WO2014171532A1

WO2014171532A1 - Novel anti-human tweak antibody

Info

Publication number: WO2014171532A1
Application number: PCT/JP2014/061026
Authority: WO
Inventors: 孝則佐々木; 雅人佐藤
Original assignee: アステラス製薬株式会社
Priority date: 2013-04-19
Filing date: 2014-04-18
Publication date: 2014-10-23
Also published as: JP2016000003A; TW201506039A

Abstract

[Problem] To provide an anti-human TWEAK antibody having superior activity as compared to conventional anti-human TWEAK antibodies, and a means for preventing or treating various diseases in which human TWEAK is involved in the pathogenesis using this antibody. [Solution] Anti-human TWEAK antibody containing each of: a heavy-chain variable region comprising an amino acid sequence of amino acids nos. 1 to 118 of SEQ ID NO: 1, an amino acid sequence of amino acids nos. 1 to 125 of SEQ ID NO: 5, or an amino acid sequence of amino acids nos. 1 to 121 of SEQ ID NO: 9; and a light-chain variable region comprising an amino acid sequence of amino acids nos. 1 to 108 of SEQ ID NO: 3, an amino acid sequence of amino acids nos. 1 to 108 of SEQ ID NO: 7, or an amino acid sequence of amino acids nos. 1 to 108 of SEQ ID NO: 11.

Description

New anti-human TWEAK antibody

The present invention relates to a novel anti-human TWEAK antibody. Specifically, the novel anti-human TWEAK antibody of the present invention is an anti-human TWEAK antibody having superior activity against both human sTWEAK and human mTWEAK compared to conventional anti-human TWEAK antibodies.

TWEAK (TNF-like weak inducer оf apoptosis) is a single-transmembrane protein that is widely expressed in cells and tissues including immunocompetent cells such as macrophages and T cells and renal mesangial cells. The receptor for TWEAK is Fn14 protein, which is activated when TWEAK forms a complex with Fn14 protein, and transmits a signal into the cell. Activation of the TWEAK signal induces the expression of inflammatory chemokines, such as monocyte chemotactic factor (MCP-1), for example via activation of the NF-κB transcription factor and elicits an inflammatory response (Nat Rev Drug Discov. 2008; 7 (5): 411-425).

TWEAK exists as a membrane type TWEAK (mTWEAK) on the cell membrane, and when the extracellular region is cleaved by a furin type serine protease, it becomes a soluble type TWEAK (sTWEAK) (J Biol Chem. 2010; 285 (23 ): 17432-17441). Any TWEAK forms a complex with the Fn14 protein and transmits a signal into the cell, but the maximum activity of the transmitted signal is stronger in mTWEAK than in sTWEAK (J Immunol. 2010; 185 (3): 1593-1605, Br J Pharmacol. 2013; 170 (4): 748-764).

TWEAK is a systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, psoriasis, scleroderma and other autoimmune diseases, stroke, arteriosclerosis, ischemic acute kidney injury, chronic renal failure It is known to be involved in diseases such as cancer and muscular dystrophy.

Systemic lupus erythematosus is an autoimmune disease characterized by systemic inflammatory lesions caused by tissue deposition of immune complexes such as DNA-anti-DNA antibodies. Symptoms often have a chronic course of remission and hatred. In addition, many organs are affected, so the clinical findings are diverse, including renal disorders, central nervous lesions, vascular lesions, blood abnormalities, joint symptoms, and skin lesions. In systemic lupus erythematosus, various cells undergo apoptosis to release DNA and RNA from the cells. However, mTWEAK molecules expressed in autoreactive T cells may promote monocyte apoptosis. It has been reported (J Immunol. 2002; 169 (10): 6020-6029).

Lupus nephritis is a nephritis that develops in many patients with systemic lupus erythematosus and is a high-risk disease. In a mouse lupus nephritis pathological model, it has been reported that administration of anti-TWEAK antibody improves proteinuria and suppresses the production of chemokines such as MCP-1 and cytokines in the kidney (J Immunol. 2007; 179 (11): 7949-7958). Furthermore, it has been reported that TWEAK and Fn14 are involved in the pathological state of lupus nephritis, because an effect of improving renal pathological conditions such as proteinuria is also observed in the pathological model of lupus nephritis in Fn14 genetically deficient animals (knockout mice). (J Immunol. 2007; 179 (11): 7949-7958). Furthermore, the stimulation of TWEAK induces the expression of inflammatory mediators such as MCP-1 in human mesangial cell lines, and the production of cytokines by TWEAK stimulation is via the activation of NF-κB, and anti-TWEAK antibody Has also been reported to inhibit cytokine production (Cytokine 2009; 46 (1): 24-35). Furthermore, in patients with lupus nephritis, increased TWEAK gene expression in the kidney and increased secretion of TWEAK protein in the urine have been reported (Nephrology (Carlton). 2011; 16 (4): 426-432, J Autoimmun. 2006 Dec; 27 (4): 242-50).

Rheumatoid arthritis is a disease that causes chronic inflammation in the joint due to abnormal growth of synovial tissue present in the joint, and is an autoimmune disease that causes various functional disorders such as joint destruction by progression. It has been reported that TWEAK and Fn14 are involved in the pathological condition of rheumatoid arthritis because an improvement effect on the arthritis score and the like is observed in a test using an antagonist against TWEAK or an antagonist against Fn14 ( J Immunol.2006; 177 (4): 2610-2620, Rheumology (Oxford) .2008; 47 (4): 442-450).

Inflammatory bowel disease is an intractable disease whose cause is unknown, mainly related to intestinal tract, associated with immune abnormalities in the intestinal tract. Symptoms such as abdominal pain, diarrhea, bloody stool, diarrhea, fever, or weight loss are included, and representative examples include Crohn's disease and ulcerative colitis. It has been reported that a TWEAK signal is involved in a test using a TWEAK knockout mouse and an antagonist to TWEAK because an improvement effect on inflammation is observed (Gastroenterology. 2009; 136 (3): 912). 923).

Multiple sclerosis is a central nervous system disease characterized by inflammation and loss of myelin sheaths. In the mouse EAE model (experimental autoimmune encephalitis model), TWEAK has been reported to be involved in multiple sclerosis pathology since an antagonistic effect on TWEAK has an effect of improving symptoms (Clin Immunol. 2005; 117 (1): 15-23).

Stroke is a disease that causes neuronal necrosis and damage in the brain due to oxygen deprivation and secondary events due to ischemia. In a mouse cerebral ischemia model, TWEAK has been reported to be improved by antagonists to TWEAK, and therefore, involvement of TWEAK in stroke has been reported (J Cereb Blood Flow Metab. 2007; 27 (3): 534-544). .

Acute kidney injury is a serious condition that develops due to various factors such as traumaticity, bleeding from surgery, rapid decrease in blood pressure, dehydration, side effects of drugs, and renal function decreases within a few days. It is roughly divided into cell damage and ischemic damage. Fn14 has been reported to increase in expression in human ischemic kidneys, and in a mouse acute kidney injury model, renal function and survival rate were examined in a test using an antagonist against TWEAK or an antagonist against Fn14. It has been reported that TWEAK and Fn14 are involved in acute kidney injury because of the improvement effect (Kidney Int. 2011; 79 (2): 179-188, J Am Soc Nephrol. 2008; 19 (4): 695-703).

It is known that the expression of TWEAK and Fn14 increases in various carcinomas (PLOS ONE. 2013; 8 (3) e57436). It has also been reported that cells overexpressing TWEAK have a transforming ability (Cancer Res. 2004; 64 (24): 8968-8972). Furthermore, it has been reported that antagonists to TWEAK suppress cancer cell proliferation in the Xenograft model (cancer-bearing transplant model) (Clin Cancer Res. 2010; 16 (2): 497-508).

TWEAK is known to be deeply involved in muscular atrophy. For example, it has been reported that muscle atrophy is reduced in a TWEAK gene-deficient mouse in a muscle atrophy model, or conversely, muscle atrophy is enhanced in a TWEAK overexpressing mouse (J Clin Invest. 2007; 117: 889-901, International myoniconic consortium meeting 9: O-39, 2013, Am J Pathol. 2012; 180 (4): 1603-1613).

Therefore, if a monoclonal antibody that specifically binds to TWEAK and inhibits the action of TWEAK can be developed, it is expected that TWEAK is useful for the treatment and prevention of various diseases related to pathogenesis.

Antibodies that have a function-inhibiting action against human TWEAK that have been studied so far include mouse monoclonal antibody mP2D10 and its humanized monoclonal antibodies huP2D10-1 and huP2D10-2 (Patent Document 1), A mouse monoclonal antibody MTW-1 (Non-patent Document 1) has been reported. Further, TW212, which is a hamster monoclonal antibody, and its humanized monoclonal antibody (Patent Document 2), and TW305, which is a rabbit monoclonal antibody, and its humanized monoclonal antibody (Patent Document 3) have also been reported. However, it cannot be said that conventional antibodies have sufficient neutralizing activity against human TWEAK.

In addition, the main factors that define the effective dose of antibody drugs include binding activity and neutralization activity to the antigen of the antibody, and the amount of antigen present in the body, which improves binding activity and neutralization activity. This leads to a reduction in dosage, and as a result, can be said to be a very beneficial improvement that also leads to a reduction in the patient's economic burden and medical costs.

For these reasons, it is necessary to obtain an anti-human TWEAK antibody superior in activity compared to conventional anti-human TWEAK antibodies in order to administer it to humans and use it for the treatment and prevention of various diseases.

International Publication No. 2006/130374 International Publication No. 2010/115555 International Publication No. 2012/045671

An object of the present invention is to provide an anti-human TWEAK antibody having superior activity compared to conventional anti-human TWEAK antibodies.

As a result of repeated considerable inventive studies in the production of anti-human TWEAK antibodies, the present inventors have obtained 1) a heavy chain variable region comprising amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 1 and amino acids of SEQ ID NO: 3 An anti-human TWEAK antibody comprising a light chain variable region comprising the amino acid sequence of Nos. 1 to 108, 2) a heavy chain variable region comprising the amino acid sequence of amino acid Nos. 1 to 125 of SEQ ID No. 5, and the amino acid of SEQ ID No. 7 An anti-human TWEAK antibody comprising a light chain variable region comprising the amino acid sequence of Nos. 1 to 108, and 3) a heavy chain variable region comprising the amino acid sequence of amino acid Nos. 1 to 121 of SEQ ID No. 9, and An anti-human TWEAK antibody containing a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 108 was prepared (Example 1). 8) These antibodies are capable of binding to human sTWEAK (Example 9), binding activity to human mTWEAK (Example 10), human sTWEAK-stimulated NF-κB activation inhibitory activity (Example 11), human mTWEAK-stimulated NF It was found to have -κB activation inhibitory activity (Example 12), human sTWEAK-stimulated MCP-1 expression inhibitory activity (Example 13), and human mTWEAK-stimulated MCP-1 expression inhibitory activity (Example 14). As a result, the anti-human TWEAK antibody was provided and the present invention was completed.

That is, this invention includes the following invention as a medically or industrially useful substance and method.
(1) An anti-human TWEAK antibody selected from any one of 1) to 3) below.
1) an anti-human TWEAK antibody comprising a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 3;
2) an anti-human TWEAK antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 5 from amino acid numbers 1 to 125 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 7 to amino acid numbers 1 to 108;
3) An anti-human TWEAK antibody comprising a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 9 and a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 11.
(2) The heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1 from amino acid number 1 to 118 and the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 3 from amino acid number 1 to 108 (1) An anti-human TWEAK antibody according to 1.
(3) including the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 5 from amino acid number 1 to 125 and the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 7 to amino acid number 1 to 108 (1) An anti-human TWEAK antibody according to 1.
(4) The above (1), comprising a heavy chain variable region comprising the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 11 An anti-human TWEAK antibody according to 1.
(5) The anti-human TWEAK antibody according to any one of (1) to (4) above, wherein the heavy chain constant region of the antibody is a human Igγ1 constant region.
(6) The anti-human TWEAK antibody according to any one of (1) to (4) above, wherein the light chain constant region of the antibody is a human Igκ constant region.
(7) The anti-human TWEAK according to any one of (1) to (4) above, wherein the heavy chain constant region of the antibody is a human Igγ1 constant region and the light chain constant region of the antibody is a human Igκ constant region. antibody.
(8) The anti-human TWEAK antibody according to (2) above, comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3.
(9) The anti-human TWEAK antibody according to (3) above, comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 5 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 7.
(10) The anti-human TWEAK antibody according to (4) above, comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 11.
(11) A polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TWEAK antibody according to any one of (1) to (4) above.
(12) A polynucleotide comprising a base sequence encoding the light chain variable region of the anti-human TWEAK antibody according to any one of (1) to (4) above.
(13) An expression vector comprising the polynucleotide according to (11) and / or (12) above.
(14) A host cell transformed with the expression vector according to (13) above, which is selected from the group consisting of the following (a) to (d):
(A) a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TWEAK antibody according to any one of (1) to (4) above and a base sequence encoding the light chain variable region of the antibody A host cell transformed with an expression vector comprising a polynucleotide;
(B) an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TWEAK antibody according to any one of (1) to (4) above and a light chain variable region of the antibody A host cell transformed with an expression vector comprising a polynucleotide comprising the base sequence;
(C) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TWEAK antibody according to any one of (1) to (4) above; and (d ) A host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain variable region of the anti-human TWEAK antibody according to any one of (1) to (4) above.
(15) A host cell transformed with the expression vector according to (13) above, which is selected from the group consisting of the following (a) to (d):
(A) a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human TWEAK antibody according to any of (8) to (10) above and a polynucleotide comprising a base sequence encoding the light chain of the antibody A host cell transformed with an expression vector comprising;
(B) an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human TWEAK antibody according to any of (8) to (10) above, and a base sequence encoding the light chain of the antibody A host cell transformed with an expression vector comprising the polynucleotide;
(C) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human TWEAK antibody according to any of (8) to (10) above; and (d) the above (8) A host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain of the anti-human TWEAK antibody according to any one of (8) to (10).
(16) A method for producing an anti-human TWEAK antibody, comprising culturing the host cell according to (14) or (15) above and expressing the anti-human TWEAK antibody.
(17) An anti-human TWEAK antibody produced by the method according to (16) above.
(18) consisting of an amino acid sequence of amino acid numbers 1 to 447 of SEQ ID NO: 1, comprising a heavy chain in which glutamine of amino acid number 1 is modified with pyroglutamic acid, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3. Anti-human TWEAK antibody.
(19) A pharmaceutical composition comprising the anti-human TWEAK antibody according to any one of (1) to (10), (17) and (18) above and a pharmaceutically acceptable excipient.
(20) A pharmaceutical composition for preventing or treating systemic lupus erythematosus or lupus nephritis, comprising the anti-human TWEAK antibody according to any one of (1) to (10), (17) and (18) above.
(21) Preventing systemic lupus erythematosus or lupus nephritis comprising the step of administering a therapeutically effective amount of the anti-human TWEAK antibody according to any one of (1) to (10), (17) and (18) above Or a method for treating.
(22) The anti-human TWEAK antibody according to any one of (1) to (10), (17), and (18) for use in the prevention or treatment of systemic lupus erythematosus or lupus nephritis.
(23) Use of the anti-human TWEAK antibody according to any one of (1) to (10), (17) and (18) in the manufacture of a pharmaceutical composition for preventing or treating systemic lupus erythematosus or lupus nephritis .

The present invention provides an anti-human TWEAK antibody having superior activity against both human sTWEAK and human mTWEAK as compared with conventional anti-human TWEAK antibodies. The anti-human TWEAK antibody of the present invention has an action of inhibiting the functions of both human sTWEAK and human mTWEAK, and is useful for the prevention or treatment of various diseases in which human TWEAK is involved in pathogenesis. Such an anti-human TWEAK antibody of the present invention provides excellent improvement in clinical application such as reduction of dosage, expansion of administration interval, improvement of administration method (for example, subcutaneous injection), therapeutic efficacy and This greatly contributes to improving patient compliance.

Inhibitory activity against human mTWEAK-stimulated NF-κB activity. Inhibitory activity against human sTWEAK-stimulated MCP-1 expression. Inhibitory activity against human mTWEAK-stimulated MCP-1 expression.

Hereinafter, the present invention will be described in detail.

The basic structure of the antibody molecule is common to each class, and consists of a heavy chain with a molecular weight of 50,000 to 70,000 and a light chain with 20,000 to 30,000. The heavy chain is usually composed of a polypeptide chain containing about 440 amino acids, has a characteristic structure for each class, and corresponds to IgG, IgM, IgA, IgD, IgE, γ, μ, α, δ, ε. It is called a chain. Furthermore, IgG includes IgG1, IgG2, IgG3, and IgG4, and the heavy chains corresponding to them are called γ1, γ2, γ3, and γ4. The light chain is usually composed of a polypeptide chain containing about 220 amino acids, and two types of L-type and K-type are known and are called λ and κ chains, respectively. The peptide structure of the basic structure of an antibody molecule has a molecular weight of 150,000 to 190,000, in which two heavy chains and two light chains that are homologous are linked by a disulfide bond (SS bond) and a non-covalent bond, respectively. . The two light chains can be paired with any heavy chain. Each antibody molecule always consists of two identical light chains and two identical heavy chains.

There are four intrachain S—S bonds in the heavy chain (five and five in the ε chain) and two in the light chain, forming one loop for every 100 to 110 amino acids. The structure is similar between loops and is called a structural unit or domain. A domain located on the amino-terminal (N-terminal) side of both heavy chain and light chain is called a variable region because its amino acid sequence is not constant even if it is a specimen from the same class (subclass) of the same species. Each domain is called a heavy chain variable region (V _H ) and a light chain variable region (V _L ). The amino acid sequence on the carboxy terminus (C terminus) side of the variable region is almost constant for each class or subclass and is called a constant region.

The antigenic determinant site of an antibody is composed of V _H and V _L , and the specificity of binding depends on the amino acid sequence of this site. On the other hand, biological activities such as binding to complement and various cells reflect differences in the structure of the constant region of each class Ig. It has been found that the variability of the light chain and heavy chain variable regions is almost limited to the three small hypervariable regions present in both chains, these regions being complementarity determining regions (CDRs; each N-terminal). From the side, they are called CDR1, CDR2, CDR3). The remaining part of the variable region is called the framework region (FR) and is relatively constant.

<Anti-human TWEAK antibody of the present invention>
The anti-human TWEAK antibody of the present invention includes an anti-human TWEAK antibody having any of the following features 1) to 3).
1) An anti-human TWEAK antibody comprising a heavy chain variable region comprising the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 3.
2) An anti-human TWEAK antibody comprising a heavy chain variable region comprising the amino acid sequence of amino acid numbers 1 to 125 of SEQ ID NO: 5 and a light chain variable region comprising the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 7.
3) An anti-human TWEAK antibody comprising a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 9 and a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 11.

Preferably, the anti-human TWEAK antibody of the present invention has any of the characteristics 1) to 3) above and further comprises a heavy chain constant region and a light chain constant region. As the constant region, any subclass constant region (for example, a constant region of γ1, γ2, γ3 or γ4 as a heavy chain constant region and a constant region of λ or κ chain as a light chain constant region) may be selectable. However, the human Igγ1 constant region is preferred as the heavy chain constant region, and the human Igκ constant region is preferred as the light chain constant region.

Examples of the human Igγ1 constant region include a human Igγ1 constant region comprising the amino acid sequence of amino acid numbers 119 to 448 of SEQ ID NO: 1.

Examples of the human Igκ constant region include a human Igκ constant region consisting of the amino acid sequence from amino acid numbers 109 to 214 of SEQ ID NO: 3.

The anti-human TWEAK antibody of the present invention has the characteristics of any one of 1) to 3) above, wherein the heavy chain constant region is a human Igγ1 constant region and the light chain constant region is a human Igκ constant region. Human TWEAK antibody is more preferred.

In one embodiment, the anti-human TWEAK antibody of the present invention is an anti-human TWEAK antibody having any of the following characteristics i) to iii):
i) An anti-human TWEAK antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3.
ii) An anti-human TWEAK antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 5 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 7.
iii) An anti-human TWEAK antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 11.

It is known that when an antibody is expressed in cells, the antibody is modified after translation. Examples of post-translational modifications include cleavage of lysine at the C-terminus of the heavy chain by carboxypeptidase, modification of glutamine or glutamate at the heavy and light chain N-terminus to pyroglutamate, etc. It is known that the heavy chain C-terminal lysine is deleted and that most of the heavy chain N-terminal glutamine is modified with pyroglutamic acid (Journal of Pharmaceutical Sciences, 2008, Vol. 97, p. 2426). It is also known in the art that such post-translational modifications do not affect antibody activity (Analytical Biochemistry, 2006, Vol. 348, p. 24-39).

The antibody of the present invention includes not only an antibody having a full-length heavy chain, but also an antibody having a heavy chain lacking the C-terminal lysine, glutamine or glutamic acid at the N-terminal heavy chain modified to pyroglutamic acid by pyroglutamylation. Also included are antibodies that have been post-translationally modified upon expression in the cell, such as received antibodies.

For example, the anti-human TWEAK antibodies of the present invention described in i) to iii) include the anti-human TWEAK antibodies described in 1) to 3) below.
1) A heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein the glutamine of amino acid number 1 of SEQ ID NO: 1 is modified with pyroglutamic acid and / or lacks the lysine of amino acid number 448 of SEQ ID NO: 1 An anti-human TWEAK antibody comprising a chain and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3.
2) A heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 5, provided that the glutamic acid of amino acid number 1 of SEQ ID NO: 5 is modified to pyroglutamic acid and / or lacks the lysine of amino acid number 455 of SEQ ID NO: 5 An anti-human TWEAK antibody comprising a chain and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 7.
3) A heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 9, provided that glutamine of amino acid number 1 of SEQ ID NO: 9 is modified with pyroglutamic acid and / or lacks the lysine of amino acid number 451 of SEQ ID NO: 9 An anti-human TWEAK antibody comprising a chain and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 11.

The present invention also includes an anti-human TWEAK antibody having any of the following features 1) to 3).
1) CDR1 consisting of the amino acid sequence of SEQ ID NO: 1 from amino acid numbers 31 to 35, CDR2 consisting of the amino acid sequence of SEQ ID NO: 1 from amino acid numbers 50 to 66, and amino acid sequence of SEQ ID NO: 1 from amino acid numbers 99 to 107 A heavy chain variable region comprising CDR3 consisting of: CDR1, consisting of amino acid sequences of amino acid numbers 24 to 34 of SEQ ID NO: 3, CDR2 consisting of amino acid sequences of amino acid numbers 50 to 56 of SEQ ID NO: 3, and SEQ ID NO: An anti-human TWEAK antibody comprising a light chain variable region comprising CDR3 consisting of an amino acid sequence of 3 amino acid numbers 89 to 97.
2) CDR1 consisting of the amino acid sequence of SEQ ID NO: 5 from amino acid number 31 to 35, CDR2 consisting of the amino acid sequence of SEQ ID NO: 5 to amino acid number 50 to 66, and amino acid sequence of SEQ ID NO: 5 from amino acid number 99 to 114 A heavy chain variable region comprising CDR3 consisting of: CDR1, consisting of amino acid sequences of amino acid numbers 24 to 34 of SEQ ID NO: 7, CDR2 consisting of amino acid sequences of amino acid numbers 50 to 56 of SEQ ID NO: 7, and SEQ ID NO: An anti-human TWEAK antibody comprising a light chain variable region comprising CDR3 consisting of the amino acid sequence of amino acids Nos. 89 to 97 of 7.
3) CDR1 consisting of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 9, CDR2 consisting of the amino acid sequence of amino acid numbers 50 to 66 of SEQ ID NO: 9, and amino acid sequences of amino acid numbers 99 to 110 of SEQ ID NO: 9 A heavy chain variable region comprising CDR3 consisting of: CDR1, consisting of amino acid sequences of amino acid numbers 24 to 34 of SEQ ID NO: 11, CDR2 consisting of amino acid sequences of amino acid numbers 50 to 56 of SEQ ID NO: 11, and SEQ ID NO: An anti-human TWEAK antibody comprising a light chain variable region comprising CDR3 comprising the amino acid sequence of 11 amino acid numbers 89 to 97.

The anti-human TWEAK antibody of the present invention is an antibody that binds to human mTWEAK and human sTWEAK. Whether or not the antibody binds to human mTWEAK or human sTWEAK can be confirmed using a known binding activity measurement method. Examples of the method for measuring the binding activity include methods such as Enzyme-Linked ImmunoSorbant Assay (ELISA) and Fluorescence Activated Cell Sorting (FACS).

Measurement of the binding activity to human mTWEAK can be carried out using, for example, FACS. In an exemplary method, cells expressing human mTWEAK (consisting of the amino acid sequence shown in SEQ ID NO: 16) or human mTWEAK mutant (comprising the amino acid sequence shown in SEQ ID NO: 13) are separated (eg, HEK293 cells). Add the test antibody to the poured tube and allow to react. After the reaction, it is washed and reacted with a secondary antibody such as an anti-IgG antibody labeled with a fluorescent protein such as phycoerythrin (PE), and the fluorescence value is measured using BD FACSArray Bioanalyzer (BD Biosciences) or the like. Whether or not the test antibody binds to human mTWEAK can be confirmed.

Measurement of the binding activity to human sTWEAK can be performed using, for example, ELISA. In an exemplary method, human sTWEAK protein (Peprotech, 310-06) (SEQ ID NO: 15; methionine is added to the N-terminus of the amino acid sequence of amino acid numbers 97 to 249 of wild-type human mTWEAK shown in SEQ ID NO: 16. Is immobilized on an ELISA plate, and a test antibody is added thereto for reaction. After the reaction, a secondary antibody such as anti-IgG antibody labeled with an enzyme such as horseradish peroxidase (HRP) is reacted, washed, and then a reagent for detecting its activity (for example, in the case of HRP labeling, BM-Chemiluminescence ELISA Substrate). Whether or not the test antibody binds to human sTWEAK can be confirmed by measuring the activity using (POD) (Roche Diagnostics).

The anti-human TWEAK antibody of the present invention includes mTWEAK and sTWEAK derived from other animals in addition to binding to human mTWEAK and human sTWEAK as long as they bind to human mTWEAK and human sTWEAK (for example, monkey mTWEAK and monkey sTWEAK). ) Are also included.

Preferably, the anti-human TWEAK antibody of the present invention binds to human mTWEAK and human sTWEAK and has neutralizing activity against human mTWEAK and human sTWEAK. Neutralizing activity against human mTWEAK and human sTWEAK means an activity that inhibits any biological activity mediated by human TWEAK by binding to human mTWEAK and / or human sTWEAK, and one or more of human TWEAK Biological activity can be evaluated as an index. Examples of such neutralizing activity include NF-κB activation inhibitory activity by human TWEAK stimulation and MCP-1 expression inhibitory activity by human TWEAK stimulation. Specific methods for evaluating the activity are described below. Methods such as those described in Examples 11-14 can be used.

The anti-human TWEAK antibody of the present invention uses a method known in the art based on the sequence information of the heavy chain variable region and the light chain variable region of the anti-human TWEAK antibody of the present invention disclosed in the present specification. And can be easily produced by those skilled in the art. The anti-human TWEAK antibody of the present invention is not particularly limited, and can be produced, for example, according to the method described in <Method for producing anti-human TWEAK antibody of the present invention> described later.

The anti-human TWEAK antibody of the present invention is further purified if necessary and then formulated according to a conventional method, such as systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, psoriasis, scleroderma, etc. Prevention of diseases associated with pathogenesis of human TWEAK such as autoimmune diseases, stroke, arteriosclerosis, acute kidney injury due to ischemia, chronic renal failure, cancer, Duchenne muscular dystrophy, myotonic dystrophy, inclusion body myositis Can be used for treatment.

<Polynucleotide of the present invention>
The polynucleotide of the present invention comprises a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TWEAK antibody of the present invention, and a base sequence encoding the light chain variable region of the anti-human TWEAK antibody of the present invention. Polynucleotides to contain are included.

In one embodiment, the polynucleotide comprising the base sequence encoding the heavy chain variable region of the anti-human TWEAK antibody of the present invention encodes the heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 1. A polynucleotide comprising a nucleotide sequence encoding a heavy chain variable region comprising the amino acid sequence of amino acid numbers 1 to 125 of SEQ ID NO: 5, or an amino acid of amino acid numbers 1 to 121 of SEQ ID NO: 9 A polynucleotide comprising a base sequence encoding a heavy chain variable region comprising a sequence.

Examples of the polynucleotide containing the base sequence encoding the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 1 from amino acid Nos. 1 to 118 include, for example, a polynucleotide comprising the base sequence of SEQ ID NO: 2 from base Nos. 1 to 354 Nucleotides are mentioned. Examples of the polynucleotide containing the base sequence encoding the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 5 from amino acid Nos. 1 to 125 include, for example, the polynucleotide containing the base sequence of SEQ ID NO: 6 from base Nos. 1 to 375 Is mentioned. Examples of the polynucleotide containing the base sequence encoding the heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 9 include, for example, the polynucleotide containing the base sequence of base numbers 1 to 363 of SEQ ID NO: 10 Is mentioned.

In a preferred embodiment, the polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TWEAK antibody of the present invention is a polynucleotide comprising a base sequence encoding a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1, A polynucleotide containing a base sequence encoding a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 5 or a polynucleotide containing a base sequence encoding a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 9.

Examples of the polynucleotide containing the base sequence encoding the heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1 include the polynucleotide containing the base sequence shown in SEQ ID NO: 2. As a polynucleotide containing the base sequence which codes the heavy chain which consists of an amino acid sequence shown by sequence number 5, the polynucleotide containing the base sequence shown by sequence number 6 is mentioned, for example. As a polynucleotide containing the base sequence which codes the heavy chain which consists of an amino acid sequence shown by sequence number 9, the polynucleotide containing the base sequence shown by sequence number 10 is mentioned, for example.

In one embodiment, the polynucleotide comprising the base sequence encoding the light chain variable region of the anti-human TWEAK antibody of the present invention encodes the light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 3. A polynucleotide comprising a nucleotide sequence encoding a light chain variable region comprising the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 7, or an amino acid of amino acid numbers 1 to 108 of SEQ ID NO: 11 A polynucleotide comprising a nucleotide sequence encoding a light chain variable region comprising a sequence.

Examples of the polynucleotide containing the base sequence encoding the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 3 from amino acid number 1 to 108 include, for example, the polynucleotide comprising the base sequence of base number 1 to 324 of SEQ ID NO: 4 Is mentioned. Examples of the polynucleotide containing the base sequence encoding the light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 7 include, for example, the polynucleotide containing the base sequence of base numbers 1 to 324 of SEQ ID NO: 8 Is mentioned. Examples of the polynucleotide containing the nucleotide sequence encoding the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 11 from amino acid number 1 to 108 include, for example, the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 12 from base number 1 to 324 Is mentioned.

In a preferred embodiment, the polynucleotide comprising a base sequence encoding the light chain variable region of the anti-human TWEAK antibody of the present invention is a polynucleotide comprising a base sequence encoding a light chain consisting of the amino acid sequence represented by SEQ ID NO: 3, A polynucleotide containing a base sequence encoding a light chain consisting of the amino acid sequence shown in SEQ ID NO: 7 or a polynucleotide containing a base sequence encoding a light chain consisting of the amino acid sequence shown in SEQ ID NO: 11.

Examples of the polynucleotide containing the base sequence encoding the light chain consisting of the amino acid sequence shown in SEQ ID NO: 3 include the polynucleotide containing the base sequence shown in SEQ ID NO: 4. As a polynucleotide containing the base sequence which codes the light chain which consists of an amino acid sequence shown by sequence number 7, the polynucleotide containing the base sequence shown by sequence number 8 is mentioned, for example. As a polynucleotide containing the base sequence which codes the light chain which consists of an amino acid sequence shown by sequence number 11, the polynucleotide containing the base sequence shown by sequence number 12 is mentioned, for example.

The polynucleotide of the present invention can be easily prepared by those skilled in the art using a method known in the art based on the base sequence. For example, the polynucleotide of the present invention can be synthesized using gene synthesis methods known in the art. Examples of such gene synthesis methods include various methods known to those skilled in the art, such as antibody gene synthesis methods described in WO90 / 07861.

<Expression vector of the present invention, transformed host cell of the present invention, method for producing the anti-human TWEAK antibody of the present invention, and anti-human TWEAK antibody produced by the method>
The expression vector of the present invention comprises a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TWEAK antibody of the present invention and / or a base sequence encoding the light chain variable region of the anti-human TWEAK antibody of the present invention. An expression vector containing the polynucleotide is included.

Preferred expression vectors of the present invention include an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human TWEAK antibody of the present invention, and a base sequence encoding the light chain of the anti-human TWEAK antibody of the present invention. And an expression vector comprising a polynucleotide, or an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human TWEAK antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain of the antibody. .

Expression vectors used for expressing the polynucleotide of the present invention include various host cells such as eukaryotic cells (eg, animal cells, insect cells, plant cells, yeast) and / or prokaryotic cells (eg, E. coli). Expressing a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TWEAK antibody of the present invention and / or a polynucleotide comprising a base sequence encoding the light chain variable region of the anti-human TWEAK antibody of the present invention; As long as the polypeptide encoded by can be produced, it is not particularly limited. Examples of such expression vectors include plasmid vectors and viral vectors (eg, adenovirus, retrovirus), and preferably, pEE6.4 or pEE12.4 (Lonza) can be used. Alternatively, an antibody gene can be expressed by introducing a variable region gene fragment into an expression vector having a human Ig constant region gene in advance, such as AG-γ1 and AG-κ (for example, see WO94 / 20632).

The expression vector of the present invention may contain a promoter operably linked to the polynucleotide of the present invention. Examples of promoters for expressing the polynucleotide of the present invention in animal cells include virus-derived promoters such as CMV, RSV, SV40, actin promoter, EF (longation factor) 1α promoter, heat shock promoter, and the like. Examples of the promoter for expression in bacteria (for example, Escherichia) include trp promoter, lac promoter, λPL promoter, tac promoter, and the like. Examples of promoters for expression in yeast include GAL1 promoter, GAL10 promoter, PH05 promoter, PGK promoter, GAP promoter, and ADH promoter.

The transformed host cell of the present invention includes a host cell transformed with the expression vector of the present invention selected from the group consisting of the following (a) to (d).
(A) transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TWEAK antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody; Host cells;
(B) an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TWEAK antibody of the present invention and an expression vector comprising a polynucleotide comprising the base sequence encoding the light chain variable region of the antibody Transformed host cells;
(C) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TWEAK antibody of the present invention;
(D) A host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain variable region of the anti-human TWEAK antibody of the present invention.

In one embodiment, the transformed host cell of the present invention is a host cell transformed with the expression vector of the present invention selected from the group consisting of the following (a) to (d).
(A) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human TWEAK antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain of the antibody;
(B) transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human TWEAK antibody of the present invention and an expression vector comprising a polynucleotide comprising the base sequence encoding the light chain of the antibody Host cell;
(C) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human TWEAK antibody of the present invention;
(D) A host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain of the anti-human TWEAK antibody of the present invention.

When animal cells, insect cells, or yeast are used as host cells, the expression vector of the present invention may contain a start codon and a stop codon. In this case, the expression vector of the present invention comprises an enhancer sequence, the antibody of the present invention or the 5 ′ and 3 ′ untranslated regions of the gene encoding the heavy chain variable region or light chain variable region thereof, secretory signal sequence, splicing It may contain a junction, a polyadenylation site, or a replicable unit. When using Escherichia coli as a host cell, the expression vector of the present invention may contain a start codon, a stop codon, a terminator region, and a replicable unit. The expression vector of the present invention may contain a selection marker (for example, tetracycline resistance gene, ampicillin resistance gene, kanamycin resistance gene, neomycin resistance gene, dihydrofolate reductase gene) that is usually used depending on the purpose.

Preferred transformed host cells of the present invention include a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human TWEAK antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain of the antibody. A host cell transformed with an expression vector, and an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human TWEAK antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain of the antibody And a host cell transformed with an expression vector.

The host cell to be transformed is not particularly limited as long as it is compatible with the expression vector to be used and can be transformed with the expression vector to express the antibody. Examples of host cells to be transformed include various cells (for example, animal cells (for example, CHOK1SV cells), insect cells (for example, natural cells or artificially established cells) commonly used in the technical field of the present invention. Sf9), bacteria (such as Escherichia), yeast (such as Saccharomyces and Pichia)), and preferably, cultured cells such as CHOK1SV cells, CHO-DG44 cells, 293 cells, and NS0 cells are used. be able to.

The method for transforming the host cell is not particularly limited, and for example, a calcium phosphate method, an electroporation method, or the like can be used.

The method for producing an anti-human TWEAK antibody of the present invention includes a method for producing an anti-human TWEAK antibody, comprising culturing the transformed host cell of the present invention and expressing the anti-human TWEAK antibody. .

The anti-human TWEAK antibody of the present invention also includes an anti-human TWEAK antibody produced by the method for producing the anti-human TWEAK antibody of the present invention.

The method for producing the anti-human TWEAK antibody of the present invention is not particularly limited as long as it includes the step of culturing the transformed host cell of the present invention and expressing the anti-human TWEAK antibody. Preferred host cells used in the method include the above-described preferred transformed host cells of the present invention.

The cultured host cells can be cultured by a known method. The culture conditions such as temperature, medium pH, and culture time are appropriately selected. When the host cell is an animal cell, examples of the medium include MEM medium containing about 5 to 20% fetal bovine serum (Science, 1959, Vol. 130, No. 3373, p. 432-7), DMEM medium ( Virology, 1959, Vol. 8, p. 396), RPMI 1640 medium (J. Am. Med. Assoc., 1967, Vol. 199, p. 519), 199 medium (Exp. Biol. Med., 1950, Vol. 73, p.1-8) and the like can be used. The pH of the medium is preferably about 6 to 8, and the culture is usually carried out at about 30 to 40 ° C. for about 15 to 72 hours with aeration and stirring as necessary. When the host cell is an insect cell, for example, Grace's medium containing fetal bovine serum (Proc. Natl. Acad. Sci. USA, 1985, Vol. 82, p. 8404) can be used as the medium. . The pH of the medium is preferably about 5 to 8, and the culture is usually carried out at about 20 to 40 ° C. for about 15 to 100 hours with aeration and agitation as necessary. In the case where the host cell is Escherichia coli or yeast, as the medium, for example, a liquid medium containing a nutrient source is appropriate. The nutrient medium preferably contains a carbon source, inorganic nitrogen source or organic nitrogen source necessary for the growth of the transformed host cell. Examples of the carbon source include glucose, dextran, soluble starch, and sucrose. Examples of the inorganic nitrogen source or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, and meat extract. , Soybean meal, potato extract and the like. If desired, other nutrients (eg, inorganic salts (eg, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (eg, tetracycline, neomycin, ampicillin, kanamycin, etc.)) Good. The pH of the medium is preferably about 5-8. When the host cell is Escherichia coli, preferred media include LB medium, M9 medium (Mol. Clo., Cold Spring Harbor Laboratory, Vol. 3, A2.2) and the like. Cultivation is usually carried out at about 14 to 43 ° C. for about 3 to 24 hours with aeration or agitation as necessary. When the host cell is yeast, for example, a Burkholder minimum medium (Proc. Natl. Acad. Sci. USA, 1980, Vol. 77, p. 4505) can be used as the medium. Cultivation is usually carried out at about 20 to 35 ° C. for about 14 to 144 hours with aeration and agitation, if necessary. By culturing as described above, the anti-human TWEAK antibody of the present invention can be expressed.

The method for producing the anti-human TWEAK antibody of the present invention includes the step of culturing the transformed host cell of the present invention and expressing the anti-human TWEAK antibody, and further, anti-human TWEAK antibody from the transformed host cell. A step of recovering, preferably isolating or purifying the human TWEAK antibody can be included. Isolation or purification methods include, for example, methods using solubility such as salting out and solvent precipitation, methods using differences in molecular weight such as dialysis, ultrafiltration and gel filtration, ion exchange chromatography and hydroxylapatite chromatography. Methods that use charge such as chromatography, methods that use specific affinity such as affinity chromatography, methods that use hydrophobicity differences such as reversed-phase high-performance liquid chromatography, and isoelectric points such as isoelectric focusing The method of using the difference of these is mentioned. Preferably, the antibody accumulated in the culture supernatant can be purified by various chromatography, for example, column chromatography using a protein A column or a protein G column.

<Pharmaceutical composition of the present invention>
The pharmaceutical composition of the present invention includes a pharmaceutical composition comprising the anti-human TWEAK antibody of the present invention and a pharmaceutically acceptable excipient. The pharmaceutical composition of the present invention can be prepared by an ordinarily used method using an excipient usually used in the art, that is, a pharmaceutical excipient or a pharmaceutical carrier. Examples of dosage forms of these pharmaceutical compositions include parenteral agents such as injections and infusions, and can be administered by intravenous administration, subcutaneous administration, or the like. In the formulation, excipients, carriers, additives and the like corresponding to these dosage forms can be used within a pharmaceutically acceptable range.

The pharmaceutical composition of the present invention may contain a plurality of types of anti-human TWEAK antibodies of the present invention. For example, a heavy chain C-terminal lysine deletion antibody, an antibody that has undergone N-terminal post-translational modification, an antibody that has lost the heavy chain C-terminal lysine and has undergone N-terminal post-translational modification, and / or a heavy chain C-terminal lysine A pharmaceutical composition containing an antibody having N-terminal post-translational modification is also included in the present invention.

For example, the anti-human TWEAK antibody described in i) above (that is, an anti-human TWEAK antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3) The contained pharmaceutical composition of the present invention also includes a pharmaceutical composition containing two or more anti-human TWEAK antibodies among the following (1) to (4).
(1) An anti-human TWEAK antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 1 from amino acid numbers 1 to 447 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 3.
(2) An anti-human TWEAK antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein the glutamine of amino acid No. 1 is modified with pyroglutamic acid, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3.
(3) consisting of an amino acid sequence from amino acid numbers 1 to 447 of SEQ ID NO: 1, comprising a heavy chain in which glutamine of amino acid number 1 is modified with pyroglutamic acid, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3; Anti-human TWEAK antibody.
(4) An anti-human TWEAK antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3.
The same applies to the pharmaceutical composition of the present invention containing the anti-human TWEAK antibody described in ii) and iii).

The amount of the anti-human TWEAK antibody of the present invention added in the formulation varies depending on the degree and age of the patient's symptoms, the dosage form of the formulation to be used, the antibody binding titer, etc., for example, 0.001 mg / kg to 100 mg. / Kg or so can be used.

The pharmaceutical composition of the present invention can be used as a pharmaceutical composition for preventing or treating diseases in which human TWEAK is involved in pathogenesis, such as systemic lupus erythematosus or lupus nephritis.

The present invention includes a pharmaceutical composition for preventing or treating systemic lupus erythematosus or lupus nephritis comprising the anti-human TWEAK antibody of the present invention. The present invention also includes a method for preventing or treating systemic lupus erythematosus or lupus nephritis comprising the step of administering a therapeutically effective amount of the anti-human TWEAK antibody of the present invention. The present invention also includes the anti-human TWEAK antibody of the present invention for use in the prevention or treatment of systemic lupus erythematosus or lupus nephritis. The present invention also includes the use of the anti-human TWEAK antibody of the present invention in the manufacture of a pharmaceutical composition for preventing or treating systemic lupus erythematosus or lupus nephritis.

Although the present invention has been generally described, specific examples referred to for further understanding are provided herein for purposes of illustration and not limitation.

For parts using commercially available kits or reagents, experiments were conducted according to the attached protocol unless otherwise specified.

(Example 1: Acquisition of human mTWEAK-expressing HEK293 cells)
The present inventors obtained a human mTWEAK expression vector and a human mTWEAK expression cell. A human mTWEAK mutant gene (SEQ ID NO: 14 (base sequence encoding the amino acid sequence shown in SEQ ID NO: 13)) was recombined into a pcDNA3.1 vector (Life Technologies) which is a vector for mammalian cell expression (in this specification) And also referred to as “human mTWEAK expression vector”). This vector was introduced into a human kidney cell line HEK293 cell (ATCC: CRL-1573) using Fugene HD (Promega) as a transfection reagent. The human mTWEAK variant shown in SEQ ID NO: 13 is human mTWEAK from which the amino acid sequence (cleavage site by protease) of amino acid numbers 90 to 93 of wild-type human mTWEAK shown in SEQ ID NO: 16 is deleted. It has been confirmed that the TWEAK signal is activated even when the amino acid is deleted. The cells were selectively cultured in a Hygromycin-containing DMEM medium and then monocloned by the limiting dilution method. Thereafter, clones showing high protein expression were selected by flow cytometry measurement using an anti-human TWEAK antibody (PE-labeled CARL-1; BD).

(Example 2: Production of anti-human TWEAK antibody-producing hybridoma)
In order to obtain an anti-human TWEAK antibody, the present inventors produced an antibody using a human monoclonal antibody development technology “Velocimmune antibody technology: Regeneron (US Pat. No. 6,596,541)”. Verosimune mice were immunized with human sTWEAK protein (Peprotech, 310-06) and human mTWEAK-expressing HEK293 cells obtained in Example 1 together with an adjuvant that elicits an immune response. Mice were immunized several times, and an increase in blood antibody titer was confirmed, and final immunization was performed. According to a conventional method, the spleen and lymph nodes of the immunized mouse were removed, lymphocytes were collected, and this was cell-fused with mouse myeloma cells SP2 / 0 to prepare a hybridoma. A hybridoma limiting dilution sample was prepared and monocloned. About each clone, after expanding culture, the medium was changed to CD hybridoma medium (Life Technologies) which is a serum-free medium, and cultured for 5 days. The antibody was purified from the obtained culture supernatant using Protein G Purification kit (Generon).

(Example 3: Cell ELISA binding inhibition assay)
In order to measure the antigen-binding inhibitory activity of an antibody, the present inventors have compared human colon cancer cell line HT29 cells (ATCC: HTB-38) (human TWEAK receptor is endogenously expressed) with an antibody. The binding inhibition of sTWEAK protein was evaluated. HT29 cells were seeded at 1 × 10 ⁴ cells per well in a 384 well plate (Greiner). After removing the medium, 30 μL of 10 μL of biotin-labeled human sTWEAK protein (biotinylated Peprotech human sTWEAK protein; final concentration 200 ng / mL) and hybridoma culture supernatant were added. After incubating for 1 hour in a CO ₂ incubator set at 37 ° C., it was washed with a washing solution (HEPES buffer solution containing 0.1% bovine serum), and diluted 2000 times with a diluted solution (HEPES buffer solution containing 1% bovine serum). 30 μL of diluted horseradish peroxidase-labeled streptavidin (HRP-Streptavidin; Life Technologies) was added. The plate was incubated for 1 hour in a CO ₂ incubator set at 37 ° C., and then washed with a washing solution. POD (Roche), which is a chemiluminescence detection reagent, was added, and the amount of chemiluminescence was measured with an EnVision counter (Perkin Elmer).

Example 4: Antigen ELISA binding assay
We used an antigen ELISA to measure the antigen-specific binding activity of the antibody. Human sTWEAK protein (Peprotech) 0.5 μg / mL was immobilized on a 384 well plate (Nunc). After adding a blocking agent (Blocking One; Nacalai Tesque) and allowing to stand at room temperature for 1 hour, a washing solution (TBS-T: Tris buffer physiological saline (TBS) (0.05% Tween-20) (pH 7.4) ) And 30 μL of the hybridoma culture supernatant was added. Horseradish peroxidase-labeled goat anti-mouse Ig antibody that was incubated at room temperature for 1 hour, washed with a washing solution, and diluted 2000 times with a diluent (Blocking One diluted 2 times with a phosphate buffer) 30 μL of (HRP-goat anti-mouse Ig antibody; DAKO) was added. After incubating at room temperature for 1 hour, it was washed with a washing solution. POD (Roche), which is a chemiluminescence detection reagent, was added, and the amount of chemiluminescence was measured with an EnVision counter (Perkin Elmer).

(Example 5: Human sTWEAK-stimulated NF-κB activation inhibition assay)
From the stimulation of TWEAK, NF-κB is activated and the expression of chemokines such as MCP-1 is induced to induce an inflammatory response (Nat Rev Drug Discov. 2008; 7 (5): 411-425). The present inventors evaluated the neutralizing activity of an antibody against human sTWEAK using NF-κB activation inhibition as an index.

HEK293 cells (ATCC: CRL-1573) (which endogenously expresses the human TWEAK receptor) are cultured in DMEM medium (Life Technologies) so that they become 4 × 10 ⁶ cells / 10 cm dish 3 days before the experiment. Inoculated in a 10 cm dish (IWAKI) at 17 mL / dish, and 1 hour later, pGL4.32 [luc2P / NF-κB-RE / Hygro] Vector (Promega) was used with Fugene HD (Promega) Introduced. Thereby, the activation of NF-κB can be evaluated using the activity of luciferase as an index. The next day, the cells were collected and seeded at 30 μL with DMEM medium in a 384 well plate (Greiner) at 1 × 10 ⁴ cells / well. A 15 μL hybridoma-derived antibody solution (a solution obtained by purifying a hybridoma culture supernatant with a protein G column (Generon) and diluted with DMEM medium) was added, and the mixture was allowed to stand in a CO ₂ incubator set at 37 ° C. for 5 minutes. Furthermore, 15 μL of human sTWEAK protein (Peprotech) solution prepared in DMEM medium was added (final concentration 100 ng / mL), and cultured in a CO ₂ incubator set at 37 ° C. for 5 hours. After removing 20 μL of the culture supernatant, 40 μL of One-Glo Luciferase Assay solution (Promega) was added and stirred at room temperature for 10 minutes. Thereafter, the amount of chemiluminescence was measured with an EnVision counter (Perkin Elmer).

(Example 6: human mTWEAK-stimulated NF-κB activation inhibition assay)
Stimulation of TWEAK activates NF-κB to induce the expression of chemokines such as MCP-1 and elicit an inflammatory response, and the maximum activity of the transduction signal is higher in mTWEAK than sTWEAK ( J Immunol. 2010; 185 (3): 1593-1605, Br J Pharmacol. 2013; 170 (4): 748-764), the present inventors have used human mTWEAK as an indicator of inhibition of NF-κB activation. The neutralizing activity of the antibody against was evaluated.

HEK293 cells (ATCC: CRL-1573) were seeded in a 10 cm dish (IWAKI) at 17 mL / dish in DMEM medium (Life Technologies) so that 4 × 10 ⁶ cells / 10 cm dish were obtained 3 days before the experiment. After 1 hour, pGL4.32 [luc2P / NF-κB-RE / Hygro] Vector (Promega) was introduced into the cells using Fugene HD (Promega). Thereby, the activation of NF-κB can be evaluated using the activity of luciferase as an index. Further, 3 days before the experiment, HEK293 cells (ATCC: CRL-1573) were added to a 4 × 10 ⁶ cell / 10 cm dish in a DMEM medium (Life Technologies) in a 10 cm dish (IWAKI) at 17 mL / dish. One hour after seeding, the human mTWEAK expression vector prepared in Example 1 was introduced into cells using Fugene HD (Promega). The next day, HEK293 cells into which pGL4.32 [luc2P / NF-κB-RE / Hygro] Vector was introduced were collected and seeded at 30 μL in DMEM medium at 384 well plates (Greiner) at 1 × 10 ⁴ cells / well. A 15 μL hybridoma-derived antibody solution (a solution obtained by purifying a hybridoma culture supernatant with a protein G column (Generon) and diluted with DMEM medium) was added, and the mixture was allowed to stand in a CO ₂ incubator set at 37 ° C. for 5 minutes. Further, HEK293 cells into which the human mTWEAK expression vector was introduced were suspended in DMEM medium, added at 15 μL to 5 × 10 ⁴ cells / well, and cultured in a CO ₂ incubator set at 37 ° C. for 5 hours. After removing 20 μL of the culture supernatant, 40 μL of One-Glo Luciferase Assay solution (Promega) was added and stirred at room temperature for 10 minutes. Thereafter, the amount of chemiluminescence was measured with an EnVision counter (Perkin Elmer).

As a result of the evaluation of the antibodies in Examples 3, 4, 5, and 6, it was confirmed that antibodies named TBA3-25, TBA3-35, and TBA4-1 (chimeric antibodies) had high binding activity against human sTWEAK and human mTWEAK and It became clear that it had neutralizing activity.

Example 7: Antibody sequencing
For the antibodies identified by the aforementioned assay, we cloned genes encoding the heavy and light chains of the antibody from the hybridoma. RNA was extracted from each hybridoma, and cDNA was prepared using a cDNA amplification kit (SMARTer RACE cDNA Amplification kit; Clontech). PCR was then used to extend and amplify the heavy and light chain variable regions. The PCR product was directly sequenced with a sequencer (ABI PRISM 3100; Applied Biosystems). The PCR product was recombined into a PCR product subcloning vector such as pCR2.1-TOPO (Life Technologies), and then the gene sequence was analyzed and sequenced.

After the antibody sequencing, in order to improve the physical properties and stability of the antibody, the above-mentioned antibody TBA3-35 is introduced with amino acid mutations in the variable regions of its heavy chain and light chain to produce a modified antibody (TBA3- 35.1) was produced.

(Example 8: Production of fully human antibody)
The aforementioned antibody is an antibody in which the variable region is derived from human and the constant region is derived from mouse. Therefore, the present inventors constructed an expression vector containing both heavy chain and light chain genes using a GS vector (Lonza) to produce a fully human antibody. Specifically, a signal sequence (Nigel White et al., Protein Engineering 1987; 1 (6): 499-) on the 5 ′ side of the heavy chain variable region gene of each antibody of TBA3-25, TBA3-35.1 and TBA4-1. 505.) and a constant region gene of human Igγ1 (consisting of the nucleotide sequence from nucleotide numbers 355 to 1347 of SEQ ID NO: 2) to the 3 ′ side, and this heavy chain gene was connected to the GS vector pEE6. 4 was inserted. Further, a gene encoding a signal sequence (Nigel White et al., Supra) is located 5 ′ of the light chain variable region gene of each antibody, and a constant region gene of human κ chain (base number of SEQ ID NO: 4) is located 3 ′. The light chain genes were inserted into the GS vector pEE12.4.

The base sequence of the heavy chain of the produced fully human antibody of TBA3-25 (fully human TBA3-25) is SEQ ID NO: 2, the amino acid sequence encoded thereby is SEQ ID NO: 1, and the light chain base of the antibody The sequence is shown in SEQ ID NO: 4, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 3, respectively. The variable region of the heavy chain shown in SEQ ID NO: 1 consists of the amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 1, and the CDR1, CDR2, and CDR3 of the heavy chain are amino acid numbers 31 to 35 of SEQ ID NO: 1, respectively. It consists of an amino acid sequence from 50 to 66, 99 to 107. The variable region of the light chain shown in SEQ ID NO: 3 consists of the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 3, and the CDR1, CDR2, and CDR3 of the light chain are amino acid numbers 24 to 34 of SEQ ID NO: 1, respectively. It consists of an amino acid sequence from 50 to 56, 89 to 97.

The base sequence of the heavy chain of the produced TBA3-35.1 fully human antibody (fully human TBA3-35.1) is SEQ ID NO: 6, the amino acid sequence encoded thereby is SEQ ID NO: 5, The base sequence of the light chain is shown in SEQ ID NO: 8, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 7. The variable region of the heavy chain shown in SEQ ID NO: 5 consists of the amino acid sequence of amino acid numbers 1 to 125 of SEQ ID NO: 5, and the CDR1, CDR2, CDR3 of the heavy chain are amino acid numbers 31 to 35 of SEQ ID NO: 5, respectively. It consists of an amino acid sequence from 50 to 66, 99 to 114. The variable region of the light chain shown in SEQ ID NO: 7 consists of the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 7, and the CDR1, CDR2, and CDR3 of the light chain are amino acid numbers 24 to 34 of SEQ ID NO: 7, respectively. It consists of an amino acid sequence from 50 to 56, 89 to 97.

The base sequence of the heavy chain of the fully human antibody of TBA4-1 thus prepared (fully human TBA4-1) is shown in SEQ ID NO: 10, the amino acid sequence encoded thereby is shown in SEQ ID NO: 9, and the light chain base of the antibody The sequence is shown in SEQ ID NO: 12, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 11, respectively. The variable region of the heavy chain shown in SEQ ID NO: 9 consists of the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 9, and the CDR1, CDR2, and CDR3 of the heavy chain are amino acid numbers 31 to 35 of SEQ ID NO: 9, respectively. It consists of an amino acid sequence from 50 to 66, 99 to 110. The variable region of the light chain shown in SEQ ID NO: 11 consists of the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 11, and the CDR1, CDR2, and CDR3 of the light chain are amino acid numbers 24 to 34 of SEQ ID NO: 11, respectively. It consists of an amino acid sequence from 50 to 56, 89 to 97.

Using the above-described GS vector into which the heavy chain and light chain genes of each antibody were inserted, antibody expression was performed by two methods, transient expression and constitutive expression. For transient expression, both the heavy chain and light chain expression described above were applied to FreeStyle 293 cells (Life Technologies) cultured at about 1 × 10 ⁶ cells / mL in FreeStyle 293 Expression medium (Life Technologies). The vector was transfected with 293fectin (Life Technologies) as a transfection reagent and cultured for 7 days. Or, with respect to about 1 × 10 ⁷ of thousands of CHO-K1SV cells, both expression vectors of the above heavy and light chains were transfected using electroporation methods, CD-CHO medium (Life Technologies, Inc.) Incubated for 14 days. The culture supernatant was purified using protein A or protein G (GE Healthcare Japan) to obtain purified antibodies of each fully human antibody. For constitutive expression, the above-mentioned GS vector into which the heavy and light chain genes of each antibody were inserted was cleaved with NotI and PvuI, and Ligation-Convenience Kit (NIPPONGENE) or Ligation-high (TOYOBO) ) Was used to construct a GS vector in which both heavy chain and light chain genes were inserted. This expression vector encodes full-length heavy and light chains and glutamine synthetase, and antibodies were expressed by transfection in CHO-K1SV cells. The culture supernatant was purified with a protein A or protein G column (GE Healthcare Japan) to obtain a purified antibody of each fully human antibody. Analysis of the amino acid modification of purified fully human TBA3-25 revealed that most of the purified antibodies had heavy chain N-terminal glutamine modification to pyroglutamic acid and heavy chain C-terminal lysine deletion. .

(Example 9: Evaluation of binding activity of fully human antibody to human sTWEAK)
The present inventors evaluated the binding activity to human sTWEAK for each fully human antibody. In this example, huP2D10-2 (Patent Document 1) was used as a comparative antibody.

Human sTWEAK protein (Peprotech) was prepared to 0.5 μg / mL with phosphate buffered saline (PBS (−)) (phosphate buffered saline without calcium and magnesium), and 384 well plate (Nunc). ). 120 μl of a blocking agent (Blocking One; Nacalai Tesque) was added and allowed to stand at room temperature for 1 hour, and then washed with a washing solution (TBS-T (pH 7.4)). Prepare a dilution series of fully human antibody (fully human TBA3-25, fully human TBA3-35.1 or fully human TBA4-1) or huP2D10-2 with PBS (-) and add 30 μL (final concentration) Is 15 steps in the range of 80 nM to 0.0000003 nM). Horseradish peroxidase-labeled goat anti-mouse Ig antibody that was incubated at room temperature for 1 hour, washed with a washing solution, and diluted 2000 times with a diluent (Blocking One diluted 2 times with a phosphate buffer) 30 μL of (HRP-goat anti-mouse Ig antibody; DAKO) was added. After incubating at room temperature for 1 hour, it was washed with a washing solution. 30 μL of POD (Roche), which is a chemiluminescence detection reagent, was added, the chemiluminescence amount was measured with an EnVision counter (Perkin Elmer), and the EC50 value was calculated (Table 1).
Table 1: Binding activity of fully human antibodies to human sTWEAK

As a result, it is clear that all of fully human TBA3-25, fully human TBA3-35.1, and fully human TBA4-1 have a binding activity to human sTWEAK that is equal to or higher than that of comparative antibody huP2D10-2. It became.

(Example 10: Evaluation of binding activity of fully human antibody to human mTWEAK)
The present inventors evaluated the binding activity to human mTWEAK for each fully human antibody. In this example, huP2D10-2 was used as a comparative antibody.

Specifically, the EC50 value was calculated by a binding assay using a BD FACSArray Bioanalyzer (Becton Dickinson). Three days before the experiment, HEK293 cells (ATCC: CRL-1573) were seeded in a DMEM medium (Life Technologies) at 17 mL / dish in DMEM medium (Life Technologies) so as to be 4 × 10 ⁶ cells / 10 cm dish. One hour later, the human mTWEAK expression vector prepared in Example 1 was introduced into cells using Fugene HD (Promega). On the day of the experiment, HEK293 cells into which the human mTWEAK expression vector was introduced were dispensed at 1 × 10 ⁵ cells / well. Fully human antibodies (fully human TBA3-25, fully human TBA3-35.1 or fully human TBA4-1) with FACS buffer (phosphate buffer with 0.1% sodium azide and 10% bovine serum) ) Or a dilution series of huP2D10-2 was prepared and added at 30 μL (final concentrations ranging from 80 nM to 0.001355 nM in 11 steps). After incubation at 4 ° C. for 6 hours, 50 μL of PE-labeled goat anti-human IgG (PE-goat anti-human IgG antibody; Jackson) diluted 100-fold with FACS buffer was added at 4 ° C. Incubated for hours. Using a BD FACSArray Bioanalyzer, MFI (median fluorescence intensity) values were measured, and EC50 values were calculated (Table 2).
Table 2: Binding activity of fully human antibodies to human mTWEAK

As a result, it is clear that all of fully human TBA3-25, fully human TBA3-35.1, and fully human TBA4-1 have a binding activity for human mTWEAK that is equal to or higher than that of comparative antibody huP2D10-2. It became.

(Example 11: human sTWEAK-stimulated NF-κB activation inhibition assay)
The present inventors evaluated the neutralizing activity of the antibody against human sTWEAK using HEK293 cells for each fully human antibody. In this example, huP2D10-2 was used as a comparative antibody. In this example, instead of the hybridoma-derived antibody solution, each fully human antibody (fully human TBA3-25, fully human TBA3-35.1 or fully human TBA4-1) prepared in DMEM medium or huP2D10- The procedure was as described in Example 5, except that 15 μL of the dilution series 2 was added (7 steps with a final concentration ranging from 10000 ng / mL to 2.44 ng / mL).

After measuring the amount of chemiluminescence, IC50 values for human sTWEAK-stimulated NF-κB activity of each antibody were calculated (Table 3).
Table 3: Human sTWEAK-stimulated NF-κB activation inhibitory activity

As a result, all of fully human TBA3-25, fully human TBA3-35.1, and fully human TBA4-1 are higher than the comparative antibody huP2D10-2 with respect to human sTWEAK-stimulated NF-κB activity. It became clear that it has an inhibitory effect.

(Example 12: Human mTWEAK-stimulated NF-κB activation inhibition assay)
The present inventors evaluated the neutralizing activity of the antibody against human mTWEAK using HEK293 cells for each fully human antibody. In this example, huP2D10-2 was used as a comparative antibody. In this example, instead of the hybridoma-derived antibody solution, each fully human antibody (fully human TBA3-25, fully human TBA3-35.1 or fully human TBA4-1) prepared in DMEM medium or huP2D10- The procedure was as described in Example 6, except that 15 μL of the dilution series 2 was added (7 steps in the final concentration range of 2000 ng / mL to 0.48 ng / mL).

After measuring the amount of chemiluminescence, the IC50 value for the human mTWEAK stimulated NF-κB activity of each antibody was calculated (Table 4).
Table 4: Human mTWEAK-stimulated NF-κB activation inhibitory activity

As a result, as shown in Table 4 and FIG. 1, the comparative antibody huP2D10-2 only partially inhibited human mTWEAK-stimulated NF-κB activity, whereas fully human TBA3-25, fully human Both TBA3-35.1 and fully human TBA4-1 were found to completely inhibit human mTWEAK-stimulated NF-κB activity.

(Example 13: human sTWEAK-stimulated MCP-1 expression inhibition assay)
The present inventors evaluated the neutralizing activity of an antibody against human sTWEAK using inhibition of expression of the MCP-1 gene as an index. In this example, huP2D10-2 and TW305 humanized antibodies (Antibody 34 described in Table 6B of Patent Document 3) were used as comparative antibodies.

Human proximal tubule epithelial cell line HK-2 cells (ATCC: CRL-2190) (which endogenously expresses the human TWEAK receptor) were transferred to 1 × 10 ⁵ cells / well on the day of the experiment. A 96-well plate (IWAKI) was seeded at 120 μL / well in RPMI 1640 medium (Life Technologies). A dilution series of fully human TBA3-25, huP2D10-2, or Antibody34 was prepared in RPMI1640 medium, and 15 μL was added (6 steps in a final concentration range of 10000 ng / mL to 3.2 ng / mL). 15 μL of human sTWEAK protein (Peprotech) solution prepared in RPMI 1640 medium was added (final concentration 300 ng / mL), and cultured in a CO ₂ incubator set at 37 ° C. for 8 hours. After removing the culture supernatant, a reverse transcription product was prepared using a Cells-to-CT kit (Life Technologies). The amount of MCP-1 gene (ccl2) was measured for the reverse transcription product using Taqman Gene Expression system (Life Technologies) and Prism (Life Technologies). As an endogenous control, the amount of glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene was measured, and the amount of MCP-1 gene was corrected. Here, Hs00234140_m1 was used as a primer for ccl2, and Hs02758991_g1 (both Life Technologies) was used as a primer for gapdh.

After measuring the amount of MCP-1 gene, the IC50 value for human sTWEAK-stimulated MCP-1 expression of each antibody was calculated (Table 5).
Table 5: Human sTWEAK-stimulated MCP-1 expression inhibitory activity

As a result, as shown in Table 5 and FIG. 2, fully human TBA3-25 has a higher inhibitory effect on human sTWEAK-stimulated MCP-1 expression than comparative antibodies huP2D10-2 and Antibody34. Became clear.

(Example 14: Human mTWEAK-stimulated MCP-1 expression inhibition assay)
The present inventors evaluated the neutralizing activity of an antibody against human mTWEAK using inhibition of expression of the MCP-1 gene as an index. In this example, huP2D10-2 and Antibody34 were used as comparative antibodies.

Three days before the experiment, HEK293 cells (ATCC: CRL-1573) were seeded in a 10 cm dish (IWAKI) at 17 mL / dish in RPMI 1640 medium (Life Technologies) so that the concentration was 4 × 10 ⁶ cells / 10 cm dish. One hour later, the human mTWEAK expression vector prepared in Example 1 was introduced into cells using Fugene HD (Promega). HK-2 cells (ATCC: CRL-2190) were seeded on a 96-well plate (IWAKI) at 120 μL / well in RPMI 1640 medium (Life Technologies) at 1 × 10 ⁵ cells / well on the day of the experiment. did. A dilution series of fully human TBA3-25, huP2D10-2, or Antibody34 was prepared in RPMI1640 medium, and 15 μl was added (six concentrations in a final concentration range of 10000 ng / mL to 3.2 ng / mL). HEK293 cells introduced with the human mTWEAK expression vector were suspended in RPMI 1640 medium, added with ¹⁵ μL to 5 × 10 ⁵ cells / well, and cultured in a CO ₂ incubator set at 37 ° C. for 8 hours. After removing the culture supernatant, a reverse transcription product was prepared using a Cells-to-CT kit (Life Technologies). The amount of MCP-1 gene (ccl2) was measured for the reverse transcription product using Taqman Gene Expression system (Life Technologies) and Prism (Life Technologies). In addition, the amount of gapdh gene was measured as an endogenous control, and the amount of MCP-1 gene was corrected. Here, Hs00234140_m1 was used as a primer for ccl2, and Hs02758991_g1 (both Life Technologies) was used as a primer for gapdh.

After measuring the amount of MCP-1 gene, the IC50 value for human mTWEAK-stimulated MCP-1 expression of each antibody was calculated (Table 6).
Table 6: Human mTWEAK-stimulated MCP-1 expression inhibitory activity

As a result, as shown in Table 6 and FIG. 3, the comparative antibodies huP2D10-2 and Antibody34 only partially inhibited human mTWEAK-stimulated MCP-1 expression, whereas fully human TBA3-25 It was revealed that mTWEAK-stimulated MCP-1 expression was completely inhibited.

The anti-human TWEAK antibody of the present invention is useful for the prevention or treatment of various diseases in which human TWEAK is involved in pathogenesis.

The description of “Artificial Sequence” is described in the numerical heading <223> of the following sequence listing. Specifically, the base sequences represented by SEQ ID NOs: 2 and 4 in the sequence listing are the heavy and light chain base sequences of fully human TBA3-25, respectively, and the amino acid sequences represented by SEQ ID NOs: 1 and 3 are: The heavy and light chain amino acid sequences encoded by SEQ ID NOs: 2 and 4, respectively. The nucleotide sequences shown in SEQ ID NOs: 6 and 8 in the sequence listing are the heavy chain and light chain nucleotide sequences of fully human TBA3-35.1, respectively. The amino acid sequences shown in SEQ ID NOs: 5 and 7 are the sequences The heavy and light chain amino acid sequences encoded by numbers 6 and 8. The base sequences represented by SEQ ID NOs: 10 and 12 in the sequence listing are the heavy chain and light chain base sequences of fully human TBA4-1, respectively. The amino acid sequences represented by SEQ ID NOs: 9 and 11 are respectively represented by SEQ ID NO: 10 And the amino acid sequences of the heavy and light chains encoded by. The base sequence represented by SEQ ID NO: 14 in the sequence listing is the base sequence of the human mTWEAK variant, and the amino acid sequence represented by SEQ ID NO: 13 is the amino acid sequence of the human mTWEAK variant. The amino acid sequence shown in SEQ ID NO: 15 in the sequence listing is the amino acid sequence of the human sTWEAK protein used in this example.

Claims

An anti-human TWEAK antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1 from amino acid number 1 to 118 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 3 to amino acid number 1 to 108.
The anti-human TWEAK antibody according to claim 1, wherein the heavy chain constant region of the antibody is a human Igγ1 constant region.
The anti-human TWEAK antibody according to claim 1, wherein the light chain constant region of the antibody is a human Igκ constant region.
The anti-human TWEAK antibody according to claim 1, wherein the heavy chain constant region of the antibody is a human Igγ1 constant region, and the light chain constant region of the antibody is a human Igκ constant region.
The anti-human TWEAK antibody according to claim 1, comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3.
A polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TWEAK antibody according to claim 1.
A polynucleotide comprising a base sequence encoding the light chain variable region of the anti-human TWEAK antibody according to claim 1.
An expression vector comprising the polynucleotide according to claim 6 and / or 7.
A host cell transformed with the expression vector according to claim 8, which is selected from the group consisting of the following (a) to (d):
(A) an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TWEAK antibody according to claim 1 and a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody; Transformed host cells;
(B) an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TWEAK antibody of claim 1 and a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody A host cell transformed with an expression vector;
(C) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TWEAK antibody according to claim 1, and (d) the anti-cell according to claim 1. A host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain variable region of human TWEAK antibody.
A host cell transformed with the expression vector according to claim 8, which is selected from the group consisting of the following (a) to (d):
(A) transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human TWEAK antibody according to claim 5 and a polynucleotide comprising a base sequence encoding the light chain of the antibody Host cell;
(B) an expression vector comprising a polynucleotide comprising the base sequence encoding the heavy chain of the anti-human TWEAK antibody according to claim 5 and an expression vector comprising a polynucleotide comprising the base sequence encoding the light chain of the antibody Transformed host cells;
(C) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human TWEAK antibody according to claim 5; and (d) the anti-human TWEAK according to claim 5. A host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding an antibody light chain.
A method for producing an anti-human TWEAK antibody comprising culturing the host cell according to claim 9 or 10 and expressing the anti-human TWEAK antibody.
An anti-human TWEAK antibody produced by the method according to claim 11.
An anti-human TWEAK comprising an amino acid sequence of amino acid numbers 1 to 447 of SEQ ID NO: 1, comprising a heavy chain in which glutamine of amino acid number 1 is modified with pyroglutamic acid, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3 antibody.
A pharmaceutical composition comprising the anti-human TWEAK antibody according to claim 1, 12 or 13, and a pharmaceutically acceptable excipient.