JP2004121001A - HIGH-AFFINITY MONOCLONAL ANTIBODY TO TRANSFORMING GROWTH FACTOR-betaTYPE II RECEPTOR - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a high-affinity antihuman transforming growth factor (TGF)-β type II receptor monoclonal antibody useful for an efficient treatment of tissue fibrosis in various organs caused by actions of human TGF-β, diseases accompanied with the tissue fibrosis (nephrosclerosis, pulmonary fibrosis, hepatocirrhosis, etc.), rheumatoid arthritis, vascular restenosis and/or skin keloid, etc. <P>SOLUTION: Intensive studies made on the preparation of the monoclonal antibody to the human TGF-β type II receptor useful for the treatment of various diseases such as the tissue fibrosis led to success in obtaining of the monoclonal antibody having an ultrahigh binding affinity and an ultrahigh neutralizing activity for the human TGF-β type II receptor. <P>COPYRIGHT: (C)2004,JPO

Description

【０１３６】
「配列表フリーテキスト」
配列番号：３
他の情報：人工配列についての記載：人工的に合成したプライマー配列。
配列番号：４
他の情報：人工配列についての記載：人工的に合成したプローブ配列。
配列番号：５
他の情報：人工配列についての記載：人工的に合成したプローブ配列。
【０１３７】
【図面の簡単な説明】
【図１】ＴＧＦβ刺激により誘導されるメサンギウム細胞からのＰＡＩ−Ｉ分泌亢進に対する抗ヒトＴＧＦβヒトモノクローナル抗体の阻害活性を示す図。
【図２】ＵＵＯ処置されたサルの腎臓において腎不全症の進行に伴って起こるＴｙｐｅ　Ｉ　ｃｏｌｌａｇｅｎのｍＲＮＡの発現上昇に対する抗ヒトＴＧＦβヒトモノクローナル抗体の阻害活性を示す図。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention binds to a type II receptor of human transforming growth factor-β (Transforming Growth Factor-β; TGF-β) (hereinafter sometimes referred to as “TGF-β type II receptor” or “TβRII”). The present invention relates to a monoclonal antibody (particularly, a human monoclonal antibody) or a part thereof, a cell producing the monoclonal antibody, and a pharmaceutical composition comprising the monoclonal antibody.
[0002]
[Prior art]
In a search for growth factors of normal rat fibroblasts, two growth factors found as molecules that promote the transformation of normal cells were transformed growth factor-α (Transforming Growth Factor-α; TGF-α). And Transforming Growth Factor-β (TGF-β). Subsequent studies have shown that TGF-α is a member of the epidermal growth factor (EGF) family, while TGF-β is produced by most types of cells, and its receptors are also found in organs and cells. (Biol. Signals., Vol. 5, p. 232, 1996 and Pulmonary Fibrosis, Vol. 80 of Lung Biology in Health and Disease Series, ed. York, 1995). TGF-β has an activity of controlling cell differentiation and proliferation. The cell growth activity of TGF-β varies greatly depending on the type of cell (Roberts et al, The transforming growth factor-βs, In Peptide Growth Factors and Therbiter Repartners, Part. AB, Springer-Verlag, Berlin, 1990, 419-472). For example, while having an activity of promoting cell proliferation on mesenchymal cells such as fibroblasts and vascular smooth muscle cells, it has an activity on various types of cells such as epithelial cells, vascular endothelial cells, and blood cells. Was found to act as a factor that suppresses growth rather than promotes growth. Furthermore, it is clear that TGF-β has various functions such as not only control of cell proliferation but also control of the immune system and promotion of accumulation of extracellular matrix (ECM; Extracellular Matrix) such as collagen, fibronectin and tenisin. (Adv. Immunol., Vol. 55, p. 181, 1994 and Seminars in Cell Biol., Vol. 5, p. 389, 1994).
[0003]
Recently, it has been revealed that the mechanism of this versatile function of TGF-β stems from the specificity of TGF-β receptor structure and signaling.
TGF-β is a protein having a molecular weight of about 25 kD, and forms a dimer with two peptides. There are five isoforms of TGF-β, TGF-β1, TGF-β2 and TGF-β3 in mammals, TGF-β4 in chickens, and TGF-β5 in frogs. These isoforms have about 70% homology to each other.
[0004]
Of TGF-β, the function of TGF-β1 has been particularly analyzed. TGF-β1 plays a very important role in the healing process of wounds in living tissues (New Engl. J. Med., Vol. 331, p. 1286, 1994 and J. Cell. Biol., Vol. 119, p. .1017, 1992). At the wound site of the tissue, a rapid and dynamic biological reaction such as infiltration of inflammatory cells and fibroblasts, proliferation and angiogenesis of ECM, and cell proliferation for tissue regeneration occurs, and the wound is repaired.
[0005]
At the time of wounding, bleeding occurs first, and then TGF-β and platelet-derived growth factor (PDGF) are produced from platelets, and inactive TGF-β bound to ECM at the wound site is activated. The cells that have migrated or the cells at the wound site are exposed to the high concentration of TGF-β thus produced, so that FGF (fibroblast growth factor), TNF (Tumor necrosis factor) and IL-1 (Interleukin-1). ) Secrete growth factors and cytokines, and fibroblasts undergo ECM synthesis and secretion.
Furthermore, for example, in fibroblasts and mesenchymal cells, they are also referred to as platelet-derived growth factor (PDGF) or connective tissue growth factor (Connective Tissue Growth Factor (CTGF); Hcs24. J. Cell Biology, J. Cell Biology. Vol.114, No.6, p.1285-1294, 1991; Int.J.Biochem.Cell Biol., Vol.29, No.1, p.153-161, 1997; Circulation, Vol.95, No.95. 4, p.831-839, 1997; Cell Growth Differ., Vol.7, No.4, p.469-480, 1996; J. Invest. Dermatol., Vol. J. Invest. Dermatol., Vol. 105, No. 2, p. 280-284, 1995; J. Invest. Dermatol., Vol. 105, No. 106, No. 4, p. 1, p.128-132, 1995; and International Patent Application Publication No. WO 96/38172) and increased production of fibronectin.
TGF-β1 further suppresses the production of proteolytic enzymes, increases its enzyme inhibitors, promotes the synthesis of integrins involved in the adhesion of ECM to cells, promotes the growth and deposition of ECM, and repairs wounds. Is thought to do. In addition, TGF-β also has an immunosuppressive effect by suppressing the functions of T lymphocytes and B lymphocytes and inhibiting the synthesis of TNF and IL-1.
[0006]
Although the mechanism of the regulation of TGF-β expression is still unknown, it is thought that the expression of TGF-β is regulated by binding to the ECM proteoglycan (Nature, Vol. 346). , P. 281, 1990 and Nature, Vol. 360, p. 361, 1992). That is, it is considered that TGF-β is negatively controlled by the ECM promoted by TGF-β itself, and suppresses the overexpression of TGF-β together with the healing of the wound. Therefore, it is considered that when an abnormality occurs in the negative control, TGF-β is overexpressed and a pathological state such as tissue fibrosis (fibrosis) occurs.
[0007]
On the other hand, in pulmonary fibrosis and renal sclerosis, TGF-β is maintained at a high concentration even though the deposition of ECM has sufficiently occurred, and such a pathological condition such as fibrosis may occur. Further proceed (Kidney Int., Vol. 45, p. 916, 1994 and J. Clin. Invest., Vol. 92, p. 632, 1993). In pulmonary fibrosis or renal sclerosis, whether the signal of TGF-β expression is always on due to the continuous tissue damage, or whether the negative control signal of TGF-β expression as described above does not enter , Or both are presumed to work synergistically.
[0008]
Renal sclerosis is the final picture of many renal diseases, such as chronic glomerulonephritis and diabetic nephropathy, and is characterized by mesangial cell proliferation and increased ECM. Abnormal expression of TGF-β has been observed in kidneys with renal sclerosis (J. Clin. Invest., Vol. 90, p. 1, 1992 and Proc. Natl. Acad. Sci. USA, Vol. 86). , P. 1056, 1989). In an experimental nephritis model induced by anti-Thy-1 antibody, administration of anti-TGF-β antibody was shown to suppress the progression of nephritis, indicating that TGF-β is involved in the pathogenesis of renal sclerosis. Has been suggested (Nature, Vol. 346, p. 371, 1990).
[0009]
On the other hand, in the lung, TGF-β is expressed at a high level in pulmonary fibrosis and idiopathic embryonic fibrosis induced by bleomycin administration, suggesting that TGF-β is involved in the formation of pulmonary fibrosis. Have been.
Moreover, in biopsy tissues of patients with chronic hepatitis or cirrhosis, the expression of TGF-β has been observed at the collagen deposition site. In addition, ECM deposition and TGF-β expression have been reported in arthritis such as vascular restenosis, rheumatoid arthritis, and skin keloids.
[0010]
From these facts, it has been suggested that TGF-β may be involved in the pathogenesis of fibrosis (fibrosis) in various tissues, and the function of TGF-β is suppressed by antisense drugs or gene therapy. Thus, attempts to treat tissue fibrosis have been made experimentally (Kidney Int., Vol. 50, p. 148, 1996).
The above-mentioned expression of various functions of TGF-β and formation of various pathological conditions such as tissue fibrosis caused by TGF-β are caused by transmission of a signal into a cell by binding of TGF-β to a TGF-β receptor. Is started by
[0011]
As a receptor for TGF-β in mammals such as humans and rats, a type I receptor (molecular weight: about 53 kD; GenBank Accession No: L11695; Cell, Vol. 75, No. 4, p. 681, 1993) A TGF-β type I receptor (TGF-β Type I receptor) or “Tβ RI”; a type II receptor (molecular weight of about 70 kD; GenBank Accession No: M85079; Cell, Vol. 68, No. , P.775, 1992; hereinafter also referred to as “TGF-βII type receptor” (TGF-βTypeII receptor) or “TβRII”) and type III receptor (molecular weight of about 200-300 kD; GenBank Accession No. : L0 Cell, Vol.67, No. 4, p.785, 1991; Cell, Vol.67, No.4, p.797, 1991; and Biochem.Biophys.Res.Commun., Vol.189, No. 594; 1, p. 356, 1992; hereinafter, may be referred to as “TGF-β type III receptor” (TGF-β Type III receptor) or “TβRIII”. Adv. Imm., Vol. 55, p. 181, 1994).
[0012]
Regarding the role of these receptors, functional analysis of these receptors using artificially established mutants of the TGF-β resistant mink lung epithelial cell line Mv1Lu revealed TGF-β type I receptor and TGF-β type II receptor have been shown to be important and essential for TGF-β signal transduction (J. Biol. Chem., Vol. 265, p. 18518, 1990). And J. Biol. Chem., Vol.266, p.9108, 1991). On the other hand, the TGF-β type III receptor is not essential for TGF-β signal transduction, but is thought to play an indirect role.
[0013]
The TGF-β type III receptor is a transmembrane protein consisting of 849 amino acids, and consists of an extracellular domain (761 amino acids), a transmembrane domain (24 amino acids) and an intracellular domain (43 amino acids). Be composed. The intracellular region of the TGF-β type III receptor is rich in serine (Ser) / threonine (Thr) residues, accounting for over 40%.
TGF-β type I receptor is a transmembrane protein consisting of 503 amino acids, and consists of an extracellular domain (101 amino acids), a transmembrane domain (22 amino acids) and an intracellular domain (356 amino acids). Be composed.
[0014]
The TGF-β type II receptor is also a transmembrane protein consisting of 567 amino acids, including a signal sequence (23 amino acids), an extracellular region (136 amino acids), and a cell transmembrane region (30 amino acids) And an intracellular region (378 amino acids).
The TGF-β type I receptor and TGF-β type II receptor are single-transmembrane proteins having a relatively short extracellular region and a serine / threonine kinase structure having two kinase insertion sites. In addition, an aspartic acid (Asp) -linked sugar chain is bound to the intracellular region, and 10 cysteine (Cys) residues are found in the TGF-βI type receptor and 12 in the TGF-βII type receptor. It has a characteristic protein tertiary structure.
[0015]
In the intracellular region of the TGF-β type I receptor, a region rich in glycine (Gly), serine (Ser) and threonine (Thr) called a GS region is found adjacent to the kinase region. The TGF-β type II receptor has a similar structure to the TGF-β type I receptor, but does not have a GS region.
The kinase regions of the TGF-β type I and TGF-β type II receptors have about 43% homology, and both have been shown to be serine / threonine-specific protein kinases. It has been clarified that both serine / threonine kinase domains need to act in order for β signals to be transmitted into cells (J. Biol. Chem., Vol. 269, p. 30753, 1994).
[0016]
For transmission of TGF-β signal into cells, TGF-β type I receptor and TGF-β type II receptor are essential. The TGF-β type II receptor alone binds to TGF-β, but the TGF-β type I receptor alone does not bind to TGF-β. For transmission of a TGF-β signal into a cell, it is important that both the TGF-β type I receptor and the TGF-β type II receptor form a complex on the cell surface.
[0017]
When TGF-β binds to the TGF-β type II receptor, the TGF-β type II receptor has been shown to form a heterotetrameric complex with the TGF-β type I receptor on the cell surface (J. Biol. Chem., Vol.269, p.20172, 1994). That is, the TGF-β type I receptor acts as a substrate for the TGF-β type II receptor, and when both form a complex in the presence of TGF-β, the TGF-β type II receptor becomes a TGF-β type I receptor. It is thought that phosphorylation of the GS region activates the TGF-β type I receptor, and consequently phosphorylation of other intracellular substrates leads to further transmission of TGF-β signals. (Nature, Vol. 370, p. 341, 1994).
[0018]
Although the details of the pathway of transmission of the TGF-β signal to the downstream of the TGF-β type I receptor still remain unclear, recently, TGF-β located downstream of the TGF-β type Signaling molecules (Nature, Vol. 381, p. 620, 1996; Cell, Vol. 86, p. 543), consisting of eight isoforms that mediate β signaling, and are collectively referred to as Smads (S Mothers against Dpp). Nature, 383, p. 168, 1996; and Nature, Vol. 383, p. 832, 1996), and a signaling molecule (Science, Vol. 270) called TAK1 (TGF-β activated kinase-1). , P. 2008, 1995). There. Smad2 and Smad3 bind to the TGF-β type I receptor activated by forming a complex with the TGF-β type II receptor, and are phosphorylated by the kinase of the TGF-β type II receptor. It has been shown that it separates from the type II receptor, forms a complex with Smad4 (DPC4), and translocates into the nucleus (Cell, Vol. 87, p. 1215, 1996 and EMBO J., Vol. 16). , No. 17, 1997). It is believed that the Smad2 / Smad4 or Smad3 / Smad4 complex that has translocated into the nucleus binds to a DNA-binding protein (transcription factor) and acts as a transcription activation factor to regulate gene expression (Nature, Vol. 383, p. 832, 1996 and Nature, Vol. 390, p. 465, 1997).
TAK1 has been shown to be involved in TGF-β signaling by acting as a MAPKKK on the MAP kinase cascade.
[0019]
As described above, TGF-β is deeply involved in the formation of various tissue fibrosis such as renal sclerosis, pulmonary fibrosis and cirrhosis, and also various pathologies such as chronic hepatitis, rheumatoid arthritis, vascular restenosis and skin keloids. Thus, it is considered that the formation of these pathological conditions is caused by transmission of TGF-β signal into cells via TGF-β receptor.
Therefore, it is suggested that by controlling, especially suppressing the transmission of TGF-β signal into cells, it is possible to treat or prevent those pathological conditions.
Regarding attempts to treat those conditions by suppressing TGF-β signal transduction, target TGF-β and suppress the function of TGF-β using an antibody against TGF-β or antisense to the TGF-β gene. Attempts have been made to suppress disease states such as nephritis.
[0020]
However, TGF-β receptor, which is a binding partner of TGF-β, suppresses the function of TGF-βII type receptor, in other words, targets TGF-βII type receptor, and targets TGF-βII type receptor. Almost no reports have been made on the treatment of diseases by inhibiting the signaling of TGF-β into cells.
[0021]
Under these circumstances, the present inventors have intensively studied a human monoclonal antibody against human TGF-β type II receptor, succeeded in producing various human monoclonal antibodies binding to human TGF-β type II receptor, It is the first in the world that the human monoclonal antibody inhibits signal transduction of TGF-β into cells via TGF-β type II receptor by in vitro and in vivo tests, and is useful for treating renal diseases such as nephritis. It was reported for the first time (Kidney Int., Vol. 60, No. 5, p. 1745-55, 2001; and International Application Publication WO 01-36642).
[0022]
[Problems to be solved by the invention]
In the treatment of disease using a monoclonal antibody, one of the important factors is the high binding affinity of the monoclonal antibody to the target antigen and the strength of the neutralizing activity of the activity of the target antigen.
Therefore, various diseases (for example, various tissue fibrosis such as renal sclerosis, pulmonary fibrosis and cirrhosis, etc.) using monoclonal antibodies to human TGF-β type II receptor (especially human monoclonal antibodies) aimed at by the present inventors, Efficient treatment or prevention of chronic hepatitis, rheumatoid arthritis, vascular restenosis, skin keloids, etc. (Efficiency in various aspects: medical, pharmaceutical, manufacturing technical, economic, social) For example, it is preferable to prepare an anti-TGF-β type II receptor monoclonal antibody having the highest possible antigen affinity and antigen-neutralizing activity.
[0023]
That is, the present invention provides an anti-TGF-β type II receptor monoclonal antibody (particularly a human monoclonal antibody) having high affinity / high neutralization activity, which enables efficient treatment and prevention of such various diseases. An object of the present invention is to provide a pharmaceutical composition comprising a monoclonal antibody and a method for treating the disease using the same.
[0024]
[Means for Solving the Problems]
The present inventors have conducted intensive studies on a human monoclonal antibody against the human TGF-β type II receptor to achieve the above-mentioned object. As a result, the present inventors have found that extremely high binding affinity for the human TGF-β type II receptor and It was the first in the world to succeed in producing a monoclonal antibody having a high neutralizing activity.
[0025]
Furthermore, the monoclonal antibody of the present invention not only significantly inhibits TGF-β signaling through TGF-β type II receptor into cells, but also inhibits various diseases (eg, renal diseases (eg, nephritis, renal fibrosis, Disease, renal sclerosis, etc.), various tissue fibrosis such as renal fibrosis and pulmonary fibrosis, cirrhosis, rheumatoid arthritis, cutaneous keloid, etc.) and have completed the present invention. .
[0026]
A human monoclonal antibody, which is one embodiment of the monoclonal antibody included in the present invention, is a human-derived antibody, and thus poses a major problem in the treatment of an antibody drug composed of an antibody derived from a non-human mammal such as a mouse-derived antibody. The antigenicity to humans, which was a point (side effect), that is, no serious immune rejection of the host caused by HAMA (Human anti-mouse antigenicity) was caused at all, so the value of the antibody as a drug was dramatically increased. It is to let.
[0027]
The pharmaceutical composition comprising the monoclonal antibody or a part thereof of the present invention can be used for various diseases caused by the action of TGF-β, for example, renal diseases (eg, renal fibrosis, nephritis, renal failure, and renal sclerosis). , Lung diseases (eg, pulmonary fibrosis, pneumonia, etc.), liver diseases (eg, liver tissue fibrosis, cirrhosis, hepatitis, etc.), skin diseases (eg, wounds, scleroderma, psoriasis, keloids, etc.), arthritis ( For example, rheumatoid arthritis, osteoarthritis, etc.), vascular diseases (eg, vascular restenosis, rheumatic vasculitis, etc.), tissue fibrosis in various organs (including tissue fibrosis complicated by various cancers), It is useful as a pharmaceutical for suppressing or preventing the onset and / or progress of arteriosclerosis (including concurrent tissue fibrosis) and treating or preventing the disease.
[0028]
That is, the invention is as described in the following (1) to (34) of the invention.
(1) A monoclonal antibody that binds to a human TGF-β type II receptor, wherein the DNA of the V region encoding the variable region of the heavy chain is derived from the human immunoglobulin heavy chain V gene segment 3-23. Or a part thereof.
(2) A monoclonal antibody that binds to a human TGF-β type II receptor, wherein the DNA of the V region encoding the variable region of the light chain is derived from the human immunoglobulin light chain V gene segment A30. Monoclonal antibodies or parts thereof.
(3) a monoclonal antibody that binds to a human TGF-β type II receptor, wherein the DNA of the V region encoding the variable region of the heavy chain is derived from the human immunoglobulin heavy chain V gene segment 3-23, and A monoclonal antibody or a part thereof, wherein the DNA of the V region encoding the variable region of the light chain is derived from the human immunoglobulin light chain V gene segment A30.
(4) A monoclonal antibody which binds to a human TGF-β type II receptor, wherein the variable region of the heavy chain comprises the following amino acid sequence (a) or (b), or a monoclonal antibody or a part thereof: :
(A) the amino acid sequence of amino acids 2 to 115 of SEQ ID NO: 6;
(B) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of amino acids 2 to 115 of SEQ ID NO: 6.
(5) A monoclonal antibody which binds to a human TGF-β type II receptor, wherein the variable region of the light chain comprises the following amino acid sequence of (c) or (d), or a monoclonal antibody thereof. :
(C) the amino acid sequence of amino acids 2 to 117 of SEQ ID NO: 8;
(D) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 2 to 117 of SEQ ID NO: 8.
(6) A monoclonal antibody that binds to a human TGF-β type II receptor, wherein the heavy chain variable region comprises the following amino acid sequence (a) or (b), and the light chain variable region comprises the following ( A monoclonal antibody or a part thereof comprising the amino acid sequence of c) or (d):
(A) the amino acid sequence of amino acids 2 to 115 of SEQ ID NO: 6;
(B) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 2 to 115 of SEQ ID NO: 6;
(C) the amino acid sequence of amino acids 2 to 117 of SEQ ID NO: 8;
(D) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 2 to 117 of SEQ ID NO: 8.
(7) A monoclonal antibody that binds to a human TGF-β type II receptor, wherein the variable region of the heavy chain has the following amino acid sequence (a) or (b), or a monoclonal antibody or a part thereof: :
(A) the amino acid sequence of amino acids 2 to 458 of SEQ ID NO: 6;
(B) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acid Nos. 2 to 458 of SEQ ID NO: 6.
(8) A monoclonal antibody that binds to a human TGF-β type II receptor, wherein the variable region of the light chain has the following amino acid sequence (c) or (d), or a part thereof: :
(C) the amino acid sequence of amino acids 2 to 236 of SEQ ID NO: 8;
(D) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 2 to 236 of SEQ ID NO: 8.
(9) A monoclonal antibody which binds to a human TGF-β type II receptor, wherein the variable region of the heavy chain has the following amino acid sequence (a) or (b), and the variable region of the light chain is A monoclonal antibody having the amino acid sequence of (c) or (d) or a part thereof:
(A) the amino acid sequence of amino acids 2 to 458 of SEQ ID NO: 6;
(B) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 2 to 458 of SEQ ID NO: 6;
(C) the amino acid sequence of amino acids 2 to 236 of SEQ ID NO: 8;
(D) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 2 to 236 of SEQ ID NO: 8.
(10) A monoclonal antibody that binds to a human TGF-β type II receptor, and has a dissociation constant (Kd) of 10 × 10 ^-10 (M) A monoclonal antibody or a part thereof having a smaller numerical value.
(11) The monoclonal antibody or a part thereof according to any one of (1) to (10), wherein the monoclonal antibody is a human monoclonal antibody.
(12) A cell that produces the monoclonal antibody according to any one of (1) to (11).
(13) The cell according to (12), wherein the cell is a fused cell obtained by fusing a mammalian B cell with a mammalian myeloma cell for the ability to produce the human monoclonal antibody. The cells as described.
(14) a gene transformed by introducing the DNA into the cell, either the DNA encoding the heavy chain of the monoclonal antibody or the DNA encoding the light chain thereof, or both DNAs; The cell according to the above (12), which is a recombinant cell.
(15) A pharmaceutical composition comprising the monoclonal antibody or a part thereof according to any of (1) to (11), and a pharmaceutically acceptable carrier.
(16) The above (15), wherein the pharmaceutical composition is used for inhibiting transmission of a signal generated by binding of human TGF-β to human TGF-β type II receptor into cells. 8. The pharmaceutical composition according to item 1.
(17) The pharmaceutical composition according to (15), wherein the pharmaceutical composition is used for suppressing tissue fibrosis.
(18) The pharmaceutical composition according to (17), wherein the fibrosis of the tissue is fibrosis in lung, liver, kidney or skin.
(19) The pharmaceutical composition according to (17), wherein the fibrosis of the tissue is fibrosis in the kidney.
(20) The pharmaceutical composition according to (15), wherein the pharmaceutical composition is used for treating or preventing a kidney disease.
(21) The pharmaceutical composition is used for suppressing or treating renal sclerosis, pulmonary fibrosis, cirrhosis, vascular restenosis, arteriosclerosis, psoriasis, scleroderma, atopy, keloid or arthritis. The pharmaceutical composition according to the above (15), wherein
(22) A polypeptide comprising the amino acid sequence of amino acids 2 to 115 of SEQ ID NO: 6.
(23) A polypeptide having the amino acid sequence of amino acids 2 to 458 of SEQ ID NO: 6.
(24) An immunoglobulin heavy-chain polypeptide having the amino acid sequence of amino acid Nos. 2 to 458 of SEQ ID NO: 6 in which one or several amino acids have been deleted, substituted, inserted, or added.
(25) A polypeptide comprising the amino acid sequence of amino acids 2 to 117 of SEQ ID NO: 8.
(26) a polypeptide having the amino acid sequence of amino acids 2 to 236 of SEQ ID NO: 8;
(27) An immunoglobulin light chain polypeptide having an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of amino acids 2 to 236 of SEQ ID NO: 8.
(28) A DNA encoding the polypeptide according to any of (22) to (27).
(29) A DNA comprising the nucleotide sequence of base numbers 96 to 440 of SEQ ID NO: 7.
(30) a DNA comprising the nucleotide sequence of base numbers 96 to 1472 of SEQ ID NO: 7;
(31) a DNA comprising the nucleotide sequence of base numbers 39 to 389 of SEQ ID NO: 9;
(32) a DNA comprising the nucleotide sequence of base numbers 39 to 749 of SEQ ID NO: 9;
(33) A vector comprising the DNA according to any of (29) to (32).
(34) A cell transformed with a vector containing the DNA according to any of (29) to (32).
[0029]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail by clarifying the meanings of phrases used in the present invention.
The `` mammal '' in the present invention means human, cow, goat, rabbit, mouse, rat, hamster, guinea pig, and the like, preferably human, rabbit, rat, hamster or mouse, and particularly preferably, A human, rat, hamster or mouse.
The terms “non-human mammal” and “non-human mammal” used in the present invention are synonymous with each other, and refer to any mammal other than a human in the above-defined mammals.
[0030]
The term `` amino acid '' used in the present invention means any naturally occurring amino acid, and is preferably represented as follows according to the three-letter or single-letter notation of the alphabet used to represent amino acids. Amino acids.
Glycine (Gly / G), alanine (Ala / A), valine (Val / V), leucine (Leu / L), isoleucine (Ile / I), serine (Ser / S), threonine (Thr / T), asparagine Acid (Asp / D), glutamic acid (Glu / E), asparagine (Asn / N), glutamine (Glu / Q), lysine (Lys / K), arginine (Arg / R), cysteine (Cys / C), methionine (Met / M), phenylalanine (Phe / F), tyrosine (Tyr / Y), tryptophan (Trp / W), histidine (His / H), proline (Pro / P).
[0031]
As used herein, the term “human TGF-β type II receptor” (sometimes referred to as “TβRII”) refers to a human TGF-β type II receptor having the structure and function reported in the previous report as described above. Body (human TGF-βtype II receptor) (for example, Cell, Vol. 68, No. 4, p. 775-785, 1992; Adv. Immunol., Vol. 55, p. 181-220, 1994; GenBank). Accession No .: M85079).
The natural human TGF-β type II receptor has the following structural features and properties.
The human TGF-β type II receptor is a transmembrane protein consisting of 567 amino acids, and has an extracellular domain (136 amino acids), a transmembrane domain (30 amino acids) and an intracellular domain (378 amino acids) Consists of The TGF-β type II receptor is a single transmembrane protein having a serine / threonine kinase structure having a relatively short extracellular region and two kinase insertion sites. In addition, aspartic acid (Asp) -linked sugar chains are bound to the intracellular region, and twelve cysteine (Cys) residues are observed, thereby taking a characteristic tertiary protein structure.
[0032]
The kinase region of the TGF-β type II receptor is a protein kinase specific for serine / threonine and has about 43% homology to the kinase region of the TGF-β type I receptor. This kinase region is essential for the transmission of TGF-β signals into cells (J. Biol. Chem., Vol. 269, p. 30753, 1994).
[0033]
The TGF-β type II receptor is a very important molecule that cooperates with the TGF-β type I receptor to transmit TGF-β signals into cells. The TGF-β type II receptor alone binds to TGF-β. When TGF-β binds to the TGF-β type II receptor, the TGF-β type II receptor has been shown to form a heterotetrameric complex with the TGF-β type I receptor on the cell surface (J. Biol. Chem., Vol.269, p.20172, 1994). When the complex is formed in the presence of TGF-β, the TGF-β type II receptor phosphorylates the GS region (described above) of the TGF-β type I receptor and activates the TGF-β type I receptor. Become As a result, it is considered that the TGF-β signal is transmitted further downstream by phosphorylating other intracellular substrates (Nature, Vol. 370, p. 341, 1994).
[0034]
Further, the “human TGF-β type II receptor” (also referred to as “TβRII”) referred to in the present invention has substantially the same amino acid sequence as that of the above-mentioned natural type protein primary structure (amino acid sequence). Mutants of a natural human TGF-β type II receptor having the same amino acid sequence are also included.
[0035]
Here, “a mutant of a natural human TGF-β type II receptor having substantially the same amino acid sequence” means the following mutant protein.
That is, a plurality of amino acids in the amino acid sequence, preferably 1 to 10 amino acids, particularly preferably 1 to 10 amino acids, as long as they have biological properties substantially equivalent to those of the natural human TGF-β type II receptor. A mutant protein having an amino acid sequence in which five amino acids have been substituted, deleted and / or modified, and a plurality of amino acids, preferably one to one in the amino acid sequence of the natural human TGF-β type II receptor A mutant protein having an amino acid sequence in which 10 amino acids, particularly preferably 1 to 5 amino acids are added.
Furthermore, a mutant having a plurality of such substitutions, deletions, modifications and additions may be used.
The human TGF-β type II receptor in the present invention can be obtained by appropriately using a known method known in the technical field such as a chemical synthesis method, a cell culture method or the like or a modification method thereof in addition to a gene recombination technique. Can be manufactured.
[0036]
Further, the “human TGF-β type II receptor” (also referred to as “TβRII”) in the present invention includes “a part” of the human TGF-β type II receptor. Here, “part” means a polypeptide containing any partial sequence in the amino acid sequence of the human TGF-β type II receptor defined above.
Preferably, it refers to the extracellular region of the human TGF-β type II receptor as defined above or any part thereof.
[0037]
The “part” of the human TGF-β type II receptor (preferably, the extracellular region of the human TGF-β type II receptor or any part thereof) is obtained by a known method known in the art described below or a modification thereof. According to the method, it can be produced by a gene recombination technique or a chemical synthesis method, or can be produced by appropriately cleaving human TGF-β type II receptor isolated by a cell culture method using a proteolytic enzyme or the like. can do.
[0038]
The “human monoclonal antibody” of the present invention is a human monoclonal antibody that binds to the human TGF-β type II receptor defined above.
More specifically, the variable region (Variable region) of the heavy chain (H chain) and the constant region of the H chain (Constant Region) constituting the immunoglobulin and the variable region of the light chain (L chain) and the constant region of the L chain are defined. The entire region including the human immunoglobulin is derived from the gene encoding human immunoglobulin. Examples of the L chain include a human κ chain and a human λ chain.
[0039]
The "human monoclonal antibody" of the present invention can be prepared by immunizing any of the following immunogens (antigens) to a human antibody-producing transgenic non-human mammal described below.
(A) Natural cells or artificially established cell lines that express the human TGF-β type II receptor as defined above on the cell surface.
(B) A transgenic cell produced by a transgenic technique so as to express the human TGF-β type II receptor as defined above on the cell surface.
(C) A cell lysate obtained by solubilizing the cells of (a) or (b), or a polypeptide fragment of human TGF-β type II receptor purified from the cell lysate.
[0040]
(D) It is prepared using a gene recombination technique so as to express a part of the human TGF-β type II receptor defined above (particularly preferably, its extracellular region or any peptide thereof) as a soluble polypeptide. Genetically modified cells.
(E) A culture supernatant obtained by culturing the genetically modified cells of (d) or an extracellular domain polypeptide of human TGF-β type II receptor purified from the culture supernatant (soluble human TGF-β type II) Receptor).
(F) Part of the chemically synthesized human TGF-β type II receptor (particularly preferably, its extracellular region or any peptide thereof).
[0041]
Furthermore, the monoclonal antibody of the present invention is a recombinant antibody obtained by transforming a host cell with cDNAs encoding each of the heavy and light chains of the monoclonal antibody of the present invention using a gene recombination technique. The cells can also be obtained from the culture supernatant by culturing the “recombinant cells” of the present invention that produce a recombinant monoclonal antibody.
The monoclonal antibody of the present invention may be a human monoclonal antibody having any isotype of IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD or IgE. Preferably, it is IgG (IgG1, IgG2, IgG3, IgG4), preferably IgG1, IgG2 or IgG4, and particularly preferably IgG4.
[0042]
The human monoclonal antibody of the present invention immunizes a human antibody-producing transgenic non-human mammal, such as a human antibody-producing transgenic mouse described below, with any of the immunogens (antigens) described in (a) to (f) above. The monoclonal antibody can be produced according to an existing general production method.
That is, for example, the human antibody-producing transgenic non-human mammal is immunized with the antigen together with Freund's Adjuvant as necessary. Polyclonal antibodies can be obtained from serum obtained from the immunized animal. In addition, a monoclonal antibody is prepared by preparing a fusion cell (hybridoma) from the antibody-producing cell obtained from the immunized animal and a myeloma cell (myeloma cell) having no autoantibody-producing ability, cloning the hybridoma, It is produced by selecting a clone that produces a monoclonal antibody showing a specific affinity for the antigen used for immunization.
[0043]
More specifically, it can be produced as follows. That is, the human antibody-producing transgenic non-human mammal (particularly preferably described below) is combined with the immunogen of any one of the above (a) to (c) together with Freund's Adjuvant, if necessary, together with Freund's Adjuvant. Immunization is carried out by subcutaneous, intramuscular, intravenous, intravenous, intraperitoneal or intraperitoneal injection or transplantation of human antibody-producing transgenic mice "). Usually, immunization is performed 1 to 4 times about every 1 to 14 days after the initial immunization, and antibody producing cells are obtained from the immunized mammal about 1 to 5 days after the final immunization. The number of immunizations and the time interval can be appropriately changed depending on the nature of the immunogen used.
[0044]
Preparation of a fusion cell (hybridoma) secreting a monoclonal antibody can be performed according to the method of Koehler and Milstein et al. (Nature, Vol. 256, p. 495-497, 1975) and a modification method analogous thereto. That is, spleen, lymph node, bone marrow or tonsil, etc., obtained from a human antibody-producing transgenic non-human mammal immunized as described above, and antibody-producing cells preferably contained in the spleen, preferably mice, rats, It is prepared by cell fusion with mammals such as guinea pig, hamster, rabbit or human, more preferably mouse, rat or human derived myeloma cells having no autoantibody-producing ability.
[0045]
Examples of myeloma cells used for cell fusion include mouse-derived myeloma P3 / X63-AG8.653 (ATCC No. CRL-1580), P3 / NSI / 1-Ag4-1 (NS-1), and P3 / X63-Ag8. . U1 (P3U1), SP2 / 0-Ag14 (Sp2 / O, Sp2), PAI, F0 or BW5147, rat-derived myeloma 210RCY3-Ag. 2.3. And human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11 or CEM-T15.
Screening for cells producing monoclonal antibodies (eg, hybridomas) involves culturing the cells, eg, in a microtiter plate, and using the culture supernatant of the wells in which growth has been observed against the immunizing antigen used in the immunization described above. The reactivity can be measured by, for example, measuring an enzyme immunoassay such as RIA (radio immunoassay) or ELISA (enzyme-linked immuno-solvent assay).
[0046]
Production of the monoclonal antibody from the hybridoma is carried out in vitro on the hybridoma or in vivo in a mouse, rat, guinea pig, hamster or rabbit or the like, preferably in a mouse or rat, more preferably in ascites of a mouse, and the obtained culture supernatant. Alternatively, it can be performed by isolating from ascites of a mammal.
[0047]
In addition, a gene encoding a monoclonal antibody is cloned from the hybridoma or the `` recombinant cell '' of the present invention that produces a recombinant monoclonal antibody described below, and the gene is converted into an endogenous gene by using a transgenic animal production technique. It is also possible to produce an integrated transgenic cow, goat, sheep or pig, and obtain a large amount of a monoclonal antibody derived from the monoclonal antibody gene from the milk of the transgenic animal (Nikkei Science, 1997). April issue, pages 78 to 84).
When the monoclonal antibody-producing cells are cultured in vitro, the hybridoma is grown, maintained, and stored in accordance with various conditions such as the characteristics of the cell type to be cultured, the purpose of the test and research, and the culture method. It can be carried out using a known nutrient medium as used for producing monoclonal antibodies or any nutrient medium derived and prepared from a known basal medium.
[0048]
As the basic medium, for example, a low calcium medium such as Ham'F12 medium, MCDB153 medium or low calcium MEM medium and a high calcium medium such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium or RD medium, etc. The basal medium may contain, for example, serum, hormones, cytokines, and / or various inorganic or organic substances, depending on the purpose.
For isolation and purification of the monoclonal antibody, the above-mentioned culture supernatant or ascites is obtained by using a saturated ammonium sulfate, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), anti-immunoglobulin column or It can be performed by subjecting it to affinity column chromatography such as a protein A column.
[0049]
The monoclonal antibody of the present invention includes a heavy chain and / or a heavy chain having an amino acid sequence in which one or several amino acids are deleted, substituted or added in each amino acid sequence of the heavy chain and / or light chain constituting the antibody. Alternatively, a human monoclonal antibody comprising a light chain is also included.
[0050]
Here, "several amino acids" means a plurality of amino acids, specifically 1 to 10 amino acids, and preferably 1 to 5 amino acids.
The above-mentioned partial modification (deletion, substitution, insertion, addition) of an amino acid into the amino acid sequence of the human monoclonal antibody of the present invention is introduced by partially modifying the base sequence encoding the amino acid sequence. can do. This partial modification of the nucleotide sequence can be carried out by a conventional method using a known site-specific mutagenesis method (Site specific mutagenesis) (Proc. Natl. Acsd. Sci. USA, Vol. 81, p. 5662-5666, 1984).
In the present invention, “derived from the human immunoglobulin light chain V gene segment” means that the DNA of the V region encoding the heavy chain or light chain variable region of the monoclonal antibody is an immunoglobulin heavy chain locus (Ig heavy chain). gene locus or any of a number of V gene segments present at the light chain locus (Ig light chain gene locus) due to transcription of the nucleic acid sequence of one of the V genes, which is completely identical to the nucleic acid sequence of the V gene Or one or several (specifically, 1 to 10) bases are different from the nucleic acid sequence of the V gene (here, it means deletion, substitution and / or insertion). Means
[0051]
The “human antibody-producing transgenic non-human mammal” of the present invention, a particularly preferred embodiment of a human antibody-producing transgenic mouse, can be produced according to a previous report (Nature Genetics, Vol. 7, p. 13-21, 1994). Nature Genetics, Vol. 15, p. 146-156, 1997; Japanese Patent Publication No. 4-504365; Japanese Patent Publication No. 7-509137; Nikkei Science, June, pp. 40-50, 1995. International Application Publication No. WO 94/25585; Nature, Vol. 368, p. 856-859, 1994;
Specifically, the human antibody-producing transgenic mouse can be produced, for example, by using a technique comprising the following steps. Other human antibody-producing transgenic non-human mammals can be produced in a similar manner.
[0052]
(1) The mouse endogenous immunoglobulin heavy chain gene is functionally disabled by substituting at least a part of the mouse endogenous immunoglobulin heavy chain locus with a drug resistance marker gene (such as a neomycin resistance gene) by homologous recombination. The step of producing an activated knockout mouse.
(2) By replacing at least a part of the mouse endogenous immunoglobulin light chain locus with a drug resistance marker gene (such as a neomycin resistance gene) by homologous recombination, the mouse endogenous immunoglobulin light chain gene (particularly the κ chain gene) B) producing knockout mice that have been functionally inactivated.
(3) The desired region of the human immunoglobulin heavy chain locus is integrated into the mouse chromosome using a vector capable of carrying a large gene, such as a yeast artificial chromosome (YAC) vector. Producing a transgenic mouse.
(4) A transgenic mouse in which a desired region of a human immunoglobulin light chain (particularly κ chain) locus is integrated into a mouse chromosome using a vector capable of carrying a large gene represented by YAC or the like. The process of making.
(5) By crossing the knockout mouse and the transgenic mouse of (1) to (4) in any order, both the mouse endogenous immunoglobulin heavy chain locus and the mouse endogenous immunoglobulin light chain locus function. Producing a transgenic mouse that has been specifically inactivated and has both the desired region of the human immunoglobulin heavy chain locus and the desired region of the human immunoglobulin light chain locus integrated on the mouse chromosome.
[0053]
The knockout mouse is constructed so that the appropriate region of the mouse endogenous immunoglobulin locus is replaced by a foreign marker gene (such as a neomycin resistance gene) by homologous recombination so that the locus cannot be rearranged (rearranged). It can be produced by activating. For the inactivation using the homologous recombination, for example, a method called positive negative selection (Positive Negative Selection; PNS) can be used (Nikkei Science, May, p. 52-62). , 1994).
Functional inactivation of the immunoglobulin heavy chain locus can be achieved, for example, by introducing a disorder into a portion of the J or C region (eg, the Cμ region). Functional inactivation of an immunoglobulin light chain (for example, a κ chain) can be achieved by, for example, introducing a disorder into a part of the J region or the C region, or a region including a region spanning the J region and the C region. It is.
[0054]
The transgenic mouse can be prepared by a conventional method commonly used in the production of transgenic animals (for example, see the latest manual for animal cell experiments, published by LCI, Chapter 7, pp. 361 to 408, 1990). ). Specifically, for example, an HPRT-negative (lacking the hypoxanthine guanine phosphoribosyltransferase gene) ES cell (embryonic stem cell) derived from a normal mouse blastcyst is blasted with the human immunoglobulin heavy chain. The gene encoding the locus or the light chain locus or a part thereof and the yeast containing the YAC vector into which the HPRT gene has been inserted are fused by spheroplast fusion. ES cells in which the exogenous gene has been integrated on the mouse endogenous gene are selected by the HAT selection method. Next, the selected ES cells are microinjected into a fertilized egg (blastocyst) obtained from another normal mouse (Proc. Natl. Acad. Sci. USA, Vol. 77, No. 12, pp. 7380-7384). , 1980; U.S. Pat. No. 4,873,191). The blastocyst is implanted into the uterus of another normal mouse as a foster mother. Thus, a chimeric transgenic mouse is born from the foster parent mouse. By crossing the chimeric transgenic mouse with a normal mouse, a heterotransgenic mouse is obtained. By crossing the heterogeneic transgenic mice, a homogenic transgenic mouse can be obtained according to Mendel's law.
[0055]
The "part of the monoclonal antibody" in the present invention means a partial region of the above-described human monoclonal antibody of the present invention, and specifically, F (ab ') ₂ , Fab ′, Fab, Fv (variable fragment of antibody), sFv, dsFv (disulphide stabilized Fv) or dAb (single domain opinion opponent). Ther. Patents), Vol. 6, No. 5, pp. 441-456, 1996).
[0056]
Here, "F (ab ') ₂ "And" Fab '"are produced by treating an immunoglobulin (monoclonal antibody) with a protease such as pepsin or papain to form a disulfide bond existing between two H chains in the hinge region. Antibody fragments produced by digestion before and after. For example, when IgG is treated with papain, it is cleaved upstream of the disulfide bond existing between the two heavy chains in the hinge region, resulting in V.V. _L (L chain variable region) and C _L (L chain constant region) and V chain _H (H chain variable region) and C _H Two homologous antibody fragments in which an H chain fragment composed of γ1 (the γ1 region in the H chain constant region) is linked by a disulfide bond at the C-terminal region can be produced. Each of these two homologous antibody fragments is called Fab '. When IgG is treated with pepsin, it is cleaved downstream of a disulfide bond existing between two H chains in the hinge region to produce an antibody fragment slightly larger than the two Fab's connected by the hinge region. Can be. This antibody fragment is expressed as F (ab ') ₂ That.
[0057]
The “cell producing the monoclonal antibody” of the present invention or the “genetically modified cell” of the present invention producing a recombinant human monoclonal antibody means any cell producing the above-described monoclonal antibody of the present invention.
Specifically, for example, cells described in any of the following (1) to (3) can be mentioned, but are not limited thereto.
(1) Monoclonal antibody obtained by immunizing the above-mentioned non-human mammal (including a human antibody-producing transgenic non-human mammal) with the immunogen (antigen) defined above and being collected from the immunized animal Producing B cells.
(2) The above-mentioned fused cells (hybridomas) obtained by cell fusion of the thus obtained monoclonal antibody-producing B cells with mammalian myeloma cells. (3) A gene encoding the monoclonal antibody (either a gene encoding a heavy chain or a gene encoding a light chain, or both, isolated from the monoclonal antibody-producing B cell or the monoclonal antibody-producing fusion cell (hybridoma)) Gene for producing a recombinant monoclonal antibody obtained by transforming a cell other than the B cell and the hybridoma (for example, CHO (Chinese hamster ovarian) cell, BHK (Baby hamster childney) cell, etc.) Recombinant cells.
Here, the genetically modified cells producing the genetically modified monoclonal antibody according to (3) above include the genetically modified monoclonal antibody produced by the B cell or hybridoma of (1). Genetically modified cells to be produced are meant.
[0058]
The “binding rate constant (ka)” in the present invention means a value indicating the strength (degree) of the binding of the monoclonal antibody to the target antigen, calculated based on the kinetics of the antigen-antibody reaction. The “dissociation rate constant (kd)” means a value indicating the strength (degree) of dissociation of the monoclonal antibody from the target antigen, calculated based on the antigen-antibody reaction kinetics. The “dissociation constant (Kd)” is a value obtained by dividing the “dissociation rate constant (kd)” value by the “dissociation rate constant (ka)” value. These constants are used as indices indicating the affinity of the monoclonal antibody for the antigen and the neutralizing activity of the antigen.
[0059]
The constants can be analyzed according to various methods. However, a commercially available measurement kit, BiacoreX (manufactured by Amersham Pharmacia) or a similar kit, can be easily used according to the instruction manual and experimental operation method attached to the kit. Can be analyzed. The ka value, kd value and Kd value determined using the kit are respectively 1 / M. It is expressed by units of Sec, 1 / Sec and M (mole). The higher the ka value of the tested monoclonal antibody, the stronger the antigen binding activity, and the lower the Kd value, the stronger the neutralizing activity.
[0060]
The human monoclonal antibody of the present invention preferably has a dissociation constant (Kd) for human TGF-β type II receptor of 10 × 10 ^-10 Monoclonal antibodies (in particular, human monoclonal antibodies) having a numerical value smaller than (M) are included.
It should be noted that the values of ka, kd, and Kd described above are expected to vary slightly depending on various conditions at the time of measurement, but may vary as an error range. It is.
[0061]
The “pharmaceutical composition” in the present invention is useful as a pharmaceutical comprising the monoclonal antibody or a part thereof that binds to the human TGF-β type II receptor of the present invention as an active ingredient, and a “pharmaceutically acceptable carrier”. Means a composition.
Here, "pharmaceutically acceptable carrier" refers to excipients, diluents, extenders, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, coloring agents, sweeteners, thickeners. , A flavoring agent, a solubilizer or other additives.
By using one or more of such carriers, medicines in the form of tablets, pills, powders, granules, injections, liquids, capsules, troches, elixirs, suspensions, emulsions, syrups, etc. A composition can be prepared.
These pharmaceutical compositions can be administered orally or parenterally. Other forms for parenteral administration include topical solutions containing one or more active substances and formulated in a conventional manner, suppositories and pessaries for enteral administration.
[0062]
The dosage varies depending on the age, sex, weight and condition of the patient, therapeutic effect, administration method, treatment time, or the type of active ingredient (such as the protein or antibody) contained in the pharmaceutical composition. The dose can be administered in the range of 10 μg to 1000 mg (or 10 μg to 500 mg) at a time per person. However, since the dose varies depending on various conditions, a dose smaller than the above dose may be sufficient, or a dose exceeding the above range may be required.
In particular, in the case of an injection, the concentration is 0.1 μg antibody / ml carrier to 10 mg antibody / ml carrier in a non-toxic pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection. Can be produced by dissolving or suspending as described above.
[0063]
The injection thus produced is applied to a human patient in need of treatment at a rate of 1 μg to 100 mg per kg body weight, preferably 50 μg to 50 mg per day, in a single administration. It can be administered once to several times. Examples of the administration form include medically appropriate administration forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and intraperitoneal injection. Preferably it is an intravenous injection.
Injectables can also be prepared as non-aqueous diluents (eg, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, etc.), suspensions and emulsions. .
Such injections can be sterilized by filtration sterilization through a bacteria retaining filter, blending of a bactericide or irradiation. Injectables can be manufactured in the form of preparation for use. That is, it can be used as a sterile solid composition by freeze-drying or the like, dissolved in sterile distilled water for injection or another solvent before use.
[0064]
INDUSTRIAL APPLICABILITY The pharmaceutical composition of the present invention is useful for inhibiting transmission of a TGF-β signal involved in the development of various disease states and diseases into cells via a TGF-β type II receptor.
Furthermore, the pharmaceutical composition of the present invention provides various diseases caused by the action of TGF-β, for example, renal diseases (renal fibrosis, nephritis, renal failure, renal sclerosis, etc.), lung diseases (for example, pulmonary fibrils). Disease, pneumonia, etc.), liver diseases (eg, liver tissue fibrosis, cirrhosis, hepatitis, etc.), skin diseases (eg, wounds, scleroderma, psoriasis, keloids, etc.), arthritis (eg, rheumatoid arthritis, deformity) Arthrosis), vascular disease (eg, vascular restenosis, rheumatic vasculitis, etc.), tissue fibrosis in various organs (including tissue fibrosis associated with various cancers), and arteriosclerosis (concurrent tissue) The present invention is useful as a pharmaceutical for suppressing or preventing the onset and / or progression of the disease (including fibrosis) and treating or preventing the disease.
In particular, since the human monoclonal antibody or the pharmaceutical composition thereof of the present invention is composed of a human-derived monoclonal antibody, it does not cause immune rejection of the host caused by HAMA (human anti-mouse antibody) and can be used as an antibody drug. Extremely useful.
[0065]
In addition, the therapeutic effect of the pharmaceutical composition of the present invention for various disease symptoms can be tested and examined by administering to a known disease model animal according to a conventional method.
For example, when examining the therapeutic effect of renal fibrosis, which is a type of tissue fibrosis and also a type of kidney disease, one of the ureters of a mouse or monkey is ligated to filter the blood or the like of the kidney. The pharmaceutical composition of the present invention is administered to a model (UUO model, Unilateral urethane obstruction) in which renal dysfunction has been induced by stopping the disease, and an index of the onset of nephritis and renal fibrosis induced by the renal dysfunction. A method of measuring the degree of suppression of the increase in the production of hydroxyproline or the suppression of the increase in the expression of mRNA of Type I collagen can be used.
[0066]
As yet another method, a pharmaceutical composition of the present invention is administered to a rat model of nephritis induced by administering an antibody against Thy-1, a cell surface marker, to a rat, and the nephritis caused by the renal dysfunction is induced. And a method for measuring the amount of protein excreted in urine, the amount of creatinine in serum, and the degree of reduction in production of extracellular matrix (fibronectin, type I collagen, etc.) which are indicators of the onset of renal fibrosis. (Nephron, Vol. 78, p. 453-463, 1998; Kidney Blood Press Res., Vol. 22, p. 5-12, 1999). The reduction in urine protein, serum creatinine, fibronectin or type I collagen indicates that the pharmaceutical composition is effective for treating the renal disease.
[0067]
Renal diseases, such as microglomerular abnormalities (eg, nephrotic microchange group (MCNS)), focal glomerulosclerosis (FGS), membranous glomerulonephritis (membranous nephropathy, MN), IgA nephropathy, For mesangial proliferative nephritis, acute glomerulonephritis after streptococcal infection (APSGN), crescentic (extravascular) nephritis, interstitial nephritis, acute renal failure, etc., use model animals detailed in the previous report (“Testing and experimental methods for production of disease-specific animal models and development of new drugs”, pp. 34-46, 1993, Technical Information Association (published)).
For skin diseases, for example, wounds, keloids, atopy, dermatitis, scleroderma, psoriasis, etc., model animals detailed in the previous report can be used ("Production of disease-specific model animals and development of new drugs"). Tests and Experimental Methods, "p.229-235, 1993, Technical Information Association (published).
[0068]
For liver diseases, for example, hepatitis (eg, viral hepatitis (type A, B, C, E, etc.)), cirrhosis or drug-induced liver injury, it is possible to use model animals detailed in the previous report. (“Test and Experimental Methods for the Preparation of Disease-Specific Animal Models and New Drug Development”, pp. 119-129 and 349-358, 1993, Technical Information Association (published)).
For example, when examining the effects on arteriosclerosis and vascular restenosis, a restenosis model created by inserting a balloon catheter into the rat aorta, performing PTCA, and simulating the restenosis can be used.
[0069]
The DNA encoding the specific polypeptide used in the present invention may be prepared by a conventional method such as a method of cloning cDNA from mRNA encoding the polypeptide, a method of isolating genomic DNA and splicing, a method of cDNA sequence or mRNA. It can be obtained by a method of preparing by PCR using a sequence as a template, or a method of chemical synthesis.
Furthermore, DNA encoding a specific part of the polypeptide or a fusion polypeptide of the part with another polypeptide can be obtained by cleaving (digesting) the DNA thus prepared with an appropriate restriction enzyme, The obtained DNA fragment can be prepared by ligating it with a linker DNA or a tag (Tag), if necessary, using an appropriate DNA polymerase or the like.
[0070]
The following method is exemplified as a method for cloning cDNA encoding the desired polypeptide from mRNA.
First, mRNA encoding a target polypeptide is prepared from a tissue or cell that expresses / produces the target polypeptide. For preparation of mRNA, total RNA prepared by a known method such as a guanidine thiocyanate method (Biochemistry, Vol. 18, p. 5294, 1979), a hot phenol method or an AGPC method is used to prepare oligo (dT) cellulose or poly-U. -It can be carried out by subjecting to affinity chromatography using Sepharose or the like.
Next, a known method such as, for example, using reverse transcriptase using the obtained mRNA as a template, for example, the method of Okayama et al. (Mol. Cell. Biol., Vol. 2, p. 161, 1982 and the same journal, Vol. 3, p. 280, 1983) or the method of Hoffman et al. (Gene, Vol. 25, p. 263, 1983) and the like, and the cDNA is converted into double-stranded cDNA. This cDNA is incorporated into a plasmid vector, a phage vector or a cosmid vector, and transformed into Escherichia coli, or after in vitro packaging, transfected into Escherichia coli to prepare a cDNA library.
[0071]
The plasmid vector used here is not particularly limited as long as it can be replicated and maintained in the host, and any phage vector can be used as long as it can be propagated in the host. Examples of commonly used cloning vectors include pUC19, λgt10, λgt11 and the like. However, when subjected to the immunological screening described below, a vector having a promoter capable of expressing a gene encoding a target polypeptide in a host is preferable.
Methods for incorporating cDNA into a plasmid include, for example, the method described in Maniatis et al. (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, p. 1.53, 1989). Examples of the method for incorporating cDNA into a phage vector include the method of Hyunh et al. (DNA Cloning, a practical approach, Vol. 1, p. 49, 1985). For convenience, a commercially available cloning kit (for example, a product of Takara Shuzo Co., Ltd.) can also be used. The thus obtained recombinant plasmid or phage vector is introduced into a suitable host such as a prokaryotic cell (for example, E. coli: HB101, DH5α, Y1090, DH10B or MC1061 / P3).
[0072]
Examples of a method for introducing a plasmid into a host include a calcium chloride method or a calcium chloride / rubidium chloride method described in (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, p. 1.74, 1989). Ration method and the like. Examples of a method for introducing a phage vector into a host include a method in which phage DNA is packaged in vitro and then introduced into a grown host. In vitro packaging can be easily performed by using a commercially available in vitro packaging kit (eg, manufactured by Stratagene, Amersham, etc.).
The method of isolating the cDNA encoding the polypeptide of interest from the cDNA library prepared by the above method can be performed by combining general cDNA screening methods.
[0073]
For example, after separately synthesizing an oligonucleotide that is considered to correspond to the amino acid sequence of the target polypeptide, ³² Labeled with P to form a probe, a known colony hybridization method (Crunstein et al., Proc. Natl. Acid. Sci. USA, Vol. 72, p. 3961, 1975) or a plaque hybridization method (Molecular) Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, p. 2.108, 1989), a method for screening a clone containing a cDNA of interest, a PCR primer, and a PCR method for a specific region of the polypeptide of interest. And selecting a clone having a DNA fragment encoding the region.
[0074]
When a cDNA library prepared using a vector capable of expressing cDNA (for example, a phage vector such as λgt11) is used, an antigen-antibody reaction using an antibody reactive with a target polypeptide is used. The desired clone can be selected. When a large number of clones are processed, it is preferable to use a screening method using a PCR method.
The nucleotide sequence of the DNA thus obtained was determined by the Maxam-Gilbert method (Maxam et al., Proc. Natl. Acad. Sci. USA., Vol. 74, p. 560, 1977) or a dideoxynucleotide using phage M13. Synthetic chain termination (Sanger et al., Proc. Natl. Acad. Sci. USA., Vol. 74, p. 5463-5467, 1977). The gene encoding the polypeptide of interest can be obtained by cutting out all or part of the gene from the clone obtained as described above using restriction enzymes and the like.
[0075]
In addition, as a preparation method by isolating a DNA encoding a target polypeptide from genomic DNA derived from a cell expressing the target polypeptide as described above, for example, the following method is exemplified.
The cells are preferably lysed using SDS or proteinase K and the like, and DNA extraction is performed by repeating extraction with phenol. The RNA is digested, preferably by ribonuclease. The obtained DNA is partially digested with an appropriate restriction enzyme, and the obtained DNA fragment is amplified with an appropriate phage or cosmid to prepare a library. Then, a clone having the target sequence is detected by, for example, a method using a radiolabeled DNA probe, and all or a part of the gene encoding the target polypeptide is cut out from the clone with a restriction enzyme or the like and obtained.
[0076]
The DNA encoding the target polypeptide can be prepared by PCR using a known mRNA or cDNA of the target polypeptide as a template ("Gene amplification PCR method: basic and new developments", Kyoritsu Shuppan) Inc., 1992).
The production of DNA encoding the target polypeptide by chemical synthesis can be carried out according to a conventional method based on the base sequence of the target polypeptide.
[0077]
The target polypeptide or a part thereof used in the present invention may be prepared by using a DNA (DNA or genomic DNA containing an intron) containing the DNA encoding the target polypeptide prepared by the method exemplified above as appropriate. A DNA fragment encoding the target polypeptide was obtained by digestion with an enzyme, and these fragments were ligated with a linker DNA or a tag (Tag) as necessary using an appropriate DNA polymerase or the like. A recombinant protein can be prepared from DNA by a conventional method using a commonly used gene recombination technique.
Specifically, it is as exemplified below. That is, the DNA prepared as described above is inserted into a vector as described in detail below to prepare an expression vector, and the expression vector is used to transform a host cell as described below to obtain a transformant. . The target polypeptide is produced in the culture supernatant by culturing the transformant. The target polypeptide in the culture supernatant can be easily purified using column chromatography or the like.
[0078]
The expression vector used to produce the target polypeptide is not particularly limited as long as it can replicate and maintain or self-propagate in various prokaryotic and / or eukaryotic host cells, and includes plasmid vectors and phage vectors. (Cloning Vectors: A Laboratory Manual, Elsview, New York, 1985).
The expression vector can be simply prepared by ligating a DNA encoding the polypeptide of interest to a vector for recombination (plasmid DNA and bacterial phage DNA) available in the art by a conventional method. Specific examples of the recombination vector used include plasmids derived from Escherichia coli such as pBR322, pBR325, pUC12, pUC13, and pUC19; plasmids derived from yeasts such as pSH19 and pSH15; and plasmids derived from Bacillus subtilis such as pUB110, pTP5, and pC194. And the like. Examples of the phage include bacteriophage such as λ phage, and animal and insect viruses (pVL1393, manufactured by Invitrogen) such as retrovirus, vaccinia virus, and nucleopolyhedrovirus.
[0079]
For the purpose of producing the target polypeptide, a plasmid vector is useful. The plasmid vector is not particularly limited as long as it has a function of expressing DNA encoding the target polypeptide in various prokaryotic and / or eukaryotic host cells and producing these polypeptides. For example, pMAL C2, pcDNA3.1 (-), pEF-BOS (Nucleic Acid Research, Vol. 18, p. 5322, 1990, etc.) or pME18S (Experimental Medical Supplement "Genetic Engineering Handbook", 1992, etc.) and the like can be mentioned. be able to.
[0080]
When a bacterium, particularly Escherichia coli, is used as a host cell, the expression vector generally comprises at least a promoter-operator region, an initiation codon, a DNA encoding the protein of the present invention, a stop codon, a terminator region, and a replicable unit.
When yeast, animal cells or insect cells are used as the host, the expression vector preferably contains at least a promoter, an initiation codon, DNA encoding the target polypeptide, and a termination codon. It may also contain a signal peptide-encoding DNA, an enhancer sequence, a 5′- and 3′-side untranslated region of the target polypeptide, a splicing junction, a polyadenylation site, a selection marker region or a replicable unit. Good. Further, it may contain a gene amplification gene (marker) usually used depending on the purpose.
[0081]
The promoter-operator-region for expressing the polypeptide of interest in bacteria includes a promoter, an operator, and a Shine-Dalgarno (SD) sequence (eg, AAGG). For example, when the host is a bacterium belonging to the genus Escherichia, those containing a Trp promoter, a lac promoter, a recA promoter, a λPL promoter, an lpp promoter, a tac promoter and the like are preferably exemplified.
Examples of a promoter for expressing a target polypeptide in yeast include a PH05 promoter, a PGK promoter, a GAP promoter, and an ADH promoter. When the host is a bacterium belonging to the genus Bacillus, a SL01 promoter, a SP02 promoter, a penP promoter, and the like are exemplified. Can be
When the host is a eukaryotic cell such as a mammalian cell, examples thereof include an SV40-derived promoter, a retrovirus promoter, and a heat shock promoter. However, it is not particularly limited to these. Use of an enhancer is also an effective method for expression.
[0082]
A suitable initiation codon is, for example, a methionine codon (ATG).
Examples of the stop codon include common stop codons (for example, TAG, TAA, and TGA).
As the terminator region, a commonly used natural or synthetic terminator can be used.
A replicable unit refers to DNA capable of replicating its entire DNA sequence in a host cell, including native plasmids, artificially modified plasmids (DNA fragments prepared from native plasmids) and Includes synthetic plasmids and the like. Suitable plasmids include E. coli. In E. coli, plasmid pBR322 or an artificially modified product thereof (a DNA fragment obtained by treating pBR322 with an appropriate restriction enzyme) is used. In yeast, yeast 2μ plasmid or yeast chromosomal DNA is used. In mammalian cells, plasmid pRSVneo ATCC 37198 is used. And plasmid pSV2dhfr ATCC 37145, plasmid pdBV-MMTneo ATCC 37224, plasmid pSV2neo ATCC 37149, plasmid pSV2bsr, and the like.
As the enhancer sequence, polyadenylation site and splicing junction site, those commonly used by those skilled in the art, such as those derived from SV40, for example, can be used.
[0083]
As the selection marker, a commonly used marker can be used by a conventional method. Examples thereof include antibiotic resistance genes such as tetracycline, ampicillin, and kanamycin.
Examples of the gene amplification gene include a dihydrofolate reductase (DHFR) gene, a thymidine kinase gene, a neomycin resistance gene, a glutamate synthase gene, an adenosine deaminase gene, an ornithine decarboxylase gene, a hygromycin-B-phosphotransferase gene, and an aspartate transcarbamylase gene. And the like.
[0084]
The expression vector of the present invention can be prepared by ligating at least the above-mentioned promoter, start codon, DNA encoding the protein of the present invention, stop codon and terminator region to an appropriate replicable unit in a continuous and circular manner. it can. At this time, if necessary, an appropriate DNA fragment (for example, a linker or another restriction enzyme site) can be used by a conventional method such as digestion with a restriction enzyme or ligation using T4 DNA ligase.
The transformed cell of the present invention can be prepared by introducing the above-described expression vector into a host cell.
[0085]
The host cell used in the present invention is not particularly limited as long as it is compatible with the above-mentioned expression vector and can be transformed, and natural cells or artificially established cells usually used in the technical field of the present invention are used. Various cells such as recombinant cells (for example, bacteria (genus Escherichia, genus Bacillus), yeast (genus Saccharomyces, genus Pichia), animal cells or insect cells, etc.) are exemplified.
Escherichia coli or animal cells are preferred, and specifically, Escherichia coli (DH5α, DH10B, TB1, HB101, XL-2Blue, etc.), and mouse-derived cells (COP, L, C127, Sp2 / 0, NS-1, or NIH3T3, etc.) , Rat-derived cells, hamster-derived cells (such as BHK and CHO), monkey-derived cells (such as COS1, COS3, COS7, CV1 and Velo) and human-derived cells (Hela, cells derived from diploid fibroblasts, myeloma cells) And Namalwa etc.).
Introduction (transformation (transfection)) of an expression vector into a host cell can be performed using a conventionally known method.
[0086]
For example, in the case of bacteria (E. coli, Bacillus subtilis, etc.), for example, the method of Cohen et al. (Proc. Natl. Acad. Sci. USA., Vol. 69, p. 2110, 1972), the protoplast method (Mol. Gen.). Genet., Vol. 168, p. 111, 1979) or the competent method (J. Mol. Biol., Vol. 56, p. 209, 1971), in the case of Saccharomyces cerevisiae, for example, Hinnen et al. Natl. Acad. Sci. USA., Vol. 75, p. 1927, 1978) or the lithium method (J. Bacteriol., Vol. 153, p. 163, 1983). For example, the method of Graham (Virology, Vol. 52, p. 456, 1973), and in the case of insect cells, for example, the method of Summers et al. (Mol. Cell. Biol., Vol. 3, p. 2156-2165, 1983).
[0087]
The target polypeptide can be produced by culturing a transformed cell containing the expression vector prepared as described above (hereinafter referred to as including a transfectant) in a nutrient medium.
The nutrient medium preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the host cell (transformant). Examples of the carbon source include glucose, dextran, soluble starch, and sucrose, and examples of the inorganic nitrogen source or the organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, and so on. Examples include soybean meal and potato extract. If desired, other nutrients (eg, inorganic salts (eg, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (eg, tetracycline, neomycin, ampicillin, kanamycin, etc.)) may be contained. .
The culturing is performed by a method known in the art. Culture conditions, such as temperature, pH of the medium, and culture time are appropriately selected so that the protein of the present invention is produced in large quantities.
[0088]
Specific examples of the culture medium and culture conditions used according to the host cell are shown below, but the invention is not limited thereto.
When the host is a bacterium, an actinomycete, a yeast or a filamentous fungus, for example, a liquid medium containing the above-mentioned nutrients is suitable. Preferably, the medium has a pH of 5 to 8.
If the host is E. coli. In the case of E. coli, preferred mediums include LB medium, M9 medium (Miller et al., Exp. Mol. Genet, Cold Spring Harbor Laboratory, p. 431, 1972), YT medium and the like. In such a case, the cultivation can be carried out usually at 14 to 43 ° C. for about 3 to 24 hours with aeration and stirring as necessary.
[0089]
When the host is a bacterium belonging to the genus Bacillus, the reaction can be carried out usually at 30 to 40 ° C. for about 16 to 96 hours, if necessary, with aeration and stirring.
When the host is yeast, examples of the medium include Burkholder minimum culture (Bostian, Proc. Natl. Acad. Sci. USA, Vol. 77, p. 4505, 1980). Desirably. The cultivation is usually performed at about 20 to 35 ° C. for about 14 to 144 hours, and if necessary, aeration and stirring can be performed.
When the host is an animal cell, for example, a MEM medium containing about 5 to 20% fetal bovine serum (Science, Vol. 122, p. 501, 1952) or a DMEM medium (Virology, Vol. 8, p. 396). 1959), RPMI 1640 medium (J. Am. Med. Assoc., Vol. 199, p. 519, 1967), 199 medium (Proc. Soc. Exp. Biol. Med., Vol. 73, p. 1, 1950). , HamF12 medium and the like can be used. The pH of the medium is preferably about 6 to 8, and the culture is usually performed at about 30 to 40 ° C. for about 15 to 72 hours, and if necessary, aeration and stirring can be performed.
[0090]
When the host is an insect cell, for example, Grace's medium containing fetal bovine serum (Proc. Natl. Acad. Sci. USA, Vol. 82, p. 8404, 1985), etc., and the pH thereof is about 5 to 8 It is preferred that The cultivation is usually performed at about 20 to 40 ° C. for 15 to 100 hours, and if necessary, aeration and stirring may be performed.
The target polypeptide can be produced by culturing the above-mentioned transformed cells (especially animal cells or Escherichia coli) and secreting them into the culture supernatant. That is, the obtained culture is filtered or centrifuged to obtain a culture filtrate (supernatant), and the polypeptide of interest is obtained from the culture filtrate according to a conventional method generally used for purifying and isolating a natural or synthetic protein. Is purified and isolated.
[0091]
Isolation and purification methods include, for example, a method utilizing specific affinity such as affinity column chromatography, a method utilizing solubility such as salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, sodium dodecyl sulfate -Methods utilizing differences in molecular weight such as polyacrylamide gel electrophoresis, methods utilizing charges such as ion exchange chromatography and hydroxylapatite chromatography, methods utilizing differences in hydrophobicity such as reverse phase high performance liquid chromatography, etc. A method using a difference between isoelectric points such as electric point electrophoresis may be used.
[0092]
On the other hand, when the target polypeptide is present in the periplasm or cytoplasm of the cultured transformant, the culture is subjected to a conventional method such as filtration or centrifugation to collect the cells or cells, and the cells are collected in an appropriate buffer. After suspending and breaking cell walls and / or cell membranes such as cells by a method such as ultrasonication, lysozyme and freeze-thaw, a membrane fraction containing the target polypeptide is obtained by a method such as centrifugation or filtration. The membrane fraction is solubilized using a surfactant such as Triton-X100 to obtain a crude solution. Then, the crude solution can be isolated and purified by using a conventional method as exemplified above.
[0093]
【Example】
Hereinafter, the present invention will be described in more detail with reference to Examples, but it is needless to say that the present invention is not limited to only the embodiments described in the Examples.
In the following examples, “TGF-β type II receptor” may be referred to as “TβRII”.
[0094]
Example 1 Preparation of immunogen
CDNA (SEQ ID NO: 1) encoding human TβRII (SEQ ID NO: 2) was prepared by a conventional method using PCR.
Specifically, using cDNA prepared from mRNA obtained from human kidney as a template, it was designed based on cDNA of human TβRII (Cell, Vol. 68, p. 775-758, 1992; GenBank Accession No .: M85079). It was synthesized by PCR using primers according to a conventional method.
[0095]
The obtained human TβRII cDNA containing the translation region was inserted into a commercially available plasmid to prepare an expression vector. CHO-K1 cells (Dainippon Pharmaceutical) were transformed with this vector by electroporation. Transformed cells were cultured in Ham's F12 nutrient medium to express recombinant human TβRII. The expression of human TβRII was confirmed by Western blotting using an anti-human soluble TβRII polyclonal antibody (R & D).
A cell membrane fraction was prepared from the recombinant cells according to a conventional method and used as an immunogen.
[0096]
Example 2 Preparation and selection of hybridoma producing anti-human TβRII human monoclonal antibody
The preparation of the monoclonal antibody in this example is described in Experimental Medicine (separate volume), Cell Engineering Handbook (edited by Toshio Kuroki et al., Published by Yodosha, pp. 66-74, 1992) and an introduction to monoclonal antibody experimental procedures (Tamie Ando) , Published by Kodansha, 1991).
[0097]
As the human TβRII as an immunogen (antigen), the cell membrane fraction of the recombinant human TβRII-expressing recombinant cells prepared above was used.
As the animal to be immunized, a transgenic mouse producing a human IgG2 antibody produced by the method described above was used (Nature Genetics, Vol. 7, p. 13-21, 1994; Nature Genetics, Vol. 15, p. 146-156, 1997; Japanese Patent Application Laid-Open No. 4-504365; Japanese Patent Application Laid-Open No. 7-509137; Nikkei Science, June, pp. 40-50, 1995, etc.).
The cell culture operation was performed using a multiwell microplate.
[0098]
The above-mentioned human antibody-producing transgenic mice (6 mice) were immunized with the antigen for the first time (day 0) by injecting the antigen together with complete Freund's adjuvant (Freund's Complete Adjuvant, ICN / CAPPEL) into the hood pad and peritoneal cavity. . Every week after the initial immunization, the antigen was boosted three times by injection into the hood pad and the peritoneal cavity together with incomplete Freund's Incomplete Adjuvant (ICN / CAPPEL), and further boosted with lymph node cells described below. Two days before the acquisition, the same antigen was used for the final immunization. The antigen and the adjuvant were mixed at a ratio of about 1: 1 for several minutes to about 1 hour.
[0099]
Lymphocytes obtained from lymph nodes and spleens isolated from each animal and mouse myeloma cells P3 / X63-AG8.653 (ATCC No .: CRL-1580) were mixed at a ratio of 5: 1, and polyethylene glycol 1500 was used as a fusion agent. A number of hybridomas were prepared by cell fusion using (Roche). The fused cells were washed twice with an RPMI1640 medium (containing 1% penicillin-streptomycin; manufactured by SIGMA), suspended in an ASF104 medium containing 10% FBS and HAT (manufactured by Kojin Bio Inc.), and seeded on a 96-well microplate.
[0100]
Hybridomas producing an anti-human TβRII human monoclonal antibody were screened from the hybridomas prepared above by the following two types of ELISA.
<1> Cell ELISA
One week after the cell fusion, the medium was replaced with a new medium, and the culture supernatant after 3 days was removed, and the hybridomas were primarily screened by Cell ELISA.
The CHO-K1 cells (Dainippon Pharmaceutical Co., Ltd.) and the recombinant CHO cells expressing human TβRII prepared above were washed with PBS and then treated with Trypsin-EDTA solution (GIBCO BRL) to collect each cell. Cells are placed in a 96-well microplate at 3-20 × 10 ³ The cells were seeded at cell / well (100 μl / well) and cultured for 2 days until confluence.
After the culture, the culture supernatant was discarded, a primary antibody solution (diluted purified anti-human TβRII antibody or supernatant of each hybridoma; 50 μl / well) was added, and the mixture was allowed to stand at room temperature for 1.5 hours. The plate was washed twice with ASF104 medium containing 1% fetal calf serum ("wash buffer"; manufactured by Niken Biomedical Research Institute) and diluted with a 1,000- to 10,000-fold HRP (horse radiation) secondary antibody. (peroxidase) -labeled anti-human IgFc antibody (50 μl / well; manufactured by American Qualex) or HRP-labeled mouse IgFc antibody (50 μl / well; manufactured by Amersham) was added, and the mixture was allowed to stand at room temperature for 1 hour. The plate was washed twice with a washing buffer (200 μl / well), and TMB peroxidase substrate solution (100 μl / well; manufactured by Bio-Rad) as a substrate was added, followed by color development for 20 minutes, followed by 0.5 M HH. ₂ SO ₄ (50 μl / well) was added to stop the reaction, and the absorbance at a wavelength of 450 nm was measured with a microplate reader (manufactured by Bio-Rad).
[0101]
Positive hybridomas were selected by comparing with the value in Cell ELISA using wild-type CHO-K1 cells as a control.
As a primary antibody negative control, an anti-KLH human monoclonal antibody (JMab23) prepared by immunizing the above-described human antibody-producing transgenic mouse with KLH (keyhole limpet hemocyanin, manufactured by PIERCE) was used. .
This Cell ELISA technique was also used for analysis of cross-reactivity to mouse TβRII and rat TβRII and isotyping of the monoclonal antibody of the present invention.
For the former Cell ELISA, mouse TβRII-highly expressing CHO cells or rat TβRII-highly expressing CHO cells prepared in the same manner as described above were used. In the latter Cell ELISA, the following antibodies for isotyping were used.
1. HRP-labeled anti-human IgFc antibody (EY Lab)
2. HRP-labeled anti-human IgM antibody (manufactured by American Qualex)
3. HRP-labeled anti-human Igκ antibody (manufactured by TAGO)
4. HRP-labeled anti-mouse Igλ antibody (manufactured by Solution Biotechnology Associates)
[0102]
<2> ELISA
A human TβRII-Fc chimeric molecule (25 ng / well; 50 μl / well; Genzyme) or a human B7h-Fc chimeric molecule (negative control; see International Patent Publication WO01 / 87981) is placed on an ELISA microplate (Nunc). In addition, the mixture was left standing at room temperature for 1 hour to perform coating, Block Ace (200 μl / well) was added, and the mixture was left standing at room temperature for 2 hours or more to block unreacted portions. Then, after washing twice with PBS-T (200 ml / well), the primary antibody (diluted anti-human TβRII antibody (positive control; anti-TβRII human monoclonal antibody produced by clone TR2B19; see International Patent Publication WO 01-36642). ) Or the culture supernatant of each hybridoma; 50 μl / well) was added, and the mixture was allowed to stand at room temperature for 1.5 hours. Next, the plate was washed three times with PBS-T (200 μl / well), and a secondary antibody, an HRP-labeled anti-human Igκ antibody (2,000-fold dilution: 50 μl / well; manufactured by TAGO) was added. Let stand for hours. Next, the plate was washed four times with PBS-T, and TMB peroxidase substrate solution (100 μl / well; manufactured by Bio Rad) as a substrate was added, followed by color development for 15 minutes. ₂ SO ₄ (50 μl / well) was added to stop the reaction, and the absorbance at a wavelength of 450 nm was measured with a microplate reader (manufactured by Bio-Rad).
Positive hybridomas were selected by comparison with the values obtained by ELISA using a human B7h-Fc chimera molecule as a control.
The above-mentioned anti-KLH human monoclonal antibody (JMab23) was used as a primary antibody negative control.
[0103]
Screening by the Cell ELISA described above revealed that all 480 of the 480 clones were positive. In the above-mentioned ELISA screening, 27 out of 480 clones positive for Cell ELISA were positive. In addition, 7 out of 480 clones were human IgG and human Igκ in the above-mentioned isotyping Cell ELISA.
Based on a series of screening results, hybridoma clones JMab195, JMab197, and JMab198 that were positive in the above two types of screening and whose isotypes were human IgG2 and human Igκ were selected.
The well containing the selected clones was replaced with a separately prepared cloning medium ASF104 medium (1% penicillin-streptomycin; 1 × HT; 10% fetal bovine serum; 1% non-essential amino acid; and 1% T-STIM), and further replaced. The cells were cultured and cloned by the limiting dilution method.
In addition, as a result of the cross-reactivity test by the Cell ELISA, none of the anti-antibodies produced by the three clones showed cross-reactivity with either rat or mouse TβRII.
[0104]
Example 3 Inhibitory Activity of Anti-TGFβ Human Monoclonal Antibody on Cell Growth Inhibition Induced by TGFβ Stimulation
The biological activity of the anti-human TβRII human monoclonal antibody produced by each of the hybridomas selected above was analyzed as follows.
TGF-β has a cell growth promoting activity on mesenchymal cells such as fibroblasts and vascular smooth muscle cells of various tissues, for example, various types of cells such as epithelial cells, vascular endothelial cells and blood cells. Has the activity of suppressing cell proliferation.
In this test, the inhibitory effect of the anti-human TβRII human monoclonal antibody produced by each of the hybridoma clones selected above on the suppression of proliferation of epithelial cells by TGF-β stimulation was analyzed as follows.
[0105]
A 96-well microtiter plate was seeded with human-derived epithelial keratinocytes (200 cells / 100 μl DMEM-high glucose / well containing 0.1% FBS), and CO was seeded. ₂ The cells were cultured in an incubator for 2 days. The medium was removed, each well was washed twice with the same culture medium, and a new culture medium was added. Next, an anti-TβRII human monoclonal antibody (0.23; 0.47; 0.93; 1.88; 3.75; 7.5) purified from the culture supernatant of the hybridomas (JMab195, JMab197 and JMab198) prepared above. 15; 30; 60 μg / ml) at 37 ° C. ₂ The cells were cultured in an incubator for 3 hours. After the culture, human TGFβ1 (0.3 ng / ml; 50 μl / well; manufactured by Genzyme TECHNE) was added and CO 2 was added. ₂ The cells were cultured in an incubator for 2 days.
[0106]
Then, in each well, methyl- [ ³ [H] -thymidine (0.5 μCi / well; manufactured by Amersham-Pharmacia) was added, and the cells were further cultured for 6 hours. Cells were trapped on GF / C filters using a cell harvester (PACKARD). After the filter was dried at 40 ° C. for 2 hours or more, Microscinti O (manufactured by PACKARD) was added, and the filter was trapped by the filter. ³ The radioactivity of H was measured using a β counter (TOP COUNT) and used as an index of cell proliferation activity.
In addition, as a control test, the test was performed in the same manner as described above without adding human TGF-β1. As the negative control antibody, the aforementioned anti-KLH human monoclonal antibody (JMab23) was used.
The test was performed three times for each clone, and the IC of each monoclonal antibody was determined. ₅₀ The average value of (μg / ml) was as follows.
JMab195: IC ₅₀ (Μg / ml) = 3.03 ± 0.86
JMab197: IC ₅₀ (Μg / ml) = 4.60 ± 0.89
JMab198: IC ₅₀ (Μg / ml) = 4.14 ± 0.93
From these results, it was shown that any of the anti-human TGFβ human monoclonal antibodies significantly suppressed the suppression of proliferation of human-derived keratinocytes (keratinocytes) induced by TGFβ1 stimulation.
[0107]
Example 4 Inhibitory Activity of Anti-Human TGFβ Human Monoclonal Antibody on Increased PAI-I Secretion from Mesangial Cells Induced by TGFβ Stimulation
225cm ₂ Using a flask, normal human mesangial cells (CC-2559; Sanko Junyaku) were converted to RPMI 1640 medium (Niken Bio) containing 1% Penicillin-Streptmycin (GIBCO BRL) and 20% fetal bovine serum (FBS; MORGATE). CO at 37 ° C ₂ The cells were cultured in an incubator. When the cells reached 90% confluence, they were used in the following tests.
[0108]
Cells (3 × 10 ⁴ cells / well), and on the following day, the medium was replaced with a PRMI1640 medium containing no FBS (containing 1% Penicillin-Streptmycin) (hereinafter referred to as FBS (-) / RPMI1640), followed by 24 hours of starvation (Starvation). . The medium was removed, FBS (−) / RPMI1640 (500 μl) and purified anti-TGFβ human monoclonal antibody (250 μl) derived from each of the hybridomas (JMab195, JMab197 and JMab198) selected above were added to each well, and incubated for 1 hour. Thereafter, cells were stimulated by adding human TGFβ1 (250 μl of 0.4 ng / ml; manufactured by R & D Systems). After culturing for 24 hours, the culture supernatant (250 μl / well) was collected in a 96-well microplate. The remaining culture supernatant was discarded, and FBS (−) / RPMI1640 (500 μl) containing 5% WST-1 (Roche) was added to the cells, followed by incubation for 4 hours. The culture supernatant (100 μl) was collected in a 96-well microplate, and the absorbance at a wavelength of 450 to 650 nm was measured using a microplate reader.
[0109]
The concentration of PAI-I (plasmogen activator inhibitor) secreted into the culture supernatant was measured using a commercially available ELISA kit, TintElize PAI-I kit (manufactured by Biopool International), according to the operation manual attached to the kit. Various solutions (40; 20; 10; 5; 2.5; 0 ng / ml) of Standard PAI-I Plasma, a reagent of the kit, were prepared as human PAI-I standard substances.
PET Buffer (100 μl / well) was added to the microplate in the kit, and the plate was slightly shaken for 1 minute. The culture supernatant of each cell obtained above (diluted 2-fold with distilled water; 20 μl) or a PAI-I standard (20 μl) was added to the N well and the A well. Further, a conjugate (50 μl / well) was added and the mixture was gently shaken for 2 hours. Next, the solution in each well was removed by decanting, and washed (4 times) by adding PET Buffer (250 μl). After washing, a substrate (200 μl / well) was added, and the mixture was gently shaken for 15 minutes. Next, sulfuric acid (50 mol / well of 0.5 mol / l) was added, and the mixture was allowed to stand for 10 minutes under light shielding, and then the absorbance at a wavelength of 490 nm was measured using a microplate reader. The concentration of PAI-I secreted into each cell culture supernatant was determined from the obtained absorbance according to a calibration curve.
[0110]
As the negative control, the above-mentioned anti-KLH human monoclonal antibody (JMab23) and anti-human CETP monoclonal antibody (JMab109; see International Application Publication No. WO96 / 34948) were used. In addition, as comparative controls, separately prepared anti-human TβRII human monoclonal antibodies (anti-TβRII human monoclonal antibodies produced by clone TR2B19; see WO01-36642) and anti-TGFβ1 monoclonal antibodies (JMab83; ATCC No. HB- Monoclonal antibody 1D11.16.8) produced by 9849 hybridoma was used.
[0111]
The results are shown in FIG.
Inhibitory activity of each anti-human TβRII human monoclonal antibody on the secretion of PAI-I from mesangial cells induced by TGF stimulation (IC ₅₀ (Μg / ml)) was as follows.
JMab195: 2.2 μg / ml
JMab197:> 100 μg / ml
JMab198:> 100 μg / ml
Control (JMab148 / TR2B19): 22.3 μg / ml
[0112]
As a result, the PAI-I production inhibitory activity of JMab195 was much stronger than that of other anti-human TβRII human monoclonal antibodies (JMab197 and JMab198), and the inhibitory activity of control anti-human TβRII human monoclonal antibody (JMab148 / TR2B19). It was found that the strength was 10 times or more stronger than that of the first embodiment.
In addition, as a result of analyzing the inhibitory activity of each antibody on TGFβ-induced PAI-I production using mesangial cells of monkeys (cynomolgus monkey; Cary) in the same manner as described above, the anti-TβRII human monoclonal antibody JMab195 of the present invention The secretion of PAI-I from salmesangial cells induced by TGFβ stimulation was significantly inhibited in an antibody concentration-dependent manner. This result indicated that JMab195 not only reacts with monkey TβRII but also has its neutralizing activity.
[0113]
Example 5 Measurement of Affinity and Neutralizing Activity of Anti-Human TGF-βII Type Receptor Human Monoclonal Antibodies (JMab195 and JMab148) for Antigen (Human TGF-βII Type Receptor)
The anti-human TGF-β type II receptor human monoclonal antibody JMab195 which showed the strongest inhibitory activity in the TGFβ-induced mesangial cell PAI-I production inhibitory activity test and the anti-human TGF-βII type human monoclonal antibody as a positive control The binding rate constant (ka), dissociation rate constant (kd), and dissociation constant of JMab148 (an anti-TβRII human monoclonal antibody produced by clone TR2B19; see WO01-36642) with the human TGF-βII type receptor ( Kd) was measured using a commercially available measurement kit Biacore X (manufactured by Amersham-Pharmacia).
Operations other than the immobilization of the antigen (human TβRII-Fc chimera molecule) described below on the sensor chip were performed according to the instruction manual attached to the commercially available measurement kit Biacore X (manufactured by Amersham-Pharmacia) and the experimental operation method.
[0114]
A running buffer (D-PBS (-); manufactured by Nikken Biomedical Research Laboratories) is flowed through the flow cell 1 (Flow Cell-1) set in the kit at 20 μl / min, and then 100 mM NaOH (10 μl). And 1% Triton X-100 (manufactured by Sigma) were added to wash the cell. After running the running buffer for 10 minutes and confirming that the baseline was stabilized, the flow rate of the running buffer was adjusted to 5 μl / min. A solution (35 μl) consisting of 5 mM NHS (N-hydroxysuccinimide) and 200 mM EDC (N-Ethyl-N ′-(dimethylaminopropyl) carbodiimide) was added to activate the carboxyl group of CM coated on the sensor chip surface. .
Next, 30 μl of a solution of a human TβRII-Fc chimeric molecule (Genzyme) (5 μg / ml; dissolved in a 10 mM sodium acetate buffer (pH 5.0)) was added, and the human TβRII-Fc was used as a sensor chip. Immobilized.
Then, unreacted activated carboxyl groups were blocked by adding 35 μl of 1 M ethanolamine hydrochloride. This operation of immobilizing the antigen was performed at 25 ° C. The same immobilization operation was performed a plurality of times for the following multiple tests.
In the immobilization operation, the amount of the immobilized antigen was 292.3 to 564.4 RU (resonance unit). RU indicates the mass per area, and 1 RU = 1 pg / mm ² It is.
[0115]
Flow cell 2 (Flow Cell-2) as a reference was capped by treating in the same manner as above without adding the human TβRII-Fc.
A phosphate buffer solution is flowed through the flow cell (sensor chip) at a flow rate of 20 μl / min, and the purified anti-human TGF-β type II receptor human monoclonal antibody JMab195 or JMab148 (6-16 μg / ml, 60 μl) prepared above is added. did.
The measurement was performed using the binding phase for 2 minutes and the dissociation phase for 2 minutes as standard conditions. The amount of the antibody bound to the antigen and the amount of the antibody dissociated from the antigen were measured over time to obtain a sensorgram.
[0116]
Based on the obtained sensorgram data, the binding rate constant (ka), dissociation rate constant (kd), and dissociation constant (Kd; Kd; Kd = kd / ka) were determined using analysis software (BIAevaluation 3.0) attached to the kit. Was calculated.
The anti-human TβRII human monoclonal antibody JMab195 of the present invention has a dissociation constant (average of six tests) of 8.06 × 10 6 ^-10 (M), the dissociation constant of the control anti-human TβRII human monoclonal antibody JMab148 (mean of four tests) 1.91 × 10 ^-9 It had significantly higher antigen affinity and neutralizing activity than (M).
Surprisingly, the dissociation constant of JMab195 is extremely rare even in the field of monoclonal antibody research and development. ^-10 The antigen affinity was remarkably high, on the order of (M) or more.
[0117]
Example 6 Gene sequence of anti-human TβRII human monoclonal antibody JMab195 and
Determination and analysis of amino acid sequence
A cDNA sequence encoding the variable region of the heavy chain (Heavy Chain) and the cDNA sequence encoding the variable region and the constant region of the light chain (Light Chain) constituting the human monoclonal antibody JMab195 against human TβRII prepared in the above Examples. Was determined as described below, and the structural characteristics of the gene were analyzed.
After culturing the hybridoma producing JMab 195 produced in the above-described example, centrifugation is performed to collect the precipitate. ⁺ Stored at -80 ° C until RNA extraction.
[0118]
<1> Preparation of mRNA
PolyA from hybridoma ⁺ RNA extraction and purification were performed as follows using a commercially available QuickPrep mRNA Purification Kit (manufactured by Amersham Pharmacia). According to the experimental operation manual attached to the kit, PolyA was obtained from the frozen hybridoma cells. ⁺ PolyA purified by RNA extraction and purification twice ⁺ Collected as RNA. PolyA obtained ⁺ The concentration of RNA was determined by measuring the absorbance at a wavelength of 260 nm.
[0119]
<2> cDNA synthesis
PolyA obtained ⁺ Using RNA as a template, a double-stranded cDNA was synthesized by a reverse transscriptase method using a commercially available cDNBA synthesis kit SuperScript Choice System for cDNA synthesis (manufactured by GIBCO BRL) and a synthetic oligonucleotide NotI-T primer (SEQ ID NO: 3).
That is, the purified PolyA ⁺ RNA (about 5 μg) and NotI-T primer (400 pmol) were mixed, heated at 70 ° C. for 10 minutes, ice-cooled, 5 × 1st strand buffer (4 μl), 10 mM dNTP solution (1 μl) and 0.1 M A DTT solution (2 μl) was added and mixed. After performing pre-incubation at 37 ° C. for 2 minutes, SuperScriptII Reverse Transcriptase (5 μl) was added, and the reaction was carried out at 37 ° C. for 1 hour. After the reaction, DEPC-treated water (93 μl), 10 mM dNTP (3 μl), 5 × 2 nd strand buffer (30 μl), DNA polymerase I (4 μl), RNaseH (1 μl) and E. coli. Coli DNA ligase (1 μl) was added, mixed, and reacted at 16 ° C. for 2 hours. After the reaction, T4 DNA polymerase (2 μl) was added, and the mixture was further reacted for 2 minutes. Then, phenol / chloroform (150 μl) was added, and the mixture was centrifuged at 14,500 rpm for 5 minutes at room temperature to collect an aqueous layer (130 μl). To the collected aqueous layer, 3M NaOAc (13 μl) and ethanol (325 μl) were added, mixed, cooled at −80 ° C., centrifuged at 14,500 rpm for 15 minutes, and the supernatant was discarded to collect a pellet. After the pellet was washed with 70% ethanol, the supernatant was discarded, dried under reduced pressure, and dissolved in TE buffer to obtain a cDNA solution.
[0120]
<3> Preparation of cDNA adapter
24adp (100 pmol / μl; 50 μl) and 5′-end phosphorylated 20 adp (100 pmol / μl; 50 μl) were added to the above cDNA solution, mixed, and 3M NaOAc (10 μl) and ethanol (250 μl) were added. And cooled overnight. After cooling, the mixture was centrifuged (14,500 rpm; 4 ° C .; 15 minutes), the supernatant was discarded, and the pellet was collected. The pellet was washed with 70% ethanol, the supernatant was discarded, and the pellet was dried. Add an annealing buffer (10 mM Tris-HCl; pH 8.0; 0.1 mM EDTA; 5 mM MgCl) to the dried pellet. ₂ 200 mM NaCl), and the mixture was heated for 1 hour and then returned to room temperature to obtain a cDNA adapter solution.
[0121]
<4> Preparation of cDNA library
The adapter solution (6 μl) was added to the cDNA solution (48 μl) obtained above, and Ligation High (27 μl; Toyobo) was further added and mixed. After mixing, the mixture was reacted at 16 ° C. for about 1.5 hours, and then heated at 70 ° C. for 10 minutes to stop the reaction, followed by cooling immediately. TE buffer (19 μl), 3 M NaOAc and ethanol (250 μl) were added to the ice-cooled sample, mixed, and cooled to −80 ° C. Then, the mixture was centrifuged (14,500 rpm; 15 minutes; 4 ° C.), the supernatant was discarded, and the pellet was washed with 70% ethanol and dried under reduced pressure. After drying, sterile distilled water and 10XH buffer (Takara Shuzo) were added to dissolve the pellet. Next, a restriction enzyme NotI (3 μl) was added and reacted at 37 ° C. for 2 hours. Next, 10 mM ATP (2 μl) and T4 kinase (2 μl) were added to the reaction solution, and the mixture was reacted at 37 ° C. for 30 minutes. After the reaction, 3M NaOAc (15 μl) and ethanol (375 μl) were added, cooled to −80 ° C., centrifuged (14,500 rpm; 4 ° C .; 15 minutes), discarded the supernatant, washed with 70% ethanol, and dried under reduced pressure. And dissolved in TE buffer (20 μl). This was used as a complete cDNA solution.
[0122]
A loading buffer (4 μl; manufactured by Takara Shuzo) was added to the complete cDNA solution, a half of which was applied to a polyacrylamide gel (manufactured by Daiichi Pure Chemicals), and electrophoresed. The gel was immersed in an ethidium bromide solution to stain the nucleic acid (15 minutes) and irradiated with ultraviolet light to take a photograph. DNA from 0.5 to 3 kb in size was recovered from the gel. Each of the recovered DNAs was ligated to a commercially available lambda phage vector λEXcell (manufactured by Amersham Pharmacia) using a commercially available DNA Ligase (manufactured by TOYOBO). Next, the DNA reaction product was used as a lambda phage using a commercially available lambda phage packaging kit Gigapack III Gold (manufactured by STRATAGENE), and a cDNA library was prepared using Escherichia coli NM522 attached to the vector λEXcell as a host.
[0123]
The cDNA library was screened by a conventional method using a synthetic oligo DNA as a probe to prepare cDNAs encoding the antibody heavy chain and the antibody light chain, respectively. As the probe relating to the antibody heavy chain, the synthetic oligo DNA described in SEQ ID NO: 4 was used. As the probe relating to the antibody light chain, the synthetic oligo DNA shown in SEQ ID NO: 5 was used.
The nucleotide sequence of each of the obtained cDNAs was determined by using commercially available Applied Biosystems 3100 Genetic Analyzer (manufactured by Applied Biosystems) and PRISM377 DNA Sequencer (manufactured using various DNA-based primers using various primers made by PE-Applied Biosystems).
The cDNA sequence containing the open reading frame (ORF) of the heavy chain of a human monoclonal antibody against human TβRII produced by hybridoma JMab195 is shown in SEQ ID NO: 6, and the full length of the heavy chain encoded by the open reading frame (the signal sequence is (SEQ ID NO: 7), the cDNA sequence containing the open reading frame (ORF) of the light chain in SEQ ID NO: 8, and the full length of the heavy chain encoded by the open reading frame (signal sequence SEQ ID NO: 9).
[0124]
Based on each of the determined DNA sequences, a human immunoglobulin of a variable region of human immunoglobulin prepared by Tomlinson et al. Using gene sequence analysis software ABI PRISM DNA sequencing software and ABI PRISM AutoAssembler (manufactured by Applied Biosystems). V BASE / V GOLD (Tomlinson, IM, et al; V BASE: "A Data of Human Immunoglobulin Variable Region Genes"; MRC Center for Medicine, England, Russia, England, England, 2nd Edition;
As a result, it was revealed that the V region, D region, J region, and C region of each of the heavy and light chains of the human monoclonal antibody had the highest homology to the following segments.
<Heavy chain>
V region: 3-23; D region: DN4; J region: JH5b; C region: human G2
<Light chain>
V region: A30; J region: JK1; C region: human κ
[0125]
As a result of further analysis, the heavy chain V region DNA of JMab195 was compared with the human immunoglobulin heavy chain V gene segment 3-23 (SEQ ID NO: 12; the amino acid sequence was SEQ ID NO: 13). It was found that the gene had a mutation of three bases, and as a result, three amino acid mutations occurred.
GTC (Val) → ATC (Ile)
GCT (Ala) → GTT (Val)
GGT (Gly) → GTT (Val)
[0126]
On the other hand, the light chain V region DNA of JMab195 has a deletion of 3 bases and a mutation of 4 bases described below as compared with the human immunoglobulin heavy chain V gene segment A30. The mutation was found to have occurred.
CAA (Gln) → CTA (Leu)
AGC (Ser) → AAC (Asn)
CAG (Gln) → CAA (Gln)
AGT (Ser) → ACT (Thr)
[0127]
Example 7 Therapeutic effect of anti-human TβRII monoclonal antibody JMab195 on kidney disease and tissue fibrosis
The therapeutic effect of the anti-human TβRII human monoclonal antibody JMab195 of the present invention on renal disease and tissue fibrosis was examined using a disease model in which cynomolgus monkey kidney had artificial dysfunction and fibrosis.
The disease model includes various diseases or pathological conditions, for example, kidney diseases (renal insufficiency, nephritis, renal fibrosis, etc.), various tissue fibrosis (renal fibrosis, pulmonary fibrosis, fibrosis in liver tissue, skin Fibrosis in tissues, such as fibrosis in synovial tissue associated with rheumatoid arthritis, fibrosis associated with various cancers), skin diseases (scleroderma, psoriasis, atopy, etc.), liver diseases (cirrhosis, fibers in liver tissue) Disease, hepatitis, etc.), pulmonary diseases (pulmonary fibrosis, pneumonia, etc.), rheumatoid arthritis, arteriosclerosis, and other pathological features or clinical findings . Thus, the therapeutic effects observed in this study are representative of the therapeutic effects of such a series of diseases or pathological conditions.
[0128]
Cynomolgus monkey (male, 3 years old, 3 animals per group, Cary) intramuscularly with ketamine (1 ml / body; Sankyo) and intravenous sodium pentobarbital (5-10 mg / kg; Dainippon Pharmaceutical) Administered and anesthetized. After shaving the periphery of the left flank and disinfecting with 70% ethanol, the left flank is incised and the ureter on the left kidney side is ligated at two places (a position close to the kidney and a position close to the bladder). Cleavage (UUO, Unilateral urethane obstruction). Immediately after the ureter was cut, the abdominal wall was sutured, and ampicillin sodium / cloxacillin sodium (0.5 ml / body; manufactured by Meiji Seika) was intramuscularly administered.
Immediately after the suture, one of the following test substances or control substances was intravenously administered. As a solvent for the substance, PBS (−) / 0.01% polysorbate 80 was used.
[0129]
1. Test substance
Various concentrations of anti-human TβRII human monoclonal antibody JMab195
(1) 30 mg / kg; 5 mg / ml to 6 ml / kg
(2) 10 mg / kg; 1.67 mg / ml to 6 ml / kg
(3) 3 mg / kg; 0.5 mg / ml to 6 ml / kg
2. Positive control substance
Various concentrations of anti-human TGFβ1 monoclonal antibody JMab83 (ATCC No. HB-9849)
Monoclonal antibody 1D11.16.8 produced by the hybridoma
(1) 30 mg / kg; 5 mg / ml to 6 ml / kg
(2) 10 mg / kg; 1.67 mg / ml to 6 ml / kg
(3) 3 mg / kg; 0.5 mg / ml to 6 ml / kg
3. Negative control substance
Various concentrations of anti-KLH human monoclonal antibody JMab23 (described above)
(1) 30 mg / kg; 5 mg / ml to 6 ml / kg
(2) 10 mg / kg; 1.67 mg / ml to 6 ml / kg
(3) 3 mg / kg; 0.5 mg / ml to 6 ml / kg
[0130]
After the administration, the test monkeys were bred in a breeding room by an ordinary method.
Ketamine (1 ml / body; manufactured by Sankyo) was intramuscularly administered and sodium pentobarbital (5 to 10 mg / kg; manufactured by Dainippon Pharmaceutical) was intravenously administered to each of the test monkeys on day 3 from the one-kidney ligation procedure. After anesthesia, the animals were sacrificed by exsanguination. The abdomen was then opened along the midline, and the left kidney was removed as a UUO-treated kidney and the right kidney as a control normal kidney. After measuring the wet weight of each kidney, a part of the renal cortex was collected as a sample for mRNA measurement performed below, frozen with liquid nitrogen, and stored at -80 ° C.
[0131]
According to a conventional method, Type I collagen mRNA and GAPDH mRNA expressed in each renal cortex sample were quantified as follows.
Total RNA of each renal cortex sample was obtained using a commercially available RNeasy Mini Kit (manufactured by QIAGEN) in accordance with the experimental operation manual attached to the kit. RNA was quantified using Ultraspec300 (manufactured by Pharmacia Biotech).
Using the obtained total RNA as a template, M-MLV Reverse Transcriptase (manufactured by Invitrogen), RNase-Free DNase Set (manufactured by QUIAGEN), 5 × 1st strand buffer and 0.1M DTT, 10M dNTP (manufactured by Invitrogen) cDNA was prepared using a primer (manufactured by Invitrogen) according to a conventional method.
[0132]
The obtained cDNA was added to RNase Free Water (manufactured by QUIAGEN), 10 × TaqMan buffer A (manufactured by Applied Biosystems), 25 mM MgCl ₂ , Primesense, Primer antisense, Fluorescent Probe, and AmpliTaq Gold (manufactured by Applied Biosystems) were added and reacted, and the ABI PRISM 7700 Sequence Detector was used for each of the samples obtained by using the samples obtained by using the samples obtained by using the samples obtained by using the samples obtained from the sampler. Was measured.
The expression level of Type I collagen mRNA in each sample was analyzed by dividing the copy number of Type I collagen mRNA measured above by the copy number of GAPDH mRNA.
[0133]
The results are shown in FIG.
As a result, the anti-TβRII human monoclonal antibody JMab195 of the present invention significantly suppressed the expression of mRNA of Type I collagen, which is a typical parameter of kidney disease and tissue fibrosis, as compared with the negative control. Also, surprisingly, the inhibitory effect of the JMab195 was even higher than that of a monoclonal antibody against TGFβ1, a TβRII ligand (positive control; 1D11).
This indicated that the anti-TβRII human monoclonal antibody JMab195 of the present invention had significant inhibitory and therapeutic effects on kidney disease and tissue fibrosis.
[0134]
【The invention's effect】
Since the monoclonal antibody of the present invention that binds to the human TGF-β type II receptor has extremely high binding affinity and neutralizing activity for the human TGF-β type II receptor, it contains the monoclonal antibody or a part thereof. Can be used for various diseases caused by the action of TGF-β, such as renal diseases (renal fibrosis, nephritis, renal failure, renal sclerosis, etc.), lung diseases (eg, pulmonary fibrosis, pneumonia, etc.) ), Liver diseases (eg, liver tissue fibrosis, cirrhosis, hepatitis, etc.), skin diseases (eg, wounds, scleroderma, psoriasis, keloids, etc.), arthritis (eg, rheumatoid arthritis, osteoarthritis, etc.) Vascular disease (eg, vascular restenosis, rheumatic vasculitis, etc.), tissue fibrosis in various organs (including tissue fibrosis complicated by various cancers), and arteriosclerosis (including complicated tissue fibrosis). )Such Onset and / or inhibiting the progression, prevent, it is useful as a medicament for the treatment or prevention of the disease.
[0135]
[Sequence list]

[0136]
"Sequence List Free Text"
SEQ ID NO: 3
Other information: description of artificial sequence: artificially synthesized primer sequence.
SEQ ID NO: 4
Other information: Description of artificial sequence: artificially synthesized probe sequence.
SEQ ID NO: 5
Other information: Description of artificial sequence: artificially synthesized probe sequence.
[0137]
[Brief description of the drawings]
FIG. 1 shows the inhibitory activity of an anti-human TGFβ human monoclonal antibody on the enhancement of PAI-I secretion from mesangial cells induced by TGFβ stimulation.
FIG. 2 is a graph showing the inhibitory activity of anti-human TGFβ human monoclonal antibodies on the increased expression of Type I collagen mRNA that occurs with the progression of renal insufficiency in the kidneys of monkeys treated with UUO.

Claims (34)

A monoclonal antibody which binds to a human TGF-β type II receptor, wherein the DNA of the V region encoding the variable region of the heavy chain is derived from the human immunoglobulin heavy chain V gene segment 3-23. Antibody or part thereof. A monoclonal antibody that binds to a human TGF-β type II receptor, wherein the DNA of the V region encoding the variable region of the light chain is derived from the human immunoglobulin light chain V gene segment A30. Part of that. A monoclonal antibody that binds to a human TGF-β type II receptor, wherein the DNA of the V region encoding the variable region of the heavy chain is derived from the human immunoglobulin heavy chain V gene segment 3-23, and A monoclonal antibody or a part thereof, wherein the DNA of the V region encoding the variable region is derived from human immunoglobulin light chain V gene segment A30. A monoclonal antibody that binds to a human TGF-β type II receptor, wherein the heavy chain variable region comprises the following amino acid sequence (a) or (b), or a part thereof:
(A) the amino acid sequence of amino acids 2 to 115 of SEQ ID NO: 6;
(B) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of amino acids 2 to 115 of SEQ ID NO: 6. A monoclonal antibody that binds to a human TGF-β type II receptor, wherein the variable region of the light chain comprises the following amino acid sequence (c) or (d), or a part thereof:
(C) the amino acid sequence of amino acids 2 to 117 of SEQ ID NO: 8;
(D) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 2 to 117 of SEQ ID NO: 8. A monoclonal antibody that binds to a human TGF-β type II receptor, wherein the heavy chain variable region comprises the following amino acid sequence (a) or (b), and the light chain variable region comprises the following (c) or A monoclonal antibody or a part thereof comprising the amino acid sequence of (d):
(A) the amino acid sequence of amino acids 2 to 115 of SEQ ID NO: 6;
(B) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 2 to 115 of SEQ ID NO: 6;
(C) the amino acid sequence of amino acids 2 to 117 of SEQ ID NO: 8;
(D) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 2 to 117 of SEQ ID NO: 8. A monoclonal antibody that binds to a human TGF-β type II receptor, wherein the heavy chain variable region has the following amino acid sequence (a) or (b):
(A) the amino acid sequence of amino acids 2 to 458 of SEQ ID NO: 6;
(B) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acid Nos. 2 to 458 of SEQ ID NO: 6. A monoclonal antibody that binds to a human TGF-β type II receptor, wherein the variable region of the light chain has the following amino acid sequence (c) or (d), or a part thereof:
(C) the amino acid sequence of amino acids 2 to 236 of SEQ ID NO: 8;
(D) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 2 to 236 of SEQ ID NO: 8. A monoclonal antibody that binds to a human TGF-β type II receptor, wherein the variable region of the heavy chain has the amino acid sequence of the following (a) or (b), and the variable region of the light chain is the following (c) Or a monoclonal antibody having the amino acid sequence of (d) or a part thereof:
(A) the amino acid sequence of amino acids 2 to 458 of SEQ ID NO: 6;
(B) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 2 to 458 of SEQ ID NO: 6;
(C) the amino acid sequence of amino acids 2 to 236 of SEQ ID NO: 8;
(D) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 2 to 236 of SEQ ID NO: 8. A monoclonal antibody that binds to human TGF-β type II receptor, wherein the dissociation constant (Kd) with human TGF-β type II receptor is smaller than 10 × 10 ⁻¹⁰ (M). Monoclonal antibodies or parts thereof. The monoclonal antibody according to any one of claims 1 to 10, or a part thereof, wherein the monoclonal antibody is a human monoclonal antibody. A cell that produces the monoclonal antibody according to any one of claims 1 to 11. 13. The cell according to claim 12, wherein the cell is a fused cell obtained by fusing a mammalian B cell and a mammalian myeloma cell with the ability to produce the human monoclonal antibody. Genetically modified cells wherein the cells are transformed by introducing either the DNA encoding the heavy chain of the monoclonal antibody or the DNA encoding the light chain thereof, or both DNAs into the cells The cell according to claim 12, which is: A pharmaceutical composition comprising the monoclonal antibody according to any one of claims 1 to 11, or a part thereof, and a pharmaceutically acceptable carrier. The medicament according to claim 15, wherein the pharmaceutical composition is used for inhibiting transmission of a signal generated by binding of human TGF-β to a human TGF-β type II receptor into cells. Composition. The pharmaceutical composition according to claim 15, wherein the pharmaceutical composition is used for suppressing tissue fibrosis. 18. The pharmaceutical composition according to claim 17, wherein the tissue fibrosis is lung, liver, kidney or skin fibrosis. 18. The pharmaceutical composition according to claim 17, wherein the tissue fibrosis is kidney fibrosis. The pharmaceutical composition according to claim 15, wherein the pharmaceutical composition is used for treating or preventing a kidney disease. The pharmaceutical composition is used for controlling or treating renal sclerosis, pulmonary fibrosis, cirrhosis, vascular restenosis, arteriosclerosis, psoriasis, scleroderma, atopy, keloid or arthritis. Item 16. The pharmaceutical composition according to Item 15, A polypeptide comprising the amino acid sequence of amino acids 2 to 115 of SEQ ID NO: 6. A polypeptide having the amino acid sequence of amino acids 2 to 458 of SEQ ID NO: 6. An immunoglobulin heavy chain polypeptide having the amino acid sequence of amino acids 2 to 458 of SEQ ID NO: 6, with one or several amino acids deleted, substituted, inserted, or added. A polypeptide comprising the amino acid sequence of amino acids 2 to 117 of SEQ ID NO: 8. A polypeptide having the amino acid sequence of amino acids 2 to 236 of SEQ ID NO: 8. An immunoglobulin light chain polypeptide having an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added in the amino acid sequence of amino acids 2 to 236 of SEQ ID NO: 8. A DNA encoding the polypeptide according to any one of claims 21 to 26. A DNA comprising the nucleotide sequence of base numbers 96 to 440 of SEQ ID NO: 7. A DNA comprising the nucleotide sequence of base numbers 96 to 1472 of SEQ ID NO: 7. A DNA comprising the nucleotide sequence of nucleotides 39 to 389 of SEQ ID NO: 9. A DNA comprising the nucleotide sequence of base numbers 39 to 749 of SEQ ID NO: 9. A vector comprising the DNA according to any one of claims 29 to 32. A cell transformed with the vector containing the DNA according to any one of claims 29 to 32.

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