression vector of the water-soluble FcRn was intended to produce two genes into one expression vector using a multisite gateway system. Table 1 is the αFcRn and β2m genes having the signal sequence of human growth hormone, respectively, and the restriction enzyme sequences of αFcRn 5 'terminal NheI / 3' terminal XhoI and β2m 5 'terminal NheI / 3' terminal XbaⅠ, respectively. The amplification product was cloned into the pcDNA3.1 (+) vector after gene amplification by the PCR method described in the primer (primer).

Specifically, plasmid DNA was extracted from clones (αFcRn: hMU008093 / B2m: hMU005156) purchased from Korea Research Institute of Bioscience and Biotechnology. 2 μl 200 ng template, 2 μl 10 pmole N-primer, 2 μl 10 pmole C-primer, 25 μl 2X Solgent mixture and 19 μl distilled water were added to make 50 μl of reaction solution.

PCR reaction was performed 25 times from the first denaturation 95 ℃ 2 minutes, secondary denaturation 95 ℃ 20 seconds, primer conjugation 60 ℃ 40 seconds, elongation reaction 72 ℃ 1 minute in the course of secondary denaturation to elongation reaction and then the final enzyme The reaction proceeded at 72 ° C. for 5 minutes. The amplification of the hGH signal sequence was performed in the same composition. After each PCR reaction, DNA was extracted from a PCR band on an agarose gel and used as a template for a PCR reaction. hGH leader αFcRn gene amplification products are Nhe I and Xho I enzymes, hGH leader β2m gene amplification products are Nhe I and Xba I enzymes for more than 4 hours at 37 ℃, pcDNA3.1 (+) vector is also Nhe I / Xho I and Nhe I / Xba I The enzymes were then electrophoresed at 37 ° C. for at least 4 hours. DNA fragment bands sized on agarose gels were recovered using an agarose gel extraction kit. The DNA fragment and the vector fragment were mixed to a total of 10 ul in a ratio of 1: 5, and then incubated at room temperature for 2 hours by adding 10 μl NEB ligase buffer and 1 μl ligase. 5 μl of the Ligation mixture was placed in DH5α competent cells, transformed by heat shock at 42 ° C. for 1 minute, and cultured in LB solid medium containing ampicillin. To secure colonies. The colonies were incubated for 24 hours at 37 ° C. in LB liquid medium containing ampicillin, and then plasmid DNA was isolated and analyzed by DNA sequencing. Thus, each of the natural leader αFcRn, hGH leader αFcRn, natural leader β2m, and hGH leader β2m genes were pcDNA3. It was confirmed that 1 (+) was inserted.

Using the Jump-In ^™ fast gateway cloning kit, two plasmid DNAs cloned into the pcDNA3.1 (+) vector were used as a template, according to the manufacturer's instructions. PCR was performed using a PCR primer inserted with a site. Through this process, the final expression vector in which the hGH leader αFcRn / β2m was inserted into the pJTI ^TM Fast DEST vector was prepared. The produced expression vector was confirmed the nucleotide sequence through genetic analysis.

Example 2 Preparation of Water-soluble FcRn-expressing Cell Line

Plasmid DNA to be used for transfection (transfection) should have a high concentration and purity, so prepared by QIAGEN plasmid purification (plasmid purification). Confirmation by Gene Sequencing A bacterial clone of the hGH leader FcRn pJTI ^™ Fast DEST vector was inoculated into a 100 ml LB flask containing ampicillin. Bacteria cells were harvested by centrifugation at 3600 rpm for 15 minutes. After removing the media, mix the 10 ml P1 solution and completely dissolve the pellets by vortexing, then gently invert 5 times by adding 10 ml P2 solution at room temperature. Incubate for 5 minutes. 10 ml P3 solution was then inverted five times and incubated for 30 minutes on ice. After incubation was centrifuged for 30 minutes at 4 ℃ 15,000 rpm. Supernatant was transferred to a new 50 ml centrifuge tube and centrifuged for an additional 15 minutes. The supernatant was loaded into a column equilibrated with 10 ml QBT solution. After loading of the solution by gravity was washed twice with 30 ml QC solution. A 50 ml centrifuge tube was placed under the column and eluted with 15 ml QF solution. The eluent solution was mixed with 10.5 ml isopropanol solution and centrifuged at 4 ° C. 15,000 rpm for 30 minutes. After checking the DNA pellet visually, the supernatant was carefully removed, centrifuged for 15 minutes by adding 5 ml 70% ethanol, the supernatant was removed so that the pellet did not fall, and the pellet was dissolved in 150 μl of LAR water. When the pellets melted, they were filtered through a 0.22 μm filter. DNA concentration is checked using a Nanodrop instrument, and purity can be used as DNA for transfection if the A260 value is between 0.1 and 1.0.

24 hours before the transfection process, the cells were prepared by inoculating at 6-7 X 10 ⁵ cells / ml. After 24 hours, cell viability and cell number were measured. To remove clumps of cells, transfer to a 50 ml tube by volume measured and vortex for 20 to 30 seconds. Transfer to a new flask with 3 x 10 ⁷ viable cells in a total of 28 ml of medium.

20 μg of each of the FcRn DEST vector and phiC31 integrase vector with hGH leader sequence were diluted in 1 ml OptiMEM ^® I medium. Subsequently, 80 µg of 293 pectin was diluted in 1 ml of OptiMEM ^® I medium and allowed to stand at room temperature for 5 minutes. When the leaving time is more than 5 minutes, the activity of pectin decreases so that it is not left for a long time. At the end of the stand time, each 1 ml dilution solution was mixed and allowed to stand at room temperature for 30 minutes to form a complex of DNA and pectin. When the mixture was left to stand, it was placed in a flask containing 3 X 10 ⁷ viable cells. The flask was mounted on an orbital shaker in an 8% CO ₂ , 37 ° C. incubator and shaken at 125 rpm. After 24 hours of transfection, the cultures were exchanged with fresh 293 medium.

By selectively killing the cells that were not integrated through the process for selectively selecting cells in which the FcRn expression vector was permanently integrated (chromosome), the expression amount relative to the cell number could be increased. 48 hours after transfection, Hygromycin B was added to initiate selection. Media exchange and antibiotic treatment were performed once every three days, and cell viability and cell number were counted over time to analyze and prepare expression cell lines.

Expression of the prepared FcRn stable cell line was confirmed by Western blotting analysis using a primary antibody against αFcRn and β2m. Quantitative analysis was performed using commercially available human β2-microglobulin as a standard, and the results were analyzed through image analysis.

The medium during FcRn stable cell line culture was sampled and centrifuged at 15,000 rpm for 10 minutes. 20 μl of the supernatant was transferred, and 4 μl of 5 × reducing sample loading dye was added thereto and left at 95 ° C. for 5 minutes. Thus prepared samples were loaded with 12 μl on a 12% NuPAGE Bis-Tris gel. Human β2-microglobulin standard material was loaded at 5 ng and 10 ng. The PAGE gel was run at 250 volts for 35 minutes and transferred to a PVDF membrane at 30 volts for 90 minutes. Blocking was performed for 1 hour with 10% skim milk. Primary antibodies to αFcRn and β2m were incubated for 1 hour at room temperature by diluting 1: 1,000 in TBST solution, respectively. Secondary antibodies were diluted 1: 5,000 in TBST solution and incubated at room temperature for 30 minutes. After washing with TBST solution for more than 30 minutes. ECL solution A and B were mixed 1: 1 and sprinkled on the membrane, and reacted for 1 minute. Every 10 seconds color development was by manual increasement and the results proceeded to image analysis.

Example 3 Water-soluble FcRn Expression and Purification

FcRn expressed from the prepared stable cell line is in a form secreted into the medium. An experiment was conducted to determine the timing for obtaining water-soluble FcRn from the culture of the stable cell line. 30X10 ⁴ cell / ml and 60X10 ⁴ A 30 ml flask of cells / ml was prepared and samples were collected for 7 days from the date of cell inoculation. Samples were analyzed by western blotting (WB) and the date of maximum expression after inoculation was determined as the timing of sample media. After cell inoculation, the medium was centrifuged for 10 minutes at 4 ℃ 3,600rpm after incubation. Only the media supernatant (supernatant media) was collected so as not to drop the cell pellet, and then filtered using a 0.22 μm filter. In order to purify the 800 ~ 1000 ml of the obtained sample, a buffer change (concentration) process was performed by ultra filtration (UF). First, about 2 L of distilled water was poured to remove NaOH in the filter, and washed with distilled water sufficiently, and then, 200 mM Bis-Tris, 150 mM NaCl pH 6.0 buffer, which is a buffer to be used, was flowed. At this time, the degree of NaOH removal in the filter can be checked by measuring and comparing the pH of the buffer and the permeate to the feed reservoir. When the pH of the permeate became equal to pH 6.0, the sample was injected into the source and concentrated to 200 ml. To change the buffer of the medium sample to 20 mM Bis-Tris, 150 mM NaCl pH 6.0 buffer, about 2 L or more of about 10-fold was filtered. The pressure sensor was connected to P1, the feed / inlet, and operated in manual mode. Trans-membrane pressure (TMP) was between 0.15 and 0.20 and no more than 0.5. At the end of the buffer exchange, 200 ml of sample medium was concentrated to 50 ml. The concentrated sample is centrifuged for 15 minutes at 3,600rpm at 4 ℃, only the supernatant is transferred.

Approximately 50 ml sample prepared by the UF were purified by affinity chromatography (affinity chromatography) using IgG Sepharose 6 fast flow. IgG Sepharose 6 fast flow was filled into an XK16 column and used as a column volume of 8 ml, and affinity chromatography was performed at a linear velocity of 150 cm / hr (5 ml / min). After injecting 50 ml of concentrated sample at 2 ml / min flow rate, at least 5 CV of washing buffer (20 mM Bis-Tris, 150 mM NaCl pH 5.8) was flowed at the same flow rate. Proceed to the point of recovery. Thereafter, an elution buffer (50 mM Tris-Cl, pH8.0) was flowed at a flow rate of 4 ml / min, and fractions were collected by 2 ml. Protein concentration was measured using the Bradford quantitative method, and purity analysis was performed by Coomassie staining after electrophoresis.

Example 4 Polyphage and Monophage Screening by Phage Display Method

The isolated shFcRn (soluble human FcRn) was used as an antigen to select polyphages that bind to the FcRn α chain but not to β2m in a pH range from pH 6.0 to pH 7.4 from the human antibody cDNA library. (See FIG. 1) Eight monophage among them were subjected to screening gene analysis. The detailed method is as follows.

A library having a variety of 2.7 × ^{10 10} was incubated at 30 ° C. for 16 hours, concentrated with PEG (polyethyleneglycol), and prepared in PBS buffer. Panning up to 3rd with shFcRn antigen confirmed that colony titer of phage to antigen was amplified by 10 to 100-fold in the 3rd, and monoclonal in 3rd polyclonal phage antibody group Phage antibody groups were prepared and their primary binding ability was confirmed by their respective binding capacity to antigens. Monoclonal phage antibodies were obtained through finger priniting and sequencing. Cells obtained by tertiary panning from library cells were infected with helper phage and cultured, and then ELSIA was prepared using immuno-plates with poly ScFv-phages present in the supernatant. Was performed. As a result, the binding ability to the antigen was confirmed to be enhanced (enrichment) in the tertiary poly ScFv-phage (see Fig. 1). In order to sequence each of the monophage clones identified by fingerprinting with Bst N1, DNA separation of monoclonal cells was performed to obtain DNA, and to view DNA analysis results. The CDR regions of V _H and V _L were identified, and the similarity between these antibodies and germ line antibody groups was determined by NCBI's web page (http://www.ncbi.nlm.nih.gov/igblast/). Using the Ig BLAST program of), it was possible to select and obtain phage antibodies specific for eight shFcRn. The variable region sequences of the eight antibodies selected and the nucleotide sequences encoding them are listed in Tables 2 and 3. In addition, the CDR sequences in each variable region are listed in Table 4.

Example 5 Human Antibody Production and Production

From the selected monoclonal scFv phage, the variable regions of the heavy chain and the light cahin were respectively amplified and cloned into pNATAB vectors for human antibody expression. An Fc gene is included in the pNATAB vector for heavy chain expression, and a light chain constant region has already been constructed in the pNATAB vector for light chain expression. IgG) form. Human antibodies were obtained by transient transfection of two types of plasmid DNA expressing heavy and light chains into 293E cells to purify the antibody secreted in the medium using a protein A column. Detailed production method is as follows.

The day before transfection, 5 × 10 ⁶ male 293E cells were seeded in a 100 mm culture dish. After confirming that the confluence was 90% or more, transfection was performed. 5 μg of heavy and light chain plasmid DNA were prepared, respectively, and then placed in 500 μl serum free DMEM medium. Into the medium containing the plasmid DNA was added 20 μg of polyethylenimine (polyscience, cat #. 23966), a transfection reagent, mixed with a pipette and incubated at room temperature for 15 minutes. DNA and transfection reagent mixtures were then added to the medium in which 293E cells were growing. The next day, the medium was replaced with fresh serum-free medium, and the medium was harvested with medium replacement every two days. The medium in which the antibody is expressed is concentrated using a Pellicon 3 filter (Millipore, cat #. P3C030C01), and then a Hi-Trap protein A FF column (protein A FF) in an AKTA purifier system. human antibody was purified using column) (GE healthcare, cat #. 17-5079-01). The antibody eluted using 100 mM glycine-HCl (pH3.3) buffer was quantified by dialysis in PBS and then absorbance at 280 nm.

Example 6 Determination of Avidity of Antibody through SPR

The binding ability of the antibody through the SPR proceeded by a method of measuring affinity by immobilization of the shFcRn to the Proteon GLC chip (Bio-Rad) as a ligand.

Analysis using a Proteon XPR36 (Bio-Rad) SPR instrument was performed to determine the K _D of the anti-FcRn 161 antibody that binds to FcRn. The analysis of ligand and analyte was performed using Proteon GLC chip and the interaction was analyzed at pH 6.0 and pH 7.4, respectively. Immobilization of the ligand was activated by injecting 1X EDAC (EDC) and 1X Surfo-NHS in a 1: 1 mixture for 5 minutes. The ligand was diluted with 25 μg / ml of shFcRn in 10 mM sodium acetate, pH 4.5, and injected for 5 minutes at a flow rate of 30 μl / min. Deactivation was performed by ethanolamine (Ethanolamine) HCl at 30 μl / min 5 minutes at a flow rate was confirmed that the shFcRn is fixed to 1200 ~ 1500 RU. The analyte antibody is flowed at 30 μl / min at the immobilized chip. Assay antibody was injected by diluting the assay antibody diluted in 0.05% Tween 20 / PBS at pH 6.0 or pH 7.4 to 100 nM, 50 nM, 20 nM, 10 nM, 5 nM, 2.5 nM. The association time of the antibody of analysis was 240 seconds and the dissociation time was 600 seconds. Regeneration of the chip was allowed to flow 10 mM Glycine, pH 2.5 at 100 μl / min flow rate for 18 seconds. Kinetics analysis was performed based on sensorgrams from six concentrations. Kinetics analysis was performed using Proteon XPR36 software. Kinetic parameters of the antibodies resulting from the SPR analysis are shown in Table 5.

Example 8 Preparation of Full Length FcRn Expression Vector

Cross-reactivity of human FcRn-expressing cell lines with FcRn in monkeys, mice, and rats to determine whether eight antibodies inhibit the binding of hFcRn to human cells and their interaction with IgG Fc In order to investigate the expression vector for the production of cell lines overexpressing a variety of FcRn on a variety of cell surface was prepared.

1) Human FcRn

Plasmid DNA was extracted from the clone (FcRn: hMU008093 / B2m: hMU005156) purchased from the Korea Research Institute of Bioscience and Biotechnology. 2 μl 200 ng template, 2 μl 10 pmole N-primer 2 μl 10 pmole C-primer, 25 μl 2X Solgent mixture, 19 μl distilled water were added to make 50 μl of reaction solution. The sequences of the primers used in the experiments are shown in Table 1. PCR reaction was performed 25 times from the first denaturation 95 ℃ 2 minutes, secondary denaturation 95 ℃ 20 seconds, primer conjugation 60 ℃ 40 seconds, elongation reaction 72 ℃ 1 minute in the course of secondary denaturation to elongation reaction and then the final enzyme The reaction proceeded for 5 minutes at 72 ° C. αFcRn gene amplification products are Nhe I and Xho I enzymes, β2m gene amplification products are Nhe I and Xba I enzymes for more than 4 hours at 37 ℃, pcDNA3.1 (+) vector also Nhe I / Xho I and Nhe I / Xba I enzymes at 4 ℃ 37 Electrophoresis was performed after the treatment for more than an hour. DNA fragment bands sized on agarose gels were recovered using an agarose gel extraction kit. The DNA fragment and the vector fragment were mixed to a total of 10 μl at a ratio of 1: 5, and incubated for 2 hours at room temperature by adding 10 μl NEB ligase buffer and 1 μl ligase. 5 μl of the ligation mixture was put into DH5α competent cells and heat shock treated at 42 ° C. for 1 minute to transform, and cultured in LB solid medium containing ampicillin to secure colonies. The colonies were incubated for 24 hours at 37 ° C. in LB liquid medium containing ampicillin and plasmid DNA was isolated to confirm that each of the αFcRn / β2m genes was inserted into pcDNA3.1 (+) by DNA sequencing.

Two plasmid DNA cloned into pcDNA3.1 (+) vector as a template using Jump-In ^™ fast gateway cloning kit was used at the 5 'and 3' ends. PCR was performed using a PCR primer containing an attB site. Through this process, the final expression vector in which the hGH leader αFcRn / β2m was inserted into the pJTI ^TM Fast DEST vector was prepared. The produced expression vector was confirmed the nucleotide sequence through genetic analysis.

2) Production of full-length FcRn expression vector of mouse, rat, and monkey

Mouse, rat and monkey cDNA libraries were purchased from BioChain and used. Mouse cDNA (C1334149), rat cDNA (C1434149), monkey cDNA (C1534150-cy) were used as templates. 1 μl template, 2 μl 10 pmole N-primer of each species described in Table 1, 2 μl 10 pmole C-primer, 25 μl 2X solvent mixture, 20 μl distilled water were added to make 50 μl of reaction solution. Primer sequences used in the experiments are listed in Table 1. PCR reaction was performed 25 times from the first denaturation 95 ℃ 2 minutes, the second denaturation 95 ℃ 20 seconds, primer conjugation 56 ℃ 40 seconds, elongation reaction 72 ℃ 1 minute in the course of secondary denaturation to elongation reaction and then the final enzyme The reaction proceeded at 72 ° C. for 5 minutes. αFcRn and β2m gene amplification products were subjected to electrophoresis after treatment with Kpn I and Xho I enzymes for at least 4 hours at 37 ° C. and pcDNA3.1 (+) vector at 37 ° C. for at least 4 hours, respectively. DNA fragment bands sized on agarose gels were recovered using DNA agarose gel extraction kits. The DNA fragment and the vector fragment were mixed at a ratio of 1: 5 to 10 ul in total, and incubated at room temperature for 2 hours by adding 10 μl NEB ligase buffer and 1 μl ligase. 5 μl of the ligation mixture was transformed into DH5α competent cells by heat shock treatment at 42 ° C. for 1 minute, and cultured in LB solid medium containing ampicillin to secure colonies. The colonies were incubated for 24 hours at 37 ° C. in LB liquid medium containing ampicillin and plasmid DNA was isolated to confirm that each of the FcRn / β2m genes of each species was inserted through DNA sequencing. PCR primers containing attB sites at the 5 'and 3' ends of two plasmid DNA cloned into the pcDNA3.1 (+) vector as a template using the Jump-In ^™ Fast Gateway Cloning Kit. PCR was carried out using. Through this process, the final expression vector into which the αFcRn / β2m of each species having the hGH leader was inserted was constructed in the pJTI ^TM Fast DEST vector. The produced expression vector was confirmed the nucleotide sequence through genetic analysis.

Example 9 Preparation of Full Length FcRn Expression Cell Line

Plasmid DNA to be used for transfection should be prepared by QIAGEN plasmid purification because it should have a high concentration and purity. Confirmation by Gene Sequencing Bacterial clones of the hGH leader FcRn pJTI ^™ Fast DEST vector were inoculated into 100 ml LB flasks containing ampicillin. Bacteria cells were harvested by centrifugation at 3600 rpm for 15 minutes. After removal of the medium, 10 ml P1 solution is mixed and the pellets are completely released by vortexing. 10 ml P2 solution was added to gently invert 5 times and incubated for 5 minutes at room temperature. It was then inverted five times by the addition of 10 ml P3 solution and incubated for 30 minutes on ice. After incubation was centrifuged for 30 minutes at 4 ℃ 15,000rpm. The supernatant was transferred to a new 50 ml centrifuge tube and then centrifuged for an additional 15 minutes. The supernatant was loaded into a column equilibrated with 10 ml QBT solution. After loading of the solution by gravity it was washed twice with 30 ml QC solution. A 50 ml centrifuge tube was placed under the column and eluted with 15 ml QF solution. The elution solution was mixed with 10.5 ml isopropanol solution and centrifuged at 4 ° C. 15,000 rpm for 30 minutes. After checking the DNA pellet visually, the supernatant was carefully removed and centrifuged for 15 minutes by the addition of 5ml 70% ethanol. Remove the supernatant so that the pellet does not fall off, and dissolve the pellet in 150 μl of LAR water. When the pellets melted, they were filtered through a 0.22 μm filter. DNA concentration was checked using a nanodrop device, and the purity was used for transfection of DNA having an A260 value of 0.1 to 1.0.

Cell lines expressing human, rat, mouse and monkey FcRn are as follows. HEK293 cells were used in humans, 3T3L1 cells were used in mice, Rat-2 fibroblast cells were used in rats, and COS-7 cell lines were used in monkeys. Each cell line was inoculated to be 80% confluent in a T75 flask 24 hours before the transfection process. After 24 hours it was replaced with fresh medium.

5 μg of each FcRn DEST vector and phiC31 Integrase vector were diluted in 500 μl of OptiMEM ^® I medium. Subsequently, 20 μl of Lipofectamine 2000 was diluted in 500 μl of OptiMEM ^® I medium and allowed to stand at room temperature for 5 minutes. At the end of the standing time, each 500 μl of the diluted solution was mixed and allowed to stand at room temperature for 30 minutes to form a complex of DNA and lipofectamine. At the end of the mixing time, the mixture was placed in a flask containing cells. After 6 hours of incubation in an incubator at 5 ° C. CO ₂ , 37 ° C., the cells were replaced with fresh 10% FBS / DMEM medium.

In order to selectively select cells in which the FcRn expression vector is permanently integrated with a chromosome, the following process was performed. By selectively killing unintegrated cells through this process, the expression level can be increased. 48 hours after transfection, selection was started by the addition of Hygromycin B. Media exchange and antibiotic treatment were performed once for 3 days of culture, and colonies formed over time were removed and cultured. Thus, a cloned stabilized cell line formed from a single clone was constructed by clonal selection.

Selected stabilized cell line construction was confirmed through FACS analysis. Human FcRn stabilized cell lines were identified by shifting populations by binding Alexa488 labeled hIgG1 at pH 6.0. Similarly, the monkey stabilized cell line was confirmed by a change in population by binding Cyanomolgus monkey IgG1 labeled Alexa488 at pH 6.0. The mouse FcRn stabilized cell line bound mouse IgG1 at pH 6.0 for 1 hour, and then the amount of mouse IgG1 bound using FITC labeled anti-mouse IgG1 was measured by FACS. This confirmed the change in population. The rat FcRn stabilized cell line bound anti-rat FcRn mouse antibody at pH 7.4 for 1 hour, and then measured the amount of rat IgG bound using FACS using an FITC-labeled anti-Rat IgG antibody to measure the change in population. Confirmed.

Example 10 FcRn Binding Analysis of Antibodies Using FACS

HEK293 stabilizing cells expressing human FcRn were analyzed for pH-specific binding to FcRn using the FACS system. 100,000 HEK293 stabilized cells were washed in PBS and pelleted by centrifugation at 4500 rpm for 5 minutes in a desktop microcentrifuge. 1 μg of antibody was added to 100 μl of pH 6.0 or pH 7.4 PBS / 10 mM EDTA. The pellet was resuspended with 100 μl of antibody solution and incubated on ice for 60 minutes. These cells were washed once with 150 μl of pH-specific buffer and pelletized. Alexa488-labeled anti-human antibody goat antibody (1 mg / ml) was diluted to 1/20 in pH-specific buffer, the pellet was resuspended in 100 μl, and then incubated on ice for 60 minutes. These cells are washed once with 150 μl of pH-specific buffer, pelleted, and then resuspended in 150 μl of pH-specific buffer and transferred to FACS analysis tubes. These cells were analyzed by FACS using BD FACSDivaTM v6.1.3 software (BD Bioscience). The result is the average fluorescence intensity: was shown as (M ean F luorescence I ntensity MFI) (see Fig. 2). Of the eight selected antibodies, it was confirmed that HL161-1A, HL161-11G, and HL161-11H bind stronger than the comparative IgG1 and bind to cell surface hFcRn.

Example 11 Blocking Function Analysis of Antibodies Using FACS

Eight kinds of antibodies having confirmed binding ability to cell surface hFcRn were treated to HEK293 cells expressing hFcRn on the cell surface, and the blocking function of the antibody was confirmed to decrease binding of Alexa-Fluo-488 fluorescently labeled hIgG1. The analysis process is described in detail as follows.

1) Labeling of Human IgG1 with Alexa-Fluor-488

Human IgG1 (Calbiochem, cat # .400120) was labeled with Alexa Fluor 488 Protein Labeling Kit (Molecular Probed / Invitrogen, Carlsbad, Calif.) According to the manufacturer's suggested protocol. Specifically, 50 μl of 1 M sodium bicarbonate, pH 9.0, was added to 500 μl of 2 mg / ml IgG solution in PBS. The protein solution was then added to Alexa-Fluor-488 succinimidyl ester (dry powder) and incubated for 1 hour at room temperature with stirring. The protein was purified by size-exclusion chromatography using a kit component column (Bio-Rad Biogel P-30 Fine size exclusion purification resin). Samples were loaded onto the column and eluted with PBS. The first colored band contains the labeled protein. The degree of labeling was determined by measuring the absorbance of the eluted IgG at A280 (absorbance at 280 nm and A494 (absorbance at 494 nm).) The protein molar concentration was determined using the formula below.

(M) = [A280-(A494 x 0.11) x Dilution factor] / 203,000

In addition, the formula used to derive the moles of dye per mole of protein is as follows.

(M) = A494 x dilution factor / 71,000 x protein concentration

Typically 4-5 moles of IgG molar Alexa-Fluor-488 were incorporated.

2) Cell competition analysis using FcRn expressing cells

HEK293 stabilizing cells expressing human FcRn were analyzed for blocking of antibodies against FcRn using FACS. Antibodies were diluted in binding buffer (PBS pH 6.0, 10 mM EDTA) to 50 μl at 2,000 nM, 400 nM, 80 nM concentrations and aliquoted into tubes. To this was added 1 μM Alexa488 labeled hIgG1 (pH 6.0) to each tube at 10 μl. HEK293 stabilized cells were diluted to 2,500,000 / ml in binding buffer, and 40 μl of the cell suspension was added to the tube to which the antibody and the labeled antibody were added. Therefore, 100,000 cells, 100 nM of labeled antibody, 1000 nM, 500 nM, and 250 nM competition antibody were present in the final volume of 100 μl. These cells are washed once with 150 μl of pH 6.0 buffer, pelletized, and then resuspended in 150 μl in pH-specific buffer and transferred to FACS analysis tubes. These cells were analyzed by FACS using BD FACSDivaTM v6.1.3 software (BD Bioscience). The results are expressed as mean fluorescence intensity (MFI).

MFI of the experimental group was treated after subtracting the MFI value (background signal) measured only through the cells. MFI of the control tube (Alexa Fluor 488 alone and no contention interest) was converted to 100% to convert the percentage of MFI of the contention-containing tube.

It was determined that the contention rate of the competition antibody was high when the content of the human IgG1 was lower than the MFI of the containing tube. Referring to the results of FIG. 3, it was found that HL161-1A, HL161-11G, and HL161-11H have a blocking function.

Example 12 Cross-Reactivity Confirmation Using FACS

The cross-reactivity of each species was confirmed to see if three human antibodies with blocking functions could bind to other species of FcRn and block the binding of IgG of each species to FcRn. Detailed experiment contents are as follows.

1) Confirmation of Cross-Reactivity to Mouse FcRn

3T3-L1 cells stably expressing mouse FcRn were cultured in 10% FCS DMEM medium, and then removed from the culture plate by trypsin treatment. After washing three times with cold binding buffer (PBS, pH 6.0 10 mM EDTA), cells were prepared by diluting the cell number to 10 ⁵ / ml with binding buffer. A 1.5 ml tube 10 ⁴ cells were dispensed to each 100 μl into the next, it was put HL161 antibody (1 mg / ml) 1 μl to the frequency divider cell. Then incubate on ice for 1 hour. As an experimental control antibody, 1 μl of rabbit antibody (Santacruz, sc-66893) and human IgG (Abcam, cat #. 409120) that bind mouse FcRn were used. Cells were recovered by centrifugation at 4 ° C. and 4000 rpm for 5 minutes, washed once with binding buffer, and then resuspended with 100 μl binding buffer. 1 μl of anti-human IgG goat antibody (Invitrogen, Cat # .A11013) was added to the cells as a secondary antibody conjugated with FITC fluorescent material. In case of experiment of anti-mouse FcRn rabbit antibody, the anti-rabbit antibody was incubated on ice for 1 hour, then centrifuged for 5 minutes at 4 ° C and 700g for 5 minutes to recover the cells, and then washed once with binding buffer. Finally, cells were resuspended in 400 ul binding buffer, and then FACS analyzed to confirm cross-reactivity of HL161 antibodies against mouse FcRn (see FIG. 4). The pH 7.4 was carried out in the same manner as the experimental procedure using the pH 6.0 binding buffer by maintaining the pH of the binding buffer to 7.4. The degree of binding of each antibody to mouse FcRn is expressed in relative MFI based on human IgG. At both pH 6.0 and pH 7.4, the HL161 antibodies HL161-1A and hl161-11G bound to mouse FcRn, but not HL161-11H (see FIG. 4).

2) Cross Reactivity Confirmation for Rat FcRn

Rat fibroblast, Rat-2 (KCTC #. AC28203) cells stably expressing rat FcRn were incubated in 10% FBS DMEM medium and then detached from the culture plate by trypsin treatment. After washing three times with cold binding buffer (PBS pH 6.0, 10 mM EDTA), cells were prepared by diluting the cell number to 10 ⁵ / ml with binding buffer. A 1.5 ml tube 10 ⁴ cells is dispensed to each 100 μl into the next, was put HL161 antibody (1 mg / ml) 1 μl to the frequency divider cell. Then incubate on ice for 1 hour. As a control antibody of the experiment, 1 μl of mouse 1G3 antibody and human IgG (Abcam, cat #. 409120) that bind to rat FcRn were used. The 1G3 antibody was used after purification from mouse hybridoma cells (ATCC #. CRL2434). Cells were recovered by centrifugation at 4 ° C. and 4000 rpm for 5 minutes, washed once with binding buffer, and then resuspended with 100 μl binding buffer. 1 μl of anti-human IgG goat antibody (Invitrogen, cat #. A11013) was added to the cells as a secondary antibody conjugated with FITC fluorescent material. For experiments treated with anti-rat FcRn mouse antibody, the cells were incubated on ice for 1 hour with anti-mouse IgG goat antibody (Invitrogen, cat #. A11001), and then centrifuged at 4 ° C. and 4000 rmp for 5 minutes. After recovery, the cells were washed once with binding buffer and finally resuspended in 400 μl binding buffer, followed by FACS analysis to confirm cross-reactivity of HL161 antibodies against mouse FcRn (see FIG. 4). The pH 7.4 was carried out in the same manner as the experimental procedure using the pH 6.0 binding buffer by maintaining the pH of the binding buffer to 7.4. The degree of binding of each antibody to rat FcRn is expressed in relative MFI based on human IgG. At both pH 6.0 and pH 7.4 conditions, the HL161 antibodies HL161-1A and HL161-11G bound to rat FcRn, but not HL161-11H (see FIG. 4).

3) Cross Reactivity Confirmation on Monkey FcRn

Cos7 cells stably expressing monkey FcRn were incubated in 10% FBS DMEM medium and then detached from the culture plate by trypsin treatment. After washing three times with cold binding buffer (PBS pH 6.0, 10 mM EDTA), cells were prepared by diluting the cell number to 10 ⁵ / ml with binding buffer. A 1.5 ml tube 10 ⁴ cells is dispensed to each 100 μl into the next, was put HL161 antibody (1 mg / ml) 1 μl to the frequency divider cell. Then incubate on ice for 1 hour. As a control antibody of the experiment, 1 μg of cynomolgus monkey IgG (Equitech-Bio Inc, cat # .SLCM66 0100) and human IgG (Abcam, cat #. 409120) were used. Cells were harvested by centrifugation at 700 ° C. for 5 minutes at 4 ° C., washed once with binding buffer, and then resuspended with 100 μl binding buffer. 1 μl of anti-human IgG goat antibody (Invitrogen, cat #. A11013), a secondary antibody bound to FITC fluorescent material, was added to the cells, and then incubated on ice for 1 hour. Thereafter, cells were recovered by centrifugation at 4 ° C. and 700 g for 5 minutes, washed once with binding buffer, and finally resuspended in 400 μl binding buffer, followed by FACS analysis for HL161 antibody against mouse FcRn. Cross-reactivity was confirmed (see FIG. 4). The pH 7.4 was carried out in the same manner as the experimental procedure using the pH 6.0 binding buffer by maintaining the pH of the binding buffer to 7.4. The degree of binding of each antibody to monkey FcRn is expressed in relative MFI based on human IgG. The HL161 antibodies HL161-1A, HL161-11G and HL161-11H bound to monkey FcRn at both pH 6.0 and pH 7.4 (see FIG. 4).

Example 13 Effect of Human Antibody on mFcRn − / − hFCRN Transformed 276 Mice

Tg276 (hFcRn + / +, hβ2m + / +, mFcRn − / −, mβ2m − / −) mice expressing hFcRn by selecting two of three human antibodies that bind hFcRn and block hIgG1 binding by FACS analysis Jackson Laboratory) was injected to human IgG and HL161_1A, HL161_11H and human IgG was administered to determine whether it affects catabolism (human catabolism). Two HL161 candidates (HL161_1A and HL161_11H), and human IgG (Greencross, IVglobulinS) were prepared at 1 mg / mL for aliquots and stored for 4 days.Phosphate buffered saline (PBS) at pH 7.4 was used as a vehicle. Was used. hFcRn Tg276 mice were acclimated for about 7 days, water and feed were freely ingested, and the temperature (23 ± 2 ° C), humidity (55 ± 5%) and contrast cycle (12 hours) were automatically adjusted. Each group was experimented with three dogs, the delivery group was tested with one dog. Biotin-conjugated hIgG was prepared using kit (Pierce, Cat #. 21327) to use human IgG as a tracer. It was prepared according to the manufacturer's protocol and the prepared biotin-IgG was intraperitoneally injected at a dose of 5 mg / kg. The drug was administered intraperitoneally at a dose of 10 mg / kg 24, 48, 72, 96 hours after the administration of Biotin-IgG. Blood collection was lightly anesthetized using an enamel solution (Isoflurane, JW pharmaceutical), and then heparinized micro-hematocrit capillary tube (Fisher) was used to collect blood from the orbital vein. After 6, 12, 24, 48, 72, 96, 120, 168 hours after the administration of biotin-IgG, the drug was administered after the blood collection progressed at 24, 48, 72, 96 hours. After receiving 0.1 mL of whole blood in an Eppendorf tube, plasma was separated by centrifugation and stored at -70 ° C in a deep freezer (Thermo) until analysis.

The amount of biotin-hIgG1 in the blood was analyzed by the ELISA method as follows.

100 μl Neutravidin (Pierce, 31000) was injected into a 96 well plate (Costar, Cat. No. 2592) at a concentration of 1.0 μg / ml, and the cells were fixed at 4 ° C. for 16 hours. The plate was washed three times with Buffer A (0.05% Tween-20, 10 mM PBS, pH 7.4), and then reacted with PBS (pH 7.4) solution containing 1% BSA at room temperature for 2 hours. After washing the plate three times with Buffer A, neutravidin plate was prepared with PBS (pH 7.4) solution containing 0.5% BSA to correspond to 1 μg / ml. Sequential dilution of blood samples from 500 to 1000 fold in each well using Buffer B (100 mM MES, 150 mM NaCl, 0.5% BSA IgG-free, 0.05% Tween-20, pH 6.0) serial dilution) and injected into the plate each 150 μl. The injected sample was reacted at room temperature for 1 hour. After washing the plate three times with Buffer A, HRP conjugated anti-human IgG goat antibody was prepared in 1 nM, and 200 μl of each was reacted at 37 ° C. for 2 hours. After washing the plate three times with Ice cold buffer B, the substrate solution 3,3 ', 5,5'-tetramethylbenzidine (RnD, Cat.No: DY999) was used. After 100 μl injection, the reaction was performed at room temperature for 15 minutes. 50 μl of 1.0 M sulfuric acid solution (Samjeon, Cat. No: S2129) was injected to stop the reaction, and the absorbance was measured at 450 nm.

After 6 hours (the approximate T _max of biotin-IgG in mice, the time before biotin-IgG catabolism occurs), the concentration of biotin-IgG was fixed at 100%, and the remaining time was expressed in%. Is shown in FIG.

The half-life of the carrier group was 97.55 hours, and the half-life of the hIgG group for the IVIG effect was 75 hours. The half-lives of HL161, HL161-1A and HL161-11H, were 72 and 57 hours, respectively. In the case of HL161-1A, the half-life of the HL161-1A group was shorter than that of the carrier group, but similar to that of the hIgG group. In the case of HL161-11H, the half-life was shortened by more than 40 hours compared to the carrier group, and about 20 hours shorter than the hIgG group. This confirmed that the pH-independent, Fc non-competitive antibody to hFcRn is effective in increasing the catabolism of endogenous antibodies.

<110> Hanall Biopharma Co., Ltd. <120> FcRn specific human antibody and composition for treatment of          autoimmune diseases <160> 32 <170> Kopatentin 1.71 <210> 1 <211> 345 <212> DNA <213> Unknown <220> <223> HL161-1A Heavy chain <400> 1 caggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggcaggtc cctgagactc 60 tcctgtgcag cctctggttt cagttttggt gaatatggca tgcactgggt ccggcaagct 120 ccagggaagg gcctggagtg ggtctcaggt gttagttgga acagtggtag cattgcctat 180 gcggactctg tgaggggccg attcaccatc tccagagaca acagcaaaaa ctccctgtat 240 ctgcaaatga acagtctgag agccgaggac acggccgtgt attactgtgc gagaggtaga 300 agtatggacg tctggggcca agggaccacg gtcaccgtct cctca 345 <210> 2 <211> 115 <212> PRT <213> Unknown <220> <223> HL161-1A Heavy chain <400> 2 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Gly Glu Tyr              20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Gly Val Ser Trp Asn Ser Gly Ser Ile Ala Tyr Ala Asp Ser Val      50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser 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 Gly Arg Ser Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr             100 105 110 Val Ser Ser         115 <210> 3 <211> 348 <212> DNA <213> Unknown <220> <223> HL161-2A Heavy chain <400> 3 cagatgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcaac aactatgctg tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaagg atcatcccta tccttggtat agcaaactac 180 gcacagacat tccagggcag agtcacgatt accgcggaca aatccacgac cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aagagatcgt 300 tacggtatgg acgtctgggg ccaagggacc acggtcaccg tctcctca 348 <210> 4 <211> 116 <212> PRT <213> Unknown <220> <223> HL161-2A Heavy chain <400> 4 Gln Met 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 Asn Asn Tyr              20 25 30 Ala Val 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 Thr Phe      50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Thr 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 Asp Arg Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val             100 105 110 Thr Val Ser Ser         115 <210> 5 <211> 369 <212> DNA <213> Unknown <220> <223> HL161-2D Heavy chain <400> 5 caggtgcagc tggtggagtc tgggggaggc ttggtacagc cagggcggtc cctgagactc 60 tcctgtacag cttctggatt cacctttggt gattatgcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtaggtttc attagaagca aagcttatgg tgggacaaca 180 gaatacgccg cgtctgtgaa aggcagattc accatctcaa gagatgattc caaaagcatc 240 gcctatctgc aaatgaacag tctgagagcc gaggacacgg ccgtgtatta ctgtgcgaga 300 gaggggctgt tcctgcccct gggaggtttt gatttatggg gcctagggac aatggtcacc 360 gtctcctca 369 <210> 6 <211> 123 <212> PRT <213> Unknown <220> <223> HL161-2D Heavy chain <400> 6 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg   1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr              20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Ala      50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile  65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                  85 90 95 Tyr Cys Ala Arg Glu Gly Leu Phe Leu Pro Leu Gly Gly Phe Asp Leu             100 105 110 Trp Gly Leu Gly Thr Met Val Thr Val Ser Ser         115 120 <210> 7 <211> 348 <212> DNA <213> Unknown <220> <223> HL161-6C Heavy chain <400> 7 caggtgcagc tggtggagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60 tcctgcaagg catctggata caccttcacc agctactata tgcactgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaata atcaacccta gtggtggtag cacaagctac 180 gcacagaagt tccagggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aagagggggg 300 ggggcttttg atatctgggg ccaagggaca atggtcaccg tctcctca 348 <210> 8 <211> 116 <212> PRT <213> Unknown <220> <223> HL161-6C Heavy chain <400> 8 Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met          35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe      50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val 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 Gly Gly Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val             100 105 110 Thr Val Ser Ser         115 <210> 9 <211> 369 <212> DNA <213> Unknown <220> <223> HL161-9F Heavy chain <400> 9 caggtgcagc tggtggagtc tgggggaggc ttggtacagt cagggcggtc cctgagactc 60 tcctgtacag cttctggatt cacctttggt gattatgcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtaggtttc attagaagca aagcttatgg tgggacaaca 180 gaatacgccg cgtctgtgaa aggcagattc accatctcaa gagatgattc caaaagcatc 240 gcctatctgc aaatgaacag tctgagagcc gaggacacgg ccgtgtatta ctgtgcgaga 300 gaggggctgt tcctgcccct gggaggtttt gatttatggg gcctagggac aatggtcacc 360 gtctcctca 369 <210> 10 <211> 123 <212> PRT <213> Unknown <220> <223> HL161-9F Heavy chain <400> 10 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Arg   1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr              20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Ala      50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile  65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                  85 90 95 Tyr Cys Ala Arg Glu Gly Leu Phe Leu Pro Leu Gly Gly Phe Asp Leu             100 105 110 Trp Gly Leu Gly Thr Met Val Thr Val Ser Ser         115 120 <210> 11 <211> 363 <212> DNA <213> Unknown <220> <223> HL-161 10E Heavy chain <400> 11 caggtgcagc tggtggagtc tgggggaggc ttagtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggctt cagattcagc aactttgcca tgacctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcaact cttagtggta gtggtggtag tatacaccac 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagtctgag agccgaggac acggccgtgt attactgtgc gaaagggccc 300 ttgaggggac agccggccta ccttgacccc tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 12 <211> 121 <212> PRT <213> Unknown <220> <223> HL161-10E Heavy chain <400> 12 Gln Val Gln Leu Val 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 Phe Arg Phe Ser Asn Phe              20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Thr Leu Ser Gly Ser Gly Gly Ser Ile His His 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 Lys Gly Pro Leu Arg Gly Gln Pro Ala Tyr Leu Asp Pro Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 13 <211> 345 <212> DNA <213> Unknown <220> <223> HL161-11G Heavy chain <400> 13 cagatgcagc tggtggagtc ggggggaggc gtggtccagc ctgggaggtc tctgagactc 60 tcctgtgtag ggtctggatt caacttcaac agttatggca tacactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtgggagga atattttatg atggaagtca agtaaagtat 180 gcagactccg tgaagggccg agtctccatc tatgccatga attccaagaa cacagcgtat 240 ctgcaaatga acagtctgag agccgaggac acggccgtgt attactgtgc gcgacgaaac 300 ctcctggact actggggcca gggaacggtg gtcaccgtct cctca 345 <210> 14 <211> 115 <212> PRT <213> Unknown <220> <223> HL161-11G Heavy chain <400> 14 Gln Met Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg   1 5 10 15 Ser Leu Arg Leu Ser Cys Val Gly Ser Gly Phe Asn Phe Asn Ser Tyr              20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Gly Ile Phe Tyr Asp Gly Ser Gln Val Lys Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Val Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala 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 Arg Asn Leu Leu Asp Tyr Trp Gly Gln Gly Thr Val Val Thr             100 105 110 Val Ser Ser         115 <210> 15 <211> 357 <212> DNA <213> Unknown <220> <223> HL-161 11H Heavy chain <400> 15 cagatgcagc tggtagagtc tgggggaggt ttggtacagc cgggcaggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agctatgcta tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagcaa taaatactac 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag atctgaggac acggccgtgt attactgtgc gagaggtagt 300 ggtggtcgtg acgcttttga tgtctggggc caaggaacaa tgatcaccgt ctcctca 357 <210> 16 <211> 119 <212> PRT <213> Unknown <220> <223> HL161-11H Heavy chain <400> 16 Gln Met Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr              20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn 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 Ser Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ser Arg Gly Ser Gly Gly Arg Asp Ala Phe Asp Val Trp Gly Gln Gly             100 105 110 Thr Met Ile Thr Val Ser Ser         115 <210> 17 <211> 330 <212> DNA <213> Unknown <220> <223> HL161-1A Light chain <400> 17 aattttatgc tgactcagcc cgcctccgtg tctgggtctc ctggacagac gatcaccatc 60 tcctgcactg gaagcagcag cgacgttggt ggttataact atgtctcctg gtaccaacag 120 cacccaggca aagcccccca actcatcatt tatgatgtca ctaagcggcc ctcaggggtt 180 tctaatcgct tctccggctc caagtctggc aactcggcct ccctgaccat ctctggactc 240 caggctgagg acgaggctga ttattactgc agctcataca gcagcagcac tttttacgtc 300 ttcggaactg ggaccaaggt caccgtccta 330 <210> 18 <211> 110 <212> PRT <213> Unknown <220> <223> HL161-1A Light chain <400> 18 Asn Phe Met Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln   1 5 10 15 Thr Ile Thr Ile Ser Cys Thr Gly Ser Ser Ser Asp Val Gly Gly Tyr              20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Gln Leu          35 40 45 Ile Ile Tyr Asp Val Thr Lys Arg Pro Ser Gly Val Ser Asn Arg Phe      50 55 60 Ser Gly Ser Lys Ser Gly Asn Ser 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 Ser Ser Ser                  85 90 95 Thr Phe Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu             100 105 110 <210> 19 <211> 330 <212> DNA <213> Unknown <220> <223> HL161-2A Light chain <400> 19 cagctcgtgc tgactcagcc accctcaacg tctgagaccc ccgggcagag ggtcaccatc 60 tcttgttctg gaagcagctc caacatcgga agtaattatg tatactggta ccagcaactc 120 ccaggaacgg cccccaaact cctcatctat aggaataatc agcggccctc aggggtccct 180 gaccgattct ctggctccaa gtctggcact tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtgca tcatgggatg acagcctgag tggtgtggtt 300 ttcggcggag ggaccaagct gaccgtccta 330 <210> 20 <211> 110 <212> PRT <213> Unknown <220> <223> HL161-2A Light chain <400> 20 Gln Leu Val Leu Thr Gln Pro Pro Ser Thr Ser Glu 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 Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Arg 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 Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Asp Ser Leu                  85 90 95 Ser Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 21 <211> 330 <212> DNA <213> Unknown <220> <223> HL161-2D Light chain <400> 21 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtcaccatc 60 tcctgcaccc gcagcagtgg cagcattgcc gccaactatg tgcactggta ccaacagcgc 120 ccgggcagtc cccccaccac tgtcatctat aacgataacc aaagaccctc tggagtccct 180 gatcggttct ctgggtccat cgacaggtcc tccaactctg cctccctcac catctctgga 240 ctgaagactg aggacgaggc tgactactac tgtcagtcct acgatagtac cacttatgca 300 ttcggcggag ggaccaagct gaccgtccta 330 <210> 22 <211> 110 <212> PRT <213> Unknown <220> <223> HL161-2D Light chain <400> 22 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys   1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ala Asn              20 25 30 Tyr Val His Trp Tyr Gln Gln Arg Pro Gly Ser Pro Pro Thr Thr Val          35 40 45 Ile Tyr Asn Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Ile Asp Arg Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly  65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser                  85 90 95 Thr Thr Tyr Ala Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 23 <211> 321 <212> DNA <213> Unknown <220> <223> HL161-6C Light chain <400> 23 gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60 atcacttgtc gggcgagtca gggtatcagc aactgggtag cctggtatca gcagaaacca 120 ggcaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagac ttcactctca ccatcagcag cctgcagcct 240 gaagattttg caatttacta ttgtcaacag ggtcacagtt tcccgtacac ttttggccaa 300 gggaccaagg tggaaatcaa a 321 <210> 24 <211> 107 <212> PRT <213> Unknown <220> <223> HL161-6C Light chain <400> 24 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 Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Trp              20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile          35 40 45 Tyr Ala Ser Ser Leu Gln Ser Gly Val Ser 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 Phe Ala Ile Tyr Tyr Cys Gln Gln Gly His Ser Phe Pro Tyr                  85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 25 <211> 330 <212> DNA <213> Unknown <220> <223> HL161-9F Light chain <400> 25 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtcaccatc 60 tcctgcaccc gcagcagtgg cagcattgcc gccaactatg tgcactggta ccaacagcgc 120 ccgggcagtc cccccaccac tgtcatctat aacgataacc aaagaccctc tggagtccct 180 gatcggttct ctgggtccat cgacaggtcc tccaactctg cctccctcac catctctgga 240 ctgaagactg aggacgaggc tgactactac tgtcagtcct acgatagtac cacttatgca 300 ttcggcggag ggaccaagct gaccgtccta 330 <210> 26 <211> 110 <212> PRT <213> Unknown <220> <223> HL161-9F Light chain <400> 26 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys   1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ala Asn              20 25 30 Tyr Val His Trp Tyr Gln Gln Arg Pro Gly Ser Pro Pro Thr Thr Val          35 40 45 Ile Tyr Asn Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Ile Asp Arg Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly  65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser                  85 90 95 Thr Thr Tyr Ala Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 27 <211> 327 <212> DNA <213> Unknown <220> <223> HL161-10E Light chain <400> 27 tcctatgagc tgacacagcc actctcggtg tcaatgtccc caggacaaac ggccaggatc 60 acctgttctg gagatgcttt gtcaaagcaa tatgcttctt ggtaccagct gaagccaggc 120 caggcccctg tggtggtgat gtataaagac actgagaggc cctcagggat ccctgaccga 180 ttctctggct ccagctccgg gacaacagtc acgttgacca tcagtggagt ccaggcagaa 240 gacgaggctg attattactg tcaatcaata acagacaaga gtggtactga tgtgatcttc 300 ggcggaggga ccaagctgac cgtccta 327 <210> 28 <211> 109 <212> PRT <213> Unknown <220> <223> HL161-10E Light chain <400> 28 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Met Ser Pro Gly Gln   1 5 10 15 Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Ser Lys Gln Tyr Ala              20 25 30 Ser Trp Tyr Gln Leu Lys Pro Gly Gln Ala Pro Val Val Val Met Tyr          35 40 45 Lys Asp Thr Glu Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser      50 55 60 Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu  65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ile Thr Asp Lys Ser Gly Thr                  85 90 95 Asp Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 <210> 29 <211> 330 <212> DNA <213> Unknown <220> <223> HL161-11G Light chain <400> 29 aattttatgc tgactcagcc cgcctccgtg tctgggtccc ctggacagtc gatcaccatc 60 tcctgcactg gaagcagcag cgacgttggt ggttataact atgtctcctg gtaccaacag 120 cacccaggca aagcccccca actcatcatt tatgatgtca ctaagcggcc ctcaggggtt 180 tctaatcgat tctccggctc caagtctggc aactcggcct ccctgaccat ctctggactc 240 caggctgagg acgaggctga ttattactgc agctcataca gcagcagcac tttttacgtc 300 ttcggaactg ggaccaaggt caccgtccta 330 <210> 30 <211> 110 <212> PRT <213> Unknown <220> <223> HL161-11G Light chain <400> 30 Asn Phe Met 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 Ser Ser Ser Asp Val Gly Gly Tyr              20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Gln Leu          35 40 45 Ile Ile Tyr Asp Val Thr Lys Arg Pro Ser Gly Val Ser Asn Arg Phe      50 55 60 Ser Gly Ser Lys Ser Gly Asn Ser 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 Ser Ser Ser                  85 90 95 Thr Phe Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu             100 105 110 <210> 31 <211> 321 <212> DNA <213> Unknown <220> <223> HL161-11H Light chain <400> 31 gacatccaga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggccagtca gagtattagt agccggttgg cctggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctataag gcatctagct tagaaactgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240 gatgattttg caacttacta ttgtcaacag acgaacagtt tccctctcac tttcggcgga 300 gggaccaagg tggagatcaa a 321 <210> 32 <211> 107 <212> PRT <213> Unknown <220> <223> HL161-11H Light chain <400> 32 Asp Ile Gln Met Thr Gln Ser Ser Ser Thr Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Arg              20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile          35 40 45 Tyr Lys Ala Ser Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Asn Ser Phe Pro Leu                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105

Claims (27)

A heavy chain variable region, or such a heavy chain variable region, of an FcRn 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 Heavy chain variable region having at least 90% sequence homology. The method of claim 1,
A heavy chain variable region of the FcRn specific antibody, or 90% or more sequence thereof, having an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16 Heavy chain variable region having homology. A light chain variable region, or such a light chain variable region, of an FcRn specific antibody comprising CDR1, CDR2 and CDR3 included in any one amino acid sequence selected from SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30 and 32 Light chain variable region having at least 90% sequence homology. The method of claim 3, wherein
The light chain variable region of the FcRn specific antibody, or such light chain variable region and at least 90% sequence, having any one amino acid sequence selected from SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, and 32 Light chain variable region having homology. An FcRn 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 FcRn specific antibody, or fragment of such an antibody, comprising a combination of variable regions selected from (a) to (h) below.
(a) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the amino acid sequence of SEQ ID NO: 2, or a heavy chain variable region having 90% or more sequence homology therewith and CDR1, CDR2 included in the amino acid sequence of SEQ ID NO: 18 And a light chain variable region comprising CDR3, or a light chain variable region having 90% or more sequence homology thereto;
(b) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the amino acid sequence of SEQ ID NO: 4 or a heavy chain variable region having 90% or more sequence homology therewith and CDR1, CDR2 included in the amino acid sequence of SEQ ID NO: 20 And a light chain variable region comprising CDR3, or a light chain variable region having 90% or more sequence homology thereto;
(c) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the amino acid sequence of SEQ ID NO: 6, or a CDR1, CDR2 included in the amino acid sequence of SEQ ID NO: 22 with a heavy chain variable region having at least 90% sequence homology therewith; And a light chain variable region comprising CDR3, or a light chain variable region having 90% or more sequence homology thereto;
(d) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the amino acid sequence of SEQ ID NO: 8, or a heavy chain variable region having 90% or more sequence homology therewith and CDR1, CDR2 included in the amino acid sequence of SEQ ID NO: 24; And a light chain variable region comprising CDR3, or a light chain variable region having 90% or more sequence homology thereto;
(e) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the amino acid sequence of SEQ ID NO: 10, or a CDR1, CDR2 included in the amino acid sequence of SEQ ID NO: 26 with a heavy chain variable region having 90% or more sequence homology therewith; And a light chain variable region comprising CDR3, or a light chain variable region having 90% or more sequence homology thereto;
(f) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the amino acid sequence of SEQ ID NO: 12, or a heavy chain variable region having 90% or more sequence homology therewith and CDR1, CDR2 included in the amino acid sequence of SEQ ID NO: 28 And a light chain variable region comprising CDR3, or a light chain variable region having 90% or more sequence homology thereto;
(g) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the amino acid sequence of SEQ ID NO: 14, or a CDR1, CDR2 included in the amino acid sequence of SEQ ID NO: 30 with a heavy chain variable region having 90% or more sequence homology therewith; And a light chain variable region comprising CDR3, or a light chain variable region having 90% or more sequence homology thereto; And
(h) a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the amino acid sequence of SEQ ID NO: 16, or a CDR1, CDR2 included in the amino acid sequence of SEQ ID NO: 32 and a heavy chain variable region having 90% or more sequence homology therewith; And a light chain variable region comprising CDR3, or a light chain variable region having 90% or more sequence homology thereto. An FcRn specific antibody, or fragment of such an antibody, comprising the heavy and light chain variable region sequences selected from (a) to (h) below.
(a) a heavy chain variable region having an amino acid sequence of SEQ ID NO: 2, or a heavy chain variable region having at least 90% sequence homology with a light chain variable region having an amino acid sequence of SEQ ID NO: 18, or at least 90% Light chain variable region having a;
(b) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4, or a heavy chain variable region having 90% or more sequence homology therewith and a light chain variable region having the amino acid sequence of SEQ ID NO: 20, or 90% or more thereof Light chain variable region having a;
(c) a heavy chain variable region having an amino acid sequence of SEQ ID NO: 6, or a heavy chain variable region having at least 90% sequence homology with a light chain variable region having an amino acid sequence of SEQ ID NO: 22, or at least 90% Light chain variable region having a;
(d) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8, or a heavy chain variable region having at least 90% sequence homology with the light chain variable region having the amino acid sequence of SEQ ID NO: 24, or at least 90% sequence homology therewith Light chain variable region having a;
(e) a heavy chain variable region having an amino acid sequence of SEQ ID NO: 10, or a heavy chain variable region having at least 90% sequence homology with a light chain variable region having an amino acid sequence of SEQ ID NO: 26, or at least 90% Light chain variable region having;
(f) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 12, or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 28 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 28, or 90% or more Light chain variable region having;
(g) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14, or a heavy chain variable region having at least 90% sequence homology with the light chain variable region having the amino acid sequence of SEQ ID NO: 30, or at least 90% sequence homology therewith Light chain variable region having; And
(h) a heavy chain variable region having an amino acid sequence of SEQ ID NO: 16, or a heavy chain variable region having at least 90% sequence homology with a light chain variable region having an amino acid sequence of SEQ ID NO: 32, or at least 90% Light chain variable region having. The method according to any one of claims 5 to 7,
The antibody or fragment of the antibody may be a single-chain antibody, diabody, tribody, tetrabody, Fab fragment, F (ab ') ₂ fragment, Fd, scFv, domain antibody, bispecific antibodies, mini, having a binding function to FcRn. Body, scap, IgD antibody, IgE antibody, IgM antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody, IgG4 antibody, derivatives of antibody constant region, and phosphorus based on protein scaffolds. FcRn-specific antibodies or fragments of such antibodies. An isolated antibody according to any one of claims 5 to 8, or a fragment of such an antibody; And pharmaceutical composition for treating autoimmune disease comprising at least one pharmaceutically acceptable carrier. The method of claim 9,
The autoimmune diseases include immune neutropenia, guillain-barre syndrome, epilepsy, autoimmune cerebral encephalitis, Isaac syndrome, nevus syndrome, vulgaris cystic duct, deciduous septum, bullous cystic duct, acquired epidermal bleb, gestational pseudovesicle, mucos Selected from membrane-like bullous, antiphospholipid syndrome, autoimmune anemia, autoimmune grave disease, goodpasture syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus and idiopathic thrombocytopenic purpura (ITP) Pharmaceutical composition, characterized in that. The method of claim 9,
The autoimmune diseases include immune neutropenia, guillain-barre syndrome, epilepsy, autoimmune cerebral encephalitis, Isaac syndrome, nevus syndrome, vulgaris cystic duct, deciduous septum, bullous cystic duct, acquired epidermal bleb, gestational pseudovesicle, mucos Selected from membrane-like bullous, antiphospholipid syndrome, autoimmune anemia, autoimmune grave disease, goodpasture syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus and idiopathic thrombocytopenic purpura (ITP) Pharmaceutical composition, characterized in that. 12. The method of claim 11,
One sequence selected from the sequences set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31, or at least 90% of such sequence A polynucleotide having a sequence selected from the sequences having homology. 9. A polynucleotide encoding the FcRn specific antibody, or fragment of such antibody, according to any one of claims 5-8. The method of claim 13,
An FcRn specific antibody, or polynucleotide encoding a fragment of such antibody, characterized in being selected from a combination of the following polynucleotide sequences.
(a) SEQ ID NO: 1 or a sequence having at least 90% sequence homology therewith, and SEQ ID NO: 17 or a sequence having at least 90% sequence homology thereto;
(b) a sequence having SEQ ID NO: 3 or at least 90% sequence homology therewith, and SEQ ID NO: 19 or a sequence having at least 90% sequence homology;
(c) a sequence having SEQ ID NO: 5 or at least 90% sequence homology therewith and SEQ ID NO: 21 or a sequence having at least 90% sequence homology;
(d) a sequence having SEQ ID NO: 7 or at least 90% sequence homology with it, and a sequence having SEQ ID NO: 23 or at least 90% sequence homology therewith;
(e) a sequence having SEQ ID NO: 9 or at least 90% sequence homology therewith, and a sequence having SEQ ID NO: 25 or at least 90% sequence homology therewith;
(f) a sequence having SEQ ID NO: 11 or at least 90% sequence homology therewith, and SEQ ID NO: 27 or a sequence having at least 90% sequence homology;
(g) a sequence having SEQ ID NO: 13 or at least 90% sequence homology therewith, and SEQ ID NO: 29 or a sequence having at least 90% sequence homology; And
(h) a sequence having SEQ ID NO: 15 or at least 90% sequence homology therewith, and a sequence having SEQ ID NO: 31 or at least 90% sequence homology with it. 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 method of claim 16,
The host cell is a host cell, characterized in that selected from animal cells, plant cells, yeast, E. coli, insect cells. The method of claim 16,
The transfected host cells are monkey kidney cells (COS7) cells, NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) ) Cells, MDCK, myeloma cell line, HuT 78 cells and HEK293 cells, E. coli, Bacillus subtilis subtilis), genus Streptomyces (Streptomyces sp.), Pseudomonas sp.), Proteus mirabilis or Staphylococcus sp.), Aspergillus sp.), Pichia pastoris pastoris , Saccharomyces cerevisiae ), genus Schizosaccharomyces sp.) and Neurospora crassa . 20. A method for producing an FcRn specific antibody, fragment or variable region of the antibody, comprising culturing the vector or host cell according to any one of claims 15-18. A composition for diagnosing autoimmune disease, comprising the isolated antibody according to any one of claims 5 to 8, or a fragment of such an antibody. The method of claim 20,
The autoimmune diseases include immune neutropenia, guillain-barre syndrome, epilepsy, autoimmune cerebral encephalitis, Isaac syndrome, nevus syndrome, vulgaris cystic duct, deciduous septum, bullous cystic duct, acquired epidermal bleb, gestational pseudovesicle, mucos Selected from membrane-like bullous, antiphospholipid syndrome, autoimmune anemia, autoimmune grave disease, goodpasture syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus and idiopathic thrombocytopenic purpura (ITP) A composition, characterized in that. A composition for detecting FcRn comprising the isolated antibody according to any one of claims 5 to 8, or a fragment of such an antibody. A method for detecting FcRn using an isolated antibody according to any one of claims 5 to 8, or a fragment of such an antibody. The method of claim 23, wherein
FcRn detection method, characterized in that performed in vitro or in vivo. The method of claim 23, wherein
The antibody or fragment of such an antibody is labeled with a detectable substance. The method of claim 25,
The labeling method for detecting FcRn, characterized in that made using one material selected from enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. The method of claim 23, wherein
At least one method selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIIA), tissue immunohistochemistry and competition immunoassay. FcRn detection method.

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