KR20110049766A - Human antibodies and fab specific for interleukin-15 and tumer necrosis factor-alpha - Google Patents

Abstract

PURPOSE: An antigen or antibody which specifically binds to interleukin-15 or TNF-alpha is provided to treat inflammatory autoimmune diseases without immunogenicity. CONSTITUTION: An antibody or antigen binding fragment which specifically binds to interleukin-15 or TNF-alpha contains heavy chain variable region and light chain variable region. The heavy chain variable region contains heavy chain CDR of sequence numbers 7, 8, and 9. The heavy chain variable region binds to a human antibody heavy chain constant region.

Description

Antibodies or antigen-binding fragments that specifically bind to interleukin-15 or tumor necrosis factor {Human antibodies and Fab specific for Interleukin-15 and Tumer necrosis factor-alpha}

The present invention relates to an antibody or antibody fragment specifically binding to interleukin-15 or tumor necrosis factor, which is effective in treating autoimmune diseases, a gene thereof, and a composition for treating autoimmune diseases containing the same.

Interleukin-15 (IL-15) is an inflammation-mediated cytokine, a 14-15 kD glycoprotein. Constituent expression has been reported in various cells and tissues including monocytes, macrophages, fibroblasts, keratinocytes and dendritic cells (Waldmann and Tagaya, 1999; Fehniger and Caligiuri, 2001). Expression is upregulated as reported in monocytes stimulated with IFN-γ and LPS, or under inflammatory symptoms caused by infection with viruses, bacteria or protozoa (Kirman et al., 1998; Waldmann et al., 1998; Waldmann and Tagyaya). , 1999; Fehniger and Caligiuri, 2001). Moreover, IL-15 produced locally in chronic inflammation, such as rheumatoid arthritis, tends to amplify inflammation by recruitment and activation of synovial T-cells. The IL-15-induced effect has been suggested to play an important role in disease pathology (Kirman et aL, 1998; McInnes et al., 1996; McInnes et al., 1997; McInnes and Liew, 1998; Fehniger and Caligiuri, 2001).

McInnes and his collaborators (Mclnnes et al., 1997; McInnes and Liew, 1998) reported the induction of TNF-α production after IL-15 stimulation in T-cells derived from patients with rheumatoid arthritis. Moreover, peripheral blood T cells activated with IL-15 have been shown to induce significant TNF-α production by macrophages through a cell-contact dependent mechanism. Because of the destructive role of TNF-α in rheumatoid arthritis, inhibition of the cytokine reduces disease activity (Bathon et al., 2000; Klippel, 2000; Lovell et al., 2000; Maini and Taylor, 2000).

TNF is a type of protein produced by various types of cells, including monocytes and macrophages. At least two TNFs, in particular TNF alpha and TNF beta (lymphotoxin), have been described previously.

These known TNFs have important pathological effects on a number of different target cells implicated in the inflammatory response. The protein causes fibroblasts and synovial cells to secrete latent collagenase and Prostagladin E2, and allows the osteocytes to perform bone resorption.

These proteins increase the surface adhesion of endothelial cells for neutral affinity. They also cause endothelial cells to secrete coagulation activity, reducing their ability to dissolve clots. In addition, they inhibit the expression of the enzyme lipoprotein lipase, thereby disengaging the activity of adipocytes from stored lipids. TNF causes hepatocytes to synthesize proteins known as "acute phase reactauts", and they act as pyrogens in the hypothalamus. Through these activities, TNF is known to play an important role in the response of the organism to stress, infection and injury (Lancet, 1988.2.27, 483p PJ Selby et al.; J. Clin. Invest. 82:1321(1988) HF Starnes, Jr. et al.; Cell 50:553 (1987) A. Oliff et al.; and Lancet, 1987.2.14, 355p A. Waage et al.).

However, despite this normal useful action, the action of TNF has been found to be harmful.

For example, TNF-α injected into an animal causes symptoms of septic shock; It was observed that the amount of endogenous TNF increased with injection of the bacterial or bacterial cell wall. TNF also causes bowel necrosis and acute lung failure, and stimulates the metabolism of muscle proteins. In addition, the ability of TNF to increase the amount of coragenases in the joints and direct the chemotaxix and migration of leukocytes and lymphocytes may also contribute to the deterioration of cartilage and proliferation of synovial tissue in this disease. Therefore, TNF can play a role as a mediator of both acute and chronic immunopathology in rheumatoid arthritis. TNF can also cause some clotting disorders through changes in endothelial cells. In addition, excessive TNF production is detected in AIDS patients, and may cause some pyrogenicity, acute reactions, and malignancies seen in such diseases and leukemias.

Under these and other circumstances in which TNF has a detrimental action, there are certain inhibitors of TNF action for clinical use. When taken systematically, TNF inhibitors are useful in the treatment of septic shock and malignancies. When applied topically, the TNF inhibitor serves to prevent tissue destruction of inflamed joints and other inflamed areas. In fact, the TNF inhibitor is more effective when taken together with interleukin-I (IL-1) inhibition.

Therefore, inflammatory cytokines (IL-15) and TNF are recognized as excellent targets in the field of developing antibodies for the treatment of autoimmune diseases and the like.

The importance of phage display technology for the development of therapeutic antibodies is further emphasized due to the possibility of obtaining fully human antibodies. Extensive clinical trials of more than a dozen human antibodies produced by phage display technology, including cancer, autoimmune disease, graft rejection, and infectious diseases are ongoing (Richert et al., 2005; Walsh, 2006; Aires da Silva et al. al., 2008)

The Molecular Diversity Library (Combinatorial Library) refers to a systematic collection of mixtures in which several to tens of unit molecules are combined to form thousands to tens of thousands of diverse molecules. A phage-displayed peptide library in which tens of millions to hundreds of millions of different phages are made by changing amino acid combinations in some peptide regions of bacteriophage envelope proteins that are easy to genetically manipulate with a biological approach. ) Is a representative example (Cwirla et al., Proceedings of National Academy of Science USA, 87: 6578, 1990). Examples of this include a mixture made by synthesizing short peptides while changing amino acid combinations, or a mixture of low-molecular substances produced by changing various combinations of substituents on a certain skeleton.

Their characteristics are composed of thousands to hundreds of millions of phages or molecules, but the general phage-display library method intended from them without testing one by one is ① coat protein pⅢ (or pVIII) of phage. Inserting an oligonucleotide of a random sequence into a gene site corresponding to the N-terminus; ② expressing a fusion protein of a part of the natural envelope protein and a polypeptide encoded by the oligonucleotide of the random sequence; ③ treating a receptor material capable of binding to the polypeptide encoded by the oligonucleotide; ④ The step of eluting the peptide-phage particles bound to the receptor using a low pH or binding competitive molecule (Biopanning); ⑤ amplifying the phage eluted by panning in the host cell; ⑥ repeating the method to obtain the desired amount; And (7) determining the sequence of an active peptide from the DNA sequences of the phage clones selected by panning.

Thus, by using the dual vector system-II developed by the present inventors, it is possible to produce an excellent human library having a gene diversity of ^{2.5 × 10 9, which is ~100 times faster and easier than the conventional library production.}

When an antibody is used for treatment, antibody molecules containing mouse-derived sequences are often degraded and do not show an effect when introduced into humans, and due to their immunogenicity, production of human anti-mouse antibodies in vivo in humans. And, if administered frequently, not only negates the expected efficacy, but also poses a threat of side effects such as anaphylactic shock. In addition, there are cases where the Fc region of the antibody does not act on human effector cells.

In addition, humanized antibodies are prepared by humanizing mouse monoclonal antibodies and their immunogenicity is reduced. However, since the CDR regions of humanized antibodies are derived from mice, the risk of immunogenicity and side effects still remains.

Under these circumstances, the present inventors have obtained complete human anti-human antibodies using phage display technology, which are fully human antibodies or fragments thereof that alone provide specific therapeutic efficacy, which are expected to be free from such risks. .

The present inventors have endeavored to develop a Fab structure that specifically binds to interleukin-15 or tumor necrosis factor and exhibits an immune disease therapeutic effect. As a result, interleukin-15 antibodies and tumors having different amino acid sequences in the CDRs from known humanized antibodies. The necrosis factor antibody was completed.

The main object of the present invention is to provide a human monoclonal antibody or antigen-binding fragment (Fab) thereof that specifically binds to interleukin-15 or tumor necrosis factor.

Another object of the present invention is to provide a gene having a DNA sequence encoding the human monoclonal antibody.

Another object of the present invention is to provide a composition for the treatment of autoimmune diseases without an immune response containing the human monoclonal antibody or antigen-binding fragment thereof.

One aspect of the present invention relates to a human antibody or antigen-binding fragment (Fab) thereof that specifically binds to interleukin-15 (IL-15).

Another aspect of the present invention relates to an antibody or antigen-binding fragment (Fab) thereof that specifically binds to tumor necrosis factor-alpha (TNF-α).

The term “antigen binding fragment (Fab; fragment antigen binding)” as used herein is a Y-shaped structure in which the N-terminal portion of the heavy chain and the light chain are bonded by an S (cysteine)-S (cysteine) bond in an IgG (Y chain). It refers to an open part, wherein the heavy and light chains are divided into a variable region (VH or VL) and a constant region (CH or CL), respectively, and each region is made by a different gene. Certain portions of the VH and VL sites constitute the antigen-binding site, and the antigen-binding sites are respectively located in the antigen-binding fragment region of the Y-shaped monomer.The antigen-binding fragment has antigen-binding but complement-binding properties, etc. Does not have the nature of.

In the process of developing an antibody that specifically binds to interleukin-15 or tumor necrosis factor, the present inventors identified the CDRs of the heavy chain variable region and the CDRs of the light chain variable region (see Table 1, FIGS. 1 and 4). .

Thus, the antibody or antigen-binding fragment thereof (Fab) specifically binding to the interleukin-15 (human interleukin-15; IL-15) is

_{Heavy chain variable regions (IL-15VH1 (V H3} )) comprising heavy chain CDRs of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 and light chain CDRs (complementary determining regions) of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 It includes a light chain variable region (HuVLk1-30 (V _{L κ1} )) containing.

_{Or, comprising the heavy chain variable region (IL-15VH1 (V H3} )) comprising the heavy chain CDRs of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 and the light chain CDRs of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 It includes a light chain variable region (HuVLk3-14 (V _{L κ3} )).

_{Or, comprising the heavy chain variable region (IL-15VH1 (V H3} )) comprising the heavy chain CDRs of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 and the light chain CDRs of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 It includes a light chain variable region (HuVLk1-13 (V _{L κ1} )).

Or, a heavy chain variable region comprising the heavy chain CDRs of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 (IL-15VH2 (V _H2 )) and a light chain variable comprising the light chain CDRs of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 Region (HuVLk1-30 (V _{L κ1} )).

In addition, the present inventors identified the variable region of the heavy chain and the variable region of the light chain, and the DNA and amino acid sequences were registered in Genebank as follows, but not yet disclosed. Therefore, more preferably, the heavy chain variable region (IL-15VH1 (V _H3 )) is characterized in that the amino acid sequence (Accession no.FJ490200) represented by SEQ ID NO: 25, and the light chain variable region (HuVLk1-30 (V _{L κ1} ), HuVLk3-14 (V _{L κ3} ), HuVLk1-13 (V _{L κ1} )), the amino acid sequence of SEQ ID NO: 26 (FJ497788), SEQ ID NO: 27 (FJ497787), characterized in that it is represented by SEQ ID NO: 28 (FJ497786) do. In addition, the heavy chain variable region (IL-15VH2 (V _H2 )) is characterized in that the amino acid sequence (FJ490201) represented by SEQ ID NO: 29, the amino acid sequence of the light chain variable region (HuVLk1-30 (V _{L κ1} )) Is characterized by being represented by SEQ ID NO: 26 (FJ497788).

In addition, the antibody or antigen-binding fragment thereof (Fab) that specifically binds to the tumor necrosis factor-alpha (TNF-α) is

A heavy chain variable region comprising the heavy chain CDRs of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 (TNF-αVH1 (V _H3 )) and a light chain variable including the light chain CDRs of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 Region HuVLk1-30 (V _{L κ1} )).

Or, a heavy chain variable region comprising the heavy chain CDRs of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 (TNF-αVH2 (V _H3 )) and a light chain comprising the light chain CDRs of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 It includes a variable region (HuVLk1-30 (V _{L κ1} )).

Alternatively, a heavy chain variable region comprising the heavy chain CDRs of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 (TNF-αVH3 (V _H2 )) and a light chain comprising the light chain CDRs of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 It includes a variable region (HuVLk1-30 (V _{L κ1} )).

In addition, Genebank registration numbers of the heavy chain variable region and light chain variable region DNA and amino acid sequences of tumor necrosis factor are as follows.

Therefore, more preferably, each of the heavy chain variable regions (TNF-αVH1 (V _H3 ), TNF-αVH2 (V _H3 ), TNF-αVH3 (V _H2 )) is SEQ ID NO: 30 (Accession no. FJ4902002), 31 (FJ4902002). ), 32 (FJ4902002), and the amino acid sequence of the light chain variable region (HuVLk1-30 (V _{L κ1} )) is represented by SEQ ID NO: 26 (FJ497788).

The amino acid sequence of the variable region also includes an amino acid sequence having 90% or more homology.

The term "homology" as used herein is to indicate the degree of similarity with the amino acid sequence of a wild type protein, and the heavy chain variable region of the present invention and its CDR and light chain variable region and the amino acid sequence encoding the CDR And preferably at least 90%, more preferably at least 95%, and even more preferably at least 98%. The comparison of homology can be performed with the naked eye or using a comparison program that is easy to purchase. Commercially available computer programs can calculate the homology between two or more sequences as a percentage (%), and the homology (%) can be calculated for adjacent sequences.

The antibody fragment may be applied in another form of an antibody fragment bound to a part of the constant region of a human immunoglobulin by a DNA recombination technique known in the art.

In addition, another aspect of the present invention relates to an antibody or antigen-binding fragment (Fab) thereof in which the heavy chain variable region and the light chain variable region are bound to a human antibody heavy chain constant region and a human antibody light chain constant region, respectively.

The amino acid sequence and DNA of the human antibody heavy chain constant region and the human antibody light chain constant region are known in the art.The amino acid sequence of each human antibody heavy chain constant region is represented by SEQ ID NO: 33, and the amino acid sequence of the human antibody light chain constant region is SEQ ID NO: It is denoted as 34.

Methods of generating interleukin-15 antibodies, or tumor necrosis factor antibodies, include recombinant phage display technology. These techniques are generally as follows.

Essentially, this method provides synthesis of recombinant immunoglobulin libraries for the interleukin-15 antigen, or tumor necrosis factor antigen, displayed on the surface of the filamentous phage, and high affinity for the interleukin-15 antigen, or tumor necrosis factor antigen. Genie may involve selection of phages that secrete antibodies.

Phage display technology replaces the conventional method of isolating antibodies produced during an immune response. Using this technique a very large percentage of the immune repertoire can be assayed, and then it is possible to use conventional methods. In addition, the phage display may be manufactured using a dual vector system-II (DVS-II) developed by the present inventors and applied for a PCT patent (application number; PCT/KR2008/005238) on September 4, 2008. . Phage display technology relies on molecular technology that efficaciously captures the entire repertoire of immunoglobulins associated with a response to a given antigen. This technique is described in Barber et al., Proc. Natl. Acad. Sci., USA, 88, 7978-7982, (1991).

Basically, the immunoglobulin heavy chain gene is PCR amplified and cloned into a vector containing the gene encoding a small amount of the coat protein of the phage in such a way that a heavy chain fusion protein is produced. These vectors contain specific primers that PCR amplify the immunoglobulin gene. These primers are based on human sequences obtained from a database. As the heavy chain fusion protein is associated, it is incorporated into the phage particle together with the light chain gene. Each recombinant phage contains, within its genome, a gene for a different antibody Fab molecule displayed on the surface. Within this library, an excess of different antibodies can be cloned and displayed.

The phage library is mounted on an antigen-coated microliter well, non-specific phage is removed by washing, and antigen-binding phage is eluted. Interleukin-15, tumor necrosis factor antigen, its gene can be cloned and sequenced, and can be easily prepared by recombinant methods. Isolation of the genome from the antigen specific clone and allowing the antibody to be expressed in the form of a soluble Fab for further characterization. If Fab is selected as an effective therapeutic candidate, it can be easily converted to whole antibody.

As used herein, the term “antibody” is used in a broad sense and includes in particular monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies). do. Antibodies can be bound to other molecules (eg toxins).

In addition, another aspect of the present invention is a composition comprising the antibody or antigen-binding fragment (Fab) thereof according to the present invention.

As a result of confirming the binding of the antibody fragment according to the present invention to interleukin-15 or tumor necrosis factor by ELISA, the antibody and fragment thereof according to the present invention were respectively bound to interleukin-15 or tumor necrosis factor, and Fab according to the present invention As a result of performing Competitive inhibition ELISA of the fragments, it was found that the fragments showed remarkable binding affinity.

Monoclonal antibodies and fragments thereof according to the present invention should act as effective immunosuppressants. This is important for the treatment of many diseases where immunosuppression is therapeutically desirable, such as autoimmune diseases, to suppress unwanted antigen-specific IgG responses, and also for the prevention of organ rejection and graft versus host disease. In essence, the antibodies of the invention will be useful in treating any disease where inhibition of an inflammatory cytokine (interleukin-15), a tumor necrosis factor is therapeutically desirable.

The main therapeutic applications with anti-interleukin-15 antibodies, anti-tumor necrosis factor antibodies of the present invention are, for example, idiopathic thrombocytopenia purpura (ITP), systemic lupus erythematosus (SLE), type 1 diabetes, multiple sclerosis. , Aplastic anemia, psoriasis and rheumatoid arthritis. Another important therapeutic application is the prevention of graft versus host disease (GVHD) during organ transplantation and bone marrow transplantation (BMT). The antibodies of the present invention can be used to induce host resistance to donor-specific alloantigens, thereby promoting engraftment and reducing the incidence of graft rejection. In a murine animal model of allogeneic heart transplantation, it has been demonstrated that intravenous administration of CTLA4-Ig can induce immunosuppression or even induction of resistance to alloantigens [Lin et al., J. Exp. Med. 178:1801, 1993; Torka et al., Proc. Natl. Acad. Sci., USA, 89:11102, 1992]. Anti-interleukin-15 antibodies, anti-tumor necrosis factor antibodies of the present invention are predicted to exhibit similar or greater activity.

Antibodies produced by methods known in the art can be purified by a combination of affinity and size exclusion chromatography for characterization in functional biological assays. These assays include determination of specificity and binding affinity, and effector functions associated with the expressed isotype, eg ADCC, or complement binding. Such antibodies can be used as passive or active therapeutics for many human diseases including B cell lymphoma, infectious diseases including AIDS, autoimmune and inflammatory diseases and transplantation. Antibodies can be used in their native form, or can be used as part of an antibody/chelate, antibody/pharmaceutical or antibody/toxin complex. Additionally, whole antibodies or antibody fragments (Fabs) can be used as imaging reagents or latent vaccines or immunogens in active immunotherapy for the development of anti-idiotypic responses.

The amount of antibody useful to produce a therapeutic effect can be determined using standard techniques well known to those of skill in the art. Antibodies will generally be provided by standard techniques in pharmaceutically acceptable buffers and may be administered by any desired route. Because of the efficacy of the claimed antibodies and their tolerance by humans, it is possible to treat various diseases or diseases in the human body by repeatedly administering these antibodies.

Anti-interleukin-15 antibodies, anti-tumor necrosis factor antibodies (or fragments thereof) of the present invention are useful for inducing immune suppression, i.e. for inducing suppression of the immune system of a human or animal. Accordingly, the present invention relates to a composition for prophylactically or therapeutically inducing immune suppression in such a person or animal by administering the antibody of the present invention in an effective and non-toxic amount to a person or other animal in need of immunosuppression. will be.

The fact that the antibodies of the present invention are useful for inducing immune suppression is that they are transplanted organs or tissues (e.g., kidney, heart, lung, bone marrow, skin, cornea, etc.) And prevention; Treatment and prevention of skin signs of autoimmune, inflammatory, proliferative and hyperproliferative diseases, immunologically mediated diseases (e.g., rheumatoid arthritis, lupus erythematosus, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis , Type 1 diabetes, uveitis, neproose syndrome, psoriasis, atopic dermatitis, contact dermatitis and further eczemic dermatitis, seborrheic dermatitis, lichen planus, pemphigus, bullous pemphigus, epidermal blisters, urticaria, angioedema, Vasculitis, redness, skin eosinophilia, alopecia areata, etc.); Reversible obstructive airway disease, intestinal inflammation and allergies (e.g., chronic digestive disorders, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease and ulcerative colitis) and food-related allergies (e.g., migraine, rhinitis, and eczema) It suggests that it is useful in the treatment of

Those of skill in the art will be able to determine, by routine experimentation, an effective and non-toxic amount of an antibody to induce immune suppression. However, in general, an effective dose will be about 0.05 to 100 mg/kg body weight per day.

The antibody or fragment thereof of the present invention can be administered to humans or other animals in an amount sufficient to produce such an effect to a therapeutic or prophylactic degree, according to the above-mentioned treatment method. Such an antibody or fragment thereof of the present invention can be administered to such humans or other animals in a conventional form prepared by combining the antibody of the present invention with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. Those skilled in the art will recognize that the form and properties of a pharmaceutically acceptable carrier or diluent are defined by the amount of active ingredient intended to be combined with it, the route of administration and other well-known variables.

For example, the composition of the present invention may be formulated or used in combination with drugs such as antihistamines, anti-inflammatory analgesics, anticancer agents, and antibiotics that have already been used.

The pharmaceutical dosage form of the composition of the present invention may be used in the form of a pharmaceutically acceptable salt thereof, and may be used alone or in combination with other pharmaceutically active compounds, as well as in a suitable set.

The composition according to the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. have. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.

In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient in the extract, such as starch, calcium carbonate, and sucrose. ) Or lactose (lactose), gelatin, etc. are mixed to prepare. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The route of administration of the antibodies (or fragments thereof) of the present invention may be oral, parenteral, inhalation or topical. As used herein, the term “parenteral” includes intravenous, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. Subcutaneous and intramuscular forms of parenteral administration are generally preferred. The preferred dosage of the composition of the present invention varies depending on the condition and weight of the patient, the degree of disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art.

The daily parenteral and oral doses of applying the compounds of the present invention to prophylactically or therapeutically induce immune suppression or to treat carcinogenic tumors therapeutically are from about 0.05 to 1 kg of body weight per day. 100 mg, preferably about 0.5 to 10 mg. Administration may be administered once a day, or may be divided several times. The above dosage does not limit the scope of the present invention in any way.

The antibodies or fragments thereof of the present invention can also be administered by inhalation. The term “inhalation” refers to intranasal and oral inhalation administration. Dosage forms suitable for such administration, such as aerosol formulations or metered dose inhalers, can be prepared by conventional techniques. The preferred dose of the compound of the present invention to be used is generally about 10 to 100 mg. Administration may be administered once a day, or may be divided several times. The above dosage does not limit the scope of the present invention in any way.

Antibodies or fragments thereof of the invention can also be administered topically. Topical administration refers to non-systemic administration, and application of an antibody (or fragment thereof) of the invention to the epidermis, oral cavity and instillation of such an antibody or fragment thereof into the ear, eye and nose, and such antibody or fragment thereof. This includes cases where it does not significantly enter the bloodstream. Systemic administration means oral, intravenous, intraperitoneal and intramuscular administration. The amount of antibody or fragments thereof required for a therapeutic or prophylactic effect will, of course, vary depending on the antibody selected, the nature and severity of the disease to be treated and the animal to be treated, and will ultimately depend on the choice of the physician. A suitable topical dose of the antibody or fragment thereof of the present invention will preferably be about 1 to 100 mg/kg body weight per day. Administration may be administered once a day, or may be divided several times. The above dosage does not limit the scope of the present invention in any way.

Antibodies and fragments thereof may be used alone, but it is preferred to provide them as pharmaceutical formulations. For topical administration, the active ingredient may be included in an amount of 10% by weight, preferably 5% by weight or less, more preferably 0.1 to 1% by weight of the formulation, but 0.001 to 10% by weight, preferably It may be included in an amount of 1 to 2% by weight.

The topical formulations of the present invention may contain the active ingredient along with one or more acceptable carrier(s) and any other therapeutic ingredient(s). The carrier(s) must be acceptable in that it is compatible with the other ingredients of the formulation and must be harmless to its recipients.

Formulations suitable for topical administration are liquid or semi-liquid preparations suitable for penetration through the skin into the area in need of treatment, e.g., medicines, lotions, creams, ointments or pastes applied, and administered to the eyes, ears or nose. It includes a dropping agent suitable for the following.

Drops according to the invention may comprise sterile aqueous or oily solutions or suspensions, by dissolving the active ingredient in an aqueous solution of an antimicrobial and/or antifungal and/or any other suitable preservative, preferably including a surface active agent. Can be manufactured. The resulting solution can be sorted by filtration, transferred to a suitable sealed container, autoclaved and maintained for sterilization. Alternatively, the solution can be sterilized by filtration and transferred to a container by aseptic technique. Antimicrobial and antifungal agents suitable for inclusion in drops are phenyl mercury nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of oily solutions include glycerol, diluted alcohol and propylene glycol.

Lotions according to the invention include those suitable for application to the skin or eyes. The lotion applied to the eye may optionally contain a sterile aqueous solution containing an antimicrobial agent, and may be prepared by a method similar to a method of preparing a drop. Lotions or liniments for application to the skin may also include agents that promote drying and cool the skin, for example alcohols or acetone, and/or wetting agents such as glycerol or oils such as castor oil or peanut oil.

Creams, ointments or pastes according to the invention are semi-solid formulations of the active ingredient for external application. They can be prepared by mixing the active ingredient alone or in a solution or suspension in an aqueous or non-aqueous fluid with a base with or without oil in particulate or powder form with the aid of a suitable machine. The base may be a hydrocarbon such as hard, soft or liquid paraffin, glycerol, beeswax or metallic soap; mucus; Natural oils such as almond, corn, peanut, castor or olive oil; Wool fat or derivatives thereof, or fatty acids such as stearic acid or oleic acid may be included together with alcohols such as propylene glycol or macrogol. The formulation may incorporate any suitable surface active agent such as anionic, cationic or nonionic surface active agent such as sorbitan ester or polyoxyethylene derivatives thereof. Suspension agents such as natural rubber, inorganic materials such as cellulose derivatives or siliceous silica, and other ingredients such as lanolin may also be included.

Anti-interleukin-15 antibodies, anti-tumor necrosis factor antibodies or fragments thereof of the invention can also be administered in combination with inflammatory cytokines or other moieties that inhibit and modulate tumor necrosis factor.

Those of skill in the art will determine that the optimal amount and interval of individual administration of the antibody or fragment thereof will depend on the nature and extent of the disease to be treated, the dosage form, the route and site of administration, and the particular animal being treated, and such optimal conditions will depend on conventional techniques. It will be appreciated that it can be determined by. In addition, those skilled in the art will recognize that the optimal course of treatment, i.e. the number of administrations of an antibody of the invention or a fragment thereof, given per day for a defined number of days, can be achieved by a person skilled in the art using conventional procedures of treatment determination testing.

Without further elaboration, it is believed that one skilled in the art will be able to use the present invention to its fullest extent using the above description.

The antibodies and pharmaceutical compositions of the present invention are particularly suitable for parenteral administration, ie subcutaneous, intramuscular or intravenous administration. Compositions for parenteral administration will typically contain an antibody of the present invention or a fragment thereof or a cocktail solution thereof dissolved in an acceptable carrier, preferably an aqueous carrier. Various aqueous carriers such as water, buffered water, 0.4% saline, 0.3% glycine and the like can be used. These solutions are sterile and generally do not contain particulates. These solutions can be sterilized by conventional and well known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary ingredients as required for approximate physiological conditions, such as pH adjusters, buffers, and the like. The concentration of the antibodies or fragments thereof of the invention in such pharmaceutical formulations can vary widely, i.e. from less than about 0.5% by weight, usually at least 1% to 15 or 20% by weight, which are mainly depending on the particular mode of administration chosen. It will be selected based on fluid volume, viscosity, etc.

Accordingly, the pharmaceutical composition of the present invention for intramuscular injection can be prepared to contain 1 mL of sterile buffered water and 50 mg of the antibody or fragment thereof of the present invention. Similarly, a pharmaceutical composition of the present invention for intravenous infusion may be prepared to contain 250 mL of sterile Ringer's solution, and 150 mg of an antibody or fragment thereof of the present invention. Practical methods for preparing compositions for parenteral administration are well known to those skilled in the art, and are described, for example, in Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania, incorporated herein by reference. have.

Antibodies (or fragments thereof) of the invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has proven to be effective when using conventional immunoglobulins, and freeze drying and reconstitution techniques known in the art can be used.

Depending on the desired result, the pharmaceutical composition of the present invention can be administered for prophylaxis and/or treatment. For treatment, the composition is administered to a patient already suffering from the disease in an amount sufficient to treat or at least partially inhibit the disease and its complications. For prophylaxis, a composition containing the antibody of the present invention or a cocktail thereof is administered to a patient who has not yet suffered a disease to improve the patient's tolerance.

Single or multiple administrations of the pharmaceutical composition can be carried out according to the dosage level and mode selected by the treating physician. In any case, the pharmaceutical composition of the present invention should provide a modified antibody (or fragment thereof) of the present invention in an amount sufficient to effectively treat the patient.

In addition, it should be noted that the antibodies of the present invention can be used for the design and synthesis of peptides or non-peptide compounds (mimetics) useful for treatment as antibodies.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

The human interleukin-15 antibody or tumor necrosis factor antibody and fragments thereof of the present invention are obtained using phage display technology, and the human interleukin-15 antibody or tumor necrosis factor antibody and fragments thereof of the present invention are completely human antibodies, without immunogenicity. It is expected that it can be used as a drug for treating inflammatory autoimmune diseases.

1 shows the protein sequence of _{the human V L} kappa gene present in the antibody library (HuDVH5L).
Fig. 2 shows E. coli producing Fab-pIII fusion molecules specific for interleukin-15 (Il-15) (Fig. 2A) or tumor necrosis factor (TNF-α) (Fig. 2B) It shows the results of monoclonal ELISA for selection of clones.
Figure 3 shows the results of measuring the antigen specificity of the anti-IL-15 (Figure 3a) or anti-TNF-α (Figure 3b) Fab-pIII molecules produced in E. coli clones using ELISA.
Figure 4 shows the protein sequence of the human V _H specific for the IL-15 (Fig. 4a) or TNF-α (Fig. 4b) selecting from the antibody library (HuDVH5L).

Hereinafter, in relation to the method of preparing a human combinatorial Fab antibody library using a dual vector system-II (DVS-II) among the descriptions, the matters repeated herein are prior patent application (application number; PCT/KR2008/005238 ) Sept. 4, 2008).

reagent( Reagents )

Oligonucleotides for PCR were synthesized from Bioneer Co., South Korea. All enzymes including polymerase and restriction enzymes used in DNA cloning were purchased from Takara.

naive V _H genes Preparation of

E. coli for preparation of V _{H repertoire} ^{The 2 x 10 6} human naive Fd (V _H +C _H1 ) gene expressed in TG1 cells (Stratagene, USA) was previously selected. And the 330 bp gene fragment corresponding to FW1-CDR1-FW2-CDR2-FW3 was subjected to PCR method (sense primer: 5'-GCTGCTGGTCTGCTGCTCCT-3' and anti-sense primer: 5'-CGCacagtaatagacggccgtgtc-3') at 94°C. It was amplified by 20 cycles at 1 minute, 56° C. 1 minute, and 72° C. 1 minute.

At the same time, the 250 bp gene fragment encoding FW3-CDR3-FW4-partial CH1 was also amplified under the above conditions using a PCR method (sense primer: 5'-gacacggccgtctattactgtGCG-3'and anti sense primer 5'-tgtaggacagccgggaaggt-3). .

The two PCR products were separated using the Wizard Clean-up kit (Promega, USA). The separated two gene fragments were amplified 8 cycles at 94°C for 1 minute, 56°C for 2 minutes, and 72°C for 2 minutes using an assembly PCR method at the same molecular ratio.

Finally 350 bp V _H gene fragment was PCR pull-through method (HuVH sense: 5'-GCAACTGC GGCCCAGCCGGCC ATGGCCSAGGTGCAGCTGKTGCAGTCTGG-3 'and HuJH anti-sense: 5'-GGG GGCCAATGTGGCC GATGAGGAGACGGTGACCAKGGTBCCTTGGCCCCA-3') (Non-complementary Sfi I restriction sites are written in italics, and degeneracy is denoted as S = G or C; K = G or T; B = G, T or C) for 1 minute at 94℃, 1 minute at 56℃, and 20 at 1 minute at 72℃ It was obtained by cycle amplification.

Heavy chain Sub library (a heavy chain sublibrary ) For configuration DNA Cloning

V _H gene and pHf1g3A-2 phagemid (Joo et al., 2008) were treated with sfi 1 restriction enzyme and then reacted with T4 DNA ligase.

After phenol/chloroform extraction and ethanol precipitation, the ligation DNA product was transformed into E.coli Electro Ten Blue cells (stratagene, USA) using Gene pluser (Biorad, USA) under the conditions of 2.5KV, 25㎌, 200W. .

The transformed cells were cultured in SOB medium (0.5% yeast extract, 2.0% tryptone, 10 mM NaCl, 2.5mM KCl, 10 mM MgCl ₂ , pH 7.0) at 37° C. for 1 hour. After that, it was transferred to 2 liter 2x YT culture solution (1% yeast extract, 1.6% tryptone, 100 mM NaCl, pH 7.0) containing 2% glucose, 50 µg/ml ampicillin, and 10 µg/ml carbenicillin, and transferred to 37°C for 6 hours. While incubated.

The cultured cells were obtained by centrifugation for 20 minutes at 3300 x g and 4°C.

The pHf1g3A-2 phagemid DNA containing the heavy chain antibody repertoire was isolated using the Wizard plus SV miniprep kit (promega).

Phage display ( combinatorial phage display ) to prepare an antibody library

To prepare pLT-2 plasmids containing 5 different human kappa light chain genes, E. coli _{TG1 cells containing pLT-2 plasmid were added to OD 600} in 2x YT culture medium. _{Incubated until = 0.5, washed with sterile H 2} O containing 10% glycerol, and transformed the pHf1g3A-2 phagemid extracted from the heavy chain sub-library. For antibiotic selection, incubation was performed at 37°C for 8 hours in 2 liter 2x YT culture medium containing 10 μg/ml tetracyclin. After centrifuging 100 ml of the culture medium selected by antibiotics at 3300 xg for 10 minutes, the cell pellet is reproduced with 400 ml of a new 2x YT culture solution containing 2% glucose and 20 MOI of Ex12 helper phage (IG Therapy, south korea). It was cloudy and incubated for 1 hour at 37°C. This is for phage rescue.

After centrifugation at 3300 xg for 10 minutes, a cell pellet was obtained, resuspended in a new 4 liter 2x YT/ATKA culture solution containing 70㎍/ml kanamycin and 0.001% arabinose (w/v), and resuspended at 27℃ O/ N was cultured. 0.45 mm after centrifugation process The supernatant containing the recombinant phage fragment was harvested by sterilization with a filter.

Before bio-panning, the phage divided by 40 ml was precipitated using PEG/NaCl solution and then resuspended in 1 ml of sterilized phosphate-buffered saline (PBS) to use concentrated phage.

Recombinant human IL -15 antigen and TNF - alpha antigen

For the preparation of recombinant human IL-15 antigen, total RNA was extracted from nerve peripheral blood lymphocytes using a Trizol reagent (gibco/BRL, USA). 1 ^st strand cDNA was synthesized using oligo dT primers and reverse transcriptase (Roche, Germany).

After that, the cDNA encoding site of IL-15 is PCR method (sense: 5'-ggg GAATTC aactgggtgaatgtaataagtgatt-3' and anti-sense: 5'-ggg GAATTC agaagtgttgatgaacatttggac-3', where the Eco RI restriction site is written in italics). It was obtained by amplification under conditions of 20 cycles at 94°C for 1 minute, 56°C for 1 minute, and 72°C for 1 minute.

The 342 bp PCR product was separated using a Wizard DNA Clean-up kit and then treated with EcoR I. pMAL-c2 (New England Biolab, USA) was also treated with the same restriction enzyme and then ligation with IL-15 cDNA.

After the Ligation reaction was transformed into TG1 cells, the transformed cells were plated on 2x YT plates containing 50 µg/ml ampicillin for antibiotic selection.

To isolate recombinant IL-15 antigen fused with maltose-binding protein, E.coli TG1 cells were incubated in 400 ml of 2x YT/A culture medium until OD ₆₀₀ = 0.5, and then 0.1 mM isopropyl-β-D-1 -thiogalactopyranoside (IPTG) was added and incubated for 4 hours by shaking.

After harvesting the cells by centrifugation, the cell membrane was destroyed by sonication.

MBP-IL-15 fusion protein was isolated from cell extracts using amylase resin (Sigma-aldrich, USA). Recombinant human TNF-alpha was purchased from ATGen (South Korea).

Biopanning ( Biopanning )

Enrichment of recombinant phage binding to a specific antigen was performed by the method described in Joo et al, 2008. Briefly, after coating 10 ㎍/ml MBP-IL-15 or TNF-alpha on MaxiSorb ELISA plates (Nunc, Denmark) using a coating buffer (0.1 M NaHCO3, pH 9.6), obtained from HuDVH5L library. 10 ¹¹ phage was added to react

In the case of panning of MBP-IL-15, 1 μM of MBP was reacted with phage in advance to remove the phage that reacted with a part of MBP.

Eluted phage was mixed with new TG1 cells and spread on 2x YT/AC plates for antibiotic screening.

After incubation at 27° C. O/N, the cells were scraped using a sterilized glass rod and harvested in 2x YT culture. Phagemid DNA was isolated using a Wizard plasmid purification kit.

200 ng of phagemid DNA was transformed into 200 µl of TG1 cell for transformation containing pLT-2 plasmid with 5 light chains using a Gene pulser.

The transformed cells were plated on 2x YT/ACT and cultured at 27° C. O/N. Cells were collected from the plate. Using the method described above, phage rescue was performed in 100ml of 2x YT/ACTKA containing Ex12 helper phage. Panning repeats the above method 3 times.

Enzyme - linked immunosobent assay ( ELISA )

ELISA was performed by the method described in Joo et al, 2008 using the E. coli culture supernatant containing water-soluble Fab-pIII fusion molecules.

To perform monoclonal ELISA, E. coli TG1 colonies obtained through the third panning were incubated at 37°C using 96 well plates (Nunc) in 100 µl of 2x YT/ACTG culture medium until _{OD 600 ㎜ = 0.5.} . Bacterial cells were centrifuged at 3300 xg for 10 minutes, and the cell pellet was resuspended in 100 µl of a new 2x YT/ACT culture solution containing 0.02% arabinose and 0.1 mM IPTG.

After culturing O/N at 27℃, the culture supernatant is 10㎍/ml of MBP-IL-15, MBP, TNF-alpha, or MaxiSorb ELISA plates coated with bovine serum albumin (BSA) (Sigma-Aldrich) diluted in coating buffer. 50 µl was added to each well.

Goat anti-human kappa L chain antibody-HRPO (Sigma-Aldrich) was added to detect Fab-pIII fusion molecules that react specifically with the antigen.

Plates were incubated for 1 hour at 37°C and washed 3 times with PBST. After that, the reaction result was measured using a 3,3'5,5' tetramethyl benzidine (TMB) substrate (Sigma-Aidrich).

Absorbance was measured at 450 nm using an ELISA reader (BioRad). The results are shown in Figs. 2A and 2B.

Fab clones that specifically bind antigen-specific anti-IL-15 and anti-TNF-alpha Fab-pIII fusion molecules were selected as the strongest response in monoclonal ELISA.

Then, a water-soluble Fab-pIII was prepared using 5 ml of 2x YT/ATG culture solution.

ELISA is 10㎍/ml glutathione-S-transferase (GST) (IG Therapy), MBP, MBP-IL-15, TNF-alpha, L-glutamate dehydrogenase (L-Glu) (Sigma-Aldrich), BSA or bovine ELISA was performed using MaxiSorb ELISA plates prepared with superoxide dismutase (SOD) (Sigma-Aidrich).

Mouse anti-IL-15 mAb (ATCC, USA) or mouse anti-TNF-alpha mAb (Ms X Hu TNF-alpha) (Chemicon, USA) was used as a positive antibody standard.

Antibody variable region gene sequence analysis ( Sequencing analysis of the antibody V genes)

The pHf1g3A-2 phagemid and pLT-2 plasmid were selected from E.coli cells producing Fab molecules that specifically respond to MBP-IL-15 or TNF-alpha using the Wizard plus SV minipreps kit (promega).

Two different sequencing primers complementary to pHf1g3A-2 and pLT-2 (5'-GTGCCGTTCTATAGCCATAGCAC-3' or 5'-GGCACTGGCTGGTTTCGCTA CCGTG-3) were used to analyze the _{V H} and V _{L genes.}

Automatic DNA sequencing was performed by Solgent Co., South Korea.

The analyzed sequence is shown in Table 1.

SEQ ID NOs: 25-32 shown in the following table show the amino acid sequences of all VH and VL including the amino acid sequences of the CDR regions of VH and VL.

Specificity Fab clones V _H ( subgroup ) V _L ( subgroup ) Frequency Anti - IL -15 4 H10 (3 E3 )





One H3



3 C1 (4 H7 )





IL- 15 V _H1 ( V _H 3 )
SEQ ID NO:25
CDR 1_SEQ ID NO: 1
CDR 2_SEQ ID NO:2
CDR 3_SEQ ID NO:3
HuV _Lk1 -30 ( V _L k1 )
SEQ ID NO:26
CDR 1_SEQ ID NO:16
CDR 1_SEQ ID NO:17
CDR 1_SEQ ID NO:18
HuV _Lk3 -14 ( V _L k3 )
SEQ ID NO:27
CDR 1_SEQ ID NO:19
CDR 1_SEQ ID NO:20
CDR 1_SEQ ID NO:21
HuV _Lk1 -13 ( V _L k1 )
SEQ ID NO:28
CDR 1_SEQ ID NO:22
CDR 2_SEQ ID NO:23
CDR 3_SEQ ID NO:24 2/6




1/6




2/6 8 C5 IL- 15 V _H2 ( V _H 2 )
SEQ ID NO:29
CDR 1_SEQ ID NO:4
CDR 2_SEQ ID NO:5
CDR 3_SEQ ID NO:6 HuV _Lk1 -30 ( V _L k1 )
SEQ ID NO:26
CDR 1_SEQ ID NO:16
CDR 2_SEQ ID NO:17
CDR 3_SEQ ID NO:18 1/6 Anti - TNF -α TNFa #One TNF- αV _H1 ( V _H 3 )
SEQ ID NO:30
CDR 1_SEQ ID NO:7
CDR 2_SEQ ID NO:8
CDR 3_SEQ ID NO:9




HuV _Lk1 -30 ( V _L k1 )
SEQ ID NO:26
CDR 1_SEQ ID NO:16
CDR 2_SEQ ID NO:17
CDR 3_SEQ ID NO:18
5/15 TNFa #2 TNF- α V _H2 ( V _H 3 )
SEQ ID NO:31
CDR 1_SEQ ID NO:10
CDR 2_SEQ ID NO:11
CDR 3_SEQ ID NO:12 9/15 TNFa #3 TNF- α V _H3 ( V _H 2 )
SEQ ID NO:32
CDR 1_SEQ ID NO:13
CDR 2_SEQ ID NO:14
CDR 3_SEQ ID NO:15 1/15

As shown in the table above, in the case of Anti-IL-15, the sequences of 4H10 and 3E3 were identical to each other, and the sequences of 3C1 and 4H7 were identical to each other. That is, Fab clones (4H10, 1H3, 3C1, 8C5) are classified into four types. Of these, 3 Fab clones (4H10, 1H3, 3C1) consist of the same V _H gene, IL-15V _H1 (Accession no. FJ490200), but only the light chain variable region HuV _{L κ1} -30 (FJ497788), HuV _{L κ3} -14 (FJ497787), HuV _{L κ1} -13 (FJ497786). On the other hand, 8C5 is composed of V _H gene IL-15V _H2 (FJ490201) and the light chain variable region HuV _{L κ1} -30. As a result of the amino acid sequence of IL-15V _H1 and IL-15V _H2 shown in FIG. 4A, it can be seen that there is little amino acid sequence homology of all heavy chain CDRs. The Fabs fragment and the gene of the variable region are shown in Table 1 as shown above.

The gene sequences of anti-TNF-α Fab clones, #1, #4, #7, #8 and #11 are identical and #2, #5, #6, #9, #10, #12, #13, # The 14 and #15 gene sequences are identical. Therefore, as a result, it is classified as TNFa#1, TNFa#2, and TNFa#3. Each of the V _H genes of Fab clones is TNF-αV _H1 (Accession no. FJ4902002), TNF-αV _H2 (FJ4902002) TNF-αV _H3 (FJ4902002), but the V _L genes are the _{same as HuV L κ1} -30 (Table 1). _{The amino acid sequence of these 3 V H} specific for TNF-α is shown in FIG. 4B.

Competitive inhibition ELISA

Competitive inhibition ELISA was performed to measure the binding affinity of Fab fragments (Lee et al, 2004). Mix the supernatant of the E.coli culture medium containing the water-soluble Fab-pIII fusion molecule with 5X10 ^-6 M-5X10 ^-9 M MBP-IL-15 antigen or TNF-alpha antigen diluted with 0.5% BSA in PBS, or only the supernatant. It was reacted at room temperature for 2 hours.

The Fab and antigen mixture were transferred to Maxisorb ELISA plates coated with 5 μg/ml of MBP-IL-15 antigen and TNF-alpha antigen, and ELISA was performed in the same manner as above. Recombinant MBP-IL-15, TNF-α and 5 antigens (GST, MBP, L-Glu, BSA, SOD) not related to this were coated on a microtiter plate. The supernatant containing water-soluble Fab-pIII molecules of MBP-IL-15 6 clones (Fig. 3a) and TNF-α 15 clones (Fig. 3b) was produced in the presence of IPTG and arabinose, showing a positive reaction in monoclonal ELISA. Goat anti-human kappa to which HRPO is bound was used as a secondary antibody. The hIL-15-M110 hybridoma supernatant (Fig. 3a) and mouse anti-TNF-α mAb (Fig. 3b) were used as positive antibodies, which were detected as Goat anti-mouse IgG to which HRPO was bound. The binding signal was confirmed with TMB substrate, and OD ₄₅₀ analyzed at _nm. Date is expressed as the average value after three experiments. _{IC 50} was calculated by analyzing the concentration of a water-soluble step-by-step antigen to inhibit 50% of Fab molecules that bind to the immobilized antigen in the absence of a competitive antigen. The titration curve was obtained by ELISA method and analysis of Klotz plots (Friguet et al., 1985).

As shown in Figure 3, the soluble Fab-pIII 6 molecules (4H10 (3E3), 1H3, 3C1 (4H7), 8C5) of MBP-IL-15 clones showed remarkable antigen binding affinity, and the water solubility of TNF-α clones Fab-pIII 15 molecules also showed remarkable antigen binding affinity.

For more in-depth analysis, 2 anti-IL-15 Fabs, 4H10, and 1H3 and 2 anti-TNF-α Fabs, TNFa#, which consistently showed remarkable binding signals to MBP-IL-15 and TNF-α during ELISA analysis. Competitive inhibition ELISA was performed by selecting 1 and TNFa#2. Between 0.5 nM and 5 μM antigen concentration, _{IC 50} was measured to _{calculate the dissociation constants (K d} s). As a result, the separation constant of 4H10 was 0.1 μM and that of 1H3 was 0.7 μM. These results show that when composed of the same light chain (IL-15V _H1 ), but composed of different light chains, the binding affinity of the Fab is affected. In the case of anti-TNF-α Fabs, the separation constant of TNFα#1 was 1 μM, and the separation constant of TNFα#2 was 0.2 μM. The results are shown in Table 2.

Specificity Fab clones Antigen K _d (μM) Anti-IL-15 4H10
1H3 MBP-IL-15
MBP-IL-15 0.1
0.7 Anti-TNF-α TNFα#1 TNF-α One TNFα#2 TNF-α 0.2

K _d s were determined by competitive inhibition ELISA.

In the field of development of therapeutic antibodies for autoimmune diseases, interleukin-15 and tumor necrosis factor are recognized as excellent targets, so the selection and commercialization of human antibodies that specifically bind to them are in the field of autoimmune disease treatment including leucatism. Will greatly contribute to

<110> Korea University Industry-Academy Cooperation Foundation <120> Human antibodies and Fab specific for Interleukin-15 and Tumer necrosis factor-alpha <160> 34 <170> KopatentIn 1.71 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-IL-15 heavy variable region (4H10), mRNA (IL-15VH1), CDR 1 <400> 1 Gly Leu Ser Ile Ser Asp Ala Trp Met His 1 5 10 <210> 2 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-IL-15 heavy variable region (4H10), mRNA (IL-15VH1), CDR 2 <400> 2 Leu Ile Thr Asn Lys Thr Gly Gly Gly Thr Ile Ile Tyr Ala Ala Pro 1 5 10 15 Val Lys Gly <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-IL-15 heavy variable region (4H10), mRNA (IL-15VH1), CDR3 <400> 3 Thr Tyr Asp Gln Gly Tyr 1 5 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-IL-15 heavy variable region, mRNA (IL-15VH2), CDR 1 <400> 4 Gly Asp Ser Val Ser Ser Asn Ser Ala Ala Trp Asn 1 5 10 <210> 5 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-IL-15 heavy variable region, mRNA (IL-15VH2), CDR 2 <400> 5 Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val 1 5 10 15 Lys Ser <210> 6 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-IL-15 heavy variable region, mRNA (IL-15VH2), CDR 3 <400> 6 His Lys Asp Pro Gly Ser Ser Trp Tyr Arg Gln Pro Phe Asp Tyr 1 5 10 15 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-TNF-alpha heavy variable region, mRNA (TNF-a VH1), CDR 1 <400> 7 Gly Phe Thr Phe Ser Ser Tyr Trp Met Ser 1 5 10 <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-TNF-alpha heavy variable region, mRNA (TNF-a VH1), CDR 2 <400> 8 Glu Ile Asn His Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 9 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-TNF-alpha heavy variable region, mRNA (TNF-a VH1), CDR 3 <400> 9 Leu Gly Trp Thr Met Val Arg Gly Val Lys Tyr Tyr Tyr Gly Met Asp 1 5 10 15 Val <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-TNF-alpha heavy variable region, mRNA (TNF-a VH2), CDR 1 <400> 10 Gly Phe Ser Phe Gly Asp Tyr Ala Met His 1 5 10 <210> 11 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-TNF-alpha heavy variable region, mRNA (TNF-a VH2), CDR 2 <400> 11 Gly Ile Ser Trp Asn Ser Arg Ile Ile Gly Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 12 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-TNF-alpha heavy variable region, mRNA (TNF-a VH2), CDR 3 <400> 12 Met Gly Gly Asp Ser Ser Gly Tyr Tyr Ser Glu Pro Tyr Tyr Tyr Phe 1 5 10 15 Asp Tyr <210> 13 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-TNF-alpha heavy variable region, mRNA (TNF-a VH3), CDR 1 <400> 13 Gly Asp Ser Val Ser Ser Asn Ser Ala Ala Trp His 1 5 10 <210> 14 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-TNF-alpha heavy variable region, mRNA (TNF-a VH3), CDR 2 <400> 14 Lys Thr Tyr Tyr Arg Ser Arg Trp Phe Tyr Asp Tyr Ala Val Ser Leu 1 5 10 15 Lys Ser <210> 15 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-TNF-alpha heavy variable region, mRNA (TNF-a VH3), CDR 3 <400> 15 Leu Ala Pro Asp Ile Ser Ala Ala Gly Phe Asp Tyr 1 5 10 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens Ig variable kappa-30 from HuDVH5L mRNA, complete cds., CDR 1 <400> 16 Arg Ala Ser Gln Ser Ile Ser Arg Tyr Leu Asn 1 5 10 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens Ig variable kappa-30 from HuDVH5L mRNA, complete cds., CDR 2 <400> 17 Gly Ala Ser Arg Leu Glu Ser 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens Ig variable kappa-30 from HuDVH5L mRNA, complete cds., CDR 3 <400> 18 Gln Gln Ser Asp Ser Val Pro Val Thr 1 5 <210> 19 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens Ig variable kappa-14 from HuDVH5L mRNA, complete cds., CDR 1 <400> 19 Arg Ala Ser Gln Ser Ile Phe Asn Tyr Val Ala 1 5 10 <210> 20 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens Ig variable kappa-14 from HuDVH5L mRNA, complete cds., CDR 2 <400> 20 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 21 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens Ig variable kappa-14 from HuDVH5L mRNA, complete cds., CDR 3 <400> 21 Gln Gln Arg Ser Lys Trp Pro Pro Thr Trp Thr 1 5 10 <210> 22 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens Ig variable kappa-13 from HuDVH5L mRNA, complete cds., CDR 1 <400> 22 Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly 1 5 10 <210> 23 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens Ig variable kappa-13 from HuDVH5L mRNA, complete cds., CDR 2 <400> 23 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 24 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens Ig variable kappa-13 from HuDVH5L mRNA, complete cds., CDR 3 <400> 24 Leu Gln Asp Tyr Asn Tyr Pro Trp Thr 1 5 <210> 25 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-IL-15 heavy variable region (4H10), mRNA (IL-15VH1) <400> 25 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Glu Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Ser Ile Ser Asp Ala 20 25 30 Trp Met His Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Leu Ile Thr Asn Lys Thr Gly Gly Gly Thr Ile Ile Tyr Ala Ala 50 55 60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Thr Thr Tyr Asp Gln Gly Tyr Trp Gly Gln Gly Thr Met 100 105 110 Val Thr Val Ser Ser 115 <210> 26 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens Ig variable kappa-30 from HuDVH5L mRNA, complete cds. <400> 26 Glu Leu Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asp Ser Val Pro Val 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 100 105 <210> 27 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens Ig variable kappa-14 from HuDVH5L mRNA, complete cds. <400> 27 Glu Leu Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Phe Asn Tyr 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Lys Trp Pro Pro 85 90 95 Thr Trp Thr Phe Gly Gln Gly Thr Arg Val Asp Ile Lys Arg 100 105 110 <210> 28 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens Ig variable kappa-13 from HuDVH5L mRNA, complete cds. <400> 28 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Pro Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asp Tyr Asn Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Gln Val Glu Ile Lys Arg 100 105 <210> 29 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-IL-15 heavy variable region, mRNA (IL-15VH2) <400> 29 Gln Val Gln Leu Leu Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Ser His Lys Asp Pro Gly Ser Ser Trp Tyr Arg Gln 100 105 110 Pro Phe Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser 115 120 125 <210> 30 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-TNF-alpha heavy variable region, mRNA (TNF-a VH1) <400> 30 Gln Val Gln Leu Leu Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Ile Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Asn Ser Val Ala Thr Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Leu Gly Trp Thr Met Val Arg Gly Val Lys Tyr Tyr Tyr Gly Met 100 105 110 Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 31 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-TNF-alpha heavy variable region, mRNA (TNF-a VH2) <400> 31 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Gly Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Arg Ile Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Met Gly Gly Asp Ser Ser Gly Tyr Tyr Ser Glu Pro Tyr Tyr 100 105 110 Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 32 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Homo sapoens anti-TNF-alpha heavy variable region, mRNA (TNF-a VH3) <400> 32 Glu Val Gln Leu Val Gln Ser Gly Pro Gly Leu Leu Lys Pro Ser Gln 1 5 10 15 Thr Leu Ala Leu Thr Arg Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Ser Ala Ala Trp His Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Asp 35 40 45 Trp Leu Gly Lys Thr Tyr Tyr Arg Ser Arg Trp Phe Tyr Asp Tyr Ala 50 55 60 Val Ser Leu Lys Ser Arg Met Thr Ile Asn Thr Asp Thr Ser Asn Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Asp His Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Leu Ala Pro Asp Ile Ser Ala Ala Gly Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 33 <211> 102 <212> PRT <213> Artificial Sequence <220> <223> constant heavychain region of human antibody <400> 33 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 1 5 10 15 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 20 25 30 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 35 40 45 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 50 55 60 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 65 70 75 80 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 85 90 95 Lys Thr Val Glu Arg Lys 100 <210> 34 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> constant lightchain region of human antibody <400> 34 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105

Claims (5)

A heavy chain variable region comprising the heavy chain CDRs of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, a heavy chain variable region comprising SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, or SEQ ID NO: 13, SEQ ID NO: 14 And a necrosis comprising a heavy chain variable region selected from the group consisting of a heavy chain variable region comprising a heavy chain CDR of SEQ ID NO: 15 and a light chain variable region comprising a light chain CDRs of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 An antibody or antigen binding fragment thereof (Fab) that specifically binds to factor (TNF-α).
The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region is represented by any one selected from the group consisting of SEQ ID NOs: 30, 31, and 32, and the light chain variable region is represented by SEQ ID NO: 26. Fab).
The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region and the light chain variable region are respectively bound to a human antibody heavy chain constant region and a human antibody light chain constant region.
The antibody or antigen-binding fragment thereof according to claim 3, wherein the human antibody heavy chain constant region is SEQ ID NO: 33, and the human antibody light chain constant region is SEQ ID NO: 34.
A gene having a DNA sequence encoding the antibody of any one of claims 1 to 2.

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