JP2009292822A - Bispecific antibody which substitutes for functional protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a bispecific antibody which substitutes for functional protein, more particularly, a bispecific antibody which substitutes for a ligand function to a heteroreceptor, a bispecific antibody which substitutes for a function of a cofactor which reinforces enzyme reaction and a medicinal composition containing the antibody as an active ingredient. <P>SOLUTION: The bispecific antibody substitutes for the ligand function to an I-type interferon receptor composed of two kind of molecules of AR1 chain and AR2 chain. In addition, the bispecific antibody substitutes for the function of a blood coagulation factor VIII/an activated blood coagulation factor VIII which reinforces the enzyme reaction by combining with both of a blood coagulation factor IX/an activated blood coagulation factor IX and a blood coagulation factor X. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a bispecific antibody that substitutes for a functional protein. Specifically, the present invention relates to a bispecific antibody that substitutes for the function of a ligand for a heteroreceptor, a bispecific antibody that substitutes for the action of a cofactor that enhances an enzyme reaction, and a pharmaceutical composition containing the antibody as an active ingredient.

Antibodies are attracting attention as pharmaceuticals because of their high stability in blood and low antigenicity. Among them are bispecific antibodies that can simultaneously recognize two types of antigens. Bispecific antibodies have long been proposed. However, only antibodies that merely connect two kinds of antigens have been reported so far, for example, for the purpose of targeting NK cells, macrophages, and T cells (see Non-Patent Document 8). For example, MDX-210, which is undergoing clinical trials, is only a bispecific antibody that targets monoclonals etc. expressing FcγRI to cancer cells expressing HER-2 / neu. Therefore, until now, there has been no example of using bispecific antibodies as an alternative to proteins that function in vivo.
One of proteins that function in vivo is a ligand for a receptor. These ligands include interleukin (IL) -2,3,4,5,6,7,9,10,11,12,13,15, erythropoietin (EPO), growth hormone (GH), granulocyte colony Stimulating factor (G-CSF), thrombopoietin (TPO), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), interferon (IFN-α, IFN-β, IFN-γ, etc.) Ciliary neurotropic factor (CNTF), leukemia inhibitory factor (LIF), Oncostatin M, cardiotropin-1 (CT-1), tumor necrosis factor (TNF), and the like.

  In these receptors, it is considered that a ligand is bound to change a distance and an angle of a receptor molecule that forms a dimer or a quantifier, thereby transmitting a signal into a cell. Thus, a suitable anti-receptor antibody can be an antibody that can mimic the dimerization or quantification of a receptor by a ligand.

  Monoclonal antibodies have already been reported which have a ligand-substituting action for TPO receptor (MPL) consisting of homodimers (see Patent Document 1 and Non-Patent Document 1), EPO receptor, and GH receptor. Each of these antibodies is considered to exhibit a platelet count recovery effect upon thrombocytopenia, an erythrocytosis effect during anemia, or a growth promoting effect on short stature, and is expected to be applied to medicine.

  However, in the case of a receptor that forms a heterodimer, it is necessary to form a complex of two or several kinds of receptor molecules, and therefore, a general antibody cannot be expected to replace the ligand function. For this purpose, the above-mentioned bispecific antibody capable of recognizing two kinds of receptor molecules with two arms is considered suitable, but no report has been made yet.

  One of the proteins that function in vivo is a cofactor. Examples of cofactors include tissue factor (TF), blood coagulation factor V (F.V), activated blood coagulation factor V (F.Va), blood coagulation factor VIII (F.VIII), and activation. Blood coagulation factor VIII (F.VIIIa), thrombomodulin (TM), protein S (PS), protein Z (PZ), heparin, complement C4b, complement regulatory factor factor H (Complement Regulatory Factor H), Examples include Membrane Cofactor Protein (MCP) and Complement Receptor 1 (CR1).

  Of these, F.M. VIII / F. VIIIa is sufficient F.V. It is a cofactor necessary for the expression of IXa activity. Scheiflinger F et al. IX / F. The IXa antibody was tested for F.I. F. by IXa. It has been found that there is an effect of promoting X activation (see Patent Document 2). However, F.A. In the measurement of clotting recovery ability of VIII-deficient plasma, the clotting recovery ability by this antibody alone has not been shown. Only when IXa is added, the coagulation recovery ability is shown.

  F. VIIIa is F.I. In addition to interacting with IXa, F.I. It is known to interact with X (see Non-Patent Documents 6 and 7). In this regard, the antibody of Scheiflinger F et al. VIII / F. It cannot be said that the function of VIIIa is sufficiently substituted, and its activity is presumed to be insufficient.

  As a result of diligent research, the present inventors have succeeded in producing a bispecific antibody that substitutes for the action of a functional protein, leading to the present invention.

US Patent Application Publication No. 98/17364 International Publication No. 01/19992 U.S. Pat. No. 4,474,893 EP 404,097 International Publication No. 93/11161 Japanese Patent Application No. 2002-112369 Japanese Patent Application No. 2003-012648 JP-A-5-304992 Japanese Patent Laid-Open No. 2-145187 JP-A-5-213775 JP-A-10-165184 JP-A-11-71288 JP 2002-518041 A Japanese National Patent Publication No. 11-506310 Japanese Patent Laid-Open No. 5-199894 JP 10-51085 A JP-A-5-184383

Deng D et al., “Blood”, 1998, Vol. 92, no. 6, p. 1981-1988 Nilsson IM et al., “J. Inter. Med.”, 1992, Vol. 235, p. 25-32 L_o ▼ fqvist T et al., “J. Inter. Med.”, 1997, Vol. 241, p. 395-400 The 24th Annual Meeting of the Japanese Society for Thrombosis and Hemostasis Academic Expert Group Hemophilia Standardization Study Group Mini Symposium, 2001, http://www.jsth.org Medical Bulletin # 193 1994 Mertens K et al., “Thromb. Haemost.”, 1999, Vol. 82, p. 209-217 Lapan KA et al., “Thromb. Haemost.”, 1998, Vol. 80, p. 418-422 Segal DM et al., “Journal of Immunological Methods”, 2001, Vol. 248, p. 1-6 Bos R and Nieuwenhuitzen W, “Hybridoma”, 1992, Vol. 11, no. 1, p. 41-51 Brennan M et al., “Science”, 1985, Vol. 229, no. 1708, p. 81-3 Karpovsky B et al., “J. Exp. Med.”, 1984, Vol. 160, no. 6, p. 1686-701 Suresh MR et al., “Methods Enzymol.”, 1986, Vol. 121, p. 210-28 Massimo YS et al., “J. Immunol. Methods”, 1997, Vol. 201, p. 57-66 Brennan M et al., “Science”, 1985, Vol. 229, p. 81 Sharaby MR et al., “J. Exp. Med.”, 1992, Vol. 175, p. 217-25 Holliner P et al., “Proc. Natl. Acad. Sci. USA”, 1993, Vol. 90, p. 6444-8 Ridgway JB et al., “Protein Eng.”, 1996, Vol. 9, p. 617-21 Hammerling U et al., “J. Exp. Med.”, 1968, Vol. 128, p. 1461-73 Kuroka Tawa et al., “Bio / Technology”, 1989, Vol. 7, p. 1163 Link BK et al., “Blood”, 1993, Vol. 81, p. 3343 Nitta T et al., “Lancet”, 1990, Vol. 335, p. 368-71 deLeij L et al., “Fundation Nationale de Transfusion Sanguine, Les Ulis France”, 1990, p. 249-53 Le Doussal JM et al., “J. Nucl. Med.”, 1993, Vol. 34, p. 1662-71 Stickney DR et al., “Cancer Res.”, 1991, Vol. 51, p. 6650-5 Weiner LM et al., “Cancer Res.”, 1993, Vol. 53, p. 94-100 Kroesen BJ et al., “Br. J. Cancer”, 1994, Vol. 70, p. 652-61 Weiner GJ et al., “J. Immunol.”, 1994, Vol. 152, p. 2385 Suresh MR et al., “Proc. Natl. Acad. Sci. USA”, 1986, Vol. 83, p. 7989-93 Milstein C and Cuello AC, “Nature”, 1983, Vol. 305, p. 537 Xiang J et al., “Mol. Immunol.”, 1990, Vol. 27, p. 809 Bebbington CR et al., “Bio / Technology”, 1992, Vol. 10, p. 169 Huse WD et al., “Science”, 1989, Vol. 246, p. 1275 McCafferty J et al., “Nature”, 1990, Vol. 348, p. 552 Kang AS et al., “Proc. Natl. Acad. Sci. USA”, 1991, Vol. 88, p. 4363

  An object of the present invention is to provide a bispecific antibody that substitutes for the action of a functional protein. More specifically, an object of the present invention is to provide a bispecific antibody that substitutes for a ligand function for a receptor including a heteroreceptor molecule, and a bispecific antibody that substitutes for a cofactor function that enhances an enzyme reaction.

  As a result of intensive studies, the present inventors succeeded in isolating a ligand function surrogate antibody against a type I interferon receptor composed of two types of molecules, an AR1 chain and an AR2 chain. That is, the present inventors have succeeded in isolating a bispecific antibody having a ligand function alternative action for a receptor composed of a heteromolecule for the first time.

  Furthermore, as a result of intensive studies, the present inventors have found that F.M. IX / F. IXa and F.I. Binds specifically to both X and F.F. VIIIa cofactor action, ie F.I. F. by IXa. We have succeeded in finding a bispecific antibody that replaces the action of promoting X activation. That is, the present inventors have succeeded in producing a bispecific antibody that recognizes both an enzyme and a substrate of the enzyme and can substitute for the function of a cofactor of the enzyme.

  Since both the ligand protein of the receptor consisting of the heteromolecule and the enzyme cofactor are functional proteins, the present inventors have for the first time developed a bispecific antibody having an alternative function of the functional protein. It can be said that.

That is, the present invention relates to a bispecific antibody that substitutes for a functional protein. More specifically, the present invention relates to a bispecific antibody having a ligand function surrogate action for a receptor containing a heteromolecule, and a bispecific antibody substituting the function of a cofactor that enhances an enzyme reaction, more specifically, ,
[1] A bispecific antibody that substitutes for the action of a functional protein,
[2] a bispecific antibody having a ligand function-substituting activity for a receptor containing a heteromolecule,
[3] The antibody according to [2], wherein the receptor containing a heteromolecule is a dimer,
[4] The antibody according to [2], wherein the receptor is a cytokine receptor,
[5] The antibody according to [4], wherein the cytokine receptor is an interferon receptor,
[6] The antibody according to [5], wherein the interferon receptor is a type I interferon receptor,
[7] The antibody according to [6], wherein the type I interferon receptor comprises an AR1 chain and an AR2 chain,
[8] The antibody according to [7], which has an action of substituting the function of interferon, which is a ligand of type I interferon receptor,
[9] The antibody according to [8], comprising a variable region of an anti-AR1 chain antibody and a variable region of an anti-AR2 chain antibody,
[10] A variable region consisting of the following amino acid sequence (a) in an anti-AR1 chain antibody and a variable region consisting of the amino acid sequence described in any of (b1) to (b10) below in an anti-AR2 chain antibody: [9] the antibody according to
(A) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 1, and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 2. (b1) The heavy chain variable region is the amino acid set forth in SEQ ID NO: 7. (B2) The H chain variable region is the amino acid sequence described in SEQ ID NO: 9, and the L chain variable region is described in SEQ ID NO: 10. Amino acid sequence (b3) The H chain variable region is the amino acid sequence described in SEQ ID NO: 19, and the L chain variable region is the amino acid sequence described in SEQ ID NO: 20. (b4) The H chain variable region is described in SEQ ID NO: 13. (B5) The H chain variable region is the amino acid sequence described in SEQ ID NO: 23, and the L chain variable region is SEQ ID NO: 24. Amino acid sequence ( b6) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 5, and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 6. (b7) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 17. And the L chain variable region is the amino acid sequence described in SEQ ID NO: 18 (b8) The H chain variable region is the amino acid sequence described in SEQ ID NO: 15, and the L chain variable region is the amino acid described in SEQ ID NO: 16. Sequence (b9) The heavy chain variable region is the amino acid sequence described in SEQ ID NO: 21 and the light chain variable region is the amino acid sequence described in SEQ ID NO: 22. (b10) The heavy chain variable region is described in SEQ ID NO: 11. An amino acid sequence, wherein the L chain variable region is the amino acid sequence of SEQ ID NO: 12 [11] The variable region comprising the amino acid sequence of (a) below in the anti-AR1 chain antibody, and the following (b1 in the anti-AR2 chain antibody): Comprising - a variable region comprising the amino acid sequence of any one of (b3), the antibody according to [9],
(A) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 3, and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 4. (b1) The heavy chain variable region is the amino acid set forth in SEQ ID NO: 25 (B2) The H chain variable region is the amino acid sequence described in SEQ ID NO: 9, and the L chain variable region is described in SEQ ID NO: 10. Amino acid sequence (b3) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 21 and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 22 [12] [2] to [11] A composition comprising the described antibody and a pharmaceutically acceptable carrier;
[13] The composition according to [12], which is a pharmaceutical composition used for the prevention and / or treatment of viral diseases, malignant neoplasms, and immune diseases,
[14] The composition according to [13], wherein the viral disease is a disease that develops and / or progresses due to hepatitis C virus infection,
[15] The composition according to [14], wherein the disease that develops and / or progresses due to hepatitis C virus infection is acute or chronic hepatitis C, cirrhosis, or liver cancer;
[16] The composition according to [13], wherein the viral disease is a disease that develops and / or progresses due to hepatitis B virus infection,
[17] The composition according to [16], wherein the disease that develops and / or progresses due to hepatitis B virus infection is acute or chronic hepatitis B, cirrhosis, or liver cancer,
[18] The malignant neoplasm is chronic myelogenous leukemia, malignant melanoma, multiple myeloma, renal cancer, glioblastoma, medulloblastoma, astrocytoma, hairy cell leukemia, AIDS-related Kaposi's sarcoma, cutaneous T cell lymphoma , The composition according to [13], which is non-Hodgkin lymphoma,
[19] The composition according to [13], wherein the immune disease is multiple sclerosis,
[20] A viral disease, malignant neoplasm or immunity comprising the step of administering the antibody according to any one of [2] to [11] or the composition according to any one of [12] to [19] Methods for preventing and / or treating sexually transmitted diseases,
[21] Use of the antibody according to any one of [2] to [11] for producing the composition according to any of [12] to [19] [22] At least [2] to [11 A kit for use in the method for prevention and / or treatment according to [20], comprising the antibody according to any one of [1] or the composition according to [12],
[23] an antibody that recognizes both an enzyme and a substrate of the enzyme, a bispecific antibody that replaces the function of a cofactor that enhances the enzymatic reaction,
[24] The antibody according to [23], wherein the enzyme is a proteolytic enzyme,
[25] The antibody according to [24], wherein the proteolytic enzyme, the substrate and the cofactor are blood coagulation / fibrinolysis-related factors,
[26] The blood coagulation / fibrinolysis-related factor enzyme is blood coagulation factor IX and / or activated blood coagulation factor IX, the substrate is blood coagulation factor X, and the cofactor is blood coagulation factor VIII and / or activity The antibody according to [25], which is factorized blood coagulation factor VIII,
[27] A complementarity determining region comprising the amino acid sequence of CDR3 of (a1) or (a2) below or a functionally equivalent complementation determining region in anti-blood coagulation factor IX / IXa antibody, [23] to [26] comprising a complementarity determining region consisting of the amino acid sequence of CDR3 according to any one of the following (b1) to (b9) in a factor X antibody or a complementarity determining region functionally equivalent thereto: ] The antibody according to any one of
(A1) The amino acid sequence of H chain CDR3 described in SEQ ID NO: 42 (a2) The amino acid sequence of H chain CDR3 described in SEQ ID NO: 46 (b1) The amino acid sequence of H chain CDR3 described in SEQ ID NO: 50 (b2 ) H chain CDR3 is the amino acid sequence described in SEQ ID NO: 54 (b3) H chain CDR3 is the amino acid sequence described in SEQ ID NO: 58 (b4) H chain CDR3 is the amino acid sequence described in SEQ ID NO: 62 (b5) H The chain CDR3 is the amino acid sequence set forth in SEQ ID NO: 66 (b6) The H chain CDR3 is the amino acid sequence set forth in SEQ ID NO: 70 (b7) The H chain CDR3 is the amino acid sequence set forth in SEQ ID NO: 74 (b8) The H chain CDR3 Is the amino acid sequence set forth in SEQ ID NO: 78 (b9) the heavy chain CDR3 is the amino acid sequence set forth in SEQ ID NO: 82 [28] anti-blood coagulation factor IX / IXa The following (a1) or (a2) complementarity determining region consisting of the amino acid sequence of CDR or a functionally equivalent complementarity determining region, and the following (b1) to (b9) in anticoagulation factor X antibody: The antibody according to any one of [23] to [26], comprising a complementarity determining region consisting of the amino acid sequence of the CDR according to any one of the above or a functionally equivalent complementarity determining region,
(A1) H chain CDR1,2,3 is the amino acid sequence described in SEQ ID NO: 40, 41,42 (a2) H chain CDR1,2,3 is the amino acid sequence described in SEQ ID NO: 44,45,46 (b1 ) H chain CDR1, 2, 3 is the amino acid sequence described in SEQ ID NO: 48, 49, 50 (b2) H chain CDR1, 2, 3 is the amino acid sequence described in SEQ ID NO: 52, 53, 54 (b3) H The chain CDR1, 2, 3 is the amino acid sequence described in SEQ ID NO: 56, 57, 58 (b4) The H chain CDR1, 2, 3 is the amino acid sequence described in SEQ ID NO: 60, 61, 62 (b5) The H chain CDR1 , 2 and 3 are amino acid sequences described in SEQ ID NOs: 64, 65 and 66 (b6) H chain CDRs 1, 2 and 3 are amino acid sequences described in SEQ ID NOs: 68, 69 and 70 (b7) H chain CDRs 1 and 2 , 3 are SEQ ID NOS: 72, 73, (B8) amino acid sequence described in SEQ ID NO: 76, 77, 78 (b9) H chain CDR1, 2, 3 is converted into SEQ ID NO: 80, 81, 82 A composition comprising the antibody according to any one of the amino acid sequences [29] [23] to [28] and a pharmaceutically acceptable carrier,
[30] The composition according to [29], which is a pharmaceutical composition used for the prevention and / or treatment of bleeding, a disease accompanied by bleeding, or a disease caused by bleeding,
[31] Bleeding, a disease accompanied by bleeding, or a disease caused by bleeding is a disease that develops and / or progresses due to decreased or deficient activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII. [30] the composition according to
[32] The composition according to [31], wherein the disease that develops and / or progresses due to decreased or deficient activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII is hemophilia A,
[33] A disease that develops and / or develops due to decreased or deficient activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII is caused by blood coagulation factor VIII and / or activated blood coagulation factor VIII The composition according to [31], which is a disease in which an inhibitor appears.
[34] The composition according to [31], wherein the disease that develops and / or progresses due to decreased or deficient activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII is acquired hemophilia ,
[35] The composition according to [31], wherein the disease that develops and / or progresses due to decreased activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII is von Willebrand disease,
[36] Bleeding, bleeding-related disease, or bleeding comprising the step of administering the antibody according to any of [23] to [28] or the composition according to any of [29] to [35] A method for preventing and / or treating a disease caused by
[37] Use of the antibody according to any one of [23] to [28] for producing the composition according to any of [29] to [35],
[38] A kit for use in the prevention and / or treatment method according to [36], comprising at least the antibody according to any of [23] to [28], or the composition according to [29],
Is to provide.

  The bispecific antibody (bispecific antibody) in the present invention is a molecule composed of two types of antibodies or antibody fragments having specificity for different antigens. The bispecific antibody is not particularly limited, but is preferably monoclonal.

  The bispecific antibody of the present invention is preferably a recombinant antibody produced using a gene recombination technique. (See, for example, Borrebaeck CAK and Larger JW, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom MACMILLAN PUBLISHERS LTD, 1990). A recombinant antibody is obtained by cloning DNA encoding the antibody from a hybridoma or antibody-producing cells such as sensitized lymphocytes that produce the antibody, incorporating the DNA into an appropriate vector, and introducing the vector into a host for production. be able to.

Furthermore, the antibody in the present invention may be an antibody fragment or an antibody modification product thereof. Examples of antibody fragments include diabody (Db), linear antibodies, single-chain antibody (hereinafter also referred to as scFv) molecules, and the like. Here, the “Fv” fragment is the smallest antibody fragment and contains a complete antigen recognition site and a binding site. An “Fv” fragment is a dimer (V _H -V _L dimer) in which one heavy (H) chain variable region (V _H ) and light (L) chain variable region (V _L ) are strongly linked by a non-covalent bond. . The three complementary determining regions (CDRs) of each variable region interact to form an antigen binding site on the surface of the V _H -V _L dimer. Six CDRs confer antigen binding sites on the antibody. However, even one variable region (or half of an Fv containing only three CDRs specific for the antigen) has a lower affinity than the entire binding site, but has the ability to recognize and bind to the antigen. .

The Fab fragment (also referred to as F (ab)) further includes a constant region of the L chain and a constant region (CH1) of the H chain. Fab ′ fragments differ from Fab fragments in that they additionally have several residues from the carboxy terminus of the heavy chain CH1 region including one or more cysteines from the antibody hinge region. Fab′-SH refers to Fab ′ in which one or more cysteine residues in the constant region have a free thiol group. F (ab ′) fragments are produced by cleavage of the disulfide bond at the cysteine at the hinge portion of the F (ab ′) ₂ pepsin digest. Other chemically conjugated antibody fragments are known to those skilled in the art.

Diabody refers to a bivalent antibody fragment constructed by gene fusion (Holliger P et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP 404,097, WO93 / 11161 etc.). A diabody is a dimer composed of two polypeptide chains, and each of the polypeptide chains cannot bind to the L chain variable region (V _L ) and the H chain variable region (V _H ) in the same chain. Are linked by a linker having a short length, for example, about 5 residues. The encoded V _L and V _H in the same polypeptide chain, to form a dimer can not be formed a single chain variable region fragments for short therebetween linker diabody binding two antigens It will have a part.

Single chain antibodies or scFv antibody fragments include the _VH and _VL regions of an antibody, and these regions are present in a single polypeptide chain. In general, Fv polypeptides further comprise a polypeptide linker between the _VH and _VL regions, which allows the scFv to form the necessary structure for antigen binding (for a review of scFv, see Pluckthun). “The Pharmacology of Monoclonal Antibodies” Vol. 113 (see Rosenburg and Moore ed (Springer Verlag, New York) pp. 269-315, 1994)). The linker in the present invention is not particularly limited as long as it does not inhibit the expression of the antibody variable region linked to both ends thereof.

  IgG type bispecific antibodies can be secreted by hybrid hybridoma (quadroma) resulting from the fusion of two hybridomas producing IgG antibodies (Milstein C et al. Nature 1983, 305: 537-540). Moreover, it can be made to secrete by co-expressing the gene of the L chain and H chain which comprise two types of target IgG, and a total of four types of genes by introduce | transducing into a cell. At this time, IgG of a heterogeneous combination with respect to the H chain can be preferentially secreted by performing an appropriate amino acid substitution in the CH3 region of the H chain (Ridgway JB et al. Protein Engineering 1996, 9: 617-621, Merchant). AM et al. Nature Biotechnology 1998, 16: 677-681).

Bispecific antibodies can also be made by chemically cross-linking Fab ′. For example, Fab ′ prepared from one antibody is maleimidized with o-PDM (ortho-phenylenedi-maleimide) and reacted with Fab ′ prepared from the other antibody to crosslink Fab ′s derived from different antibodies. Bispecific F (ab ′) ₂ can be made (Keler T et al. Cancer Research 1997, 57: 4008-4014). In addition, a method of chemically binding a Fab′-thionitrobenzoic acid (TNB) derivative and an antibody fragment such as Fab′-thiol (SH) is also known (Brennan M et al. Science 1985, 229: 8183).

A leucine zipper derived from Fos, Jun or the like can be used instead of chemical crosslinking. Fos, Jun also forms homodimers, but utilizes the preferential formation of heterodimers. The Fab ′ added with Fos leucine zipper and the other Fab ′ added with Jun are expressed and prepared. Bispecific F (ab ′) ₂ can be formed by mixing and reacting with the reduced monomers Fab′-Fos and Fab′-Jun under mild conditions (Kostelny SA et al. J of Immunology, 1992, 148: 1547-53). This method is not limited to Fab ′ and can also be applied to scFv, Fv, and the like.

Bispecific antibodies can also be made in diabodies. A bispecific diabody is a heterodimer of two cross-over scFv fragments. That is, V _H (A) -V _L (B), V _H (B)-produced by linking V _H and V _L derived from two antibodies A and B with a relatively short linker of about 5 residues. V _L (A) can be used to construct heterodimers (Hollinger P et al. Proc of the National Academy of Sciences of the USA 1993, 90: 6444-6448).

  In this case, two kinds of scFvs are connected with a flexible and relatively long linker of about 15 residues (single-chain diabody: Kipriyanov SM et al. J of Molecular Biology. 1999, 293: 41-56) By performing substitution (knobs-into-holes: Zhu Z et al. Protein Science. 1997, 6: 781-788), the target configuration can be promoted.

Sc (Fv) ₂ that can be prepared by linking two kinds of scFvs with a flexible, relatively long linker of about 15 residues can also be a bispecific antibody (Mullender WD et al. J of Biological Chemistry, 1994, 269: 199-206).

  Examples of the modified antibody include antibodies bound to various molecules such as polyethylene glycol (PEG). In the modified antibody of the present invention, the substance to be bound is not limited. In order to obtain such a modified antibody, it can be obtained by chemically modifying the obtained antibody. These methods are already established in this field.

  The origin of the antibody of the present invention is not limited to human antibodies, mouse antibodies, rat antibodies and the like. Further, it may be a genetically modified antibody such as a chimeric antibody or a humanized antibody.

  Methods for obtaining human antibodies are already known. For example, a target human antibody can be obtained by immunizing a transgenic animal having all repertoires of human antibody genes with a target antigen (International Patent Application Publication) Nos. WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735).

  Genetically modified antibodies can be produced using known methods. Specifically, for example, a chimeric antibody is an antibody comprising the H chain and L chain variable regions of an immunized animal antibody and the H and L chain constant regions of a human antibody. A chimeric antibody can be obtained by ligating DNA encoding the variable region of an antibody derived from an immunized animal with DNA encoding the constant region of a human antibody, incorporating it into an expression vector, introducing it into a host, and producing it. .

  A humanized antibody is a modified antibody, also referred to as a reshaped human antibody. Humanized antibodies are constructed by transplanting CDRs of antibodies from immunized animals into the complementarity determining regions of human antibodies. The general gene recombination technique is also known.

  Specifically, several oligonucleotides were prepared so that a DNA sequence designed to link the CDR of a mouse antibody and the framework region (FR) of a human antibody was linked to the terminal portion. It is synthesized from nucleotides by PCR. The obtained DNA is obtained by ligating with the DNA encoding the human antibody constant region, then incorporating it into an expression vector, introducing it into a host and producing it (European Patent Application Publication No. EP 239400, International Patent Application Publication). No. WO 96/02576). As the FR of a human antibody linked through CDR, a complementarity determining region that forms a favorable antigen binding site is selected. If necessary, the amino acid of the framework region in the variable region of the antibody may be substituted so that the complementarity determining region of the reshaped human antibody forms an appropriate antigen-binding site (Sato K et al, Cancer Research 1993, 53: 851-856). Further, it may be substituted with framework regions derived from various human antibodies (see International Patent Application Publication No. WO 99/51743).

  The present invention provides a bispecific antibody having an alternative function of a functional protein, more preferably a bispecific antibody having an alternative function of a functional protein. A preferred embodiment of the antibody of the present invention is an antibody having a ligand function-substituting activity for a receptor containing a heteromolecule.

  In the present invention, a receptor containing a heteromolecule refers to a receptor composed of two or more proteins (receptor molecules) having different receptors (multimers). Multimers are not limited by the number of proteins (receptor molecules) such as dimers, trimers, and tetramers, but are preferably dimers. For example, when the receptor is a dimer, the heteroreceptor indicates that the two constituent proteins (receptor molecules) are not identical.

  An antibody having a ligand function surrogate activity refers to an antibody having an agonistic action on a certain receptor. In general, when a ligand, which is an agonist, binds to a receptor, the three-dimensional structure of the receptor protein changes, and the receptor is activated (if the receptor is a membrane protein, a signal such as cell proliferation is usually given). Be emitted). In the case of a receptor that forms a dimer, the ligand function surrogate antibody can function in the same manner as a ligand by dimerizing the receptor at an appropriate distance and angle. That is, a suitable anti-receptor antibody can mimic receptor dimerization by a ligand, and can be a ligand function surrogate antibody.

  In a preferred embodiment of the present invention, cytokine heteroreceptors can be mentioned as the receptor of the present invention.

  Cytokines are generally used as a general term for physiologically active proteins that control the proliferation and differentiation of various blood cells, but may also refer to growth factors and growth inhibitory factors of cells including non-immune cells. Therefore, cytokines are a general term for protein factors that are released from cells and mediate cell-to-cell interactions such as immunity, control action of inflammatory reaction, antiviral action, antitumor action, and regulation of cell proliferation / differentiation.

  Specific examples of cytokines acting on the heteroreceptor in the present invention include IL-2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, colony stimulating factor (GM). -CSF, etc.), interferon (IFN-α, IFN-β, IFN-γ, etc.), CNTF, LIF, Oncostatin M, CT-1 and the like can be mentioned, but interferon is preferred, particularly preferably type I Interferon.

  Interferons include IFN-α, IFN-β, IFN-γ, IFN-τ, and the like. Since IFN-α and IFN-β are highly homologous, these two IFNs can react via the same receptor. IFN-α, IFN-β and IFN-τ are classified as type I interferons.

  Examples of type I interferon receptors include the AR1 chain (GenBank ACCESSION No: J03171, literature: Uze G et al. Cell 1990, 60: 225-34) and the AR2 chain (GenBank ACCESSION No: U29584, literature: Domanski P et al. And Jof Biological Chemistry 1995, 270: 21606-11, LutfaUa G et al. EMBO J 1995, 14: 5100-8).

  The method for obtaining the ligand function surrogate bispecific antibody of the present invention is not particularly limited, and may be obtained by any method. For example, when obtaining a bifunctional antibody with alternative ligand function for a heteroreceptor composed of two types of receptor molecules (A chain and B chain), an anti-A chain antibody and an anti-B chain antibody are obtained. Thereafter, a bispecific antibody comprising the H chain and L chain of the anti-A chain antibody and the H chain and L chain of the anti-B chain antibody is prepared. Here, it is desirable that a plurality of types of anti-A chain antibodies and anti-B chain antibodies are obtained, and it is preferable to prepare bispecific antibodies in as many combinations as possible. After producing the bispecific antibody, an antibody having a ligand function alternative activity is selected. Bispecific antibodies can be produced by known methods such as fusion of antibody-producing hybridomas or introduction of antibody expression vectors into cells.

  Antibodies against the receptor can be obtained by methods known to those skilled in the art. For example, it can be prepared by immunizing an immunized animal with an antigen. Antigens that immunize animals include complete antigens having immunogenicity and incomplete antigens (including haptens) having no immunogenicity. In the present invention, a receptor considered to act as a ligand function surrogate antibody of the present invention is used as the antigen (immunogen). The receptor in the present invention is not particularly limited, but is preferably a heterodimer. As an animal to be immunized, for example, mouse, hamster, rhesus monkey, or the like can be used. Those skilled in the art can immunize these animals with antigens by well-known methods. In the present invention, preferably, the variable regions of the L and H chains of the antibody are recovered from the immunized animal or cells of the animal. This operation can be performed by those skilled in the art using generally known techniques. Animals immunized with an antigen express antibodies against the antigen, especially in spleen cells. Thus, for example, mRNA can be prepared from spleen cells of an immunized animal, and the variable regions of the L chain and H chain can be recovered by RT-PCR using primers corresponding to the variable regions of the animal. .

Specifically, the animal is immunized with each of the A chain and B chain of the receptor. The receptor used as an immunogen may be the whole protein constituting the receptor or a partial peptide of the protein. In addition, as an immunogen used for immunizing an animal, an antigen that is an antigen may be bound to another molecule to form a soluble antigen, or a fragment thereof may be used in some cases. When a transmembrane molecule such as a receptor is used as an antigen, it is preferable to use these fragments (for example, the extracellular region of the receptor). Alternatively, a cell that expresses a transmembrane molecule on the cell surface can be used as an immunogen. Such a cell may be a cell derived from nature (such as a tumor cell line) or a cell configured to express a transmembrane molecule by a recombinant technique. MRNA is extracted from the spleen cells of this animal, and cDNAs of the L chain and H chain variable regions are recovered by RT-PCR using primers corresponding to the vicinity of the variable region. Primers corresponding to CDRs, primers corresponding to frameworks that are less diverse than CDRs, or primers corresponding to signal sequences and CH1 or L chain constant regions (C _L ) can be used. It is also possible to immunize lymphocytes in vitro. Using this, a library that presents scFv or Fab is constructed. The antigen-binding antibody clone is concentrated and cloned by panning, and an antibody expression vector is prepared using the variable region. A bispecific antibody can be obtained by introducing an anti-A chain antibody expression vector and an anti-B chain antibody expression vector into the same cell and expressing the antibody. In this case, it is also possible to perform screening using a similar library using mRNA derived from peripheral blood mononuclear cells, spleen, tonsil, etc. of humans or non-immunized animals.

The selection of an antibody having a ligand function alternative activity can be performed, for example, by the following method.
(1) By adding an antibody during culturing of a cell that proliferates in a ligand-dependent manner, it is used as an index whether or not the cell proliferates like a ligand. When the cells proliferate, the test multispecific antibody is determined to have a ligand function alternative action.
(2) Using as an index whether or not a reaction similar to the ligand is exhibited by adding it during the cultivation of a cell line showing the original activity (not necessarily proliferation) of the ligand. An antibody is determined to have a ligand function surrogate action when it shows a similar reaction to a ligand.

  The above cells normally express a heteroreceptor on which the antibody can act as an agonist on the cell surface, and the receptor emits a signal by binding to a ligand. The cells used in the above method are preferably cells that can proliferate in a receptor-dependent manner (ligand-dependent proliferating cells). Moreover, it is preferable that the said receptor is what normally emits a cell proliferation signal by couple | bonding with a ligand. However, when the receptor does not emit a cell proliferation signal, it is used in the above method by fusing the receptor with a receptor that emits a cell proliferation signal to form a so-called chimeric receptor. Can do. The chimeric receptor emits a cell proliferation signal by binding to a ligand. A receptor suitable for constructing a chimeric receptor by fusing with the receptor is not particularly limited as long as it is a receptor that emits a cell proliferation signal, but is usually a membrane protein, more preferably extracellular. The receptor is a receptor fragment having a ligand binding function, and the intracellular region is a receptor fragment having a signal transduction function. Specific examples of receptors used in the intracellular region include GH receptor, G-CSF receptor, MPL, EPO receptor, c-Kit, Flt-3, IL-2 receptor, IL-3 receptor, IL- 5 receptor, GM-CSF receptor and the like. As a preferable example of the ligand-dependent proliferating cell in the present invention, specifically, a ligand expressing a chimeric receptor whose extracellular region is a ligand receptor fragment and whose intracellular region is a G-CSF receptor fragment Dependent proliferating cells Ba / F3 can be shown. In addition, examples of cells that can be used in the above method include NFS60, FDC-P1, FDC-P2, CTLL-2, DA-1, and KT-3.

  The obtained antibody can be purified to homogeneity. Separation and purification of antibodies may be carried out using separation and purification methods used for ordinary proteins. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing etc. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratories, 1988), but is not limited thereto. Examples of the column used for affinity chromatography include a protein A column and a protein G column.

  When the antibody of the present invention is an antibody having a ligand function-substituting activity against, for example, a type I interferon receptor including AR1 chain and AR2 chain, preferably, the variable region in the anti-AR1 chain antibody and And a variable region in an AR2 chain antibody. The interferon function surrogate antibody was prepared by the following method. IL-3 dependent mouse pro-B cell line Ba / F3 expressing chimeric receptors in the extracellular region of each of the receptor molecules AR1 chain and AR2 chain of type I interferon receptor and in the intracellular region of G-CSF receptor Was established. Each cell was immunized into the peritoneal cavity of BALB / c.

  PolyA (+) RNA was extracted from the spleen of an immunized mouse with an increased antibody titer, and scFv was synthesized by RT-PCR to construct a scFv-displayed phage library. The bound phages were concentrated by a panning method in which a phage library derived from the spleen of an AR1 chain-expressing Ba / F3 immunized mouse and a biotinylated soluble AR1 chain were mixed and captured with streptavidin magnetic beads. Anti-AR1 chain antibody-displayed phages were selected by phage ELISA using soluble AR1 chain. Similarly, an anti-AR2 chain antibody phage was selected using a library phage derived from the spleen of soluble AR2 chain and AR2 chain expressing Ba / F3 immunized mouse. Antibodies differing in the amino acid sequence of CDR3 of the heavy chain, which is said to be most involved in antibody specificity, were selected.

scFv was inserted between a signal sequence for animal cells and CH1-hinge-CH2-CH3 to prepare an scFv-CH1-Fc expression vector. An antibody against the AR1 chain and an antibody against the AR2 chain were introduced into cells in various combinations to express bispecific antibodies.
BaF3-ARG was established by introducing an expression vector of a chimeric molecule of each of the extracellular regions of AR1 chain and AR2 chain and the intracellular region of G-CSF receptor into Ba / F3. The cells proliferated in dependence on IFN-α. A bispecific antibody in a combination of antibodies capable of supporting the growth of BaF3-ARG was selected.

Daudi cells are a human B cell line that is highly sensitive to cytostatic activity by IFN-α. The bispecific antibody selected previously was added to Daudi cells and confirmed to inhibit proliferation in the same manner as IFN-α. Examples of the antibody include, but are not limited to, an antibody comprising any of the following variable regions of an anti-AR1 chain antibody and any of the following variable regions of an anti-AR2 chain antibody. it can.
-Variable region of anti-AR1 chain antibody: AR1-41, AR1-24
-Variable region of anti-AR2 chain antibody: AR2-37, AR2-11, AR2-13, AR2-45, AR2-22, AR2-43, AR2-40, AR2-14, AR2-44, AR2-33, AR2 -31

The amino acid sequences of V _H and V _L of each of the above variable regions are shown in SEQ ID NOs: 1 to 26 (the relationship between V _H and V _L of each variable region and the SEQ ID NO is shown in Table 1).

  When the anti-AR1 chain antibody is AR1-24, the partner anti-AR2 chain antibody is preferably AR2-13, AR2-31, or AR2-44, and when the anti-AR1 chain antibody is AR1-41, the partner The anti-AR2 chain antibody is AR2-11, AR2-13, AR2-14, AR2-22, AR2-33, AR2-37, AR2-40, AR2-43, AR2-44, or AR2-45. It is preferable. AR2-13 and AR2-44 can be partners to both AR1-41 and AR1-24 antibodies. Antibodies that form pairs as described above are also included in the present invention.

  A preferred embodiment of the bispecific antibody having an alternative function to the functional protein of the present invention is a bispecific antibody that substitutes for the function of a cofactor that recognizes both the enzyme and the substrate of the enzyme.

  The cofactor in the present invention is not particularly limited as long as it can act on the enzyme and enhance the enzyme reaction. Examples of the cofactor in the present invention include a proteolytic enzyme cofactor. Specific examples of cofactors for proteolytic enzymes include cofactors (F.VIII / F.VIIIa, PZ, TM, TM / PS systems) in blood coagulation / fibrinolysis-related factors and cofactors for complement reaction (C4b, MCP). , CR1, factor H) and the like.

Specific examples of the enzyme, the enzyme substrate, and the cofactor of the enzyme in the present invention include the following combinations.
(A) Example 1 of cofactor in blood coagulation / fibrinolysis-related factor
Enzyme: F.I. IXa
Substrate: F. X
Cofactor: F. VIII / F. VIIIa
Cofactor F.I. VIIIa is F.I. IXa and F.R. By binding to both of X. F. by IXa. Enhance X activation. Said enzyme F.I. IXa, substrate F.I. Some bispecific antibodies that recognize both X. Some have the effect of enhancing the activation of X. Among such antibodies are cofactors F.I. VIII / F. It is thought that there exists what has the effect | action which substitutes the action function of VIIIa.

(B) Example 2 of cofactor in blood coagulation / fibrinolysis-related factor
Enzyme: ZPI
Substrate: F. X / F. Xa
Cofactor: PZ
Cofactor PZ is a serpin family of ZPI and F.I. By binding to Xa, F.I. Increases Xa inhibitory activity. That is, ZPI and F.I. X / F. It is considered that some bispecific antibodies that recognize both Xa have an effect of substituting the function of PZ.

(C) Example 3 of cofactor in blood coagulation / fibrinolysis-related factor
Enzyme: Thrombin Substrate: TAFI
Cofactor: TM
Cofactor TM enhances activation of TAFI by thrombin. That is, it is considered that some bispecific antibodies that recognize both thrombin and TAFI have an effect of substituting the function of TM.

(D) Example 4 of cofactor in factors related to blood coagulation and fibrinolysis
Enzyme: Thrombin Substrate: PC
Cofactor: TM / PS
The TM / PS system enhances PC activation by thrombin. That is, it is considered that some bispecific antibodies that recognize both thrombin and PC substitute for the function of the TM / PS system.

(E) Cofactor reaction cofactor example 1
Enzyme: C1s
Substrate: C2
Cofactor: C4b
C4b has an action of promoting C2 degradation by C1s. That is, it is considered that some bispecific antibodies that recognize both C1s and C2 exist that substitute for the function of C4b.

(F) Example 2 of cofactor of complement reaction
Enzyme: Complement Regulatory Factor I (Complement Regulatory Factor I)
Substrate: C3b
Cofactor: Complement Regulatory Factor H (Complement Regulatory Factor H)
Membrane Cofactor Protein (MCP)
Complement Receptor 1 (CR1)
Complementary Regulatory Factors H, MCP, and CR1 have C3b degradation promoting action by Complementary Regulatory Factor I. In other words, bispecific antibodies that recognize both Complementary Regulatory Factor I and C3b are considered to have alternatives to the functions of Complementary Regulatory Factor H, MCP, and CR1.

  Among the above cofactors, F. VIII / F. VIIIa. F. VIII / F. VIIIa is subject to limited degradation by proteolytic enzymes such as thrombin. VIII / F. As long as it has the cofactor activity of VIIIa, the form is not ask | required. In addition, mutation F. VIII / F. VIIIa or F. artificially modified by gene recombination technology. VIII / F. As for VIIIa, F.I. VIII / F. As long as it has the cofactor activity of VIIIa, VIII / F. Included in VIIIa.

  The method for obtaining the cospecific function alternative bispecific antibody of the present invention is not particularly limited, and may be obtained by any method. For example, when obtaining a cofactor function alternative bispecific antibody against enzyme A and substrate B, immunized animals are immunized with enzyme A and substrate B, respectively, and anti-enzyme A antibody and anti-substrate B antibody are obtained. Thereafter, a bispecific antibody comprising the anti-enzyme A antibody H and L chains and the anti-substrate B antibody H and L chains is prepared. Here, it is desirable that a plurality of types of anti-enzyme A antibodies and anti-substrate B antibodies are obtained, and it is preferable to prepare bispecific antibodies in as many combinations as possible. After preparing the bispecific antibody, an antibody having cofactor function alternative activity is selected.

  Antibodies against enzymes or substrates can be obtained by methods known to those skilled in the art. For example, it can be prepared by immunizing an immunized animal with an antigen. Antigens that immunize animals include complete antigens having immunogenicity and incomplete antigens (including haptens) having no immunogenicity. In the present invention, an enzyme or a substrate that is considered that the cofactor function-substitute antibody of the present invention acts as a cofactor is used as the antigen (immunogen). As an animal to be immunized, for example, a mouse, a hamster, or a rhesus monkey can be used. Those skilled in the art can immunize these animals with antigens by well-known methods. In the present invention, preferably, the variable regions of the L chain and H chain of the antibody are recovered from the immunized animal or cells of the animal. This operation can be performed by those skilled in the art using generally known techniques. Animals immunized with an antigen express antibodies against the antigen, particularly in spleen cells. Therefore, for example, mRNA can be prepared from spleen cells of an immunized animal, and the variable regions of the L chain and H chain can be recovered by RT-PCR using primers corresponding to the variable regions of the animal.

  Specifically, animals are immunized with enzymes and substrates. The enzyme or substrate used as the immunogen may be the whole protein or a partial peptide of the protein. Moreover, as an immunogen used for immunizing an animal, it is possible to bind an antigen as an antigen to other molecules to make it a soluble antigen, and in some cases, a fragment thereof may be used.

MRNA is extracted from the spleen cells of this immunized animal, and cDNAs of the L chain and H chain variable regions are recovered by RT-PCR using primers corresponding to the vicinity of the variable region. Primers corresponding to CDRs, primers corresponding to frameworks that are less diverse than CDRs, or primers corresponding to signal sequences and CH1 or L chain constant regions (C _L ) can be used. It is also possible to immunize lymphocytes in vitro. Using this, a library that presents scFv or Fab is constructed. The antigen-binding antibody clone is concentrated and cloned by panning, and an antibody expression vector is prepared using the variable region. A bispecific antibody can be obtained by introducing an anti-enzyme antibody expression vector and an anti-substrate antibody expression vector into the same cell and expressing the antibody. In this case, it is also possible to perform screening using a similar library using mRNA derived from peripheral blood mononuclear cells, spleen, tonsil, etc. of humans or non-immunized animals.

The selection of an antibody having a cofactor function alternative activity can be performed, for example, by the following method.
(1) Using a reaction system containing the enzyme and the substrate, the increase in the enzyme activity (substrate resolution) by adding the antibody is used as an index for selection.
(2) Function by adding the antibody in the absence of the cofactor using a system that measures or mimics a biological function involving the enzyme, the substrate, and the cofactor (for example, a plasma coagulation measurement system) Select using recovery activity as an index.

  The obtained antibody can be purified to homogeneity. Separation and purification of antibodies may be carried out using separation and purification methods used for ordinary proteins. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing etc. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratories, 1988), but is not limited thereto. Examples of the column used for affinity chromatography include a protein A column and a protein G column.

The bispecific antibody of the present invention has an alternative cofactor, e.g. VIII / F. VIIIa, i.e. the combination of enzyme and substrate is F. IXa, and F.I. In the case of X, preferably anti-F. Variable regions in IXa antibodies and anti-F. And a variable region in the X antibody.
F. of the present invention. VIII / F. The VIIIa function alternative bispecific antibody was prepared by the following method. Commercially available F.R. IXa and F.I. Each X was immunized subcutaneously in mice. Spleen cells were isolated from the spleen of immunized mice with increased antibody titers and fused with mouse myeloma cells to produce hybridomas. Hybridomas that bind to the antigens (F.IXa, FX) were selected, and the variable regions of the L and H chains were recovered by RT-PCR using primers corresponding to the variable regions. L chain variable region to the L chain expression vector containing a C _L, H chain variable region incorporating the respective H chain expression vector containing an H chain constant region.

  Anti-F. IXa antibody (H chain, L chain) expression vector and F.I. An X antibody (H chain, L chain) expression vector was introduced into the same cell, and the antibody was expressed to obtain a bispecific antibody.

  Regarding the obtained bispecific antibody, F.I. XIa (F.IX activating enzyme), F.IX. IX (FX activating enzyme), F.X. X, F.M. A measurement system comprising a synthetic substrate of Xa (S-2222) and a phospholipid. VIII / F. The activity of substituting VIIIa (a cofactor for F.X activation by F.IXa) was evaluated. With the result, F.M. VIII / F. Bispecific antibodies with VIIIa surrogate activity were selected.

  For the bispecific antibodies selected above, see F.C. Coagulation recovery ability was measured using a coagulation measurement system (APTT) using VIII-deficient human plasma. As a result, F.I. It was confirmed that a bispecific antibody having clotting recovery ability was obtained against human plasma lacking VIII.

  The H chain CDR3 of the antibody of the present invention is not particularly limited, and specifically, the XB12 H chain CDR3 sequence (SEQ ID NO: 42) or the XT04 H chain CDR3 sequence (SEQ ID NO: 42) described in the Examples below. 46) having a complementarity-determining region consisting of any one of the amino acid sequences or a functionally equivalent complementarity-determining region, and SB04, SB05, SB06, SB07, SB21, SB30, SB34, SB38 or SB42. The complementarity determining region comprising the amino acid sequence of any one of the heavy chain CDR3 sequences (each SEQ ID NO: 50, 54, 58, 62, 66, 70, 74, 78 or 82) or functionally equivalent thereto It has a complementarity determining region.

  Furthermore, the antibody of the present invention, as a specific example, consists of an amino acid sequence of either the XB12 heavy chain CDR sequence (SEQ ID NO: 40 to 42) or the XT04 heavy chain CDR sequence (SEQ ID NO: 44 to 46). An SB04, SB05, SB06, SB07, SB21, SB30, SB34, SB38 or SB42 heavy chain CDR sequence (SEQ ID NOs: 48 to 50) having a complementarity determining region or a functionally equivalent complementarity determining region. 52-54, 56-58, 60-62, 64-66, 68-70, 72-74, 76-78 or 80-82) complementarity determining region, or a function thereof In particular, antibodies in combination with those having equivalent complementarity determining regions can be preferably shown.

  The amino acid sequences of the heavy chain variable regions of XB12, XT04, SB04, SB05, SB06, SB07, SB21, SB30, SB34, SB38 and SB42 described in the present invention are SEQ ID NOs: 39, 43, 47, 51, 55, 59, 63, 67, 71, 75 and 79.

  When producing a full-length antibody using the variable region disclosed in the present invention, the constant region is not particularly limited, and a constant region known to those skilled in the art can be used. For example, Sequences of proteins of immunological interest. , (1991), U.S. Pat. S. Department of Health and Human Services. Public Health Service National Institutes of Health and An efficient route to human bispecific IgG, (1998). Nature Biotechnology vol. 16, 677-681, etc. can be used.

  Since one embodiment of the antibody of the present invention has a ligand function-substituting action, the antibody of the present invention is an effective drug against a disease caused by a decrease in activity (function) of a receptor on which the antibody acts. Is expected to be.

  When the ligand function that the antibody of the present invention substitutes is IFN-α / β, examples of the disease include viral diseases, malignant neoplasms, and immune diseases.

  Examples of viral diseases include diseases that develop and / or develop due to hepatitis C virus, and more specifically, acute hepatitis C, chronic hepatitis C, cirrhosis, and liver cancer can be exemplified.

  Chronic hepatitis C is a chronic inflammatory disease caused by a host immune response to hepatitis C virus-infected cells. As the symptoms progress, the liver function gradually decreases, undergoes cirrhosis, and eventually leads to liver cancer. In patients with chronic hepatitis C, treatment with interferon-α / β is performed to eliminate the hepatitis C virus, but since the half-life in blood is short, daily administration is necessary, so the burden on the patient is It is quite heavy. Therefore, a drug having the action of interferon-α / β and excellent in sustainability has been demanded.

  Another viral disease includes, for example, a disease that develops and / or progresses due to hepatitis B virus. More specifically, acute hepatitis B, chronic hepatitis B, liver cirrhosis, and liver cancer are exemplified. be able to.

Malignant neoplasms include chronic myelogenous leukemia, malignant melanoma, multiple myeloma, renal cancer, glioblastoma, medulloblastoma, astrocytoma, hairy cell leukemia, AIDS-related Kaposi sarcoma, cutaneous T-cell lymphoma, non Hodgkin lymphoma can be exemplified.
Moreover, multiple sclerosis can be illustrated as an immune disease.

  Another aspect of the antibody of the present invention is that it has an action of substituting the function of a cofactor, so that the antibody of the present invention is effective against diseases caused by a decrease in the activity (function) of the cofactor. It is expected to become a new drug. When the cofactor substituted for the antibody of the present invention is a blood coagulation / fibrinolysis-related factor, examples of the disease include bleeding, a disease accompanying bleeding, a disease caused by bleeding, and the like. In particular, F.A. VIII / F. VIIIa, F.M. IX / F. IXa, F.I. XI / F. XIa functional loss and deficiency are known to cause bleeding abnormalities called hemophilia.

  Of hemophilia, congenital F. VIII / F. Abnormal bleeding due to reduced or deficient VIIIa function is called hemophilia A. If a hemophilia A patient bleeds, Replacement therapy for the VIII formulation is performed. In the case of frequent intra-articular hemorrhage on the day of intense exercise or excursion, or when classified as severe hemophilia. Prophylactic administration of VIII preparations may be performed (see Non-Patent Documents 2 and 3). This F. Prophylactic administration of VIII preparations has become very popular in recent years in order to drastically reduce bleeding episodes in hemophilia A patients. Reducing bleeding episodes not only reduces the risk of lethal and non-lethal bleeding and the associated pain, but also obviates hemophilic joint damage due to frequent intra-articular bleeding. As a result, it greatly contributes to improving QOL of hemophilia A patients.

  F. The blood half-life of the VIII preparation is short, about 12 to 16 hours. Therefore, F. It is necessary to administer the VIII preparation about 3 times a week. This is because F.A. This corresponds to maintaining approximately 1% or more as VIII activity (see Non-Patent Documents 4 and 5). Also, in the replacement therapy at the time of bleeding, except for a case where the bleeding is mild, in order to prevent re-bleeding and perform complete hemostasis, F. Periodic additional doses of VIII need to be administered.

F.F. The VIII formulation is administered intravenously. There are technical difficulties in performing intravenous administration. Particularly in the administration to young patients, the difficulty is further increased because the vein used for administration is thin.
F. mentioned above. In the preventive administration of the VIII preparation and the emergency administration in the case of bleeding, home therapy / self-injection is often used. The necessity of frequent administration and technical difficulties during administration not only cause pain to patients during administration, but also hinder the spread of home therapy and self-injection.
Accordingly, there has been a strong demand for a drug with a wide administration interval or a drug that can be easily administered as compared with existing blood coagulation factor VIII preparations.

  Furthermore, for hemophilia A patients, especially severe hemophilia A patients, F. Antibodies against VIII may be generated. When an inhibitor is generated, F.I. The effect of the VIII formulation is hindered by inhibitors. As a result, hemostasis management for patients becomes very difficult.

  When such a hemophilia A inhibitor patient has bleeding, usually a large amount of F.I. Neutralization therapy using VIII preparations, complex concentrates or F.I. Bypass therapy using the VIIa formulation is performed. However, with neutralization therapy, a large amount of F.I. Conversely, administration of a VIII formulation may increase the inhibitor (anti-F.VIII antibody) titer. In bypass therapy, complex preparations and F.I. The short blood half-life (about 2-8 hours) of the VIIa formulation is a problem. Moreover, their mechanism of action is VIII / F. Function of VIIIa, ie F. F. by IXa. Because it is independent of the function of catalyzing X activation, there are cases where the hemostasis mechanism does not function well and becomes unresponsive. For this reason, patients with hemophilia A inhibitors often cannot obtain a sufficient hemostatic effect compared to patients with non-inhibitor hemophilia A.

  Therefore, it is independent of the presence of the inhibitor and F.I. VIII / F. There has been a strong demand for drugs that replace the function of VIIIa.

  By the way, VIII / F. As bleeding disorders related to VIIIa, hemophilia, anti-F. In addition to acquired hemophilia with VIII autoantibodies, von Willebrand disease due to vWF dysfunction or deficiency is known. vWF is not only necessary for platelets to normally adhere to the subendothelial tissue at the site of vessel wall injury, Forms a complex with VIII and It is also necessary to keep VIII levels normal. In patients with von Willebrand disease, these functions have declined, resulting in abnormal hemostasis.

  Now, (i) wide dosing intervals, (ii) simple administration, (iii) independent of the presence of inhibitors, (iv) F.F. VIII / F. In order to create a pharmaceutical that substitutes its function independently of VIIIa, a method using an antibody can be considered. The blood half-life of antibodies is generally relatively long, from days to weeks. In addition, it is generally known that antibodies migrate into the blood after subcutaneous administration. That is, the antibody drug satisfies the above (i) and (ii).

  As another embodiment of the functional protein in the present invention, a protein that binds to two types of proteins having different physiological actions and controls a plurality of different physiological actions can be mentioned. Preferable examples thereof include C4b binding protein (C4bp) binding to complement fourth component C4b and protein S (PS). C4bp has the effect of dissociating C2b from the C4b-C2b complex and abolishing the aPC factorer activity of PS. Therefore, C4bp exhibits a regulatory action on the complement system and blood coagulation system. Bispecific antibodies against C4b and PS are considered to have an action that replaces the action of C4bp. C4bp also acts as a cofactor for the degradation of C4b by factor I. Therefore, bispecific antibodies against factor I and C4b are also considered to have an action that replaces the action of C4bp.

  The present invention provides a pharmaceutical composition containing the antibody of the present invention as an active ingredient. For example, when the antibody of the present invention is an antibody having an interferon function-substituting activity for a cytokine receptor, the antibody is considered to have a cytokine-like action. Therefore, the antibody is expected to be a pharmaceutical product (pharmaceutical composition) or drug having an antiviral action, an antitumor action, and a cell growth / differentiation regulating action. On the other hand, an antibody that substitutes for the function of IL-2 is a pharmaceutical (pharmaceutical composition) or drug that has an immunostimulatory / anti-tumor action by differentiation / activation of T cells or NK cells, an antibody that substitutes for the function of IL-3 Is a medicinal product (pharmaceutical composition) or drug having an effect of promoting blood cell recovery by proliferating hematopoietic progenitor cells, and an antibody substituting the function of IL-4 is a medicinal product having an antiallergic effect by induction to Th2 (humoral immunity) (Pharmaceutical composition) or drug, an antibody that substitutes for the function of IL-5 is a pharmaceutical (pharmaceutical composition) or drug having an immunostimulatory / antitumor action by inducing proliferation / differentiation of B cells / eosinophils, IL-6 An antibody substituting for the function of the drug is a pharmaceutical product (pharmaceutical composition) or drug having a platelet production stimulating action, and an antibody substituting the function of the IL-7 is immunostimulation / antitumor by proliferation of T cells / B cells. A pharmaceutical (pharmaceutical composition) or drug having an action, an antibody substituting for the function of IL-9 is a drug (pharmaceutical composition) or drug having an action of promoting blood cell recovery by mast cell proliferation / hematopoiesis, or the function of IL-10 An alternative antibody is a pharmaceutical product (pharmaceutical composition) or drug having an immunosuppressive effect, and an antibody that replaces the function of IL-11 is a pharmaceutical product (pharmaceutical composition) or drug having a platelet production stimulating action, which replaces the function of IL-12 The antibody to be used is a pharmaceutical (pharmaceutical composition) or drug having an immunostimulatory / anti-tumor action by induction on Th1 (cellular immunity), and the antibody that substitutes for the function of IL-15 is an immunostimulatory / anti-antibody by NK cell activation. Drug (pharmaceutical composition) with tumor action, or antibody that substitutes for GM-CSF function has leukocyte recovery promoting action after chemotherapy or bone marrow transplantation Drugs (pharmaceutical compositions) or drugs, antibodies that substitute for the function of CNTF are drugs (pharmaceutical compositions) or drugs that have an anti-obesity action, and antibodies that substitute for the function of LIF have the effect of stimulating platelet production and lowering blood cholesterol A pharmaceutical (pharmaceutical composition) or drug, an antibody that substitutes for the function of Oncostatin M is a pharmaceutical (pharmaceutical composition) or drug that has a hematopoietic promoting action, and an antibody that substitutes for the function of CT-1 is a pharmaceutical that has a myocardial protective action (medicine) It is expected to be a composition) or drug.

  In addition, the antibody of the present invention is F.I. IX or F.I. IXa, and F.I. Among antibodies that recognize both X and F. In the case of an antibody that substitutes for the function of VIIIa, the antibody is expected to be a drug (pharmaceutical composition) or drug for the prevention or treatment of bleeding, bleeding-related diseases, or diseases caused by bleeding Is done.

  On the other hand, ZPI and F.I. An antibody that binds to X and substitutes for the function of PZ is a pharmaceutical (pharmaceutical composition) or drug having an antithrombotic action, and an antibody that binds to thrombin and TAFI to substitute for the function of TM has a hemostatic promoting action (medicine) It is expected to become a pharmaceutical composition (pharmaceutical composition) or drug having an action of substituting the function of the PS / TM system by binding to the composition) or drug, thrombin and PC.

  In addition, since complement C4 deficiency causes systemic lupus erythematosus (SLE), an antibody that substitutes for the action of C4b is expected to be a drug (pharmaceutical composition) or drug having an SLE onset suppressing action. Since the deficiency of factor H causes suppurative infection and autoimmune glomerulonephritis, an antibody that substitutes for the action of factor H is a pharmaceutical (pharmaceutical composition) or drug that has an inhibitory effect on the onset of these diseases. It is expected.

  Since C4bp deficiency causes Behcet's disease, an antibody that substitutes for the action of C4bp is expected to be a pharmaceutical product (pharmaceutical composition) or drug having an effect of suppressing the onset of Behcet's disease.

  A pharmaceutical composition containing the antibody of the present invention used as an active ingredient for therapeutic or prophylactic purposes is mixed with an appropriate pharmaceutically acceptable carrier, vehicle, etc. inert to them as necessary. It can be formulated. For example, sterile water, physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphoric acid, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), binders and the like. In addition, other low molecular weight polypeptides, serum albumin, proteins such as gelatin and immunoglobulin, amino acids such as glycine, glutamine, asparagine, arginine and lysine, sugars and carbohydrates such as polysaccharides and monosaccharides, and sugars such as mannitol and sorbitol Alcohol may be included. In the case of an aqueous solution for injection, for example, isotonic solutions containing physiological saline, glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and appropriate dissolution You may use together with adjuvants, such as alcohol (ethanol etc.), polyalcohol (propylene glycol, PEG, etc.), nonionic surfactant (polysorbate 80, HCO-50), etc.

  If necessary, the antibody of the present invention can be encapsulated in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly [methylmethacrylic acid]) or colloid drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles). And nanocapsules etc.) (see “Remington's Pharmaceutical Science 16th edition”, Oslo Ed. (1980), etc.). Furthermore, a method of making a drug a sustained-release drug is also known and can be applied to the antibody of the present invention (Langer et al., J. Biomed. Mater. Res. 15: 267-277 (1981); Langer, Chemtech.12: 98-105 (1982); U.S. Pat. No. 3,773,919; European Patent Application Publication (EP) 58,481; Sidman et al., Biopolymers 22: 547-556 (1983); No. 133,988).

  The dosage of the pharmaceutical composition of the present invention is determined according to the judgment of the doctor in consideration of the type of dosage form, the administration method, the age and weight of the patient, the symptoms of the patient, the type of disease and the degree of progression, etc. In general, for adults, 0.1 to 2000 mg can be orally administered in 1 to several divided doses per day. More preferred is 1-1000 mg / day, even more preferred is 50-500 mg / day, and most preferred is 100-300 mg / day. These doses vary depending on the weight and age of the patient, the administration method, etc., but those skilled in the art can appropriately select an appropriate dose. The administration period is also preferably determined appropriately according to the patient's healing progress and the like.

  It is also conceivable to perform gene therapy by incorporating a gene encoding the antibody of the present invention into a gene therapy vector. As the administration method, in addition to direct administration with naked plasmid, it can be packaged in liposomes or formed as various vectors such as retrovirus vector, adenovirus vector, vaccinia virus vector, poxvirus vector, adenovirus related vector, HVJ vector, etc. (See Adolph “Virus Genome Method”, CRC Press, Florida (1996)) or coated on a bead carrier such as colloidal gold particles (WO93 / 17706, etc.). However, the antibody may be administered by any method as long as the antibody is expressed in vivo and can exert its action. Preferably, by suitable parenteral routes (intravenous, intraperitoneal, subcutaneous, intradermal, adipose tissue, intramammary tissue, inhalation or intramuscular route, injection, infusion, or gas-induced particle bombardment (electronic A sufficient amount is administered by a method such as a gun), a method via a mucosal route such as a nasal spray, etc. Ex vivo administration to liposomes, blood cells, bone marrow-derived cells, etc. using liposome transfection, particle bombardment (US Pat. No. 4,945,050), or viral infection, and reintroducing the cells into animals According to the present invention, a gene encoding the antibody of the present invention may be administered.

The present invention also provides a method for preventing and / or treating bleeding, bleeding-related diseases, or diseases resulting from bleeding, comprising the step of administering the antibody or composition of the present invention. Administration of the antibody or composition can be carried out, for example, by the method described above.
The invention also relates to the use of the antibody of the invention for the production of the (pharmaceutical) composition of the invention.
Furthermore, the present invention provides a kit for use in the above method, comprising at least the antibody or composition of the present invention. In addition, the kit can be packaged with a syringe barrel, a needle, a pharmaceutically acceptable medium, an alcohol cotton cloth, an adhesive bandage, or instructions describing how to use the kit.

FIG. 1 is a diagram showing the insertion region of pcDNA4-g4H. FIG. 2 is a diagram showing insertion regions of pcDNA4-g4L and pIND-g4L. FIG. 3 is a diagram showing the insertion region of pIND-g4H. FIG. IXa antibody XB12 and anti-F. Anti-F.F prepared with X antibodies SB04, SB21, SB42, SB38, SB30, SB07, SB05, SB06, SB34. IXa / anti-F. X. Bispecific antibodies, F.M. The result of having measured VIIIa like activity is shown. The concentration of the antibody solution is 10 μg / mL (final concentration 1 μg / mL). As a result, F.F. The activity increased in the order of XB12 / SB04, XB12 / SB21, XB12 / SB42, XB12 / SB38, XB12 / SB30, XB12 / SB07, XB12 / SB05, XB12 / SB06, and XB12 / SB34. FIG. IXa antibody XT04 and anti-F. Anti-F.F prepared with X antibodies SB04, SB21, SB42, SB38, SB30, SB07, SB05, SB06, SB34. IXa / anti-F. X. bispecific antibody or XT04 antibody, The result of having measured VIIIa like activity is shown. The concentration of the antibody solution is 10 μg / mL (final concentration 1 μg / mL). As a result, XT04 / SB04, XT04 / SB21, XT04 / SB42, XT04 / SB38, XT04 / SB30, XT04 / SB07, XT04 / SB05, XT04 / SB06, and XT04 / SB34 are F.04. It showed an increase in VIIIa-like activity. FIG. 6 shows F. at various concentrations for XB12 / SB04, which was the most active in FIG. The result of having measured VIIIa like activity is shown. As a result, XB12 / SB04 was F.D. It showed an increase in VIIIa-like activity. FIG. 7 shows plasma clotting time (APTT) in the presence of XB12 / SB04, XB12 / SB21, XB12 / SB42, XB12 / SB38, XB12 / SB30, XB12 / SB07, XB12 / SB05, XB12 / SB06, XB12 / SB34. The measurement results are shown. The concentration of the antibody solution is 3.4 μg / mL for XB12 / SB06, and 20 μg / mL otherwise. As a result, XB12 / SB04, XB12 / SB21, XB12 / SB42, XB12 / SB38, XB12 / SB30, XB12 / SB07, XB12 / SB05, XB12 / SB06, and XB12 / SB34 were compared with the absence of antibody. Showed a shortening effect. FIG. 8 shows plasma clotting time (APTT) in the presence of XT04 / SB04, XT04 / SB21, XT04 / SB42, XT04 / SB38, XT04 / SB30, XT04 / SB07, XT04 / SB05, XT04 / SB06, and XT04 / SB34. The measurement results are shown. The concentration of the antibody solution is 10 μg / mL for XT04 / SB06, and 20 μg / mL otherwise. As a result, XT04 / SB04, XT04 / SB21, XT04 / SB42, XT04 / SB38, XT04 / SB30, XT04 / SB07, XT04 / SB05, and XT04 / SB06 have an effect of shortening the coagulation time compared to the absence of the antibody. Indicated. XT04 / SB34 did not shorten the coagulation time. FIG. 9 shows the results of measuring the coagulation time at various concentrations for XB12 / SB04, which has the highest effect of shortening the coagulation time (APTT) in FIGS. As a result, XB12 / SB04 showed an effect of shortening the coagulation time in a concentration-dependent manner. 10 to 13 show the ISRE activation ability of antibodies against pISRE-Luc-introduced K562 cells. □ indicates IFN-α2a, and ● indicates a combination of anti-AR1 chain and anti-AR2 chain bispecific antibodies in the figure. It shows that the antibody can activate ISRE in a dose-dependent manner with a specific activity per molecule comparable to that of IFN.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
[Example 1] Antigen and immunity An expression vector for a soluble receptor in which a tag of FLAG (AR1FLAG, AR2FLAG) or His6 (AR1His, AR2His) is added to the C-terminus of each extracellular region of human AR1 chain and AR2 chain. The cells were introduced into CHO cells and purified from the culture supernatant using an affinity column. A high expression cell was established by introducing an expression vector of a chimeric molecule of the extracellular region of human AR1 chain and the G-CSF receptor intracellular region into mouse pro B cell line Ba / F3. Similarly, a high-expressing cell of a chimeric molecule of the extracellular region of the human AR2 chain and the G-CSF receptor intracellular region was established. Each cell was immunized into the peritoneal cavity of BALB / c. AR1His or AR2His was intravenously injected 3 days before the removal of the spleen.

[Example 2] Separation of antibody from scFv display library (a) Panning of phage library PolyA (+) RNA was extracted from the spleen of an immunized mouse, and scFv was synthesized by RT-PCR. A phagemid library expressed as a fusion protein with gene3 was constructed (J. Immun. Methods, 201, (1997), 35-55). Library E. coli (2 × 10 ⁹ cfu) was inoculated into 50 mL 2 × YTAG (100 μg / mL ampicillin, 2 × TY containing 2% glucose) and cultured at 37 ° C. to an OD 600 of 0.4 to 0.5. 4 × 10 ¹¹ helper phage VCSM13 was added and left at 37 ° C. for 15 minutes for infection. 450 mL 2 × YTAG, 25 μL 1 mol / L IPTG was added thereto, and cultured at 26 ° C. for 10 hours. The culture supernatant was collected by centrifugation, mixed with 100 mL PEG-NaCl (10% polyethylene glycol 8000, 2.5 mol / L NaCl), and allowed to stand at 4 ° C. for 60 minutes. Phage was precipitated by centrifugation at 10,800 × g for 30 minutes, the precipitate was suspended in 40 mL of water, mixed with 8 mL PEG-NaCl, and allowed to stand at 4 ° C. for 20 minutes. Phage was precipitated by centrifugation at 10,800 × g for 30 minutes and suspended in 5 mL PBS. AR1FLAG and AR2FLAG prepared in Example 1 were labeled with biotin using No-Weighed Premeasured NHS-PEO ₄ -Biotin Microtubes (Pierce). 100 pmol of biotin-labeled AR1FLAG or AR2FLAG was added to the phage library and contacted with the antigen for 60 minutes. 600 μL of Streptavidin MagneSphere (Promega) washed with 5% M-PBS (PBS containing 5% skim milk) was added and allowed to bind for 15 minutes. The beads were washed 3 times each with 1 mL PBST (PBS containing 0.1% Tween-20) and PBS. The beads were suspended in 0.8 mL of 0.1 mol / L glycine / HCl (pH 2.2) for 5 minutes to elute the phages. The collected phage solution was neutralized by adding 45 μL 2 mol / L Tris, added to 10 mL of logarithmic growth phase (OD 600 = 0.4 to 0.5) XL1-Blue, and allowed to stand at 37 ° C. for 30 minutes. Infected. This was spread on 2 × YTAG plates and cultured at 30 ° C. Colonies were collected, inoculated into 2 × YTAG, and cultured at 37 ° C. until OD 600 = 0.4 to 0.5. 5 μL of 1 mol / L IPTG and 10 ¹¹ pfu helper phage (VCSM13) were added to 10 mL of the culture solution and allowed to stand at 37 ° C. for 30 minutes. After centrifugation, the cells were resuspended in 100 mL of 2 × YTAG containing 25 μg / mL kanamycin and cultured at 30 ° C. for 10 hours. The culture supernatant was collected by centrifugation, mixed with 20 mL PEG-NaCl, and allowed to stand at 4 ° C. for 20 minutes. Phage was precipitated by centrifugation at 10,800 × g for 30 minutes, and suspended in 2 mL PBS for the next panning. In the second panning, the beads were washed 5 times with PBST and PBS. A clone producing an AR chain-binding phage was selected by ELISA from E. coli obtained by infecting the eluted phage.

(B) Phage ELISA
The single colony was inoculated into 150 μL 2 × YTAG and cultured at 30 ° C. overnight. After inoculating 5 μL of this in 500 μL 2 × YTAG and culturing at 37 ° C. for 2 hours, 100 μL of 2 × YTAG containing 2.5 × 10 ⁹ pfu of helper phage and 0.3 μL of 1 mol / L IPTG was added and allowed to stand at 37 ° C. for 30 minutes. Subsequently, the cells were cultured overnight at 30 ° C., and the centrifuged supernatant was subjected to ELISA. StreptaWell 96 microtiter plates (Roche) were coated overnight with 100 μL of PBS containing 1.0 μg / mL biotin-labeled AR1FLAG or AR2FLAG. After washing with PBST to remove the antigen, it was blocked overnight with 200 μL of 2 w / v% M-PBS. 2% (w / v) M-PBS was removed, the culture supernatant was added here, and the mixture was allowed to stand for 40 minutes to bind the antibody. After washing, bound phages were detected with HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted with 2 w / v% M-PBS and BM blue POD substrate (Roche), and the reaction was stopped by addition of sulfuric acid. The value of was measured.

(C) Sequencing and clone selection Primers PBG3-F1 (5′-CAGCTATGAAATACCTATTGCC-3 ′ / SEQ ID NO: 27) and PBG3-R1 (5′-CTTTTCATATAATCAAAATACCACCGG-3 ′) from the phage solution of clones that were positive by ELISA / SEQ ID NO: 28) was used to amplify the scFv region by PCR and determine its nucleotide sequence. 1 μL of phage solution, 2 μL of 10 × KOD Dash buffer, 0.5 μL of 10 μmol / L primer, and 20 μL of PCR reaction solution containing 0.3 μL of KOD Dash polymerase (Toyobo, 2.5 U / μL) at 96 ° C. with Perkin Elmer 9700, Amplification was performed for 10 seconds, 55 ° C., 10 seconds, 72 ° C., 30 seconds, and 30 cycles. After PCR, 3 μL of ExoSAP-IT (Amersham) was added to 5 μL of the reaction solution, and incubated at 37 ° C. for 20 minutes, then at 80 ° C. for 15 minutes. For this sample, BigDye Terminator Cycle sequencing kit (PIG3-Terminal Cycle sequencing kit) using PBG3-F2 (5′-ATTGCCTACGCGCAGCCCGCT-3 ′ / SEQ ID NO: 29) or PBG3-R2 (5′-AAATACCCGGAACCAGAGCC-3 ′ / SEQ ID NO: 30) as a primer. The reaction was carried out using an Applied Biosystems PRISM 3700 DNA Sequencer. Forty-five clones were selected for the anti-AR1 chain and the anti-AR2 chain, each having a different CDR3 of the amino acid sequence deduced from the base sequence.

[Example 3] Bispecific antibody expression Via a SfiI site between a human signal sequence driven by the CAGG promoter and intron-CH1-Fc (human IgG4 cDNA) for expression as scFv-CH1-Fc An expression vector pCAGGss-g4CH hetero IgG4 into which scFv can be inserted was constructed. In order to express it as a hetero molecule, IgG1 knobs-intoholes (Ridgway JB et al. Protein Engineering 1996, 9: 617-621) was used to make an amino acid substitution to the CH3 portion of IgG4. Type A is a substitute for Y349C, T366W. Type B is a substitution product of E356C, T366S, L368A, Y407V. Substitution (-ppcpScp- → -ppcpPcp-) was also introduced into both hinge portions. In addition, a human IL3 signal sequence was constructed for the A type, and a human IL-6 signal sequence was constructed for the B type (pCAGG-IL3ss-g4CHPa, pCAGG-IL6ss-g4CHPb). The PCR product of the scFv region of the clone selected from the nucleotide sequence was treated with SfiI, and the anti-AR1 chain clone was subcloned into pCAGG-IL3ss-g4CHPa and the anti-AR2 chain clone was subcloned into pCAGG-IL3ss-g4CHPb. Expression vectors were transfected into HEK293 cells using Lipofectamine 2000 for all combinations of a total of 2025 types of anti-AR1 chain and anti-AR2 chain clone 45 × 45, and the culture supernatant after 3 days was collected.

[Example 4] Separation of bispecific antibody with alternative ligand function (a) Ba / F3 proliferation assay BaF3-ARG is a mouse IL-3-dependent proliferation cell Ba / F3 and the extracellular region of G1 and AR2 chain -Established by introducing an expression vector of a chimeric molecule with the intracellular region of the CSF receptor. BaF3-ARG proliferated depending on IFNα. A 96-well plate was seeded with 0.1 mL of medium containing 1 × 10 ³ cells and sample per well washed three times. After culturing for 4 days, 10 μL of viable cell count measuring reagent SF (nacalai test) was added and incubated at 37 ° C. for 2 hours, and then A450 was measured.

(B) Daudi Cell Growth Inhibition Assay Daudi cells are a human B cell line that is highly sensitive to IFN. 6.25 × 10 ³ cells per well were seeded in a 96-well plate with 0.1 mL of medium containing the sample. After culturing for 4 days, 10 μL of viable cell count measuring reagent SF (nacalai test) was added and incubated at 37 ° C. for 2 hours, and then A450 was measured.

(C) Sequence of bispecific antibody with alternative ligand function The amino acid sequence of the variable region of the antibody selected by the above screening is shown in SEQ ID NOs: 1-26. The relationship between the name of each antibody and the sequence number is shown in Table 1 above.

(D) Reporter gene assay using ISRE To 100 mL of DMRIE-C (Invitrogen) added to 3 mL of OPTI-MEM I and stirred, 40 μg of pISRE-Luc was added and allowed to stand at room temperature for 20 minutes after stirring. did. This 8x10 ⁶ / 2mL OPTI-MEM I was added to human cell K562 adjusted to, after 4 hours incubation at 37 ° C., the cells were further cultured overnight by adding 15% FCS-RPMI1640 of 10 mL. The next day, K562 recovered by centrifugation was resuspended in 10.5 mL of 10% FCS-RPMI 1640 and seeded on a 96-well flat bottom plate at 70 μL / well.
The concentration of bispecific scFv-CH in the antibody gene-introduced HEK293 culture supernatant was adjusted to 12.5 ng / mL in terms of IgG, and a 5-fold dilution series was prepared. Alternatively, the Bisspecific IgG-expressing COS7 culture supernatant was diluted 2-fold to prepare a 5-fold dilution series. This was added at 30 μL / well to the cells into which the reporter plasmid was introduced. As a positive control well, 30 μL / well of a 5-fold dilution series of IFN-α2a was dispensed. After culturing at 37 ° C. for 24 hours, 50 μL / mL of Bright-Glo Luciferase Assay System (Promega) was added and allowed to stand at room temperature for 10 minutes, and then luciferase activity was measured with Analyst HT (LJL) (FIGS. 10 and 11). FIG. 12 and FIG. 13).

[Example 5] Production of non-neutralizing antibody against Factor IXa (F.IXa) 5-1. Immunization and hybridoma production Eight BALB / c mice (male, 6 weeks old at the start of immunization, Charles River, Japan) and 5 MRL / lpr mice (male, 6 weeks old at the start of immunization, Charles River, Japan) IXaβ (Enzyme Research Laboratories, Inc.) was immunized as follows. As the initial immunization, Factor IXaβ emulsified with FCA (Freund's complete adjuvant H37 Ra (Difco laboratories)) was subcutaneously administered at 40 μg / head. Two weeks later, Factor IXaβ emulsified with FIA (Difco laboratories) was subcutaneously administered at 40 μg / head. Thereafter, booster immunization was performed 3 to 7 times at 1-week intervals. After confirming the increase in serum antibody titer against Factor IXaβ by ELISA (Enzyme linked immunosorbent assay) shown in 5-2, as final immunization, factor diluted in PBS (−) (phosphate buffered saline without calcium ion and magnesium ion) IXaβ was administered intravenously at 40 μg / head. Three days after the final immunization, mouse spleen cells and mouse myeloma cells P3X63Ag8U. 1 (ATCC CRL-1597, referred to as P3U1) was cell-fused according to a conventional method using PEG1500 (Roche Diagnostics). Fusion cells suspended in RPMI1640 medium (Invitrogen) containing 10% FBS (Invitrogen) (hereinafter referred to as 10% FBS / RPMI1640) are seeded on a 96-well culture plate, and HAT selection is performed 1, 2, 3, 5 days after the fusion. The hybridoma was selectively cultured by substituting the medium (10% FBS / RPMI1640 / 2% HAT 50x concentrated (Dainippon Pharmaceutical) / 5% BM-Condimed H1 (Roche Diagnostics)). Hybridomas having Factor IXa binding activity were selected by measuring the binding activity to Factor IXa by ELISA shown in 5-2, using the culture supernatant collected on the 8th or 9th day after the fusion. Subsequently, the neutralizing activity on the enzyme activity of Factor IXa was measured by the method shown in 5-3, and a hybridoma having no neutralizing activity on Factor IXa was selected. The hybridoma was cloned by limiting dilution twice by seeding one cell per well in a 96-well culture plate. The cells confirmed to be a single colony by microscopic observation were subjected to ELISA and neutralization activity measurement shown in 5-2 and 5-3, and clones were selected. Ascites of the cloned antibody was prepared by the method shown in 5-4, and the antibody was purified from the ascites. It was confirmed by the method shown in 5-5 that the purified antibody did not prolong APTT (activated partial thromboplastin time).

5-2. Factor IXa ELISA
Coating buffer (100mM sodium bicarbonate, pH9.6,0.02 % sodium azide) the Factor IXabeta diluted with 1 [mu] g / mL, the ^{Nunc-Immuno plate (Nunc-Immuno} TM 96MicroWell TM plates MaxiSorp TM (Nalge Nunc International)) After dispensing at 100 μL / well, the mixture was incubated at 4 ° C. overnight. Tween ^(R) including 20 PBS (-) after 3 washes, diluent buffer (50mM Tris-HCl , pH8.1,1% bovine serum albumin, 1mM MgCl 2, 0.15M NaCl, 0.05% Tween ( ^{R) The} plate was blocked for 2 hours at room temperature with 20,0.02% sodium azide). After removing the buffer, 100 μL / well of mouse antiserum or hybridoma culture supernatant diluted with diluent buffer was added to the plate, and incubated at room temperature for 1 hour. After washing the plate three times, 100 μL / well of alkaline phosphatase-labeled goat anti-mouse IgG (H + L) (Zymed Laboratories) diluted 1/2000 with a diluent buffer was added and incubated at room temperature for 1 hour. After washing the plate 6 times, 100 μL / well of the chromogenic substrate Blue-Phos ^™ Phosphate Substrate (Kirkegaad & Perry Laboratories) was added and incubated at room temperature for 20 minutes. After adding 100 μL / well of Blue-Phos ^™ Stop Solution (Kirkegaad & Perry Laboratories), the absorbance at 595 nm was measured with Microplate Reader Model 3550 (Bio-Rad Laboratories).

5-3. Measurement of Factor IXa Neutralization Activity Phospholipid (Sigma-Aldrich) was dissolved in distilled water for injection and sonicated to prepare a 400 μg / mL phospholipid solution. 40 μL of Tris buffered saline (hereinafter referred to as TBSB) containing 0.1% bovine serum albumin and 10 μL of 30 ng / mL Factor IXaβ (Enzyme Research Laboratories) and 5 μL of 400 μg / mL phospholipid solution and 100 mM CaCl ₂ , 20 mM MgCl ₂ 5 μL and 10 μL of hybridoma culture supernatant were mixed in a 96-well plate and incubated at room temperature for 1 hour. To this mixed solution, 20 μL of 50 μg / mL Factor X (Enzyme Research Laboratories) and 10 μL of 3 U / mL Factor VIIIa (American diagnostic) were added and reacted at room temperature for 30 minutes. The reaction was stopped by adding 10 μL of 0.5 M EDTA to this. To this reaction solution, 50 μL of S-2222 solution (Chromogenix) was added and incubated at room temperature for 30 minutes, and then the absorbance at a measurement wavelength of 405 nm and a control wavelength of 655 nm was measured using Microplate Reader Model 3550 (Bio-Rad Laboratories, Inc.). It was measured.

5-4. Ascites preparation and antibody purification Ascites preparation of established hybridomas was performed according to a conventional method. That is, BALB / c mice (male, experiment) in which 2 × 10 ⁶ hybridomas cultured in vitro were previously administered with pristane (2,6,10,14-tetramethylpentadecane) twice intraperitoneally. 5-7 weeks old at start, Charles River Japan) or BALB / c nude mice (female, 5-6 weeks at start of experiment, Charles River Japan and Claire Japan) were transplanted intraperitoneally. Ascites fluid was collected from mice whose abdomen was enlarged at 1 to 4 weeks after transplantation.
Antibody purification from ascites was performed using a Protein G Sepharose ^™ 4 Fast Flow (Amersham Biosciences) column. Ascites diluted 2-fold with Binding Buffer (20 mM sodium acetate, pH 5.0) was applied to the column and washed with 10 column volumes of Binding Buffer. The antibody was eluted with 5 column volumes of Elution Buffer (0.1 M glycine-HCl, pH 2.5) and neutralized with Neutralizing Buffer (1 M Tris-HCl, pH 9.0). This was concentrated with Centriprep ^™ 10 (Millipore), and the solvent was replaced with TBS (50 mM Tris-buffered Saline). The antibody concentration was calculated as A (1%, 1 cm) = 13.5 from the absorbance at 280 nm. Absorbance was measured with DU-650 (Beckman Coulter).

5-5. Measurement of APTT (activated partial thromboplastin time) APTT was measured by KC10A (Amelung) connected with CR-A (Amelung). A mixture of 50 μL of antibody solution diluted with TBSB, 50 μL of standard human plasma (Dade Behring) and 50 μL of APTT reagent (Dade Behring) was heated at 37 ° C. for 3 minutes. The coagulation reaction was started by adding 50 μL of 20 mM CaCl ₂ (Dade Behring) to the mixture, and the coagulation time was measured.

[Example 6] Production of non-neutralizing antibody against Factor X (FX) 6-1. Immunization and hybridoma production Eight BALB / c mice (male, 6 weeks old at the start of immunization, Charles River, Japan) and 5 MRL / lpr mice (male, 6 weeks old at the start of immunization, Charles River, Japan) X (Enzyme Research Laboratories) was immunized as follows. As the first immunization, Factor X emulsified with FCA was subcutaneously administered at 40 μg / head. Two weeks later, Factor X emulsified with FIA was administered subcutaneously at 20 or 40 μg / head. Thereafter, booster immunization was performed 3 to 6 times at intervals of 1 week. After confirming an increase in serum antibody titer against Factor X by ELISA shown in 6-2, Factor X diluted in PBS (−) was intravenously administered as a final immunization at 20 or 40 μg / head. Three days after the final immunization, mouse spleen cells and mouse myeloma cells P3U1 were fused according to a conventional method using PEG1500. The hybridomas were selectively cultured by seeding the fused cells suspended in 10% FBS / RPMI1640 medium in a 96-well culture plate and replacing the cells with a HAT selection medium after 1, 2, 3, and 5 days of fusion. Using the culture supernatant collected on the 8th day after the fusion, the binding activity to Factor X was measured by ELISA shown in 6-2. A hybridoma having Factor X binding activity was selected, and the neutralizing activity on the enzyme activity of Factor Xa was measured by the method shown in 6-3. A hybridoma having no neutralizing activity against Factor Xa was cloned by performing limiting dilution twice. By the method shown in 5-4, ascites of the cloned antibody was prepared, and the antibody was purified from the ascites. It was confirmed by the method shown in 5-5 that the purified antibody did not prolong APTT.

6-2. Factor X ELISA
Factor X diluted to 1 μg / mL with a coating buffer was dispensed into Nunc-Immuno plate at 100 μL / well and incubated overnight at 4 ° C. Tween ^(R) including 20 PBS (-) after 3 times with wash and 2 hours blocking at room temperature plate with Diluent buffer. After removing the buffer, a mouse antiserum or hybridoma culture supernatant diluted with a diluent buffer was added to the plate and incubated at room temperature for 1 hour. After washing the plate three times, alkaline phosphatase-labeled goat anti-mouse IgG (H + L) diluted 1/2000 with a diluent buffer was added and incubated at room temperature for 1 hour. After washing the plate 6 times, 100 μL / well of the chromogenic substrate Blue-Phos ^™ Phosphate Substrate (Kirkegaad & Perry Laboratories) was added and incubated at room temperature for 20 minutes. After adding 100 μL / well of Blue-Phos ^™ Stop Solution (Kirkegaad & Perry Laboratories), the absorbance at 595 nm was measured with Microplate Reader Model 3550 (Bio-Rad Laboratories).

6-3. Measurement of Factor Xa neutralizing activity TBCP (2.78 mM CaCl ₂ , 22.2 μM phospholipid (phosphatidyl) containing 10 μL of hybridoma culture supernatant diluted 1/5 with TBSB and 40 μL of 250 pg / mL Factor Xa (Enzyme Research Laboratories) TBSB) containing choline: phosphatidylserine = 75: 25, Sigma-Aldrich) was mixed and incubated at room temperature for 1 hour. To this mixed solution, 50 μL of TBCP containing 20 μg / mL prothrombin (Enzyme Research Laboratories) and 100 ng / mL activated coagulation factor V (Factor Va (Haematologic Technologies)) was added and reacted at room temperature for 10 minutes. The reaction was stopped by adding 10 μL of 0.5 M EDTA. To this reaction solution, 50 μL of 1 mM S-2238 solution (Chromogenix) was added and incubated at room temperature for 30 minutes, and then the absorbance at 405 nm was measured with Microplate Reader Model 3550 (Bio-Rad Laboratories).

[Example 7] Construction of chimeric bispecific antibody expression vector 7-1. Preparation of DNA fragment encoding antibody variable region from hybridoma Anti-F. IXa antibody or anti-F. From a hybridoma producing an X ^antibody, total RNA was extracted according to the method of instructions described using ^{QIAGEN (R) RNeasy (R)} Mini Kit (QIAGEN). Total RNA was dissolved in 40 μL of sterile water. Single-stranded cDNA was synthesized by RT-PCR method using SuperScript cDNA synthesis system (Invitrogen) according to the method described in the description using 1-2 μg of purified RNA as a template.

7-2. Amplification and sequence analysis of antibody H chain variable region by PCR As primers for amplification of mouse antibody H chain variable region (VH) cDNA, HB described in a report of Kreber et al. (J. Immunol. Methods 1997; 201: 35-55). A primer mixture and an HF primer mixture were prepared. Using 0.5 μL each of 100 μM HB primer mixture and 100 μM HF primer mixture, reaction solution 25 μL (2.5 μl of cDNA solution prepared in 7-1, KOD plus buffer (Toyobo), 0.2 mM dNTPs, 1.5 mM) MgCl ₂ , 0.75 units DNA polymerase KOD plus (Toyobo) was prepared. PCR was performed using a thermal cycler GeneAmp PCR system 9700 (Parkin Elmer) according to the amplification efficiency of the cDNA fragment under the condition A (heated at 98 ° C. for 3 minutes, then 98 ° C. for 20 seconds, 58 ° C. for 20 seconds, 72 ° C.). 32 cycles with a reaction of 30 seconds at 30 ° C.) to Condition B (after heating for 3 minutes at 94 ° C. for 3 minutes, 5 cycles with a reaction of 94 ° C. for 20 seconds, 46 ° C. for 20 seconds, 68 ° C. for 30 seconds, The reaction comprising 94 ° C. for 20 seconds, 58 ° C. for 20 seconds, and 72 ° C. for 30 seconds was performed under any of the following conditions: 30 cycles. After PCR, the reaction solution was subjected to 1% agarose gel electrophoresis. The amplified fragment of the desired size (about 400 bp) was purified by QIAquick Gel Extraction Kit (QIAGEN) by the method described in the attached instruction, and eluted with 30 μl of sterilized water. The base sequence of each DNA fragment was determined using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) according to the DNA sequencer ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) and the method described in the attached description. The sequence group determined by this method was subjected to comparative analysis using analysis software GENETYX-SV / RC Version 6.1 (Geneticx), and those having different sequences were selected.

7-3. Preparation of Antibody Variable Region DNA Fragment for Cloning In order to add a restriction enzyme Sfi I cleavage site for cloning to both ends of the amplified antibody variable region fragment, the following operation was performed.
For amplification of Sfi I cleavage site-added VH fragment (Sfi I-VH) (Gly4Ser) 2-linker sequence of primer HB was changed to the sequence (SEQ ID NO: 31) shown in having Sfi I cleavage site (primer) VH-5′end) was prepared. Using 0.5 μl each of 10 μM sequence-specific primer VH-5′end and 10 μM primer scfor (J. Immunol. Methods 1997; 201: 35-55), 20 μL of reaction solution (purified VH cDNA prepared in 7-2) Amplified fragment solution 1 μl, KOD plus buffer (Toyobo), 0.2 mM dNTPs, 1.5 mM MgCl ₂ , 0.5 units DNA polymerase KOD plus (Toyobo)) were prepared. PCR was performed using a thermal cycler GeneAmp PCR system 9700 (Parkin Elmer) according to the efficiency of fragment amplification under the conditions A (after heating at 98 ° C for 3 minutes, 98 ° C for 20 seconds, 58 ° C for 20 seconds, 72 ° C for 30 seconds) Or a reaction consisting of 94 ° C. for 20 seconds, 46 ° C. for 20 seconds, 68 ° C. for 30 seconds for 5 cycles, and further 94 ° C. for 20 cycles. Second reaction, 58 ° C. for 20 seconds, 72 ° C. for 30 seconds, and one cycle for 30 cycles). After PCR, the reaction solution was subjected to 1% agarose gel electrophoresis. The amplified fragment of the desired size (about 400 bp) was purified by QIAquick Gel Extraction Kit (QIAGEN) according to the method described in the attached instruction, and eluted with 30 μL of sterilized water.
For amplification of mouse antibody light chain variable region (VL) cDNA fragments, first 0.5 μL each of 100 μM LB primer mixture and 100 μM LF primer described in the report of Kreber et al. (J. Immunol. Methods 1997; 201: 35-55) Using the mixture, 25 μL of the reaction solution (2.5 μL of the c-DNA solution prepared in 7-1, KOD plus buffer (Toyobo), 0.2 mM dNTPs, 1.5 mM MgCl ₂ , 0.75 units DNA polymerase KOD plus ( Toyobo)) was prepared. PCR was performed using a thermal cycler GeneAmp PCR system 9700 (Parkin Elmer) according to the efficiency of fragment amplification, followed by heating at 94 ° C. for 3 minutes, followed by 94 ° C. for 20 seconds, 46 ° C. for 20 seconds, 68 ° C. for 30 seconds. Was carried out under the conditions of 30 cycles, one cycle consisting of 94 ° C for 20 seconds, 58 ° C for 20 seconds, 72 ° C for 30 seconds. After PCR, the reaction solution was subjected to 1% agarose gel electrophoresis. The amplified fragment of the desired size (about 400 bp) was purified by QIAquick Gel Extraction Kit (QIAGEN) by the method described in the attached instruction, and eluted with 30 μL of sterilized water. The fragment is in a state where a primer LF-derived (Gly4Ser) 3-linker sequence is added to the C-terminus. (Gly4Ser) 3-linker sequence of primer LF was changed to the sequence (SEQ ID NO: 32) shown in having Sfi I cleavage site (primer VL-3) for the purpose of adding an Sfi I cleavage site to the fragment C-terminus 'end) was prepared. For amplification of Sfi I cleavage site added VL fragment (Sfi I-VL), each 0.5 μL of 10 μM VL-3′end primer mixture and 10 μM scback primer were used, and 20 μL of reaction solution (1 μL of purified VL cDNA amplified fragment solution) , KOD plus buffer (Toyobo), 0.2 mM dNTPs, 1.5 mM MgCl ₂ , 0.5 units DNA polymerase KOD plus (Toyobo)). PCR was performed by using a thermal cycler GeneAmp PCR system 9700 (Parkin Elmer) for 3 minutes after heating at 94 ° C. for 3 minutes, followed by a reaction consisting of 94 ° C. for 20 seconds, 46 ° C. for 20 seconds, 68 ° C. for 30 seconds, The reaction consisting of 94 ° C. for 20 seconds, 58 ° C. for 20 seconds, and 72 ° C. for 30 seconds was performed under 30 cycles as one cycle. After PCR, the reaction solution was subjected to 1% agarose gel electrophoresis. The amplified fragment of the target size (about 400 bp) was purified by QIAquick Gel Extraction Kit (QIAGEN) by the method described in the attached instruction, and eluted with 30 μL of sterilized water.
Purified Sfi I-VH and Sfi I-VL fragments were prepared in Sfi I (Takara Bio) according to the method described in the attached instructions, and digested overnight at 50 ° C. Thereafter, the reaction solution was purified by QIAquick PCR Purification Kit (QIAGEN) according to the method described in the attached instruction, and eluted with 30 μL of Buffer EB attached to the kit.

7-4. Human IgG4-mouse chimeric bispecific IgG antibody expression plasmid IgG1 knobs-into-holes technology (Ridgway et al.) Is used to form heteromolecules of each H chain when producing the desired bispecific IgG antibody. al., Protein Eng. 1996; 9: 617-621), an amino acid substitution product to the CH3 portion of IgG4 was prepared. Type a (IgG4γa) is a substitution product of Y349C and T366W, and type b (IgG4γb) is a substitution product of E356C, T366S, L368A, and Y407V. Furthermore, substitution (-ppcpScp->-ppcpPcp-) was also introduced into the hinge region of both substitution products. Although this technique can be almost heterozygous, it is not limited to the L chain, and the generation of unnecessary antibody molecules may affect the subsequent activity measurement. For this reason, in this strategy, one molecule of each antibody molecule (referred to as HL molecule) is expressed separately, and the expression corresponding to each HL molecule is performed in order to efficiently produce the target bispecific IgG antibody in the cell. The vector used was a different drug that required induction.

  For expression of one arm of an antibody molecule (referred to as a right arm HL molecule for convenience), a tetracycline-inducible vector pcDNA4 (Invitrogen) is subjected to each region of the H chain or L chain (FIGS. 1 and 2), ie, an animal cell signal sequence (IL3ss ) (Proc. Natl. Acad. Sci. USA. 1984; 81: 1075) and an appropriate mouse antibody variable region (VH to VL) and human IgG4γa constant region (SEQ ID NO: 33) to κ constant region (SEQ ID NO: : 34) (pcDNA4-g4H to pcDNA4-g4L) were prepared. First, pcDNA4 was digested with restriction enzyme cleavage sites Eco RV and Not I (Takara Bio) existing at the multiple cloning site. A chimeric bispecific antibody right arm heavy chain or light chain expression unit (approx. 1.6 kb to 1.0 kb, respectively) having an appropriate antibody variable region was digested with Xho I (Takara Bio), and then QIAquick PCR Purification Kit ( QIAGEN) was purified by the method described in the attached instructions, and reacted at 72 ° C. for 10 minutes in the reaction solution composition described in the attached instructions using DNA polymerase KOD (Toyobo Co., Ltd.) to smooth the ends. The blunt end fragment was purified by QIAquick PCR Purification Kit (QIAGEN) according to the method described in the attached instruction and digested with Not I (Takara Bio). The Not I-blunt fragment (about 1.6 kb to 1.0 kb, respectively) and pcDNA4 digested with Eco RV-Not I were ligated using Ligation High (Toyobo) according to the method described in the attached instructions. It was. Escherichia coli DH5α strain (Competent high DH5α (Toyobo)) was transformed with the reaction solution. Plasmid DNAs were isolated from the resulting ampicillin resistant clones using QIAprep Spin Miniprep Kit (QIAGEN).

  The other arm (referred to as the left arm HL molecule for convenience) is transferred to the ecdysone analog-inducible vector pIND (Invitrogen) for each of the H chain or L chain (FIGS. 2 to 3), ie, an animal cell signal sequence (IL3ss) (EMBO). J. 1987; 6: 2939) incorporating an appropriate mouse antibody variable region (VH to VL) and human IgG4γb constant region (SEQ ID NO: 35) to κ constant region (SEQ ID NO: 34) (pIND) -G4H to pIND-g4L) were prepared according to the method described above, and each plasmid DNA was isolated.

7-5. Construction of bispecific antibody expression vector The tetracycline-inducible expression plasmid (pcDNA4-g4H to pcDNA4-g4L) prepared in 7-4 was digested with Sfi I, and the reaction solution was subjected to 1% agarose gel electrophoresis. A fragment (about 5 kb) from which the antibody variable region portion (VH to VL (see FIGS. 1 and 2)) originally possessed was removed was purified using QIAquick Gel Extraction Kit (QIAGEN) according to the method described in the attached instructions. And eluted with 30 μL of sterile water. This fragment and the corresponding Sfi I digested anti-F. A ligation reaction was performed on IXa antibody-derived Sfi I-VH to Sfi I-VL fragments using Quick Ligation Kit (New England Biolabs) according to the method described in the attached instructions. Escherichia coli DH5α strain (Competent high DH5α (Toyobo)) was transformed with the reaction solution. Further, from the Sfi I digested ecdysone analog-inducible expression plasmid (pIND-g4H to pIND-g4L) prepared in 7-4, the antibody variable region portion (VH to VL (FIGS. 2 to 3) is prepared in the same manner as described above. And the Sfi I digested anti-F.F prepared in 7-3 corresponding to each fragment. The X antibody-derived Sfi I-VH to Sfi I-VL fragment was incorporated in the same manner.

  Each obtained ampicillin resistant transformant was confirmed to be inserted into the target fragment by colony PCR method using a primer sandwiching the inserted fragment. First, anti-F. For the IXa antibody chimeric heavy chain or light chain expression vector, a 21-mer primer CMVF (SEQ ID NO: 36) that anneals to the CMV Forward priming site existing upstream of the insertion site and a BGH Reverse priming site present downstream of the insertion site An 18-mer primer BGHR (SEQ ID NO: 37) that anneals to synthesize was synthesized (Sigma Genosys). Anti-F. For the X antibody chimeric heavy chain or light chain expression vector, the 24-mer primer EcdF (SEQ ID NO: 38) that anneals upstream to the insertion site and the 18-mer that anneals to BGH reverse priming site that exists downstream of the insertion site. Primer BGHR (SEQ ID NO: 37) was synthesized (Sigma Genosys). For colony PCR, 20 μL of a reaction solution (0.2 μL of each 10 μM primer, KOD dash buffer (Toyobo), 0.2 mM dNTPs, 0.75 units DNA polymerase KOD dash (Toyobo)) was prepared. An appropriate amount of transformant cells were added to the reaction solution, and PCR was performed. PCR is carried out using a thermal cycler GeneAmp PCR system 9700 (Parkin Elmer), heated at 96 ° C. for 1 minute, and then subjected to 30 cycles of a reaction comprising 96 ° C. for 10 seconds, 55 ° C. for 10 seconds, and 72 ° C. for 30 seconds. It went from condition. After PCR, the reaction solution was subjected to 1% agarose gel electrophoresis, and a clone from which an amplified fragment of the desired size was obtained was selected. The PCR product was inactivated with excess primers and dNTPs using ExoSAP-IT (Amersham Biosciences) according to the attached instructions. The base sequence of each DNA fragment was determined using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) according to the method described in the attached instruction of the DNA sequencer ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). The sequence group determined by this method is analyzed with the analysis software GENETYX-SV / RC Version 6.1 (Genetix), and the target clone which does not contain insertion deletion mutation or the like for VH is used in the hybridoma for VL. Unlike the P3U1-derived pseudo-VL gene, a target clone that did not contain insertion deletion mutations was selected.

  Each plasmid DNA was isolated from the target clone using QIAprep Spin Miniprep Kit (QIAGEN) and dissolved in 100 μL of sterile water. Anti-F. IXa antibody chimeric heavy chain expression vector, anti-F. IXa antibody chimeric light chain expression vector, anti-F. X antibody chimeric heavy chain expression vector, and anti-F. The X antibody chimeric light chain expression vectors were named pcDNA4-g4IXaHn, pcDNA4-g4IXaLn, pIND-g4XHn and pIND-g4XLn, respectively. Each plasmid solution was stored at 4 ° C. until use.

[Example 8] Expression of chimeric bispecific antibody in animal cells 8-1. Preparation of DNA Solution Expression of antibody right arm HL molecule expression vectors (pcDNA4-g4IXaHn and pcDNA4-g4IXaLn) is induced by tetracycline. In order to completely suppress expression in the absence of tetracycline, the plasmid pcDNA6 / TR (Invitrogen) encoding the Tet repressor is required. Further, expression induction of the antibody left arm HL molecule expression vectors (pIND-g4XHn and pIND-g4XLn) is induced by ecdysone analog (ponasterone A) which is an insect hormone. At this time, plasmid pVgRXR (Invitrogen) encoding ecdysone receptor that reacts with ponasterone A and induces retinoid X receptor is required. Therefore, a total of 6 types of plasmid DNA mixtures were prepared for transfection of animal cells. For 1 mL of cell culture medium, 218.8 ng each of pcDNA4-g4IXaHn, pcDNA4-g4IXaLn, pIND-g4XHn and pIND-g4XLn, pcDNA6 / TR and 1312.5 ng of pVgRXR were used.

8-2. Transfection of animal cells A human embryonic kidney cancer cell-derived HEK293H strain (Invitrogen) is suspended in DMEM medium (Invitrogen) containing 10% FCS (MOREGATE) and is used for adherent cells at a cell density of 5 × 10 ⁵ cells / mL. Each well of a well plate (CORNING) was cultivated in a CO ₂ incubator (37 ° C., 5% CO ₂ ) in an amount of 1 mL. The plasmid DNA mixture prepared in 8-1 was added to a mixture of 7 μL of transfection reagent, Lipofectamine 2000 (Invitrogen) and 250 μL of Opti-MEM I medium (Invitrogen), and allowed to stand at room temperature for 20 minutes. Incubated for 4-5 hours in a CO ₂ incubator (5% CO ₂ at 37 ° C.).

8-3. Induction of bispecific IgG antibody expression Aspirate the medium from the cell culture solution transfected as described above, and 1 mL CHO-S-SFM-II (Invitrogen) containing 1 μg / mL tetracycline (Wako Pure Chemical Industries). The medium was added and cultured in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for 1 day to induce primary expression of the antibody right arm HL molecule. Thereafter, the medium was removed by suction, and once washed with 1 mL CHO-S-SFM-II medium, 1 mL CHO-S-SFM-II medium containing 5 μM ponasterone A (Invitrogen) was added, and a CO ₂ incubator ( Incubation was performed at 37 ° C., 5% CO ₂ ) for 2 to 3 days to induce secondary expression of the antibody left arm HL molecule and to secrete the bispecific IgG antibody into the medium. After the culture supernatant was collected, centrifuged (approximately 2000 g, 5 min, room temperature) to remove cells, and was concentrated if necessary by ^{Microcon (R) YM-50 (} Millipore). The sample was stored at 4 ° C. until use.

[Example 9] Quantification of human IgG concentration Goat affinity purified to human IgG Fc (Cappel) was prepared at 1 μg / mL with a coating buffer and immobilized on a Nunc-Immuno plate. After blocking with Diluent buffer (DB), D.C. B. A culture supernatant sample appropriately diluted with was added. In addition, as a standard for antibody concentration calculation, D.D. B. In the same manner, human IgG4 (humanized anti-TF antibody, see WO99 / 51743) diluted in 11 steps was added in the same manner. After washing 3 times, Goat anti-human IgG, alkaline phosphate (Biosource International) was reacted. After washing 5 times, color was developed using Sigma 104 ^(R) phosphate substrate (Sigma-Aldrich) as a substrate, and the absorbance at 405 nm was measured at a reference wavelength of 655 nm with an absorbance reader Model 3550 (Bio-Rad Laboratories). The concentration of human IgG in the culture supernatant was calculated from a standard calibration curve using Microplate Manager III (Bio-Rad Laboratories) software.

[Example 10] F.F. VIIIa (activated coagulation factor VIII) -like activity assay F. of bispecific antibodies. VIIIa-like activity was assessed by the following enzyme assay. The following reactions were all performed at room temperature. A mixture of 40 μL of 3.75 μg / mL Factor IX (Enzyme Research Laboratories) and 10 μL of the antibody solution was incubated in a 96-well plate for 1 hour. Further to the mixture, 10 ng / mL of Factor XIa (Enzyme Research Laboratories) 10μL , 50μg / mL of Factor X (Enzyme Research Laboratories) 20μL , 400μg / mL of phospholipid (see Example 5-3) 5μL, 5mM CaCl ₂ And 15 μL of TBSB (hereinafter referred to as TBSB-S) containing 1 mM MgCl ₂ was added to start the enzyme reaction. After reacting for 1 hour, the reaction was stopped by adding 10 μL of 0.5 M EDTA.
After adding 50 μL of the chromogenic substrate solution to each well, the absorbance at 405 nm (reference wavelength 655 nm) at 0 minutes and 30 minutes was measured with a Model 3550 Microplate Reader (Bio Rad Laboratories). F. The VIIIa-like activity was expressed as a value obtained by subtracting the absorbance change value for 30 minutes without addition of antibody from the absorbance change value for 30 minutes after addition of the antibody (see FIGS. 4 and 5).
TBSB, Factor XIa, Factor IX, and Factor X were used as solvents for Phospholipid, TBSB-S. The chromogenic substrate solution is a 1: 1 mixture of test team chromogenic substrate S-2222 (Chromogenix) and polybrene solution (0.6 mg / L hexadimethine bromide (Sigma)) dissolved according to the attached instructions.
Furthermore, XB12 / SB04, which had the highest activity, The concentration dependency of VIIIa-like activity was measured (FIG. 6).

[Example 11] Plasma coagulation assay To clarify whether bispecific antibodies correct the coagulation ability of hemophilia A blood. The effect of the antibody on activated partial thromboplastin time (APTT) using VIII-deficient plasma was examined. 50 μL of antibody solution of various concentrations; A mixed solution of 50 μL of VIII-deficient plasma (Biomerieux) and 50 μL of APTT reagent (Dade Behring) was heated at 37 ° C. for 3 minutes. The coagulation reaction was initiated by adding 50 μL of 20 mM CaCl ₂ (Dade Behring) to the mixture. The time until coagulation was measured with KC10A (Amelung) connected with CR-A (Amelung) (FIGS. 7 and 8).
Furthermore, the concentration dependency of XB12 / SB04, which had the highest effect of shortening the coagulation time, was measured (FIG. 9).

[Example 12] Antibody purification 10 mL of the culture supernatant obtained by the method described in Example 8 was concentrated to 1 mL with Centricon ^(R) YM-50 (Millipore). To this was added 10% BSA in ^{10μL, rProtein 1% Tween (R} ) 20 and 100μL of ^{10μL A Sepharose TM Fast Flow (Amersham} Biosciences), overnight inverted for mixing at 4 ° C.. Transfer the solution to a 0.22μm filter cup ^{Ultrafree (R) -MC (Millipore)} , washed 3 times with TBS500μL containing 0.01% ^{Tween (R)} 20, the rProtein A Sepharose ^TM resin 100 [mu] L 0. 01% ^{Tween (R)} 10mM containing 20 HCl, then was allowed to stand for 3 minutes and suspended in pH 2.0, to elute the antibody. Immediately, 5 μL of 1M Tris-HCl, pH 8.0 was added for neutralization. The concentration of human IgG in the culture supernatant was calculated from a standard calibration curve using Microplate Manager III (Bio-Rad Laboratories) software. The antibody concentration was quantified according to Example 9.

According to the present invention, a bispecific antibody having a ligand function surrogate action for a receptor containing a heteromolecule is provided.
The present invention also provides a bispecific antibody that recognizes both an enzyme and a substrate of the enzyme and that replaces the function of a cofactor that enhances the enzyme activity.
Since the bispecific antibody of the present invention is considered to have high stability in blood and low antigenicity, it is highly expected to be a pharmaceutical product.

Claims (16)

A bispecific antibody that recognizes both an enzyme and a substrate of the enzyme and that substitutes for the function of a cofactor that enhances the enzymatic reaction. The antibody according to claim 1, wherein the enzyme is a proteolytic enzyme. The antibody according to claim 2, wherein the proteolytic enzyme, the substrate and the cofactor are blood coagulation / fibrinolysis-related factors. The coagulation / fibrinolysis-related factor enzyme is blood coagulation factor IX and / or activated blood coagulation factor IX, the substrate is blood coagulation factor X, and the cofactor is blood coagulation factor VIII and / or activated blood coagulation. 4. The antibody of claim 3, which is Factor VIII. A complementarity determining region comprising the amino acid sequence of CDR3 of (a1) or (a2) below or a functionally equivalent complementarity determining region in anti-blood coagulation factor IX / IXa antibody, and anti-blood coagulation factor X antibody The complementarity determining region consisting of the amino acid sequence of CDR3 according to any one of (b1) to (b9) below or a functionally equivalent complementarity determining region according to any one of claims 1 to 4 The antibody described.
(A1) The amino acid sequence of H chain CDR3 described in SEQ ID NO: 42 (a2) The amino acid sequence of H chain CDR3 described in SEQ ID NO: 46 (b1) The amino acid sequence of H chain CDR3 described in SEQ ID NO: 50 (b2 ) H chain CDR3 is the amino acid sequence described in SEQ ID NO: 54 (b3) H chain CDR3 is the amino acid sequence described in SEQ ID NO: 58 (b4) H chain CDR3 is the amino acid sequence described in SEQ ID NO: 62 (b5) H The chain CDR3 is the amino acid sequence described in SEQ ID NO: 66 (b6) The H chain CDR3 is the amino acid sequence described in SEQ ID NO: 70 (b7) The H chain CDR3 is the amino acid sequence described in SEQ ID NO: 74 (b8) The H chain CDR3 Is the amino acid sequence set forth in SEQ ID NO: 78 (b9) The heavy chain CDR3 is the amino acid sequence set forth in SEQ ID NO: 82 A complementarity determining region consisting of the amino acid sequence of CDR of (a1) or (a2) below in anti-blood coagulation factor IX / IXa antibody or a complementarity determining region functionally equivalent thereto, and anti-blood coagulation factor X antibody The complementarity determining region comprising the CDR amino acid sequence according to any one of (b1) to (b9) below or a functionally equivalent complementarity determining region according to any one of claims 1 to 4 The antibody described.
(A1) H chain CDR1,2,3 is the amino acid sequence described in SEQ ID NO: 40, 41,42 (a2) H chain CDR1,2,3 is the amino acid sequence described in SEQ ID NO: 44, 45, 46 (b1 ) H chain CDR1, 2, 3 is the amino acid sequence described in SEQ ID NO: 48, 49, 50 (b2) H chain CDR1, 2, 3 is the amino acid sequence described in SEQ ID NO: 52, 53, 54 (b3) H The chain CDR1, 2, 3 is the amino acid sequence described in SEQ ID NO: 56, 57, 58 (b4) The H chain CDR1, 2, 3 is the amino acid sequence described in SEQ ID NO: 60, 61, 62 (b5) H chain CDR1 , 2, 3 are the amino acid sequence (b6) H chain CDR1, 2, 3 described in SEQ ID NO: 64, 65, 66 are the amino acid sequence (b7) H chain CDR1, 2 described in SEQ ID NO: 68, 69, 70 , 3 in SEQ ID NOs: 72, 73, 74 The amino acid sequence (b8) H chain CDR1,2,3 described in SEQ ID NO: 76,77,78 is the amino acid sequence (b9) H chain CDR1,2,3 described in SEQ ID NO: 80, 81,82 Amino acid sequence A composition comprising the antibody according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier. The composition according to claim 7, which is a pharmaceutical composition used for the prevention and / or treatment of bleeding, a disease accompanied by bleeding, or a disease caused by bleeding. The bleeding, bleeding-related disease, or bleeding-related disease is a disease that develops and / or progresses due to decreased or deficient activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII. A composition according to 1. The composition according to claim 9, wherein the disease that develops and / or develops due to a decrease or deficiency in the activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII is hemophilia A. Diseases that develop and / or develop due to decreased or deficient activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII appear as inhibitors to blood coagulation factor VIII and / or activated blood coagulation factor VIII The composition according to claim 9, wherein the composition is a disease. 10. The composition according to claim 9, wherein the disease that develops and / or develops due to decreased or deficient activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII is acquired hemophilia. 10. The composition according to claim 9, wherein the disease that develops and / or develops due to decreased activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII is von Willebrand disease. A method for preventing bleeding, bleeding-related diseases, or diseases caused by bleeding, comprising the step of administering the antibody according to any one of claims 1 to 6 or the composition according to any one of claims 7 to 13. / Or how to treat. Use of the antibody according to any one of claims 1 to 6 for the production of a composition according to any one of claims 7 to 13. A kit for use in the method for preventing and / or treating according to claim 14, comprising at least the antibody according to any one of claims 1 to 6, or the composition according to claim 7.

Images