Bispecific antibodies binding to CD40 and EpCAM

Abstract

The present invention aims to provide a bispecific antibody comprising an antigen-binding domain that binds to CD40 and an antigen-binding domain that binds to epithelial cell adhesion molecule (EpCAM). The present invention relates to a bispecific antibody comprising an antigen-binding domain that binds to CD40 and an antigen-binding domain that binds to EpCAM.

Description

Technical Field

[0001] The present invention relates to bispecific antibodies comprising an antigen-binding domain that binds to CD40 and an antigen-binding domain that binds to epithelial cell adhesion molecule (EpCAM), bispecific antibody fragments, DNA encoding the bispecific antibody or the bispecific antibody fragment, vectors comprising the DNA, hybridomas and transformants that produce the bispecific antibody or the bispecific antibody fragment, methods for producing the bispecific antibody or the bispecific antibody fragment, therapeutic and diagnostic agents comprising the bispecific antibody or the bispecific antibody fragment, therapeutic and diagnostic methods using the bispecific antibody or the bispecific antibody fragment, and detection or assay reagents comprising the bispecific antibody or the bispecific antibody fragment. Background Art

[0002] Antibodies are glycoproteins present in the serum and tissue fluids of all mammals, and they recognize foreign antigens within the body. Antibodies participate in the body's defense by activating the complement system and binding to cell surface receptors (FcRs). These antibodies activate effector functions of FcR-expressing cells, including phagocytosis, antibody-dependent cellular cytotoxicity, mediator release, and antigen presentation.

[0003] One molecule of antibody is composed of two identical light chains (L chains) and two identical heavy chains (H chains) and has two antigen-binding sites. The class and subclass of the antibody are determined by the H chain, and each class and subclass has different inherent functions. There are five different classes of human antibodies, IgG, IgA, IgM, IgD and IgE. IgG is further divided into the subclasses of IgG1, IgG2, IgG3 and IgG4, and IgA is divided into the subclasses of IgA1 and IgA2 (Charles A.J. et.al., Immunobiology, 1997, Current Biology Ltd / Garland Publishing Inc.).

[0004] Multivalent antibodies are antibodies with multiple antigen-binding sites within one molecule. As an example of a multivalent antibody, it has been reported that a hybridoma was first used to express the H chain and L chain from two different antibodies in one cell, thereby producing a bivalent antibody that binds to two different antigens in a monovalent manner. (Non-patent document 1). However, in this method, the combination of the H chain and L chain of the antibody produces about 10 types. Therefore, the production of multivalent antibodies with the desired H chain and L chain combination is low, and it is also difficult to selectively separate and purify such multivalent antibodies, so the yield of the desired antibody is reduced.

[0005] To overcome this problem, attempts have been reported to express multiple antigen-binding sites linked together as a single polypeptide chain, thereby reducing variations in the combination of subunits and producing antibodies with a desired combination.

[0006] As an example, antibodies comprising a single-chain Fv (scFv) in which the antigen-binding sites of the H and L chains are linked by a single polypeptide are known (Non-Patent Document 2). In addition, antibodies in which two antigen-binding sites are linked using the CH1 domain of the H chain constant region of IgG1 or a partial fragment of this domain and the L chain constant region, or a flexible linker (Gly-Gly-Gly-Gly-Ser), have also been reported (Non-Patent Document 4, Patent Document 1, Patent Document 2).

[0007] These existing multivalent antibodies have the disadvantages of being easily aggregated, having low stability and productivity. However, it has been discovered that multivalent antibodies containing multiple antigen-binding sites within a single H chain polypeptide, in which the antibody heavy chain variable regions are linked via a linker having an amino acid sequence of an immunoglobulin domain or fragment thereof, exhibit high stability and improved productivity (Patent Document 3).

[0008] CD40 is identified as an antigen expressed on the surface of human B cells (Non-Patent Document 4) and is known to be a member of the TNF receptor family. TNF receptor family molecules are defined by the presence of cysteine-rich repeats in the extracellular domain. As members of the TNF receptor family, in addition to CD40, low-affinity NGF receptors, TNF receptors, CD27, OX40, and CD30 are also known.

[0009] Signaling via the TNF receptor family is induced by the binding of homotrimeric TNF family molecules to three TNF receptor families, but signals are also transmitted intracellularly by crosslinking between specific antibodies on TNF receptor family molecules, so it is believed that signal transduction requires the association of TNF receptor family molecules (Non-Patent Documents 5, 6).

[0010] CD40 is a type I membrane glycoprotein known to be expressed in a variety of cell types, including B lymphocytes, dendritic cells, monocytes, epithelial cells, and fibroblasts, as well as in certain types of tumor cells, including neoplastic human B cells. Impaired thymus-dependent immunoglobulin class switching and germinal center formation have been observed in CD40-deficient mice, confirming the important role of CD40 in both cellular and humoral immune responses (Non-Patent Document 7).

[0011] CD40 signals are involved in immunoglobulin class switching and CTL induction, and therefore are expected to be used in the activation of tumor immunity and in pharmaceuticals as adjuvants for cancer vaccines (Non-Patent Document 8).

[0012] Existing antibodies targeting CD40 include Chi-Lob 7 / 4, HCD-122, APX005M, SEA-CD40, and CP870,893 (21.4.1) (Non-Patent Document 9). Among them, CP870,893 has a strong ability to induce CD40 signals, and clinical trials have been conducted on solid tumors with systemic immune activation as the pharmacodynamic mechanism. However, efficacy has not been demonstrated, and toxicities from systemic immune activation, such as cytokine release syndrome, increased thrombotic markers, and increased liver function parameters, have been reported (Non-Patent Document 10).

[0013] The physiological ligand of CD40 is CD40 ligand (CD154, gp39). Activated T lymphocytes express CD40 ligand, which, through binding to CD40 on the surface of B lymphocytes, plays an important regulatory role in B lymphocyte differentiation and proliferation, as well as preventing spontaneous apoptosis of B lymphocytes in the germinal center (Non-Patent Document 11).

[0014] As molecules with CD40 signal-inducing ability, molecules in which the Fc region of an antibody is fused to CD40 ligand have been reported. Furthermore, a fusion antibody composed of CD40 ligand and a single-chain antibody scFv targeting a cancer antigen was produced to achieve efficient CD40 signal induction in tumors. This fusion antibody induced CD40 signaling at a dose approximately 20-fold lower than that of CD40 ligand (Non-Patent Document 12).

[0015] As bispecific antibodies that recognize CD40, IgG-type anti-human GPC3 / anti-mouse CD40 bispecific antibodies having heterodimeric heavy chains are known (Patent Document 4). Furthermore, bispecific proteins that bind to cancer antigens such as nectin-4, PSMA, or EGFR and CD40, thereby activating CD40, are known (Patent Document 5). Furthermore, methods for activating CD40-expressing cells near cancer cells using bispecific molecules such as diabodies (bifunctional antibodies) that are specific for CD40 and cancer cell surface antigens are also known (Patent Document 6).

[0016] Epithelial cell adhesion molecules (EpCAM, CD326, GA733-2, HEA125, KS1 / 4, MK-1, MH99, MOC31, 323 / A3, 17-1A, CO-17A, ESA, EGP-2, EGP34, EGP40, KSA, KS1 / 4, TROP-1, TACST-1) are 40 kDa transmembrane glycoproteins involved in cell adhesion, cell proliferation, and tumor progression, functioning as homotypic cell adhesion factors.

[0017] Since EpCAM is highly expressed in various epithelial-derived cancer cells, it is well known as a cancer antigen and is expected to serve as a target molecule or diagnostic marker for cancer molecular target drugs and a target for cancer vaccines (Non-Patent Documents 13 and 14).

[0018] Examples of EpCAM-targeting drugs include vaccine therapy using anti-idiotypic antibodies that bind to the antigen recognition site of EpCAM protein or anti-EpCAM antibodies, and anti-EpCAM antibodies such as adecatumumab (MT201), ING-1, and 3622W94 (Non-Patent Document 15).

[0019] Furthermore, to enhance the cytotoxic activity of anti-EpCAM antibodies, Proxinium Vivendiums (VB4-845), a fusion of an anti-EpCAM antibody with Pseudomonas aeruginosa exotoxin, and EMD 273066 (huKS-IL2), a fusion of an anti-EpCAM antibody with IL-2, have been developed. Removab, a bispecific antibody that uses an anti-CD3 antibody to cross-link T cells with EpCAM-positive tumor cells, thereby toxicifying tumor cells, has been approved in Europe (Non-Patent Documents 14 and 16).

[0020] Prior art literature

[0021] Patent Literature

[0022] Patent Document 1: U.S. Patent Application Publication No. 2007 / 0071675

[0023] Patent Document 2: International Publication No. 2001 / 077342

[0024] Patent Document 3: International Publication No. 2009 / 131239

[0025] Patent Document 4: International Publication No. 2015 / 156268

[0026] Patent Document 5: International Publication No. 2017 / 205738

[0027] Patent Document 6: International Publication No. 99 / 61057

[0028] Non-patent literature

[0029] Non-patent document 1: Suresh et al., Methods Enzymol. 121, 210-228, 1986

[0030] Non-patent document 2: Kranz et al., J. Hematother. 5, 403-408, 1995

[0031] Non-patent document 3: Wu et al., Nat. Biothech. 25, 1290-1297, 2007

[0032] Non-patent document 4: Clark et al., PNAS USA 1986; 83: 4494-4498

[0033] Non-patent document 5: Chuntharapai A et al., J. Immunol. 166(8), 4891, 2001

[0034] Non-patent document 6: Ashkenazi A et al., Nat Rev Cancer 2, 420, 2002

[0035] Non-patent document 7: Kawabe et al., Immunity 1994; 1: 167-168

[0036] Non-patent literature 8: Diehl L et.al., Nat Med. 1999 Jul; 5(7): 774-9.

[0037] Non-Patent Literature 9: Beatty GL et al., Expert Rev Anticancer Ther. 2017

[0038] Feb;17(2):175-186.

[0039] Non-patent literature 10: Vonderheide RH et al., Oncoimmunology. 2013 Jan

[0040] 1;2(1):e23033.

[0041] Non-patent literature 11: Bridges et al., J Immumol 1987; 139: 4242-4549

[0042] Non-patent literature 12: 1541376565889_0 et al., Mol Cancer. 2014 Apr 17; 13:85

[0043] Non-patent document 13: van der Gun BT et.al., Br J Cancer. 2011 Jul

[0044] 12;105(2):312-9.

[0045] Non-patent document 14: Chaudry MA et al., Br J Cancer. 2007 Apr

[0046] 10;96(7):1013-9.

[0047] Non-patent literature 15: Markus M et al., Cancer Cell Int. 2010 Nov 2; 10: 44.

[0048] Non-patent document 16: Linke R et al., MAbs. 2010, Vol. 2(2), pp. 129-136. Summary of the Invention

[0049] Problems to be solved by the invention

[0050] Bispecific antibodies targeting CD40 and EpCAM and their anti-tumor effects are not yet known. Therefore, the present invention aims to provide bispecific antibodies comprising an antigen-binding domain that binds to CD40 and an antigen-binding domain that binds to EpCAM, bispecific antibody fragments, DNA encoding the bispecific antibodies or bispecific antibody fragments, vectors comprising the DNA, hybridomas and transformants producing the bispecific antibodies or bispecific antibody fragments, methods for producing the bispecific antibodies or bispecific antibody fragments, therapeutic and diagnostic agents comprising the bispecific antibodies or bispecific antibody fragments, therapeutic and diagnostic methods using the bispecific antibodies or bispecific antibody fragments, and detection or measurement reagents comprising the bispecific antibodies or bispecific antibody fragments.

[0051] Technical means to solve the problem

[0052] As means for solving the above-mentioned problems, the present invention provides bispecific antibodies comprising an antigen-binding domain that binds to CD40 and an antigen-binding domain that binds to EpCAM, or a fragment thereof.

[0053] That is, the present invention relates to the following (1) to (35).

[0054] (1) A bispecific antibody comprising an antigen-binding domain that binds to CD40 and an antigen-binding domain that binds to epithelial cell adhesion molecule (EpCAM).

[0055] (2) The bispecific antibody according to (1), which binds to CD40 and EpCAM in a bivalent manner.

[0056] (3) The bispecific antibody according to (1) or (2), wherein the antigen-binding domain that binds to CD40 comprises the heavy chain variable region (VH) and light chain variable region (VL) of an antibody that specifically binds to CD40 (anti-CD40 antibody), and the antigen-binding domain that binds to EpCAM comprises the VH and VL of an antibody that specifically binds to EpCAM (anti-EpCAM antibody).

[0057] (4) The bispecific antibody according to any one of (1) to (3), comprising:

[0058] Containing the formula VH1-X-VH2-Y from the N-terminus

[0059] {where,

[0060] VH1 represents the VH of the primary antibody,

[0061] VH2 represents the VH of the second antibody,

[0062] X and Y each represent a polypeptide comprising CH1 of an antibody (wherein at least one of X and Y further comprises the hinge region of the antibody)}

[0063] The same two heavy chains of the polypeptide shown,

[0064] and 4 light chains containing the same VL,

[0065] Either one of the first antibody and the second antibody is an anti-CD40 antibody, and the other is an anti-EpCAM antibody.

[0066] (5) The bispecific antibody according to (4), wherein the light chain is a κ chain.

[0067] (6) The bispecific antibody or bispecific antibody fragment according to (4) or (5), wherein X in the above formula is a polypeptide consisting of CH1 of human IgG, and Y is a polypeptide consisting of CH1, hinge region, CH2, and CH3 of human IgG.

[0068] (7) The bispecific antibody according to (4) or (5), wherein X in the above formula is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 75, and Y is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 77.

[0069] (8) The bispecific antibody according to (4) or (5), wherein X in the above formula is a polypeptide comprising CH1, hinge region, CH2, and CH3 of human IgG, and Y is a polypeptide comprising CH1 of human IgG.

[0070] (9) The bispecific antibody according to (4) or (5), wherein X in the above formula is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 77, and Y is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 75.

[0071] (10) The bispecific antibody according to any one of (3) to (9), wherein VL comprises complementarity determining regions (CDRs) 1 to 3 comprising the amino acid sequences shown in SEQ ID NOs: 23 to 25, respectively.

[0072] (11) The bispecific antibody according to any one of (3) to (10), wherein VL comprises the amino acid sequence shown in SEQ ID NO: 22.

[0073] (12) The bispecific antibody according to any one of (1) to (11), which has CD40 agonist activity.

[0074] (13) The bispecific antibody according to any one of (1) to (12), which does not exhibit CD40 agonist activity in the absence of cells expressing EpCAM and exhibits CD40 agonist activity only in the presence of cells expressing EpCAM.

[0075] (14) The bispecific antibody according to any one of (3) to (13), wherein the anti-CD40 antibody is an anti-CD40 antibody without agonist activity.

[0076] (15) The bispecific antibody according to any one of (4) to (14), wherein the first antibody is an anti-CD40 antibody and the second antibody is an anti-EpCAM antibody.

[0077] (16) The bispecific antibody according to any one of (4) to (14), wherein the first antibody is an anti-EpCAM antibody and the second antibody is an anti-CD40 antibody.

[0078] (17) The bispecific antibody according to any one of (3) to (16), wherein the anti-CD40 antibody is an anti-CD40 antibody comprising any one heavy chain variable region (VH) selected from the group consisting of the following (1a) to (1d).

[0079] (1a) VH comprising CDR1 to CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively;

[0080] (1b) VH comprising CDR1 to CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 33 to 35, respectively;

[0081] (1c) VH comprising CDR1 to CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 38 to 40, respectively, and

[0082] (1d) VH comprising CDR1 to CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 43 to 45, respectively.

[0083] (18) The bispecific antibody according to any one of (3) to (17), wherein the anti-CD40 antibody is an anti-CD40 antibody comprising a VH comprising an amino acid sequence represented by any one of SEQ ID NOs: 27, 32, 37, and 42.

[0084] (19) The bispecific antibody according to any one of (3) to (18), wherein the anti-EpCAM antibody is an anti-EpCAM antibody comprising any one VH selected from the group consisting of the following (2a) to (2d).

[0085] (2a) VH comprising CDR1 to CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 52 to 54, respectively;

[0086] (2b) VH comprising CDR1 to CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 57 to 59, respectively;

[0087] (2c) VH comprising CDR1 to CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 62 to 64, respectively, and

[0088] (2d) VH comprising CDR1 to CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 67 to 69, respectively.

[0089] (20) The bispecific antibody according to any one of (3) to (19), wherein the anti-EpCAM antibody is an anti-EpCAM antibody comprising a VH containing an amino acid sequence represented by any one of SEQ ID NOs: 51, 56, 61, and 66.

[0090] (21) A bispecific antibody fragment of the bispecific antibody according to any one of (1) to (20).

[0091] (22) The bispecific antibody fragment according to (21), which is a Fab-type bispecific antibody fragment or a F(ab')2-type bispecific antibody fragment.

[0092] (23) A DNA encoding the bispecific antibody according to any one of (1) to (20) or the bispecific antibody fragment according to (21) or (22).

[0093] (24) A recombinant vector containing the DNA described in (23).

[0094] (25) A transformant obtained by introducing the recombinant vector described in (24) into a host cell.

[0095] (26) A method for producing the bispecific antibody described in any one of (1) to (20) or the bispecific antibody fragment described in (21) or (22), characterized in that the transformant described in (25) is cultured in a culture medium, the bispecific antibody described in any one of (1) to (20) or the bispecific antibody fragment described in (21) or (22) is produced and accumulated in the culture, and the bispecific antibody or the bispecific antibody fragment is collected from the culture.

[0096] (27) A therapeutic and / or diagnostic agent for a disease associated with at least one of human CD40 and human EpCAM, comprising the bispecific antibody described in any one of (1) to (20) or the bispecific antibody fragment described in (21) or (22) as an active ingredient.

[0097] (28) The agent according to (27), wherein the disease associated with at least one of human CD40 and human EpCAM is cancer.

[0098] (29) A method for treating and / or diagnosing a disease related to at least one of human CD40 and human EpCAM, which uses the bispecific antibody described in any one of (1) to (20) or the bispecific antibody fragment described in (21) or (22).

[0099] (30) The method according to (29), wherein the disease associated with at least one of human CD40 and human EpCAM is cancer.

[0100] (31) The bispecific antibody according to any one of (1) to (20) or the bispecific antibody fragment according to (21) or (22), which is used for treating and / or diagnosing a disease associated with at least one of human CD40 and human EpCAM.

[0101] (32) The bispecific antibody or bispecific antibody fragment according to (31), wherein the disease associated with at least one of human CD40 and human EpCAM is cancer.

[0102] (33) Use of the bispecific antibody according to any one of (1) to (20) or the bispecific antibody fragment according to (21) or (22) in the manufacture of a therapeutic and / or diagnostic agent for a disease associated with at least one of human CD40 and human EpCAM.

[0103] (34) The use according to (33), wherein the disease associated with at least one of human CD40 and human EpCAM is cancer.

[0104] (35) A reagent for detecting or measuring at least one of EpCAM and CD40, comprising the bispecific antibody according to any one of (1) to (20) or the bispecific antibody fragment according to (21) or (22).

[0105] Effects of the Invention

[0106] According to the present invention, there are provided bispecific antibodies comprising an antigen-binding domain that binds to CD40 and an antigen-binding domain that binds to EpCAM, bispecific antibody fragments, DNA encoding the bispecific antibody or the bispecific antibody fragment, vectors comprising the DNA, hybridomas and transformants that produce the bispecific antibody or the bispecific antibody fragment, methods for producing the bispecific antibody or the bispecific antibody fragment, therapeutic and diagnostic agents comprising the bispecific antibody or the bispecific antibody fragment, therapeutic and diagnostic methods using the bispecific antibody or the bispecific antibody fragment, and detection or measurement reagents comprising the bispecific antibody or the bispecific antibody fragment. Sequence Listing <110> Kyowa Kirin Co., Ltd. <120> Bispecific antibodies that bind to CD40 and EpCAM <130> PD00975A <140> JP2017-215834 <141> 2017-11-08 <160> 86 <170> PatentIn version 3.3 <210> 1 <211> 522 <212> DNA <213> Human <220> <221> CDS <222> (1)..(522) <400> 1 gaa cca ccc act gca tgc aga gaa aaa cag tac cta ata aac agt cag 48 Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln 1 5 10 15 tgc tgt tct ttg tgc cag cca gga cag aaa ctg gtg agt gac tgc aca 96 Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr 20 25 30 gag ttc act gaa acg gaa tgc ctt cct tgc ggt gaa agc gaa ttc cta 144 Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu 35 40 45 gac acc tgg aac aga gag aca cac tgc cac cag cac aaa tac tgc gac 192 Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp 50 55 60 ccc aac cta ggg ctt cgg gtc cag cag aag ggc acc tca gaa aca gac 240 Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp 65 70 75 80 acc atc tgc acc tgt gaa gaa ggc tgg cac tgt acg agt gag gcc tgt 288 Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys 85 90 95 gag agc tgt gtc ctg cac cgc tca tgc tcg ccc ggc ttt ggg gtc aag 336 Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys 100 105 110 cag att gct aca ggg gtt tct gat acc atc tgc gag ccc tgc cca gtc 384 Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val 115 120 125 ggc ttc ttc tcc aat gtg tca tct gct ttc gaa aaa tgt cac cct tgg 432 Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp 130 135 140 aca agc tgt gag acc aaa gac ctg gtt gtg caa cag gca ggc aca aac 480 Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn 145 150 155 160 aag act gat gtt gtc tgt ggt ccc cag gat cgg ctg aga gcc 522 Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg Ala 165 170 <210> 2 <211> 174 <212> PRT <213> Human <400> 2 Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln 1 5 10 15 Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr 20 25 30 Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu 35 40 45 Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp 50 55 60 Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp 65 70 75 80 Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys 85 90 95 Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys 100 105 110 Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val 115 120 125 Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp 130 135 140 Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn 145 150 155 160 Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg Ala 165 170 <210> 3 <211> 522 <212> DNA <213> Rhesus monkey <220> <221> CDS <222> (1)..(522) <400> 3 gaa cca ccc act gca tgc aga gaa aaa cag tac cta ata aac agt cag 48 Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln 1 5 10 15 tgc tgt tct ttg tgc cag cca gga cag aaa ctg gtg agt gac tgc aca 96 Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr 20 25 30 gag ttc acc gaa aca gaa tgc ctt cct tgc ggt gaa agc gaa ttc cta 144 Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu 35 40 45 gac acc tgg aat aga gag aca cgc tgc cac cag cac aaa tac tgc gac 192 Asp Thr Trp Asn Arg Glu Thr Arg Cys His Gln His Lys Tyr Cys Asp 50 55 60 ccc aac cta ggg ctt cgg gtc cag cag aag ggc acc tca gaa aca gac 240 Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp 65 70 75 80 acc atc tgc acc tgt gaa gaa ggc ctg cac tgt acg agt gag tcc tgt 288 Thr Ile Cys Thr Cys Glu Glu Gly Leu His Cys Thr Ser Glu Ser Cys 85 90 95 gag agc tgt gtc ccg cac cgc tca tgc ttg cct ggc ttt ggg gtc aag 336 Glu Ser Cys Val Pro His Arg Ser Cys Leu Pro Gly Phe Gly Val Lys 100 105 110 cag att gct aca ggg gtt tct gat acc atc tgt gag ccc tgc ccg gtc 384 Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val 115 120 125 ggc ttc ttc tcc aat gtg tca tct gct ttt gaa aag tgt cgc cct tgg 432 Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys Arg Pro Trp 130 135 140 aca agc tgt gag acc aaa gac ctg gtt gtg caa cag gca ggc aca aac 480 Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn 145 150 155 160 aag act gat gtt gtc tgt ggt ccc cag gat cgg cag aga gcc 522 Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Gln Arg Ala 165 170 <210> 4 <211> 174 <212> PRT <213> Macaque <400> 4 Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln 1 5 10 15 Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr %20 25 30 Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu 35 40 45 Asp Thr Trp Asn Arg Glu Thr Arg Cys His Gln His Lys Tyr Cys Asp 50 55 60 Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp 65 70 75 80 Thr Ile Cys Thr Cys Glu Glu Gly Leu His Cys Thr Ser Glu Ser Cys 85 90 95 Glu Ser Cys Val Pro His Arg Ser Cys Leu Pro Gly Phe Gly Val Lys 100 105 110 Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val 115 120 125 Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys Arg Pro Trp 130 135 140 Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn 145 150 155 160 Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Gln Arg Ala 165 170 <210> 5 <211> 834 <212> DNA <213> Human <220> <221> CDS <222> (1)..(834) <400> 5 atg gtt cgt ctg cct ctg cag tgc gtc ctc tgg ggc tgc ttg ctg acc 48 Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr 1 5 10 15 gct gtc cat cca gaa cca ccc act gca tgc aga gaa aaa cag tac cta 96 Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu 20 25 30 ata aac agt cag tgc tgt tct ttg tgc cag cca gga cag aaa ctg gtg 144 Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val 35 40 45 agt gac tgc aca gag ttc act gaa acg gaa tgc ctt cct tgc ggt gaa 192 Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu 50 55 60 agc gaa ttc cta gac acc tgg aac aga gag aca cac tgc cac cag cac 240 Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His 65 70 75 80 aaa tac tgc gac ccc aac cta ggg ctt cgg gtc cag cag aag ggc acc 288 Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr 85 90 95 tca gaa aca gac acc atc tgc acc tgt gaa gaa ggc tgg cac tgt acg 336 Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr 100 105 110 agt gag gcc tgt gag agc tgt gtc ctg cac cgc tca tgc tcg ccc ggc 384 Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly 115 120 125 ttt ggg gtc aag cag att gct aca ggg gtt tct gat acc atc tgc gag 432 Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu 130 135 140 ccc tgc cca gtc ggc ttc ttc tcc aat gtg tca tct gct ttc gaa aaa 480 Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys 145 150 155 160 tgt cac cct tgg aca agc tgt gag acc aaa gac ctg gtt gtg caa cag 528 Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln 165 170 175 gca ggc aca aac aag act gat gtt gtc tgt ggt ccc cag gat cgg ctg 576 Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu 180 185 190 aga gcc ctg gtg gtg atc ccc atc atc ttc ggg atc ctg ttt gcc atc 624 Arg Ala Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile 195 200 205 ctc ttg gtg ctg gtc ttt atc aaa aag gtg gcc aag aag cca acc aat 672 Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn 210 215 220 aag gcc ccc cac ccc aag cag gaa ccc cag gag atc aat ttt ccc gac 720 Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp 225 230 235 240 gat ctt cct ggc tcc aac act gct gct cca gtg cag gag act tta cat 768 Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His 245 250 255 gga tgc caa ccg gtc acc cag gag gat ggc aaa gag agt cgc atc tca 816 Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser 260 265 270 gtg cag gag aga cag tga 834 Val Gln Glu Arg Gln 275 <210> 6 <211> 277 <212> PRT <213> Human <400> 6 Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr 1 5 10 15 Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu 20 25 30 Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val 35 40 45 Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu 50 55 60 Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His 65 70 75 80 Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr 85 90 95 Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr 100 105 110 Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly 115 120 125 Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu 130 135 140 Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys 145 150 155 160 Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln 165 170 175 Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu 180 185 190 Arg Ala Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile 195 200 205 Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn 210 215 220 Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp 225 230 235 240 Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His 245 250 255 Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser 260 265 270 Val Gln Glu Arg Gln 275 <210> (1)..(849) <211> 849 <212> DNA <213> Rhesus monkey <220> <221> CDS <222> (1)..(849) <400> 7 atg gtt cgt ctg cct ctg cag tgc gtc ctc tgg ggc tgc ttg ctg acc 48 Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr 1 5 10 15 gct gtc tat cca gaa cca ccc act gca tgc aga gaa aaa cag tac cta 96 Ala Val Tyr Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu 20 25 30 ata aac agt cag tgc tgt tct ttg tgc cag cca gga cag aaa ctg gtg 144 Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val 35 40 45 agt gac tgc aca gag ttc acc gaa aca gaa tgc ctt cct tgc ggt gaa 192 Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu 50 55 60 agc gaa ttc cta gac acc tgg aat aga gag aca cgc tgc cac cag cac 240 Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr Arg Cys His Gln His 65 70 75 80 aaa tac tgc gac ccc aac cta ggg ctt cgg gtc cag cag aag ggc acc 288 Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr 85 90 95 tca gaa aca gac acc atc tgc acc tgt gaa gaa ggc ctg cac tgt acg 336 Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Leu His Cys Thr 100 105 110 agt gag tcc tgt gag agc tgt gtc ccg cac cgc tca tgc ttg cct ggc 384 Ser Glu Ser Cys Glu Ser Cys Val Pro His Arg Ser Cys Leu Pro Gly 115 120 125 ttt ggg gtc aag cag att gct aca ggg gtt tct gat acc atc tgt gag 432 Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu 130 135 140 ccc tgc ccg gtc ggc ttc ttc tcc aat gtg tca tct gct ttt gaa aag 480 Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys 145 150 155 160 tgt cgc cct tgg aca agc tgt gag acc aaa gac ctg gtt gtg caa cag 528 Cys Arg Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln 165 170 175 gca ggc aca aac aag act gat gtt gtc tgt ggt ccc cag gat cgg cag 576 Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Gln 180 185 190 aga gcc ctg gtg gtg atc ccc atc tgc ttg ggg atc ctg ttt gtc atc 624 Arg Ala Leu Val Val Ile Pro Ile Cys Leu Gly Ile Leu Phe Val Ile 195 200 205 ctc ctc ttg gtg ctg gtc ttt atc agt gag tcc tca gaa aag gtg gcc 672 Leu Leu Leu Val Leu Val Phe Ile Ser Glu Ser Ser Glu Lys Val Ala 210 215 220 aag aag cca aac gat aag gcc ccc cac ccc aag cag gaa ccc cag gag 720 Lys Lys Pro Asn Asp Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu 225 230 235 240 atc aat ttt ctg gac gat ctt cct ggc tcc aac cct gcc gct cca gtg 768 Ile Asn Phe Leu Asp Asp Leu Pro Gly Ser Asn Pro Ala Ala Pro Val 245 250 255 cag gag act tta cat gga tgc caa ccg gtc acc cag gag gat ggc aaa 816 Gln Glu Thr Leu His Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys 260 265 270 gag agt cgc atc tca gtg cag gag aga cag tga 849 Glu Ser Arg Ile Ser Val Gln Glu Arg Gln 275 280 <210> 8 <211> 282 <212> PRT <213> Rhesus monkey <400> 8 Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr 1 5 10 15 Ala Val Tyr Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu 20 25 30 Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val 35 40 45 Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu 50 55 60 Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr Arg Cys His Gln His 65 70 75 80 Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr 85 90 95 Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Leu His Cys Thr 100 105 110 Ser Glu Ser Cys Glu Ser Cys Val Pro His Arg Ser Cys Leu Pro Gly 115 120 125 Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu 130 135 140 Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys 145 150 155 160 Cys Arg Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln 165 170 175 Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Gln 180 185 190 Arg Ala Leu Val Val Ile Pro Ile Cys Leu Gly Ile Leu Phe Val Ile 195 200 205 Leu Leu Leu Val Leu Val Phe Ile Ser Glu Ser Ser Glu Lys Val Ala 210 215 220 Lys Lys Pro Asn Asp Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu 225 230 235 24o Ile Asn Phe Leu Asp Asp Leu Pro Gly Ser Asn Pro Ala Ala Pro Val 245 250 255 Gln Glu Thr Leu His Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys 260 265 270 Glu Ser Arg Ile Ser Val Gln Glu Arg Gln 275 280 <210> 9 <211> 732 <212> DNA [[ID=4>>]<213> Human <220> <221> CDS It should be noted that in the original text, "225 230 235 24o" seems to have a typo where "24o" should probably be "240". This has been left as is in the translation to maintain consistency with the original. <222> (1)..(732) <400> 9 gca gct cag gaa gaa tgt gtc tgt gaaaac tac aag ctg gcc gcta aac 48 Only Only Gln Glu Glu Cys Val Cys Glu Asn Tyr Lys Ala Val Asn 1 5 10 15 tgc ttt gtg aat aat aat cgt caa tgc cag tgt act tca gtt gtt gt gca 96 How To Get A Clean Arg How To Get Clean How To Be Gly Ala 20 25 30 caa aat act gtc att tgc tca aag ctg gct gcc aaa tgt ttg gtg atg 144 Gln Asn Thr Val Ile Ser Lys Leu Ala Ala Lys Cys Leu Val Met 35 40 45 aag gca gaa atg aat ggc tca aaa ctt ggg aga aga gca aaa cct gaa 192 Lys Ala Glu Met Asn Gly Ser Lys Leu Gly Arg Arg Ala Lys Pro Glu 50 55 60 ggg gcc ctc cag aac aat gat ggg ctt tat gat cct gac tgc gat gag 240 Gly Ala Leu Gln Asn Asn Asp Gly Leu Tyr Asp Pro Asp Cys Asp Glu 65 70 75 80 agc ggg ctc ttt aag gcc aag cag tgc aac ggc acc tcc acg tgc tgg 288 Ser Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly Thr Ser Thr Cys Trp 85 90 95 tgt gtg aac act gct ggg gtc aga aga aca gac aag gac act gaa ata 336 Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp Lys Asp Thr Glu Ile 100 105 110 acc tgc tct gag cga gtg aga acc tac tgg atc att gaa cta aaa 384 Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile Ile Ile Glu Leu Lys 115 120 125 cac aaa gca aga gaa aaa cct tat gat agt aaa agt ttg cgg act gca 432 His Lys Ala Arg Glu Lys Pro Tyr Asp Ser Lys Ser Leu Arg Thr Ala 130 135 140 ctt cag aag gag atc aca acg cgt tat caa ctg gat cca aaa ttt atc 480 Leu Gln Lys Glu Ile Thr Thr Arg Tyr Gln Leu Asp Pro Lys Phe Ile 145 150 155 160 acg agt att ttg tat gag aat aat gtt atc act att gat ctg gtt caa 528 Thr Ser Ile Leu Tyr Glu Asn Asn Val Ile Thr Ile Asp Leu Val Gln 165 170 175 aat tct tct caa aaa act cag aat gat gtg gac ata gct gat gtg gct 576 Asn Ser Ser Gln Lys Thr Gln Asn Asp Val Asp Ile Ala Asp Val Ala 180 185 190 tat tat ttt gaa aaa gat gtt aaa ggt gaa tcc ttg ttt cat tct aag 624 Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser Leu Phe His Ser Lys 195 200 205 aaa atg gac ctg aca gta aat ggg gaa caa ctg gat ctg gat cct ggt 672 Lys Met Asp Leu Thr Val Asn Gly Glu Gln Leu Asp Leu Asp Pro Gly 210 215 220 caa act tta att tat tat gtt gat gaa aaa gca cct gaa ttc tca atg 720 Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys Ala Pro Glu Phe Ser Met 225 230 235 240 cag ggt cta aaa 732 Gln Gly Leu Lys <210> 10 <211> 244 <212> PRT[[ID=^37]] <213> Human <400> 10 Ala Ala Gln Glu Glu Cys Val Cys Glu Asn Tyr Lys Leu Ala Val Asn 1 5 10 15 Cys Phe Val Asn Asn Asn Arg Gln Cys Gln Cys Thr Ser Val Gly Ala 20 25 30 Gln Asn Thr Val Ile Cys Ser Lys Leu Ala Ala Lys Cys Leu Val Met 35 40 45 Lys Ala Glu Met Asn Gly Ser Lys Leu Gly Arg Arg Ala Lys Pro Glu 50 55 60 Gly Ala Leu Gln Asn Asn Asp Gly Leu Tyr Asp Pro Asp Cys Asp Glu 65 70 75 80 Ser Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly Thr Ser Thr Cys Trp 85 90 95 Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp Lys Asp Thr Glu Ile 100 105 110 Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile Ile Ile Glu Leu Lys 115 120 125 His Lys Ala Arg Glu Lys Pro Tyr Asp Ser Lys Ser Leu Arg Thr Ala 130 135 140 Leu Gln Lys Glu Ile Thr Thr Arg Tyr Gln Leu Asp Pro Lys Phe Ile 145 150 155 160 Thr Ser Ile Leu Tyr Glu Asn Asn Val Ile Thr Ile Asp Leu Val Gln 165 170 175 Asn Ser Ser Gln Lys Thr Gln Asn Asp Val Asp Ile Ala Asp Val Ala 180 185 190 Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser Leu Phe His Ser Lys 195 200 205 Lys Met Asp Leu Thr Val Asn Gly Glu Gln Leu Asp Leu Asp Pro Gly 210 215 220 Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys Ala Pro Glu Phe Ser Met 225 230 235 240 Gln Gly Leu Lys <210> 11 <211> 732 <212> DNA <213> Rhesus macaque <220> <221> CDS <222> (1)..(732) <400> 11 gca gct cag aaa gaa tgt gtc tgt gaa aac tac aag ctg gcc gta aac 48 Ala Ala Gln Lys Glu Cys Val Cys Glu Asn Tyr Lys Leu Ala Val Asn 1 5 10 15 tgc ttt ttg aat gac aat ggt caa tgc cag tgt act tcg att ggt gca 96 Cys Phe Leu Asn Asp Asn Gly Gln Cys Gln Cys Thr Ser Ile Gly Ala 20 25 30 caa aat act gtc ctt tgc tca aag ctg gct gcc aaa tgt ttg gtg atg 144 Gln Asn Thr Val Leu Cys Ser Lys Leu Ala Ala Lys Cys Leu Val Met 35 40 45 aag gca gaa atg aac ggc tca aaa ctt ggg aga aga gcg aaa cct gaa 192 Lys Ala Glu Met Asn Gly Ser Lys Leu Gly Arg Arg Ala Lys Pro Glu 50 55 60 ggg gct ctc cag aac aat gat ggc ctt tac gat cct gac tgc gat gag 240 Gly Ala Leu Gln Asn Asn Asp Gly Leu Tyr Asp Pro Asp Cys Asp Glu 65 70 75 80 agc ggg ctc ttt aag gcc aag cag tgc aac ggc acc tcc acg tgc tgg 288 Ser Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly Thr Ser Thr Cys Trp 85 90 95 tgt gtg aac act gct ggg gtc aga aga act gac aag gac act gaa ata 336 Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp Lys Asp Thr Glu Ile 100 105 110 acc tgc tct gag cga gtg aga acc tac tgg atc att gaa tta aaa 384 Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile Ile Ile Glu Leu Lys 115 120 125 cac aaa gca aga gaa aaa cct tat gat gtt caa agt ttg cgg act gca 432 His Lys Ala Arg Glu Lys Pro Tyr Asp Val Gln Ser Leu Arg Thr Ala 130 135 140 ctt gag gag gcg atc aaa acg cgt tat caa ctg gat cca aaa ttt atc 480 Leu Glu Glu Ala Ile Lys Thr Arg Tyr Gln Leu Asp Pro Lys Phe Ile 145 150 155 160 aca aat att ttg tat gag gat aat gtt atc act att gat ctg gtt caa 528 Thr Asn Ile Leu Tyr Glu Asp Asn Val Ile Thr Ile Asp Leu Val Gln 165 170 175 aat tct tct cag aaa act cag aat gat gtg gac ata gct gat gtg gct 576 Asn Ser Ser Gln Lys Thr Gln Asn Asp Val Asp Ile Ala Asp Val Ala 180 185 190 tat tat ttt gaa aaa gat gtt aaa ggt gaa tcc ttg ttt cat tct aag 624 Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser Leu Phe His Ser Lys 195 200 205 aaa atg gac ctg aga gta aat ggg gaa caa ctg gat ctg gat cct ggt 672 Lys Met Asp Leu Arg Val Asn Gly Glu Gln Leu Asp Leu Asp Pro Gly 210 215 220 caa act tta att tat tat gtc gat gaa aaa gca cct gaa ttc tca atg 720 Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys Ala Pro Glu Phe Ser Met 225 230 235 240 cag ggt cta aaa 732 Gln Gly Leu Lys <210> 12 <211> 244 <212> PRT <213> Rhesus monkey <400> 12 Ala Ala Gln Lys Glu Cys Val Cys Glu Asn Tyr Lys Leu Ala Val Asn 1 5 10 15 Cys Phe Leu Asn Asp Asn Gly Gln Cys Gln Cys Thr Ser Ile Gly Ala 20 25 30 Gln Asn Thr Val Leu Cys Ser Lys Leu Ala Ala Lys Cys Leu Val Met 35 40 45 Lys Ala Glu Met Asn Gly Ser Lys Leu Gly Arg Arg Ala Lys Pro Glu 50 55 60 Gly Ala Leu Gln Asn Asn Asp Gly Leu Tyr Asp Pro Asp Cys Asp Glu 65 70 75 80 Ser Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly Thr Ser Thr Cys Trp 85 90 95 Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp Lys Asp Thr Glu Ile 100 105 110 Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile Ile Ile Glu Leu Lys 115 120 125 His Lys Ala Arg Glu Lys Pro Tyr Asp Val Gln Ser Leu Arg Thr Ala 130 135 140 Leu Glu Glu Ala Ile Lys Thr Arg Tyr Gln Leu Asp Pro Lys Phe Ile 145 150 155 160 Thr Asn Ile Leu Tyr Glu Asp Asn Val Ile Thr Ile Asp Leu Val Gln 165 170 175 Asn Ser Ser Gln Lys Thr Gln Asn Asp Val Asp Ile Ala Asp Val Ala 180 185 190 Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser Leu Phe His Ser Lys 195 200 205 Lys Met Asp Leu Arg Val Asn Gly Glu Gln Leu Asp Leu Asp Pro Gly 210 215 220 Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys Ala Pro Glu Phe Ser Met 225 230 235 240 Gln Gly Leu Lys <210> 13 <211> 735 <212> DNA <213> Mus musculus <220> <221> CDS <222> (1)..(735) <400> 13 gcg gct cag aga gac tgt gtc tgt gac aac tac aag ctg gca aca agt 48 Ala Ala Gln Arg Asp Cys Val Cys Asp Asn Tyr Lys Leu Ala Thr Ser 1 5 10 15 tgc tct ctg aat gaa tat ggt gaa tgc cag tgt act tcc tat ggt aca 96 Cys Ser Leu Asn Glu Tyr Gly Glu Cys Gln Cys Thr Ser Tyr Gly Thr 20 25 30 cag aat act gtc att tgc tcc aaa ctg gcg tct aaa tgc ttg gcg atg 144 Gln Asn Thr Val Ile Cys Ser Lys Leu Ala Ser Lys Cys Leu Ala Met 35 40 45 aaa gca gaa atg act cac agc aag tct ggg agg agg ata aag ccc gaa 192 Lys Ala Glu Met Thr His Ser Lys Ser Gly Arg Arg Ile Lys Pro Glu 50 55 60 ggg gcg atc cag aac aac gat ggg ctg tac gac ccc gac tgc gac gag 240 Gly Ala Ile Gln Asn Asn Asp Gly Leu Tyr Asp Pro Asp Cys Asp Glu 65 70 75 80 cag ggg ctc ttc aaa gcc aag cag tgc aac ggc acc gcc acg tgc tgg 288 Gln Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly Thr Ala Thr Cys Trp 85 90 95 tgt gtc aac acc gcc gga gtc cga aga acc gac aag gac acg gag atc 336 Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp Lys Asp Thr Glu Ile 100 105 110 acg tgc tcc gag cgc gtg agg acc tac tgg atc atc att gaa cta aaa 384 Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile Ile Ile Glu Leu Lys 115 120 125 cac aaa gaa aga gaa agc ccc tac gac cat cag agc ttg cag act gcg 432 His Lys Glu Arg Glu Ser Pro Tyr Asp His Gln Ser Leu Gln Thr Ala 130 135 140 ctt caa gag gcg ttc aca tct cga tat aag ctg aat cag aaa ttt atc 480 Leu Gln Glu Ala Phe Thr Ser Arg Tyr Lys Leu Asn Gln Lys Phe Ile 145 150 155 160 aaa aac att atg tat gag aat aat gtt atc acc att gat ctg atg caa 528 Lys Asn Ile Met Tyr Glu Asn Asn Val Ile Thr Ile Asp Leu Met Gln 165 170 175 aac tct tct cag aaa aca caa gac gac gtg gac ata gct gat gtg gct 576 Asn Ser Ser Gln Lys Thr Gln Asp Asp Val Asp Ile Ala Asp Val Ala 180 185 190 tac tat ttt gaa aaa gat gtg aag ggg gag tcc ctg ttc cat tct tct 624 Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser Leu Phe His Ser Ser 195 200 205 aag agc atg gac ctg aga gtg aac gga gag ccg ctc gat ctg gac ccc 672 Lys Ser Met Asp Leu Arg Val Asn Gly Glu Pro Leu Asp Leu Asp Pro 210 215 220 ggg cag act ctg att tac tac gtt gat gaa aag gca ccc gag ttc tcc 720 Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys Ala Pro Glu Phe Ser 225 230 235 240 atg cag ggc ctc acg 735 Met Gln Gly Leu Thr 245 <210> 14 <211> 245 <212> PRT <213> Mus musculus <400> 14 Ala Ala Gln Arg Asp Cys Val Cys Asp Asn Tyr Lys Leu Ala Thr Ser 1 5 10 15 Cys Ser Leu Asn Glu Tyr Gly Glu Cys Gln Cys Thr Ser Tyr Gly Thr 20 25 30 Gln Asn Thr Val Ile Cys Ser Lys Leu Ala Ser Lys Cys Leu Ala Met 35 40 45 Lys Ala Glu Met Thr His Ser Lys Ser Gly Arg Arg Ile Lys Pro Glu 50 55 60 Gly Ala Ile Gln Asn Asn Asp Gly Leu Tyr Asp Pro Asp Cys Asp Glu 65 70 75 80 Gln Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly Thr Ala Thr Cys Trp 85 90 95 Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp Lys Asp Thr Glu Ile 100 105 110 Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile Ile Ile Glu Leu Lys 115 120 125 His Lys Glu Arg Glu Ser Pro Tyr Asp His Gln Ser Leu Gln Thr Ala 130 135 140 Leu Gln Glu Ala Phe Thr Ser Arg Tyr Lys Leu Asn Gln Lys Phe Ile 145 150 155 160 Lys Asn Ile Met Tyr Glu Asn Asn Val Ile Thr Ile Asp Leu Met Gln 165 170 175 Asn Ser Ser Gln Lys Thr Gln Asp Asp Val Asp Ile Ala Asp Val Ala 180 185 190 Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser Leu Phe His Ser Ser 195 200 205 Lys Ser Met Asp Leu Arg Val Asn Gly Glu Pro Leu Asp Leu Asp Pro 210 215 220 Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys Ala Pro Glu Phe Ser 225 230 235 240 Met Gln Gly Leu Thr 245 <210> 15 <211> 945 <212> DNA <213> Human <220> <221> CDS <222> (1)..(945) <400> 15 atg gcg ccc ccg cag gtc ctc gcg ttc ggg ctt ctg ctt gcc gcg gcg 48 Met Ala Pro Pro Gln Val Leu Ala Phe Gly Leu Leu Leu Ala Ala Ala Ala 1 5 10 15 acg gcg act ttt gcc gca gct cag gaa gaa tgt gtc tgt gaa aac tac 96 Thr Ala Thr Phe Ala Ala Ala Gln Glu Glu Cys Val Cys Glu Asn Tyr 20 25 30 aag ctg gcc gta aac tgc ttt gtg aat aat cgt caa tgc cag tgt 144 Lys Leu Ala Has How To Have An Arg Clean Cys Cys 35 40 45 act tca gtt gtt gca caa act gtc att tgc tca aag ctg gct gcc 192 Thr Ser Val Gly Ala Gln Asn Thr Val Ile Cys Ser Lys Leu Ala Ala Ala 50 55 60 aaa tgt ttg gtg atg aag gca gaa atg aat ggc tca aaa ctt ggg aga 240 Lys Cys Leu Val Met Lys Ala Glu Met Asn Gly Ser Lys Leu Gly Arg 65 70 75 80 aga gca aaa cct gaa ggg gcc cctc cag aac aat gat ggg ctt tat gat 288 Arg Ala Lys Pro Glu Gly Ala Leu Gln Asn Asn Asp Gly Leu Tyr Asp 85 90 95 cct gac tgc gat gag agc ggg ctc ttt aag gcc aag cag tgc aac ggc 336 Pro Asp Cys Asp Glu Ser Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly 100 105 110 acc tcc acg tgc tgg tgt gtg aac act gct ggg gtc aga aga aca gac 384 Thr Ser Thr Cys Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp 115 120 125 aag gac act gaa ata acc tgc tct gag cga gtg aga acc tac tgg atc 432 Lys Asp Thr Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile 130 135 140 atc att gaa cta aaa cac aaa gca aga gaa aaa cct tat gat agt aaa 480 Ile Ile Glu Leu Lys His Lys Ala Arg Glu Lys Pro Tyr Asp Ser Lys 145 150 155 160 agt ttg cgg act gca ctt cag aag gag atc aca acg cgt tat caa ctg 528 Ser Leu Arg Thr Ala Leu Gln Lys Glu Ile Thr Thr Arg Tyr Gln Leu 165 170 175 gat cca aaa ttt atc acg agt att ttg tat gag aat aat gtt atc act 576 Asp Pro Lys Phe Ile Thr Ser Ile Leu Tyr Glu Asn Asn Val Ile Thr 180 185 190 att gat ctg gtt caa aat tct tct caa aaa act cag aat gat gtg gac 624 Ile Asp Leu Val Gln Asn Ser Ser Gln Lys Thr Gln Asn Asp Val Asp 195 200 205 ata gct gat gtg gct tat tat ttt gaa aaa gat gtt aaa ggt gaa tcc 672 Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser 210 215 220 ttg ttt cat tct aag aaa atg gac ctg aca gta aat ggg gaa caa ctg 720 Leu Phe His Ser Lys Lys Met Asp Leu Thr Val Asn Gly Glu Gln Leu 225 230 235 240 gat ctg gat cct ggt caa act tta att tat tat gtt gat gaa aaa gca 768 Asp Leu Asp Pro Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys Ala 245 250 255 cct gaa ttc tca atg cag ggt cta aaa gct ggt gtt att gct gtt att 816 Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly Val Ile Ala Val Ile 260 265 270 gtg gtt gtg gtg ata gca gtt gtt gct gga att gtt gtg ctg gtt att 864 Val Val Val Val Ile Ala Val Val Ala Gly Ile Val Val Leu Val Ile 275 280 285 tcc aga aag aag aga atg gca aag tat gag aag gct gag ata aag gag 912 Ser Arg Lys Lys Arg Met Ala Lys Tyr Glu Lys Ala Glu Ile Lys Glu 290 295 300 atg ggt gag atg cat agg gaa ctc aat gca taa 945 Met Gly Glu Met His Arg Glu Leu Asn Ala 305 310 <210> 16 <211> 314 <212> PRT <213> Human <400> 16 Met Ala Pro Pro Gln Val Leu Ala Phe Gly Leu Leu Leu Ala Ala Ala 1 5 10 15 Thr Ala Thr Phe Ala Ala Ala Gln Glu Glu Cys Val Cys Glu Asn Tyr 20 25 30 Lys Leu Ala Val Asn Cys Phe Val Asn Asn Asn Arg Gln Cys Gln Cys 35 40 45 Thr Ser Val Gly Ala Gln Asn Thr Val Ile Cys Ser Lys Leu Ala Ala 50 55 60 Lys Cys Leu Val Met Lys Ala Glu Met Asn Gly Ser Lys Leu Gly Arg 65 70 75 80 Arg Ala Lys Pro Glu Gly Ala Leu Gln Asn Asn Asp Gly Leu Tyr Asp 85 90 95 Pro Asp Cys Asp Glu Ser Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly 100 105 110 Thr Ser Thr Cys Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp 115 120 125 Lys Asp Thr Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile 130 135 140 Ile Ile Glu Leu Lys His Lys Ala Arg Glu Lys Pro Tyr Asp Ser Lys 145 150 155 160 Ser Leu Arg Thr Ala Leu Gln Lys Glu Ile Thr Thr Arg Tyr Gln Leu 165 170 175 Asp Pro Lys Phe Ile Thr Ser Ile Leu Tyr Glu Asn Asn Val Ile Thr 180 185 190 Ile Asp Leu Val Gln Asn Ser Ser Gln Lys Thr Gln Asn Asp Val Asp 195 200 205 Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser 210 215 220 Leu Phe His Ser Lys Lys Met Asp Leu Thr Val Asn Gly Glu Gln Leu 225 230 235 240 Asp Leu Asp Pro Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys Ala 245 250 255 Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly Val Ile Ala Val Ile 260 265 270 Val Val Val Val Ile Ala Val Val Ala Gly Ile Val Val Leu Val Ile 275 280 285 Ser Arg Lys Lys Arg Met Ala Lys Tyr Glu Lys Ala Glu Ile Lys Glu 290 295 300 Met Gly Glu Met His Arg Glu Leu Asn Ala 305 310 <210> 17 <211> 945 <212> DNA <213> Rhesus monkey <220> <221> CDS <222> (1)..(945) <400> 17 atg gcg ccc ccg cag gtc ctc gcg ttc ggg ctt ctg ctt gcg gcg gcg 48 Met Ala Pro Pro Gln Val Leu Ala Phe Gly Leu Leu Leu Ala Ala Ala 1 5 10 15 act gcg agt ttt gcc gc gct cag aaa gaa tgt gtc tgt gaa aac tac 96 Thr Only Ser Phe Only Only Only Only Gln Lys Glu Cys Val Cys Glu Asn Tyr 20 25 30 aag ctg gcc gc gta aac tgc ttt ttg aat gac aat ggt caa tgc cag tgt 144 Lys Leu Ala Val Asn Cys Phe Leu Asn Asp Asn Gly Gln Cys Gln Cys 35 40 45 act tcg att ggt gca caa act gtc ctt tgc tca aag ctg gct gcc 192 Thr Ser Ile Gly Ala Gln Asn Thr Val Leu Cys Serve Lys Ala Ala Ala 50 55 60 aaa tgt ttg gtg atg aag gca gaa atg aac ggc tca aaa ctt ggg aga 240 Lys Cys Leu Val Met Lys Ala Glu Met Asn Gly Ser Lys Leu Gly Arg 65 70 75 80 aga gcg aaa cct gaa ggg gct cctc cag aac aat gat gggc ctt tac gat 288 Arg Ala Lys Pro Glu Gly Ala Leu Gln Asn Asn Asp Gly Leu Tyr Asp 85 90 95 cct gac tgc gat gag agc ggg ctc ttt aag gcc aag cag tgc aac ggc 336 Pro Asp Cys Asp Glu Ser Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly 100 105 110 acc tcc acg tgc tgg tgt gtg aac act ggg gtc aga act gac 384 Thr Ser Thr Cys Trp Cys Val Asn Thr Ala Gly Val Arg Thr Asp 115 120 125 aag gac act gaa ata acc tgc tct gag cga gtg aga acc tac tgg atc 432 Lys Asp Thr Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile 130 135 140 atc att gaa tta aaa cac aaa gca aga ga aaa cct tat gat gtt caa 480 Ile Ile Glu Lys Pro Tyr Asp Val Gln 145 150 155 160 agt ttg cgg act gca ctt gag gag gcg atc aaa acg cgt tat caa ctg 528 Ser Leu Arg Thr Ala Leu Glu Glu Ala Ile Lys Thr Thr Arg Tyr Gln Leu 165 170 175 gat cca aaa ttt atc aca aat att ttg tat gag gat aat gtt atc act 576 Asp Pro Lys Phe Is Thr Asn Has Tyre Glue Asp Asn Val Is Thr 180 185 190 att gat ctg gtt caa aat tct tct cag aaa act cag aat gat gtg gac 624 Ile Asp Leu Val Gln Asn Ser Ser Gln Lys Thr Gln Asn Asp Val Asp 195 200 205 ata gct gat gtg gct tat tat ttt gaa aaa gat gtt aaa ggt gaa tcc 672 Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser 210 215 220 ttg ttt cat tct aag aaa atg gac ctg aga gta aat ggg gaa caa ctg 720 Leu Phe His Ser Lys Lys Met Asp Leu Arg Val Asn Gly Glu Gln Leu 225 230 235 240 gat ctg gat cct ggt caa act tta att tat tat gtc gat gaa aaa gca 768 Asp Leu Asp Pro Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys Ala 245 250 255 cct gaa ttc tca atg cag ggt cta aaa gct ggt gtt att gct gtt att 816 Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly Val Ile Ala Val Ile 260 265 270 gtg gtt gtg gtg ata gca att gtt gct gga att gtt gtg ctg gtt att 864 Val Val Val Val Ile Ala Ile Val Ala Gly Ile Val Val Leu Val Ile 275 280 285 tcc aga aag aag aga atg gca aag tat gag aag gct gag ata aag gag 912 Ser Arg Lys Lys Arg Met Ala Lys Tyr Glu Lys Ala Glu Ile Lys Glu 290 295 300 atg ggt gag ata cat agg gaa ctc aat gcg taa 945 Met Gly Glu Ile His Arg Glu Leu Asn Ala 305 310 <210> 18 <211> 314 <212> PRT <213> Macaca mulatta <400> 18 Met Ala Pro Pro Gln Val Leu Ala Phe Gly Leu Leu Leu Ala Ala Ala 1 5 10 15 Thr Ala Ser Phe Ala Ala Ala Gln Lys Glu Cys Val Cys Glu Asn Tyr 20 25 30 Lys Leu Ala Val Asn Cys Phe Leu Asn Asp Asn Gly Gln Cys Gln Cys 35 40 45 Thr Ser Ile Gly Ala Gln Asn Thr Val Leu Cys Ser Lys Leu Ala Ala 50 55 60 Lys Cys Leu Val Met Lys Ala Glu Met Asn Gly Ser Lys Leu Gly Arg ​ Arg Ala Lys Pro Glu Gly Ala Leu Gln Asn Asn Asp Gly Leu Tyr Asp 85 90 95 Pro Asp Cys Asp Glu Ser Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly 100 105 110 Thr Ser Thr Cys Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp 115 120 125 Lys Asp Thr Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile 130 135 140 Ile Ile Glu Leu Lys His Lys Ala Arg Glu Lys Pro Tyr Asp Val Gln 145 150 155 160 Ser Leu Arg Thr Ala Leu Glu Glu Ala Ile Lys Thr Arg Tyr Gln Leu 165 170 175 Asp Pro Lys Phe Ile Thr Asn Ile Leu Tyr Glu Asp Asn Val Ile Thr 180 185 190 Ile Asp Leu Val Gln Asn Ser Ser Gln Lys Thr Gln Asn Asp Val Asp 195 200 205 Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser 210 215 220 Leu Phe His Ser Lys Lys Met Asp Leu Arg Val Asn Gly Glu Gln Leu 225 230 235 240 Asp Leu Asp Pro Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys Ala 245 250 255 Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly Val Ile Ala Val Ile 260 265 270 Val Val Val Val Ile Ala Ile Val Ala Gly Ile Val Val Leu Val Ile 275 280 285 Ser Arg Lys Lys Arg Met Ala Lys Tyr Glu Lys Ala Glu Ile Lys Glu 290 295 300 Met Gly Glu Ile His Arg Glu Leu Asn Ala 305 310 <210> 19 <211> 948 <212> DNA <213> Mus musculus <220> <221> CDS <222> (1)..(948) <400> 19 atg gcg ggt ccc cag gcc ctc gcg ttc ggg ctc ctg ctc gcg gtg gtc 48 Met Ala Gly Pro Gln Ala Leu Ala Phe Gly Leu Leu Leu Ala Val Val 1 5 10 15 aca gcg acg ctg gcc gcg gct cag aga gac tgt gtc tgt gac aac tac 96 Thr Ala Thr Leu Ala Ala Ala Gln Arg Asp Cys Val Cys Asp Asn Tyr 20 25 30 aag ctg gca aca agt tgc tct ctg aat gaa tat ggt gaa tgc cag tgt 144 Lys On Thr Cys Ser Ser Leu Asn Glu Tyr Gly Glu Cys Gln Cys 35 40 45 act tcc tat ggt aca cag aat act gtc att tgc tcc aaa ctg gcg tct 192 Thr Ser Tyr Gly Thr Gln Asn Thr Val Ile Cys Ser Lys Leu Ala Ser 50 55 60 aaa tgc ttg gcg atg aaa gca gaa atg act cac agc aag tct ggg agg 240 Lys Cys Leu Ala Met Lys Ala Glu Met Thr His Ser Lys Ser Gly Arg 65 70 75 80 agg ata aag ccc gaa ggg gcg atc cag aac aac gat ggg ctg tac gac 288 Arg Ile Lys Pro Glu Gly Ala Ile Gln Asn Asn Asp Gly Leu Tyr Asp 85 90 95 ccc gac tgc gac gag cag ggg ctc ttc aaa gcc aag cag tgc aac ggc 336 Pro Asp Cys Asp Glu Gln Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly 100 105 110 acc gcc acg tgc tgg tgt gtc aac acc gcc gga gtc cga aga acc gac 384 Thr Ala Thr Cys Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp 115 120 125 aag gac acg gag atc acg tgc tcc gag cgc gtg agg acc tac tgg atc 432 Lys Asp Thr Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile 130 135 140 atc att gaa cta aaa cac aaa gaa aga gaa agc ccc tac gac cat cag 480 Ile Ile Glu Leu Lys His Lys Glu Arg Glu Ser Pro Tyr Asp His Gln 145 150 155 160 agc ttg cag act gcg ctt caa gag gcg ttc aca tct cga tat aag ctg 528 Ser Leu Gln Thr Ala Leu Gln Glu Ala Phe Thr Ser Arg Tyr Lys Leu 165 170 175 aat cag aaa ttt atc aaa aac att atg tat gag aat aat gtt atc acc 576 Asn Gln Lys Phe Ile Lys Asn Ile Met Tyr Glu Asn Asn Val Ile Thr 180 185 190 att gat ctg atg caa aac tct tct cag aaa aca caa gac gac gtg gac 624 Ile Asp Leu Met Gln Asn Ser Ser Gln Lys Thr Gln Asp Asp Val Asp 195 200 205 ata gct gat gtg gct tac tat ttt gaaaaa gat gtg aag ggg gag tcc Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser 210 215 220 ctg ttc cat tct tct aagc atg gac ctg aga gtg aac gga gag ccg 720 Leu Phe His Ser Ser Lys Ser Met Asp Leu Arg Val Asn Gly Glu Pro 225 230 235 240 ctc gat ctg gac ccc ggg cag act ctg att tac tac gtt gat gaa aag 768 Mice Asp Mice Asp Pro Gly Gln Thr Mice Ile Tyr Val Asp Glu Lys 245 250 255 gca ccc gag ttc tcc atg cag ggc ctc acg gcc ggg atc atc gct gtc 816 Pro Glu Phe To Be With Gln Gly Leu Thr Gly Ile Ile Ala Val 260 265 270 att gtg gtg gtg tca tta gca gtc atc gcg ggg att gtt gtc ctg gtt 864 Ile Val Val Val Be Leu Ala Val Ile Ala Gly Ile Val Val Leu Val 275 280 285 ata tct aca agg aaa tca gca aaa tat gag agg gct gag ata aag Ile Ser Thr Arg Lys Lys Ser Ala Lys Tyr Glu Lys Ala Glu Ile Lys 290 295 300 gag atg ggt gag atc cac aga gag ctt aat gcc taa 948 Glu Met Gly Glu Ile His Arg Glu Leu Asn Ala 305 310 315 <210> 20 <211> 315 <212> PRT <213> Mus musculus <400> 20 Met Ala Gly Pro Gln Ala Leu Ala Phe Gly Leu Leu Leu Ala Val Val 1 5 10 15 Thr Ala Thr Leu Ala Ala Ala Gln Arg Asp Cys Val Cys Asp Asn Tyr 20 25 30 Lys Leu Ala Thr Ser Cys Ser Leu Asn Glu Tyr Gly Glu Cys Gln Cys 35 40 45 Thr Ser Tyr Gly Thr Gln Asn Thr Val Ile Cys Ser Lys Leu Ala Ser 50 55 60 Lys Cys Leu Ala Met Lys Ala Glu Met Thr His Ser Lys Ser Gly Arg 65 70 75 80 Arg Ile Lys Pro Glu Gly Ala Ile Gln Asn Asn Asp Gly Leu Tyr Asp 85 90 95 Pro Asp Cys Asp Glu Gln Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly 100 105 110 Thr Ala Thr Cys Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp 115 120 125 Lys Asp Thr Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile 130 135 140 Ile Ile Glu Leu Lys His Lys Glu Arg Glu Ser Pro Tyr Asp His Gln 145 150 155 160 Ser Leu Gln Thr Ala Leu Gln Glu Ala Phe Thr Ser Arg Tyr Lys Leu 165 170 175 Asn Gln Lys Phe Ile Lys Asn Ile Met Tyr Glu Asn Asn Val Ile Thr 180 185 190 Ile Asp Leu Met Gln Asn Ser Ser Gln Lys Thr Gln Asp Asp Val Asp 195 200 205 Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser 210 215 220 Leu Phe His Ser Ser Lys Ser Met Asp Leu Arg Val Asn Gly Glu Pro 225 230 235 240 Leu Asp Leu Asp Pro Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys 245 250 255 Ala Pro Glu Phe Ser Met Gln Gly Leu Thr Ala Gly Ile Ile Ala Val 260 265 270 Ile Val Val Val Ser Leu Ala Val Ile Ala Gly Ile Val Val Leu Val 275 280 285 Ile Ser Thr Arg Lys Lys Ser Ala Lys Tyr Glu Lys Ala Glu Ile Lys 290 295 300 Glu Met Gly Glu Ile His Arg Glu Leu Asn Ala 305 310 315 <210> 21 <211> 321 <212> DNA <213> artificial <220> <223> L6 DNA <220> <221> CDS <222> (1)..(321) <400> 21 gaa ata gtg ttg acg cag tct cca gcc acc ctg tct ttg tct cca ggg 48 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agc agc tac 96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 tat gat gca tcc aac agg gcc act ggc atc cca gcc agg ttc agt ggc 192 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 agt ggg tct ggg aca gac ttc act ctc acc atc agc agc cta gag cct 240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 gaa gat ttt gca gtt tat tac tgt cag cag cgt agc aac tgg cct ccg 288 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 acg ttc ggc caa ggg acc aag gtg gaa atc aaa 321 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 22 <211> 107 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 22 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <21^0> 23 <21^1> 11 <21^2> PRT <21^3> Artificial <22^0> <22^3> L6 CDR1 Amino Acid <40^0> 23 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <21^0> 24 <21^1> 7 <21^2> PRT <21^3> Artificial <22^0> It should be noted that there seems to be an inconsistent tag format in the original text (e.g., <210> vs <21^0>). The translation is done based on the original text as provided while trying to maintain the integrity of the tags.<223> L6 LCDR2 amino acids <400> twenty four Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 25 <211> 9 <212> PRT <213> Artificial <220> <223> L6 LCDR3 amino acids <400> 25 Gln Gln Arg Ser Asn Trp Pro Pro Thr 1 5 <210> 26 <211> 381 <212> DNA <213> Human <220> <221> CDS <222> (1)..(381) <400> 26 cag gtg cag cta cag cag tgg ggc gca gga ctg ttg aag cct tcg gag 48 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 acc ctg tcc ctc acc tgc gct gtc tat ggt ggg tcc ttc agt ggt tac 96 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 tac tgg agc tgg atc cgc cag ccc ccc ggg aag ggg ctg gag tgg att 144 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 ggg gaa atc aat cat ggt gga atc acc cac tac aat ccg tcc ctc aag 192 Gly Glu Ile Asn His Gly Gly Ile Thr His Tyr Asn Pro Ser Leu Lys 50 55 60 agt cga gtc acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg 240 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 aag ctg agt tct gtg acc gcc gcg gac acg gct gtg tat tac tgt gcg 288 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 aga gat aac tac tat ggt tcg ggg agt tat tat aag tac gac tac tac 336 Arg Asp Asn Tyr Tyr Gly Ser Gly Ser Tyr Tyr Lys Tyr Asp Tyr Tyr 100 105 110 ggt atg gac gtc tgg ggc caa ggg acc acg gtc acc gtt tcc tca 381 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 27 <211> 127 <212> PRT <213> Human <400> 27 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Gly Gly Ile Thr His Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Asn Tyr Tyr Gly Ser Gly Ser Tyr Tyr Lys Tyr Asp Tyr Tyr 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <s210> 28 <s211> 5 <s212> PRT <s213> Artificial <s220> <s223> R1066 HCDR1 Amino Acids <400> 28 Gly Tyr Tyr Trp Ser 1 5 <210> 29 <211> 16 <212> PRT <213> Artificial <220> <223> R1066 HCDR2 amino acid <400> 29 Glu Ile Asn His Gly Gly Ile Thr His Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 30 <211> 19 <212> PRT <213> Artificial <220> <223> R1066 HCDR3 amino acid <400> 30 Asp Asn Tyr Tyr Gly Ser Gly Ser Tyr Tyr Lys Tyr Asp Tyr Tyr Gly 1 5 10 15 Met Asp Val <210> 31 <211> 354 <212> DNA <213> Human <220> <221> CDS <222> (1)..(354) <400> 31 gaa gtg cag ctt gtg cag tcc ggg gct gaa gtg aag aag ccc ggg gaa 48 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 tcc ctg aag atc tcc tgt aag ggc tcc ggc tac tcc ttc atc atc tac 96 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ile Ile Tyr 20 25 30 tgg atc ggc tgg gtt cgc cag atg cct ggg aag ggg ctg gag tgg atg 144 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 ggg atc atc tat ccc aat gat gcc gat act cgc tac tcc ccc tcc ttc 192 Gly Ile Ile Tyr Pro Asn Asp Ala Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 cag ggg cag gtg act atc tcc gcc gac aag tcc atc tcc act gcc tat 240 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 ctg cag tgg tcc tcc ctg aag gcc tcc gat act gcc atg tac tac tgc 288 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 gtg cgc ctg ggg act cgc gat tgg ttc gat cct tgg ggg cag ggg act 336 Val Arg Leu Gly Thr Arg Asp Trp Phe Asp Pro Trp Gly Gln Gly Thr 100 105 110 ctt gtg act gtg tcc tcc 354 Leu Val Thr Val Ser Ser 115 <210> 32 <211> 118 <212> PRT <213> Human <400> 32 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ile Ile Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Asn Asp Ala Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Val Arg Leu Gly Thr Arg Asp Trp Phe Asp Pro Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 33 <211> 5 <212> PRT It should be noted that there is a possible error in the original text where "0001416" in line 29 should probably be "0001416". This has been corrected in the translation as much as possible while maintaining the integrity of the original text.<213> Artificial <220> <223> R1090S55A HCDR1 amino acid <400> 33 Ile Tyr Trp Ile Gly 1 5 <210> 34 <211> 17 <212> PRT <213> Artificial <220> <223> R1090S55A HCDR2 amino acid <400> 34 Ile Ile Tyr Pro Asn Asp Ala Asp Thr Arg Tyr Ser Pro Ser Phe Gln 1 5 10 15 Gly <210> 35 <211> 9 <212> PRT <213> Artificial <220> <223> R1090S55A HCDR3 amino acid <400> 35 Leu Gly Thr Arg Asp Trp Phe Asp Pro 1 5 <210> 36 <211> 360 <212> DNA <213> Human <220> <221> CDS <222> (1)..(360) <400> 36 gag gtc cag ctg gta cag tct ggg gct gag gtg aag aag cct ggg gcc 48 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aag gtt tcc tgc aag gct tct gga tac tcc ttc act cgc tat 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Arg Tyr 20 25 30 att atg cat tgg gtg cgc cag gcc ccc gga caa agg ctt gag tgg atg 144 With His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 gga tgg atc aac gct ggc aat ggt aac aca aaa tat tca cag aag ttc Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Served as Gln Lys Phe 50 55 60 cag ggc aga gtc acc att acc agg gac aca tcc gcg aac aca gcc tac 240 Gln Gly Arg Val Thr Has Thr Arg Asp Served Only Asn Thr Only Tyr 65 70 75 80 atg gag ctg agc agc ctg aga tct gaa gac acg gct gta tac tac tgt 288 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg aga gat gaa cca gtg gct ggt tct ttc ttt gac tac tgg gggc cag 336 Ala Arg Asp Glu Pro Val Ala Gly Ser Phe Phe Asp Tyr Trp Gly Gln 100 105 110 gga acc ctg gtc acc gtc tcc tca 360 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 37 <211> 120 <212> PRT <213> Human <400> 37 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Arg Tyr 20 25 30 Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Glu Pro Val Ala Gly Ser Phe Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 38 <211> 5 <212> PRT <213> Artificial <220> <223> R2089 HCDR1 amino acid <400> 38 Arg Tyr Ile Met His 1 5 <210> 39 <211> 17 <212> PRT <213> Artificial <220> <223> R2089 HCDR2 amino acid <400> 39 Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe Gln 1 5 10 15 Gly <210> 40 <211> 11 <212> PRT <213> Artificial <220> <223> R2089 HCDR3 amino acid <400> 40 Asp Glu Pro Val Ala Gly Ser Phe Phe Asp Tyr 1 5 10 <210> 41 <211> 384 <212> DNA <213> Human <220> <221> CDS <222> (1)..(384) <400> 41 cag gtg cag cta cag cag tgg ggc gca gga ctg ttg aag cct tcg gag 48 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 acc ctg tcc ctc acc tgc gct gtc tat ggt ggg tcc ttc agt ggt tac 96 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 tac tgg agc tgg atc cgc cag tcc cca ggg aag gga ctg gag tgg att 144 Tyr Trp Ser Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 ggg gaa atc aat cat ggt gga atc acc aac tac aat ccg tcc ctc aag 192 Gly Glu Ile Asn His Gly Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 agt cga gtc acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg 240 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 aag ctg ggc tct gtg acc gcc gcg gac acg gct gtg tat tac tgt gcg 288 Lys Leu Gly Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 aga gat aat tac tat ggt tcg ggg agt aat gat ggg gtc tac cag tac 336 Arg Asp Asn Tyr Tyr Gly Ser Gly Ser Asn Asp Gly Val Tyr Gln Tyr 100 105 110 tac ggt ata gac gtc tgg ggc caa ggg acc acg gtc acc gtc tcc tca 384 Tyr Gly Ile Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 42 <211> 128 <212> PRT <213> Human <400> 42 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Gly Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Gly Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Asn Tyr Tyr Gly Ser Gly Ser Asn Asp Gly Val Tyr Gln Tyr 100 105 110 Tyr Gly Ile Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 43 <211> 5 <212> PRT <213> Artificial <220> <223> R2178 HCDR1 amino acid <400> 43 Gly Tyr Tyr Trp Ser 1 5 <210> 44 <211> 16 <212> PRT <213> Artificial <220> <223> R2178 HCDR2 amino acid <400> 44 Glu Ile Asn His Gly Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 45 <211> 20 <212> PRT <213> Artificial <220> <223> R2178 HCDR3 amino acid <400> 45 Asp Asn Tyr Tyr Gly Ser Gly Ser Asn Asp Gly Val Tyr Gln Tyr Tyr 1 5 10 15 Gly Ile Asp Val 20 <210> 46 <211> 378 <212> DNA <213> Human <220> <221> CDS <222> (1)..(378) <400> 46 cag gtg cag ctg gtg cag tct ggg gct gag gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aag gtc tcc tgc aag gct tct gga tac acc ttc acc ggc tac 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 tat atg cac tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atg 144 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 gga tgg atc aac cct gac agt ggt ggc aca aac tat gca cag aag ttt 192 Gly Trp Ile Asn Pro Asp Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 cag ggc agg gtc acc atg acc agg gac acg tcc atc agc aca gcc tac 240 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 atg gag ctg aac agg ctg aga tct gac gac acg gcc gtg tat tac tgt 288 Met Glu Leu Asn Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg aga gat cag ccc cta gga tat tgt act aat ggt gta tgc tcc tac 336 Ala Arg Asp Gln Pro Leu Gly Tyr Cys Thr Asn Gly Val Cys Ser Tyr 100 105 110 ttt gac tac tgg ggc cag gga acc ctg gtc acc gtc tcc tca 378 Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 47 <211> 126 <212> PRT <213> Human <400> 47 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asp Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Asn Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gln Pro Leu Gly Tyr Cys Thr Asn Gly Val Cys Ser Tyr 100 105 110 Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 48 <211> 321 <212> DNA <213> Human <220> <221> CDS <222> (1)..(321) <400> 48 gac atc cag atg acc cag tct cca tct tcc gtg tct gca tct gta gga 48 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 gac aga gtc acc atc act tgt cgg gcg agt cag ggt att tac agc tgg 96 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Tyr Ser Trp 20 25 30 tta gcc tgg tat cag cag aaa cca ggg aaa gcc cct aac ctc ctg atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 tat act gca tcc act tta caa agt ggg gtc cca tca agg ttc agc ggc 192 Tyr Thr Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 agt gga tct ggg aca gat ttc act ctc acc atc agc agc ctg caa cct 240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 gaa gat ttt gca act tac tat tgt caa cag gct aac att ttc ccg ctc 288 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ile Phe Pro Leu 85 90 95 act ttc ggc gga ggg acc aag gtc cag atc aaa 321 Thr Phe Gly Gly Gly Thr Lys Val Gln Ile Lys 100 105 <210> 49 <211> 107 <212> PRT <213> Human <400> 49 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Tyr Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Thr Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ile Phe Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Gln Ile Lys 100 105 <210> 50 <211> 354 <212> DNA <213> Human <220> <221> CDS <222> (1)..(354) <400> 50 cag gtg cag ctg gtg cag tct ggg gct gag gtg aaa aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aag gtt tcc tgc aag gca tct gga tac acc ttc acc aac tac 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 tat atg cac tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atg 144 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 gga ata atc aac cct aat agt ggt agc aca ggc tac gca cag aag ttc 192 Gly Ile Ile Asn Pro Asn Ser Gly Ser Thr Gly Tyr Ala Gln Lys Phe 50 55 60 cag ggc aga gtc acc atg acc agg gac acg tcc acg agc aca gtc tac 240 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 atg gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt 288 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg aga tgg ggt tcg ggg agg ccc atg gac gtc tgg ggc caa ggg acc 336 Ala Arg Trp Gly Ser Gly Arg Pro Met Asp Val Trp Gly Gln Gly Thr 100 105 110 acg gtc acc gtc tcc tca 354 Thr Val Thr Val Ser Ser 115 <210> 51 <211> 118 <212> PRT <213> Human <400> 51 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Asn Ser Gly Ser Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Ser Gly Arg Pro Met Asp Val Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 52 <211> 5 <212> PRT <213> Artificial <220> <223> Ep59 HCDR1 amino acid <400> 52 Asn Tyr Tyr Met His 1 5 <210> 53 <211> 17 <212> PRT <213> Artificial <220> <223> Ep59 HCDR2 amino acids <400> 53 Ile Ile Asn Pro Asn Ser Gly Ser Thr Gly Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 54 <211> 9 <212> PRT <213> Artificial <220> <223> Ep59 HCDR3 amino acids <400> 54 Trp Gly Ser Gly Arg Pro Met Asp Val 1 5 <210> 55 <211> 360 <212> DNA <213> Human <220> <221> CDS <222> (1)..(360) <400> 55 gag gtg cag ctg gtg cag tct ggc gct gag gtg aag aag cct ggg gcc 48 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aag gtt tcc tgc aag gca tct gga ttc acc ttc agc agt tac 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 tat atg cac tgg gtg cga cag gcc cct gga caa ggt ctt gaa tgg atg 144 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 gga ata gtc aac cct act ggt ggt gcc aca agc tac gca cag aag ttc 192 Gly Ile Val Asn Pro Thr Gly Gly Ala Thr Ser Tyr Ala Gln Lys Phe 50 55 60 cag ggg aga gtc acc atg acc agg gac acg tcc acg agc aca gtc tac 240 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 7� 75 80 atg gag ctg agc agt ctg aga tct gag gac acg gcc gtg tat tac tgt 288 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg aaa cat ggc tac ggt gcc tcg acc ctt ttt gac tcc tgg ggc cag 336 Ala Lys His Gly Tyr Gly Ala Ser Thr Leu Phe Asp Ser Trp Gly Gln 100 105 110 gga acc ctg gtc acc gtc tcc tca 360 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 56 <211> 120 <212> PRT <213> Human <400> 56 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Val Asn Pro Thr Gly Gly Ala Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys His Gly Tyr Gly Ala Ser Thr Leu Phe Asp Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 57 <211> 5 <212> PRT <213> Artificial <220> <223> Ep203 HCDR1 amino acid <400> 57 Ser Tyr Tyr Met His 1 5 <210> 58 <211> 17 <212> PRT <213> Artificial <220> <223> Ep203 HCDR2 amino acid <400> 58 Ile Val Asn Pro Thr Gly Gly Ala Thr Ser Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 59 <211> 11 <212> PRT <213> Artificial <220> <223> Ep203 HCDR3 amino acid <400> 59 His Gly Tyr Gly Ala Ser Thr Leu Phe Asp Ser 1 5 10 <210> 60 <211> 360 <212> DNA <213> Human <220> <221> CDS <222> (1)..(360) <400> 60 gaa gtg cag ctg gtg cag tcc ggg gct gag gtg aag aag cct ggg gcc 48 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aag gtg tcc tgc aag gca tct gga tac acc ttc acc agc aac 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn 20 25 30 tat atg cac tgg gtg cgc cag gcc cct gga caa ggg ctt gag tgg atg 144 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 gga ata atc aac cct agt ggt ggt gcc aca agc tac gca cag aag ttc 192 Gly Ile Ile Asn Pro Ser Gly Gly Ala Thr Ser Tyr Ala Gln Lys Phe 50 55 60 cag ggc aga gtc acc atg acc agg gac acg tcc acg agc aca gtc tac 240 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 atg gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt 288 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg agc ggt gga tac tat ggt tcg gtg att atg gac gtc tgg ggc caa 336 Ala Ser Gly Gly Tyr Tyr Gly Ser Val Ile Met Asp Val Trp Gly Gln 100 105 110 ggg acc ctg gtc acc gtc tcc tca 360 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 61 <211> 120 <212> PRT <213> humans <400> 61 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ala Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Gly Gly Tyr Tyr Gly Ser Val Ile Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 62 <211> 5 <212> PRT <213> Artificial <220> <223> Epc051 HCDR1 Amino Acids <400> 62 Ser Asn Tyr Met His 1 5 <210> 63 <211> 17 <212> PRT <213> Artificial <220> <223> Epc051 HCDR2 Amino Acids <400> 63 Ile Ile Asn Pro Ser Gly Gly Ala Thr Ser Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 64 <211> 11 <212> PRT <213> Artificial <220> <223> Epc051 HCDR3 Amino Acids <400> 64 Gly Gly Tyr Tyr Gly Ser Val Ile Met Asp Val 1 5 10 <210> 65 <211> 354 <212> DNA <213> Human <220> <221> CDS <222> (1)..(354) <400> 65 gaa gtg cag ctg gtg cag tcc ggg act gag gtg aaa aag cct ggg gcc 48 Glu Val Gln Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aag gtt tcc tgc agg gca tct gga aac acc ttc acc aac tac 96 Ser Val Lys Val Ser Cys Arg Ala Ser Gly Asn Thr Phe Thr Asn Tyr 20 25 30 tat ata cac tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atg 144 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 gga ata atc aac cct agt ggt ggt gcc aca aac tac gca cag att ctc 192 Gly Ile Ile Asn Pro Ser Gly Gly Ala Thr Asn Tyr Ala Gln Ile Leu 50 55 60 cag ggc aga gtc acc atg acc agg gac acg tcc acg agc acc gtc tac 240 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 atg gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt 288 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg ggg ggg gga tca gca gcc acc ttt gcc tac tgg ggc cag gga acc 336 Ala Gly Gly Gly Ser Ala Ala Thr Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 ctg gtc acc gtc tcc tca 354 Leu Val Thr Val Ser Ser 115 <210> 66 <211> 118 <212> PRT <213> Human <400> 66 Glu Val Gln Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Arg Ala Ser Gly Asn Thr Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ala Thr Asn Tyr Ala Gln Ile Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Gly Gly Ser Ala Ala Thr Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 67 <211> 5 <212> PRT <213> Artificial <220> <223> Epc112 HCDR1 Amino Acids <400> 67 Asn Tyr Tyr Ile His 1 5 <210> 68 <211> 17 <212> PRT <213> Artificial <220> <223> Epc112 HCDR2 Amino Acids <400> 68 Ile Ile Asn Pro Ser Gly Gly Ala Thr Asn Tyr Ala Gln Ile Leu Gln 1 5 10 15 Gly <210> 69 <211> 9 <212> PRT <213> Artificial <220> <223> Epc112 HCDR3 Amino Acids <400> 69 Gly Gly Ser Ala Ala Thr Phe Ala Tyr 1 5 <210> 70 <211> 348 <212> DNA <213> Artificial <220> <223> 3622W94 VH DNA <220> <221> CDS <222> (1)..(348) <400> 70 cag gta cag cta gtg caa tca ggg cct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtt tcc tgc aag gct tct ggc tac acc ttc acc aac tat 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 gga atg aac tgg gta agg cag gcg cct gga cag ggg ctt gag tgg atg 144 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 ggg tgg ata aac acc tac act gga gag cca aca tat ggt gaa gat ttc 192 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Gly Glu Asp Phe 50 55 60 aag gga cgg ttt gca ttc tct cta gac aca tcc gcc agc aca gcc tat 240 Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 atg gag ctc agc tcg ctg aga tcc gag gac act gca gtc tat ttc tgt 288 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 gcg aga ttt ggt aac tac gta gac tac tgg ggt caa gga tca cta gtc 336 Ala Arg Phe Gly Asn Tyr Val Asp Tyr Trp Gly Gln Gly Ser Leu Val 100 105 110 act gtc tcc tca 348 Thr Val Ser Ser 115 <210> 71 <211> 116 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 71 Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr[[ID=1*6]] 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Gly Glu Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Phe Gly Asn Tyr Val Asp Tyr Trp Gly Gln Gly Ser Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 72 <211> 336 <212> DNA <213> Artificial <220> <223> 3622W94 VL DNA <220> <221> CDS <222> (1)..(336) <400> 72 gat att gtg atg act cag tct cca ctc tcc ctg ccc gtc acc cct gga 48 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 gag ccg gcc tcc atc tcc tgt agg tct agt aag aat ctc ctg cat agt 96 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Asn Leu Leu His Ser 20 25 30 aat ggc atc act tat ttg tat tgg tac ctg cag aag cca ggg cag tct 144 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 cca cag ctc ctg atc tat cag atg tcc aac ctt gcc tca ggg gtc cct 192 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 gac agg ttc agt agc agt gga tca ggc aca gat ttt aca ctg aaa atc 240 Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 agc aga gtg gag gct gag gat gtt ggg gtt tat tac tgt gct caa aat 288 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95 cta gag att cct cgg acg ttc ggc caa ggg acc aag gtg gag atc aaa 336 Leu Glu Ile Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 73 <211> 112 <212> PRT <213> Artificial <220> <223> synthetic construction <400> 73 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Asn Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95 Leu Glu Ile Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 74 <211> 294 <212> DNA <213> artificial <220> <223> IgG4 CH1 DNA <220> <221> CDS <222> (1)..(294)<00N2159><400> 74 gct agc acc aaa gga cct tct gta ttt cct ctt gcg cca tgc tct cgc 48 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 tct acg tca gaa tca act gcc gct ctg ggg tgc ctg gtt aaa gac tac 96 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 ttc ccg gag cct gtg aca gtg agt tgg aac tcc ggc gcc ctg aca tca 144 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 gga gtg cat aca ttt ccc gcc gtg ctt cag agc agc gga ctt tat agc 192 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 ctc agc agt gtg gtg acc gtg cca tct tcc agc ctg ggg acc aag acc 240 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 tac acc tgt aac gtg gac cac aaa ccc agc aac acc aag gtt gat aag 288 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 agg gtc 294 Arg Val <210> 75 <211> 98 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 75 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val <210> 76 <211> 981 <212> DNA <213> Artificial <220> <223> IgG4PE R409K DNA <220> <221> CDS <222> (1)..(981)<s <400> 76 gct agc acc aag ggg cca tcc gtc ttc ccc ctg gcg ccc tgc tcc agg 48 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 agc acc tcc gag agc aca gcc gcc ctg ggc tgc ctg gtc aag gac tac 96 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc 144 ]>Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc 192 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acg aag acc 240 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 tac acc tgc aac gta gat cac aag ccc agc aac acc aag gtg gac aag 288 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 aga gtt gag tcc aaa tat ggt ccc cca tgc cca cca tgc cca gca cct 336 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 gag ttc gag ggg gga cca tca gtc ttc ctg ttc ccc cca aaa ccc aag 384 Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 gac act ctc atg atc tcc cgg acc cct gag gtc acg tgc gtg gtg gtg 432 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 gac gtg agc cag gaa gac ccc gag gtc cag ttc aac tgg tac gtg gat 480 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag ttc 528 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac 576 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 tgg ctg aac ggc aag gag tac aag tgc aag gtc tcc aac aaa ggc ctc 624 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 ccg tcc tcc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga 672 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 gag cca cag gtg tac acc ctg ccc cca tcc cag gag gag atg acc aag 720 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tac ccc agc gac 768 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag 816 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc 864 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 aag cta acc gtg gac aag agc agg tgg cag gag ggg aat gtc ttc tca 912 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 tgc tcc gtg atg cat gag gct ctg cac aac cac tac aca cag aag agc 960 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 ctc tcc ctg tct ctg ggt aaa 981 Leu Ser Leu Ser Leu Gly Lys 325 <210> 77 <211> 327 <212> PRT <213> Artificial <220> <223> synthetic construction <400> 77 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 78 <211> 297 <212> DNA <213> Artificial <220> <223> IgG4PE R409K DNA includes a stop codon <400> 78 gctagcacca aaggaccttc tgtatttcct cttgcgccat gctctcgctc tacgtcagaa 60 tcaactgccg ctctggggtg cctggttaaa gactacttcc cggagcctgt gacagtgagt 120 tggaactccg gcgccctgac atcaggagtg catacatttc ccgccgtgct tcagagcagc 180 ggactttata gcctcagcag tgtggtgacc gtgccatctt ccagcctggg gaccaagacc 240 tacacctgta acgtggacca caaacccagc aacaccaagg ttgataagag ggtctga 297 <210> 79 <211> 993 <212> DNA <213> artificial <220> <223> Heterologous IgG BsAbCHa <220> <221> CDS <222> (1)..(993) <400> 79 [[ID=2,4]]gct agc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag 48 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac tac 96 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc 144 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser[[ID=|39]] 35 40 |45 ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc 192 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc 240 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 tac atc tgc aac gtg aat cac aag ccc agc aac acc aag gtg gac aag 288 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 aaa gtt gag ccc aaa tct tgt gac aaa act cac aca tgc cca ccg tgc 336 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 cca gca cct gaa gcc gcg ggg gga ccg tca gtc ttc ctc ttc ccc cca 384 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc 432 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg 480 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag 528 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg 576 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc aac 624 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa gga 672 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 cag cca cga gaa cca gaa gta gca acc ttc cca cct agt cgt gat gaa 720 Gln Pro Arg Glu Pro Glu Val Ala Thr Phe Pro Pro Ser Arg Asp Glu 225 230 235 240 ttg acc aaa aac caa gtt acc ctg gta tgt ctt gtg aca gga ttc tac 768 Leu Thr Lys Asn Gln Val Thr Leu Val Cys Leu Val Thr Gly Phe Tyr 245 250 255 ccc tca gat att gca gtt gaa tgg gag agc aat ggt caa cct gaa aac 816 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 aac tac aaa act gat cct cct ctc ttg gaa tca gac ggg agt ttc gca 864 Asn Tyr Lys Thr Asp Pro Pro Leu Leu Glu Ser Asp Gly Ser Phe Ala 275 280 285 ctt agt agc cgt ctg cgc gta gac aaa tcc cgg tgg caa cag ggg aac 912 Leu Ser Ser Arg Leu Arg Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 gta ttc tcc tgc tcc gtt atg cac gag gct ctt cac aat cat tat aca 960 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 cag aag tca ctt tct ctt tcc cct ggg aaa tga 993 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 80 <211> 330 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 80<CENSORED>Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Glu Val Ala Thr Phe Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Thr Leu Val Cys Leu Val Thr Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Asp Pro Pro Leu Leu Glu Ser Asp Gly Ser Phe Ala 275 280 285 Leu Ser Ser Arg Leu Arg Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys[[ID=I4]] 325 330 <210> 81 <211> 993 <212> DNA ^<213> Artificial <220> <223> Heterologous IgG BsAb CHb <220> <22l> CDS <222> (1)..(993) <400> 81 gct agc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag 48 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac tac 96 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc 144 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc 192 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc 240 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 tac atc tgc aac gtg aat cac aag ccc agc aac acc aag gtg gac aag 288 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 aaa gtt gag ccc aaa tct tgt gac aaa act cac aca tgc cca ccg tgc 336 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 cca gcc cct gaa gca gcc gga gga cct agc gta ttc ctt ttc ccc ccc 384 Pro Ala Pro Glu Ala Ala Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 aaa cca aag gac aca ttg atg ata agc cgt acc cct gaa gtt act tgc 432 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 gtt gtg gta gat gta tcc cac gaa gat ccc gag gta cag ttc aaa tgg 480 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp 145 150 155 160 tac gtt gac ggt gta gag gtt cat aat gct aag act aag cca cgc gag 528 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 gaa caa tat aat tcc tc agg gtt gtc tc gtc tg act gtc ctc 576 Glu Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 cac cag gac tgg ctg aac gga aag gag tac aag tgc aag gtg agc aat 624 His Gln Asp Trp Leu Asn Gly Gly Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 aaa gca ctc cca gct cct att gaa aag acc atc tcc acc acc aag aag gga 672 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly 210 215 220 cag ccc cga gag cca gct gct gtc tat acc ctt cca cca agt cgt gaa gaa 720 Gln Pro Arg Glu Pro Ala Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 atg acc aag aac cag gta aag ctc gtg tgt ttg gtg acc ggg ttt tac 768 Met Thr Asn Gln Val Lys Leu Val Cys Leu Val Thr Gly Phe Tyr 245 250 255 cct agt gat ata gca gtg gaa tgg gaa tct agc ggg caa cct gag at 816 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn 260 265 270 aac tat tat aca act cca ccc atg ttg gac tca gac ggt tct ttt agc Asn Tyr Tyr Thr Thr Pro Met Leu Asp Ser Asp Gly Ser Phe Ser 275 280 285 ctt gtc tcc tgg ctt aat gta gat aag agt cgc tgg cag caa ggg aat 912 Leu Val Ser Trp Leu Asn Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 to ttc tca tgc agt gtc atg cac gag gct ctg cac aac agg ttt aca 960 Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr 305 310 315 320 caa aaa agc ctg tcc ctg agt ccc ggc aag tga 993 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 82 <211> 330 <212> PRT <213> Artificial <220> <223> synthetic construction <400> 82 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly 210 215 220 Gln Pro Arg Glu Pro Ala Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Lys Leu Val Cys Leu Val Thr Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Tyr Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Ser 275 280 285 Leu Val Ser Trp Leu Asn Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 83 <211> 324 <212> DNA <213> Artificial <220> <223> Fab BsAb CH1-tag '<220> <221> CDS <222> (1)..(324) <400> 83 gct agc acc aag ggg cca tcc gtc ttc ccc ctg gcg ccc tgc tcc agg 48 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 agc acc tcc gag agc aca gcc gcc ctg ggc tgc ctg gtc aag gac tac 96 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc 144 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc 192 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acg aag acc 240 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 tac acc tgc aac gta gat cac aag ccc agc aac acc aag gtg gac aag 288 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 aga gtt gag tcc tac aaa gac gac gac gac aaa tga 324 Arg Val Glu Ser Tyr Lys Asp Asp Asp Asp Lys 100 105 <210> 84 <211> 107 <212> PRT <213> Artificial <220> <223> synthetic construction <400> 84 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Tyr Lys Asp Asp Asp Asp Lys 100 105 <210> 85 <211> 354 <212> DNA <213> artificial <220> <223> F(ab')2 BsAb CH1-tag <220> <221> CDS <222> (1)..(354) <400> 85 gct agc acc aag ggg cca tcc gtc ttc ccc ctg gcg ccc tgc tcc agg 48 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 agc acc tcc gag agc aca gcc gcc ctg ggc tgc ctg gtc aag gac tac 96 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc 144 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc 192 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acg aag acc 240 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 tac acc tgc aac gta gat cac aag ccc agc aac acc aag gtg gac aag 288 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 aga gtt gag tcc aaa tat ggt ccc cca tgc cca cca tgc ccc tac aaa 336 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Tyr Lys 100 105 110 gac gac gac gac aaa tga 354 Asp Asp Asp Asp Lys 115 <210> 86 <211> 117 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 86 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Tyr Lys 100 105 110 Asp Asp Asp Asp Lys 115 BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 An example of the structure of the bispecific antibody of the present invention is shown. Figure 1 (A) represents an N-terminal bispecific antibody. Figure 1 (B) shows a C-terminal bispecific antibody.

[0108] Figure 2 (A) shows the results of evaluating the expression of CD40 and EpCAM in Ramos cells by flow cytometry. Figure 2 (B) shows the results of evaluating the expression of CD40 and EpCAM in Colo205 cells by flow cytometry. Figure 2 (C) shows the results of evaluating the expression of CD40 and EpCAM in HEK293 cells using flow cytometry. Figure 2 (D) shows the results of flow cytometric evaluation of CD40 and EpCAM expression in human EpCAM / HEK293 cells. The vertical axis represents cell number, and the horizontal axis represents fluorescence intensity. The dashed line indicates binding by the anti-CD40 antibody 21.4.1, the dotted line indicates binding by the anti-EpCAM antibody 3622W94, and the hollow line indicates binding by the control antibody, an anti-DNP antibody.

[0109] Figure 3 (A) and Figure 3(B) shows the results of evaluating the CD40 signal-inducing activity of anti-CD40 monoclonal antibodies and the induction of CD95 expression on Ramos cells as indicators. The vertical axis represents fluorescence intensity, indicating the binding of anti-CD95 antibodies to Ramos cells when each antibody was added at 10, 1, or 0.1 μg / mL. For comparison, anti-DNP antibody was used as a negative control antibody, and 21.4.1 was used as an anti-CD40 agonist antibody.

[0110] Figure 4 This figure shows the CD40 signal-inducing activity of CD40-EpCAM bispecific antibodies on Ramos cells co-cultured with HEK293 cells. The vertical axis represents fluorescence intensity, indicating the binding of anti-CD95 antibodies to Ramos cells when each antibody was added at 10, 1, or 0.1 μg / mL. For comparison, anti-DNP antibody was used as a negative control antibody, 21.4.1 was used as an anti-CD40 agonist antibody, and 3622W94 was used as an anti-EpCAM antibody.

[0111] Figure 5 This figure shows the CD40 signal-inducing activity of CD40-EpCAM bispecific antibodies on Ramos cells co-cultured with human EpCAM / HEK293 cells. The vertical axis represents fluorescence intensity, indicating the binding of anti-CD95 antibodies to Ramos cells when 10, 1, or 0.1 μg / mL of each antibody was added. For comparison, anti-DNP antibody was used as a negative control antibody, 21.4.1 was used as an anti-CD40 agonist antibody, and 3622W94 was used as an anti-EpCAM antibody.

[0112] Figure 6 This figure shows the CD40 signal-inducing activity of CD40-EpCAM bispecific antibodies on Ramos cells co-cultured with Colo205 cells. The vertical axis represents fluorescence intensity, indicating the binding of anti-CD95 antibodies to Ramos cells when each antibody was added at 10, 1, or 0.1 μg / mL. For comparison, anti-DNP antibody was used as a negative control antibody, 21.4.1 was used as an anti-CD40 agonist antibody, and 3622W94 was used as an anti-EpCAM antibody.

[0113] Figure 7 (A) is a schematic diagram illustrating the effect of the CD40-EpCAM bispecific antibody on CD40-positive cells in the presence of EpCAM-positive cells. When the CD40-EpCAM bispecific antibody binds to both EpCAM-positive and CD40-positive cells, it induces CD40 signals on CD40-positive cells. Consequently, it induces CD95 expression on CD40-positive cells. Figure 7(B) is a schematic diagram illustrating the effect of the CD40-EpCAM bispecific antibody on CD40-positive cells in the absence of EpCAM-positive cells. The CD40-EpCAM bispecific antibody does not induce CD40 signals when bound only to CD40-positive cells.

[0114] Figure 8 (A) The results of fluorescent immunostaining of Ramos cells and Colo205 cells using HER2 antibody labeled with Alexa647. Figure 8 (B) shows the results of fluorescent immunostaining of Ramos cells and Colo205 cells using CtR1090S55A-Ep203 fluorescently labeled with Alexa488. Figure 8 (C) shows their overlap. The arrows indicate the positions where strong fluorescence was observed.

[0115] Figure 9 (A) to (C) show the results of toxicity tests using mice. Figure 9 The vertical axis of (A) represents the body weight (g) of mice. Figure 9 The vertical axis of (B) represents AST (Unit / L) in peripheral blood of mice. Figure 9 The vertical axis of (C) represents ALT (Unit / L) in the peripheral blood of mice. In each figure, the gray bar graph represents the measured value before administration, and the black bar graph represents the measured value 24 hours after administration. R1090(N), R1090(C) and Epc112(C) refer to R1090S55A-Ep203, CtR1090S55A-Ep203 and Ct Epc112-R1066, respectively. Anti-DNP antibody was used as a negative control antibody, and 21.4.1 was used as an anti-CD40 agonist antibody. 21.4.1 was administered at 1 mg / kg, and the other antibodies were administered at 10 mg / kg.

[0116] Figure 10 (A) to (D) show the results of toxicity tests using mice. Figure 10 (A) shows the leukocyte count of mice 24 hours after antibody administration. Figure 10 (B) shows the lymphocyte count of mice 24 hours after antibody administration. Figure 10 (C) shows the number of monocytes in mice 24 hours after antibody administration.

[0117] Figure 10 (D) shows the platelet count of mice 24 hours after antibody administration. The vertical axis of each graph represents the number of cells (1×10 3R1090(N), R1090(C), and Epc112(C) refer to R1090S55A-Ep203, CtR1090S55A-Ep203, and CtEpc112-R1066, respectively. Anti-DNP antibody was used as a negative control antibody, and 21.4.1 was used as an anti-CD40 agonist antibody. 21.4.1 was administered at 1 mg / kg, and the other antibodies were administered at 10 mg / kg.

[0118] Figure 11 (A) shows the results of evaluating the anti-tumor effect of the CD40-EpCAM bispecific antibody in mice transplanted with mEpCAM / B16F10. Figure 11 (B) shows the results of evaluating the anti-tumor effect of the CD40-EpCAM bispecific antibody in mice transplanted with B16F10. In each figure, the vertical axis represents the tumor volume (mm 3 ), with the horizontal axis representing days with the administration day being day 0. Anti-DNP antibody was used as a negative control antibody, and 21.4.1 was used as an anti-CD40 agonist antibody. 21.4.1 was administered at 1 mg / kg, and the other antibodies were administered at 10 mg / kg.

[0119] Figure 12 (A) shows the structure of a heterologous IgG-type bispecific antibody. Figure 12 (B) shows the structure of a Fab-type bispecific antibody fragment. Figure 12 (C) represents a F(ab')2 type bispecific antibody.

[0120] Figure 13 This figure shows the CD40 signal-inducing activity of CD40-EpCAM bispecific antibodies on Ramos cells co-cultured with human EpCAM / Expi293 cells. The horizontal axis represents fluorescence intensity, indicating the binding of anti-CD95 antibodies to Ramos cells when 1 μg / mL of each antibody was added. For comparison, anti-DNP antibody was used as a negative control antibody, and 21.4.1 was used as an anti-CD40 agonist antibody.

[0121] Figure 14 This figure shows the CD40 signal-inducing activity of CD40-EpCAM bispecific antibodies on Ramos cells co-cultured with Expi293 cells. The horizontal axis represents fluorescence intensity, indicating the binding of anti-CD95 antibodies to Ramos cells when 1 μg / mL of each antibody was added. For comparison, anti-DNP antibody was used as a negative control antibody, and 21.4.1 was used as an anti-CD40 agonist antibody.

[0122] Figure 15(A) shows the CD40 signal induction activity of the CD40-EpCAM bispecific antibody fragment on Ramos cells co-cultured with human EpCAM / Expi293 cells. Figure 15 (B) shows the CD40 signal-inducing activity of the CD40-EpCAM bispecific antibody fragment on Ramos cells co-cultured with Expi293 cells. In each graph, the horizontal axis represents antibody concentration. The vertical axis represents fluorescence intensity, indicating the binding of the anti-CD95 antibody to Ramos cells when each antibody was added. For comparison, anti-DNP antibody was used as a negative control antibody, and 21.4.1 was used as an anti-CD40 agonist antibody.

[0123] Specific embodiments of the invention

[0124] The present invention relates to a bispecific antibody or a fragment thereof comprising an antigen-binding domain that binds to CD40 and an antigen-binding domain that binds to EpCAM (hereinafter referred to as the bispecific antibody or the bispecific antibody fragment of the present invention).

[0125] In the present invention, CD40 is used synonymously with TNF receptor superfamily member 5 (TNFRSF5), Bp50, CDW40, MGC9013, and p50. Examples of CD40 include human CD40 comprising the amino acid sequence set forth in NCBI (http: / / www.ncbi.nlm.nih.gov / ) GenBank accession No. NP_001241 or SEQ ID NO: 6, and monkey CD40 comprising the amino acid sequence set forth in GenBank accession No. XP_005569274 or SEQ ID NO: 8. Furthermore, examples include polypeptides having the function of CD40, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence set forth in SEQ ID NO: 6, GenBank accession No. NP_001241, or GenBank accession No. XP_005569274.

[0126] The CD40 of the present invention also includes polypeptides comprising an amino acid sequence that is generally 70% or more, preferably 80% or more, and more preferably 90% or more identical to the amino acid sequence shown in SEQ ID NO: 6, GenBank accession No. NP_001241, or GenBank accession No. XP_005569274, and most preferably a polypeptide comprising an amino acid sequence that is 95%, 96%, 97%, 98%, or 99% or more identical, and having the function of CD40.

[0127] A polypeptide having an amino acid sequence in which one or more amino acid residues are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 6, GenBank accession No. NP_001241 or GenBank accession No. XP_005569274 can be synthesized by using a site-specific mutagenesis method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proceedings of the National Academy of Sciences in USA, 82, 488 (1985)], etc., for example, by introducing a site-specific mutation into a DNA encoding the amino acid sequence shown in SEQ ID NO: 6, GenBank accession No. NP_001241, or GenBank accession No. XP_005569274. The number of amino acids to be deleted, substituted, or added is not particularly limited, but is preferably 1 to several dozen, for example, 1 to 20, and more preferably 1 to several, for example, 1 to 5 amino acids.

[0128] Examples of genes encoding CD40 include the base sequence of human CD40 shown in SEQ ID NO: 5 or GenBank accession No. NM_001250, and the base sequence of monkey CD40 shown in SEQ ID NO: 7 or GenBank accession No. XM_011766922. In addition, for example, genes comprising a base sequence in which one or more bases are deleted, substituted, or added to the base sequence set forth in SEQ ID NO: 5 or GenBank accession No. NM_001250 and encoding a polypeptide having the function of CD40; genes comprising a base sequence preferably having 60% or greater identity, more preferably 80% or greater identity, and even more preferably 95% or greater identity to the base sequence set forth in SEQ ID NO: 5 or GenBank accession No. NM_001250 and encoding a polypeptide having the function of CD40; and genes comprising a DNA that hybridizes under stringent conditions with the DNA consisting of the base sequence set forth in SEQ ID NO: 5 or GenBank accession No. NM_001250 and encoding a polypeptide having the function of CD40 are also included in the genes encoding CD40 of the present invention.

[0129] Examples of DNA that hybridizes under stringent conditions include DNA that can hybridize using a probe having the base sequence shown in SEQ ID NO: 5 or GenBank accession No. NM_001250, obtained by colony hybridization, plaque hybridization, Southern blotting, or DNA microarray. Specifically, examples of DNA that can be identified include a filter or slide glass on which hybridized DNA from a colony or plaque, or a PCR product or oligoDNA having the sequence is immobilized, is hybridized at 65°C in the presence of 0.7 to 1.0 mol / L sodium chloride [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989); Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997); DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995)], followed by washing the filter or slide glass at 65°C using a 0.1 to 2-fold concentration of SSC solution (a 1-fold concentration of SSC solution consists of 150 mmol / L sodium chloride and 15 mmol / L sodium citrate). Examples of hybridizable DNA include DNA preferably having 60% or greater identity, more preferably 80% or greater identity, and even more preferably 95% or greater identity to the base sequence shown in SEQ ID NO: 5 or GenBank accession No. NM_001250.

[0130] Gene polymorphisms are often observed in the base sequences of genes encoding proteins in eukaryotes. Among the genes used in the present invention, genes that have undergone small-scale variations in base sequence due to such polymorphisms are also included in the gene encoding CD40 of the present invention.

[0131] Unless otherwise specified, the numerical values ​​of identity in the present invention may be values ​​calculated using an identity search program known to those skilled in the art. For base sequences, examples include values ​​calculated using default parameters in BLAST [J. Mol. Biol., 215, 403 (1990)], and for amino acid sequences, examples include values ​​calculated using default parameters in BLAST2 [Nucleic Acids Research, 25, 3389 (1997), Genome Research, 7, 649 (1997), http: / / www.ncbi.nlm.nih.gov / Education / BLASTinfo / information3.html].

[0132] As default parameters, G (Cost to open gap) is 5 in the case of base sequences and 11 in the case of amino acid sequences, -E (Cost to extend gap) is 2 in the case of base sequences and 1 in the case of amino acid sequences, -q (Penalty for nucleotide mismatch) is -3, -r (reward for nucleotide match) is 1, -e (expect value) is 10, -W (wordsize) is 11 residues in the case of base sequences and 3 residues in the case of amino acid sequences, -y [Dropoff (X) for blast extensions in bits] is 20 in the case of blastn and 7 in programs other than blastn, -X (X dropoff value for gapped alignment in bits) is 15, and -Z (final X dropoff value for gapped alignment in bits) is 50 in the case of blastn and 25 in programs other than blastn (http: / / www.ncbi.nlm.nih.gov / blast / html / blastcgihelp.html).

[0133] A polypeptide composed of a partial sequence of the CD40 amino acid sequence can be produced by methods known to those skilled in the art, for example, by deleting a portion of the DNA encoding the amino acid sequence set forth in SEQ ID NO: 6, GenBank accession No. NP_001241, or GenBank accession No. XP_005569274, and culturing a transformant introduced with an expression vector containing the amino acid sequence. Furthermore, based on the polypeptide or DNA produced by the above methods, a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added to a partial sequence of the amino acid sequence set forth in SEQ ID NO: 6, GenBank accession No. NP_001241, or GenBank accession No. XP_005569274 can be obtained by the same methods as described above. Furthermore, polypeptides composed of a partial sequence of the CD40 amino acid sequence, or polypeptides having an amino acid sequence in which one or more amino acids are deleted, substituted, or added to a partial sequence of the CD40 amino acid sequence, can also be produced by chemical synthesis methods such as the fluorenylmethoxycarbonyl (Fmoc) method and the tert-butyloxycarbonyl (tBoc) method.

[0134] Examples of the extracellular region of CD40 in the present invention include regions predicted from the amino acid sequence of human CD40 shown in GenBank accession No. NP_001241 using the known transmembrane region prediction programs SOSUI (http: / / sosui.proteome.bio.tuat.ac.jp / sosuiframe0.html), TMHMM ver. 2 (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ), or ExPASy Proteomics Server (http: / / Ca.expasy.org / ). Specifically, the amino acid sequence shown in SEQ ID NO: 6 or at positions 21 to 194 of GenBank accession No. NP_001241 can be mentioned.

[0135] As the function of CD40, it is possible to cite the induction of CD40 signals when a CD40 ligand or agonist binds, thereby causing various effects. For example, if a CD40 signal is induced on cancer cells, it can cause cell activation or proliferation inhibition of the cancer cells. If a CD40 signal is induced on B lymphocytes, it can, for example, induce activation of the B lymphocytes, promote expression of CD95, class switch recombination and somatic hypermutation, and induce the production of high-affinity antibodies to the antigen. If a CD40 signal is induced on dendritic cells, it can, for example, cause maturation of the dendritic cells or IL-12 production. If a CD40 signal is induced on macrophages, it can, for example, cause a decrease in surface markers of M2 macrophages and induction of expression of surface markers of M1 macrophages or the production of proinflammatory cytokines.

[0136] EpCAM in the present invention is used synonymously with CD326, GA733-2, HEA125, KS1 / 4, MK-1, MH99, MOC31, 323 / A3, 17-1A, CO-17A, ESA, EGP-2, EGP34, EGP40, KSA, KS1 / 4, TROP-1, and TACST-1.

[0137] Examples of EpCAM include human EpCAM comprising the amino acid sequence shown in Genbank Accession No. AAH14785 or SEQ ID NO: 16; monkey EpCAM comprising the amino acid sequence shown in Genbank Accession No. NP_001035118 or SEQ ID NO: 18; and mouse EpCAM comprising the amino acid sequence shown in Genbank Accession No. NP_032558 or SEQ ID NO: 20. Furthermore, examples include polypeptides having the function of EpCAM that are composed of an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 16, AAH14785, Genbank Accession No. NP_001035118, or Genbank Accession No. NP_032558.

[0138] The EpCAM of the present invention also includes polypeptides comprising an amino acid sequence that is preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more identical to the amino acid sequence shown in SEQ ID NO: 16, AAH14785, Genbank accession No. NP_001035118, or Genbank accession No. NP_032558, and most preferably an amino acid sequence that is 95%, 96%, 97%, 98%, and 99% or more identical, and having the function of EpCAM.

[0139] A polypeptide having an amino acid sequence in which one or more amino acid residues are deleted, substituted, or added to the amino acid sequence set forth in SEQ ID NO: 16, AAH14785, Genbank accession No. NP_001035118, or Genbank accession No. NP_032558 can be obtained by, for example, introducing a site-specific mutation into a DNA encoding the amino acid sequence set forth in SEQ ID NO: 16, AAH14785, Genbank accession No. NP_001035118, or Genbank accession No. NP_032558 using the above-mentioned site-specific mutation introduction method. The number of amino acids deleted, substituted, or added is not particularly limited, but is preferably 1 to several dozen, for example, 1 to 20, and more preferably 1 to several, for example, 1 to 5 amino acids.

[0140] Examples of genes encoding EpCAM in the present invention include human EpCAM genes comprising the base sequence shown in Genbank accession No. NM_002354 or sequence number 15; monkey EpCAM genes comprising the base sequence shown in Genbank accession No. XM_015433685 or sequence number 17; or mouse EpCAM genes comprising the base sequence shown in Genbank accession No. NM_008532 or sequence number 19.

[0141] In addition, for example, a gene comprising a base sequence in which one or more bases are deleted, substituted or added to the base sequence shown in SEQ ID NO: 15, Genbank accession No. NM_002354, Genbank accession No. XM_015433685 or Genbank accession No. NM_008532, and encoding a polypeptide having the function of EpCAM; a gene comprising a base sequence having 60% or more identity, preferably 80% or more identity, and more preferably 95% or more identity to the base sequence shown in SEQ ID NO: 15, Genbank accession No. NM_002354, Genbank accession No. XM_015433685 or Genbank accession No. NM_008532, and encoding a polypeptide having the function of EpCAM; and a gene comprising a base sequence comprising SEQ ID NO: 15, Genbank accession No. NM_002354, Genbank accession No. XM_015433685 or Genbank accession No. NM_008532, and encoding a polypeptide having the function of EpCAM. Genes encoding EpCAM of the present invention include genes consisting of DNA having the base sequence shown in Gen accession No. XM_015433685 or Gen accession No. NM_008532 and DNA that hybridizes under stringent conditions and encodes a polypeptide having the function of EpCAM.

[0142] Examples of the extracellular region of EpCAM in the present invention include regions predicted from the amino acid sequence of human EpCAM shown in GenBank accession No. AAH14785 using the known transmembrane region prediction programs SOSUI (http: / / sosui.proteome.bio.tuat.ac.jp / sosuiframe0.html), TMHMM ver. 2 (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ), or ExPASy Proteomics Server (http: / / Ca.expasy.org / ). Specifically, the amino acid sequence shown in SEQ ID NO: 10 or at positions 22 to 265 of GenBank accession No. AAH14785 can be cited.

[0143] Examples of the functions of EpCAM include calcium-independent cell-cell adhesion and promotion of cancer cell proliferation by upregulating C-Myc and cyclin A / E.

[0144] Antibodies are proteins derived from genes encoding all or part of the variable region and constant region of the heavy chain, as well as the variable region and constant region of the light chain, which constitute immunoglobulins (referred to as "antibody genes"). The antibodies of the present invention also include antibodies or antibody fragments of any immunoglobulin type and subclass.

[0145] The heavy chain (H chain) refers to the larger of the two polypeptides that make up the immunoglobulin molecule. The heavy chain determines the class and subclass of an antibody. IgA, IgD, IgE, IgG, and IgM have heavy chains consisting of α, δ, ε, γ, and μ chains, respectively. The constant regions of these heavy chains are characterized by distinct amino acid sequences. The light chain (L chain) refers to the smaller of the two polypeptides that make up the immunoglobulin molecule. In human antibodies, there are two types of light chains: κ and λ.

[0146] The variable region (V region) generally refers to the region of amino acid sequence located at the N-terminus of an immunoglobulin, which is rich in diversity. The region outside the variable region is referred to as the constant region (C region) due to its less diverse structure. The variable regions of the heavy and light chains combine to form the antigen-binding site, which determines the antibody's binding properties to the antigen.

[0147] In the heavy chain of a human antibody, the variable region corresponds to the amino acid sequence from No. 1 to No. 117 in the EU index of Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 1991 Fifth edition), and the constant region corresponds to the amino acid sequence from No. 118 onwards. In the light chain of a human antibody, the variable region corresponds to the amino acid sequence from No. 1 to No. 107 in the numbering system of Kabat et al. (Kabat numbering), and the constant region corresponds to the amino acid sequence from No. 108 onwards. Hereinafter, the heavy chain variable region or light chain variable region will be abbreviated as VH or VL.

[0148] The antigen binding site is the site in an antibody that recognizes and binds to an antigen, and refers to the site that forms a three-dimensional structure complementary to the antigenic determinant (epitope). Strong intermolecular interactions occur between the antigen binding site and the antigenic determinant. The antigen binding site is composed of VH and VL, each containing at least three complementary determining regions (CDRs). In the case of human antibodies, VH and VL each have three CDRs. These CDRs are referred to as CDR1, CDR2, and CDR3, respectively, from the N-terminal side.

[0149] The constant region is designated as either the heavy chain constant region or the light chain constant region, respectively, as CH or CL. CH is categorized by heavy chain subtype: α, δ, ε, γ, and μ. The CH consists of the CH1 domain, hinge domain, CH2 domain, and CH3 domain, arranged in order from the N-terminus. The CH2 and CH3 domains are collectively referred to as the Fc region. Meanwhile, CL is divided into two subtypes: Cλ and Cκ chains.

[0150] In the present invention, an anti-CD40 antibody refers to a monoclonal antibody that specifically recognizes and binds to the extracellular region of CD40. Furthermore, in the present invention, an anti-EpCAM antibody refers to a monoclonal antibody that specifically recognizes and binds to the extracellular region of EpCAM. Furthermore, antibodies in the present invention also include polyclonal and oligoclonal antibodies.

[0151] In the present invention, binding of an antibody or antibody fragment to at least one of CD40 and EpCAM can be confirmed by, for example, a method for confirming the binding of the antibody to cells expressing at least one of CD40 and EpCAM using a known immunological detection method, preferably a fluorescent cell staining method. Furthermore, known immunological detection methods such as Monoclonal Antibodies - Principles and Practice, Third Edition, Academic Press (1996), Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988), and Monoclonal Antibodies - A Laboratory Manual, Kodansha Scientific (1987) can also be used in combination.

[0152] Monoclonal antibodies are antibodies secreted by antibody-producing cells that maintain their monoclonal identity and recognize a single epitope (also called an antigenic determinant). Monoclonal antibody molecules have the same amino acid sequence (primary structure) and a single structure. Polyclonal antibodies are groups of antibody molecules secreted by antibody-producing cells from different clones. Oligoclonal antibodies are groups of antibody molecules formed by mixing multiple different monoclonal antibodies.

[0153] An epitope refers to a structural site on an antigen that an antibody recognizes and binds to. Examples of epitopes include a single amino acid sequence recognized and bound by a monoclonal antibody, a three-dimensional structure consisting of an amino acid sequence, an amino acid sequence bound to a sugar chain, and a three-dimensional structure consisting of an amino acid sequence bound to a sugar chain.

[0154] Examples of the monoclonal antibodies in the present invention include antibodies produced by hybridomas and genetically recombinant antibodies produced by transformants transformed with expression vectors containing antibody genes.

[0155] Hybridoma can be by for example preparing antigen, by obtaining the antibody producing cell with antigen specificity through the animal after this antigen immunization, and then this antibody producing cell is fused with myeloma cell and prepared.Can make this hybridoma ascites cancerization by cultivating this hybridoma or animal is cast this hybridoma, this culture fluid or ascites are separated, purified and obtain desired monoclonal antibody.As the animal that carries out immunity for antigen, as long as hybridoma can be made, then any animal can be used, be suitable for using mouse, rat, hamster and rabbit etc..In addition, also can obtain the cell with antibody production ability from such immune animal, after this cell is implemented immunity in vitro (in vitro), merge with myeloma cell, make hybridoma.

[0156] As the genetically recombinant antibodies in the present invention, for example, recombinant mouse antibodies, recombinant rat antibodies, recombinant hamster antibodies, recombinant rabbit antibodies, human chimeric antibodies (also referred to as chimeric antibodies), humanized antibodies (also referred to as CDR-grafted antibodies) and human antibodies, etc., can be listed as antibodies produced by genetic recombination technology. As genetically recombinant antibodies, it is possible to determine which animal species to use the variable regions and constant regions of the heavy and light chains, depending on the animal species or purpose of the object. For example, when the animal species as the object is human, the variable region can be set to a non-human animal derived from human or mouse, and the constant region and linker can be set to be derived from human.

[0157] Chimeric antibodies are antibodies composed of the VH and VL of antibodies from animals other than humans (non-human animals) and the CH and CL of human antibodies. As non-human animals, any non-human animal such as mice, rats, hamsters, and rabbits can be used as long as hybridomas can be produced. Chimeric antibodies can be produced by obtaining cDNA encoding VH and VL from a hybridoma derived from a non-human animal that produces a monoclonal antibody, inserting them into an animal cell expression vector having DNA encoding the CH and CL of a human antibody, constructing a chimeric antibody expression vector, and introducing it into animal cells for expression.

[0158] Humanized antibodies refer to antibodies obtained by transplanting the CDRs of VH and VL of non-human animal antibodies to the corresponding CDRs of VH and VL of human antibodies. The region outside the CDRs of VH and VL is called the framework region (hereinafter referred to as FR). Humanized antibodies can be manufactured as follows: a cDNA encoding the amino acid sequence of VH consisting of the amino acid sequence of the CDRs of VH of a non-human animal antibody and the amino acid sequence of the FRs of VH of any human antibody is constructed, and a cDNA encoding the amino acid sequence of VL consisting of the amino acid sequence of the CDRs of VL of a non-human animal antibody and the amino acid sequence of the FRs of VL of any human antibody is constructed, and an animal cell expression vector with DNA encoding CH and CL of a human antibody is inserted respectively, a humanized antibody expression vector is constructed, and introduced into an animal cell to be expressed.

[0159] Human antibodies originally refer to antibodies naturally existing in the human body, and also include antibodies obtained from human antibody phage libraries created due to recent technological advances in genetic engineering, cell engineering, and developmental engineering, and from transgenic animals that produce human antibodies.

[0160] Antibodies naturally present in the human body can be obtained, for example, by immortalizing human peripheral blood lymphocytes by infecting them with Epstein-Barr virus or the like, cloning them, culturing the antibody-producing lymphocytes, and purifying the antibodies from the culture supernatant.

[0161] Human antibody phage libraries are libraries created by inserting antibody genes produced by human B cells into phage genes, allowing the expression of antibody fragments such as Fab and scFv on the phage surface. Phage expressing antibody fragments with the desired antigen-binding activity on their surfaces can be recovered from this library, using binding activity to a substrate immobilized with an antigen as an indicator. These antibody fragments can further be converted into human antibody molecules composed of two complete H chains and two complete L chains through genetic engineering methods.

[0162] Transgenic animals that produce human antibodies are animals that have human antibody genes embedded in their cells. Specifically, for example, human antibody genes can be introduced into mouse ES cells, which are then transplanted into early mouse embryos and allowed to mature, thereby producing transgenic mice that produce human antibodies. Human antibodies derived from transgenic animals that produce human antibodies can be produced by obtaining hybridomas using hybridoma production methods commonly used in non-human animals and culturing them to produce and accumulate antibodies in the culture supernatant.

[0163] The CH of the recombinant antibody can be any CH as long as it belongs to a human immunoglobulin, preferably the human immunoglobulin G (hIgG) class. Furthermore, any of the hIgG1, hIgG2, hIgG3, and hIgG4 subclasses of the hIgG class can be used. Furthermore, the CL of the recombinant antibody can be any CL as long as it belongs to a human immunoglobulin, and either the κ or λ class can be used.

[0164] In the present invention, bispecific antibodies refer to proteins that have two antigen-binding domains with different specificities, comprising all or part of the variable and constant regions of the heavy and light chains, and the variable and constant regions of the light chains, which constitute immunoglobulins. Each antigen-binding domain of a bispecific antibody can bind to different epitopes on a single antigen or to different antigens.

[0165] In the present invention, the antigen-binding domain that binds to CD40 or EpCAM can be any antigen-binding domain as long as it specifically recognizes and binds to CD40 or EpCAM. For example, it can be any form, including antibodies, ligands, receptors, and naturally occurring interacting molecules, such as polypeptides produced by genetic recombinant techniques, protein molecules and fragments thereof, as well as low-molecular-weight forms of such protein molecules or conjugates with naturally occurring substances.

[0166] Furthermore, antigen-binding domains may be binding proteins recombined using the binding domains of known binding molecules such as antibodies, ligands, and receptors. Specific examples include recombinant proteins comprising the CDRs of antibodies that bind to respective antigens, antibody variable regions comprising CDRs, and recombinant proteins comprising antibody variable regions and the binding domains of ligands that bind to respective antigens. In the present invention, the antigen-binding domain is preferably an antibody variable region.

[0167] The bispecific antibody or bispecific antibody fragment of the present invention can bind to CD40 and EpCAM expressed on the same cell, or can bind to CD40 and EpCAM expressed on different cells.

[0168] Examples of cells expressing CD40 include antigen-presenting cells such as B cells, dendritic cells (DC), macrophages, and monocytes, and cancer cells such as Ramos cells.

[0169] Examples of cells expressing EpCAM include cancer cells of head and neck cancer, lung cancer, digestive tract cancer, breast cancer, urinary tract cancer, and the like.

[0170] Examples of the bispecific antibodies or bispecific antibody fragments of the present invention include those having CD40 agonist activity. Preferred bispecific antibodies or bispecific antibody fragments of the present invention do not exhibit CD40 agonist activity in the absence of EpCAM molecules or EpCAM-expressing cells, but exhibit CD40 agonist activity only in the presence of EpCAM molecules or EpCAM-expressing cells. Such bispecific antibodies or bispecific antibody fragments are preferred because they activate CD40 only in lesions such as cancers where EpCAM-expressing cells are present, and therefore do not produce side effects associated with systemic CD40 activation.

[0171] The CD40 agonist activity possessed by the bispecific antibody or bispecific antibody fragment of the present invention refers to the activity of inducing activation of antigen-presenting cells, inducing cell death of tumor cells, etc. by initiating signals through CD40 through the binding of the bispecific antibody or bispecific antibody fragment to CD40 on cells.

[0172] CD40 agonist activity can be confirmed by evaluating, for example, an increase in the expression level of CD95 on CD40-expressing cells such as Ramos cells.

[0173] Specifically, examples of the bispecific antibodies or bispecific antibody fragments of the present invention include bispecific antibodies or bispecific antibody fragments that, when bound to EpCAM and CD40 in the presence of cells expressing EpCAM, induce activation of antigen-presenting cells expressing CD40 and / or cell death of tumor cells.

[0174] In the present invention, CD40 antagonist activity refers to an activity that inhibits CD40 activation by a CD40 ligand or a CD40 agonist, etc. For example, it refers to an activity that inhibits signal induction by binding of a CD40 ligand or a CD40 agonist to CD40.

[0175] The CD40 antagonist activity of an antibody can be confirmed, for example, by suppressing the expression of CD95 on CD40-expressing cells such as Ramos cells induced by CD40 ligand, by the addition of the antibody.

[0176] The number of antigen-binding domains possessed by a single bispecific antibody molecule is referred to as binding valency. For example, in the present invention, when a single bispecific antibody molecule possesses two antigen-binding domains that bind to CD40 and two antigen-binding domains that bind to EpCAM, such a bispecific antibody binds to CD40 and EpCAM bivalently.

[0177] In the present invention, each bispecific antibody molecule can bind to CD40 or EpCAM with any valency, but preferably binds to CD40 and EpCAM at least twice, respectively.

[0178] Furthermore, antibodies comprising multiple antigen-binding domains linked via an appropriate linker, such as a linker comprising an immunoglobulin domain or a fragment thereof, are also encompassed by the bispecific antibodies of the present invention.

[0179] In the bispecific antibodies of the present invention, the positions of the antigen-binding domain that binds to CD40 and the antigen-binding domain that binds to EpCAM can be appropriately selected.

[0180] The bispecific antibody of the present invention can be produced by existing production techniques ([Nature Protocols, 9, 2450-2463 (2014)], International Publication No. 1998 / 050431, International Publication No. 2001 / 7734, International Publication No. 2002 / 002773, and International Publication No. 2009 / 131239) and the like.

[0181] In the bispecific antibodies of the present invention, the antigen-binding domain that binds to CD40 may be located closer to the N-terminus or closer to the C-terminus than the antigen-binding domain that binds to EpCAM.

[0182] As bispecific antibodies of the present invention, antibody V regions can be used as antigen-binding domains. Examples include antibodies containing a heavy chain comprising multiple VHs in one heavy chain and antibodies containing two heavy chains comprising one VH. Hereinafter, in antibodies containing a heavy chain comprising multiple VHs in one heavy chain, the first VH from the N-terminus is sometimes referred to as VH1, the second VH as VH2, and the nth VH as VHn. In the case of antibodies containing a heavy chain comprising two VHs in one heavy chain, the first VH from the N-terminus is referred to as VH1, and the second VH as VH2.

[0183] More specifically, bispecific antibodies comprising a heavy chain containing a plurality of VHs on one heavy chain include antibodies comprising a heavy chain comprising two or more VHs linked using a linker comprising an immunoglobulin domain or a fragment thereof.

[0184] When three or more VHs are linked, different immunoglobulin domains or fragments thereof may be used as linkers, or the same immunoglobulin domain or fragment thereof may be used. Furthermore, when two or more VHs are linked, the length or type of the immunoglobulin domain or fragment thereof may be varied so that each VH specifically binds to the antigen.

[0185] Specifically, the bispecific antibody of the present invention has at least one of the following characteristics (a) to (e).

[0186] (a) A heavy chain polypeptide comprises multiple (e.g., 2-5) different VHs, which are not adjacent to each other;

[0187] (b) VHs are linked in series (in columns) via a polypeptide linker of 10 or more amino acids. More specifically, VHs are linked using a linker comprising, for example, all or part of the amino acid sequence of an immunoglobulin domain;

[0188] (c) The light chains associate with the heavy chains to form an antigen-binding site;

[0189] (d) If Figure 1 (A) or Figure 1 (B) has a structure consisting of two heavy chain polypeptides and at least four light chain polypeptides, wherein the two heavy chain polypeptides are linked by a disulfide bond at the hinge region, and the light chain polypeptides are also linked to the heavy chain polypeptide by a disulfide bond; and

[0190] (e) The heavy chain constant region is composed of, for example, all or part of the constant region of a native antibody heavy chain (e.g., CH1 fragment, CH1, CH2, CH3, CH1-hinge, CH1-hinge-CH2, and CH1-hinge-CH2-CH3, etc.).

[0191] In the present invention, the positions of the anti-CD40 antibody VH (referring to VH derived from anti-CD40 antibody) and the anti-EpCAM antibody VH (referring to VH derived from anti-EpCAM antibody) contained in the bispecific antibody can be appropriately selected. Figure 1 (A) or Figure 1 In the bispecific antibody having the structure shown in (B), the VH of the anti-CD40 antibody may be located at the N-terminal side or the C-terminal side relative to the VH of the anti-EpCAM antibody, but is preferably located at the N-terminal side.

[0192] In the present invention, the VLs contained in the bispecific antibodies may be identical or different. The VHs of bispecific antibodies having identical VLs and capable of binding to two different antigens or two different epitopes on the same antigen may be VHs optimized or modified so that each variable region can bind to each specific antigen or epitope. For example, appropriate VHs can be selected using methods such as screening by amino acid modification or phage display.

[0193] The VL contained in the bispecific antibody of the present invention may be any VL as long as it is the VL of an anti-CD40 antibody or an anti-EpCAM antibody, but preferably contains a VL comprising CDR1, 2, and 3 of the amino acid sequences shown in SEQ ID NOs: 23, 24, and 25, respectively, and a VL comprising the amino acid sequence shown in SEQ ID NO: 22.

[0194] In the present invention, a linker refers to a chemical structure that binds multiple antigen-binding domains, preferably a polypeptide. Examples of linkers used in the bispecific antibodies of the present invention include linkers comprising the amino acid sequence of all or part of an immunoglobulin domain, and linkers comprising the amino acid sequence of all or part of a polypeptide composed of multiple immunoglobulin domains.

[0195] In the present invention, the C-terminal polypeptide refers to a polypeptide chain that binds to the C-terminus of the antigen-binding domain closest to the C-terminus among multiple antigen-binding domains. The bispecific antibodies of the present invention may or may not have a C-terminal polypeptide, but preferably have a C-terminal polypeptide. Examples of C-terminal polypeptides include polypeptides comprising the amino acid sequence of all or part of an immunoglobulin domain, and polypeptides comprising the amino acid sequence of all or part of a polypeptide composed of multiple immunoglobulin domains.

[0196] Regarding the linker or C-terminal polypeptide comprising an amino acid sequence of a portion of an immunoglobulin domain, the amino acid sequence selected from the immunoglobulin may be discontinuous or continuous, preferably a continuous amino acid sequence. Furthermore, the amino acid sequence may also include a hinge region.

[0197] In the present invention, an immunoglobulin domain refers to a peptide consisting of approximately 100 amino acid residues with at least two cysteine ​​residues and having an amino acid sequence similar to that of an immunoglobulin, as a minimum unit. In the present invention, an immunoglobulin domain also includes a polypeptide comprising multiple immunoglobulin domains of the aforementioned minimum unit. Examples of immunoglobulin domains include VH, CH1, CH2, and CH3 of an immunoglobulin heavy chain, and VL and CL of an immunoglobulin light chain.

[0198] The animal species of the immunoglobulin is not particularly limited, but is preferably human. Furthermore, the subclass of the constant region of the immunoglobulin heavy chain can be any of IgD, IgM, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and IgE, preferably derived from IgG and IgM. Furthermore, the subclass of the constant region of the immunoglobulin light chain can be either κ or λ.

[0199] In addition, immunoglobulin domains also exist in proteins other than immunoglobulins, and examples thereof include immunoglobulin domains contained in proteins belonging to the immunoglobulin superfamily, such as major histocompatibility complex (MHC), CD1, B7, and T cell receptors (TCR). As the immunoglobulin domain used in the bispecific antibodies of the present invention, any immunoglobulin domain can be applied.

[0200] In the case of human antibodies, CH1 refers to a region having an amino acid sequence of No. 118 to No. 215 as shown in the EU index. Similarly, CH2 refers to a region having an amino acid sequence of No. 231 to No. 340 as shown in the EU index of Kabat et al., and CH3 refers to a region having an amino acid sequence of No. 341 to No. 446 as shown in the EU index of Kabat et al. Between CH1 and CH2, there is a flexible amino acid region called a hinge region (hereinafter sometimes referred to as a hinge). The hinge region refers to a region having an amino acid sequence of No. 216 to No. 230 as shown in the EU index of Kabat et al.

[0201] CL refers to a region having amino acid sequences from No. 108 to No. 214 as shown in Kabat numbering in the case of a human antibody κ chain, and refers to a region having amino acid sequences from No. 108 to No. 215 in the case of a λ chain.

[0202] Examples of linkers and C-terminal polypeptides used in the bispecific antibodies of the present invention include, but are not limited to, immunoglobulin domains consisting of CH1-hinge-CH2-CH3 arranged in order from the N-terminus to the terminal, immunoglobulin domains consisting of CH1-hinge-CH2, immunoglobulin domains consisting of CH1-hinge, immunoglobulin domains consisting of CH1, N-terminal fragments of CH1, CH1 fragments consisting of 14 amino acid residues in which the amino acid residue at position 14 is Cys, and CH1 fragments consisting of amino acid residues 1 to 14 from the N-terminus of CH1, as well as sequences in which one or more amino acid residues are altered in the amino acid sequences of these immunoglobulin domain fragments.

[0203] In addition, in the present invention, as an example of a linker and a C-terminal side polypeptide, all or part of the fragments of the amino acid sequence consisting of the CH1, hinge, CH2 and CH3 of the antibody can be appropriately combined for use. In addition, these amino acid sequences can also be partially deleted or used in a reversed order. In addition, the subclass of the antibody used in the linker and the C-terminal side polypeptide is not particularly limited, preferably IgM or IgG4 or an IgG4 variant (hereinafter referred to as IgG4PE R409K) in which the Ser residue at position 228 of the heavy chain constant region of IgG4 is substituted with Pro, the Leu residue at position 235 is substituted with Asn, and the Arg residue at position 409 is substituted with Lys, more preferably IgG4 and IgG4PE R409K.

[0204] In the present invention, examples of linkers and C-terminal side polypeptides include: a polypeptide composed of 14 amino acid residues at positions 1 to 14 of the N-terminus of CH1 of IgG4 shown in sequence number 75, a polypeptide composed of CH1 of IgG4 shown in sequence number 75, and a polypeptide composed of CH (CH1, hinge, CH2 and CH3) of IgG4PE R409K shown in sequence number 77, etc., and more preferably a polypeptide composed of CH1 of IgG4 shown in sequence number 75 and a polypeptide composed of CH of IgG4PE R409K shown in sequence number 77.

[0205] The combination of the linker and the C-terminal side peptide contained in the bispecific antibody of the present invention can be any combination. As the bispecific antibody of the present invention, preferably: a bispecific antibody comprising the following linker and the following C-terminal side peptide, the linker is composed of the CH1 of IgG4 comprising the amino acid sequence shown in SEQ ID NO: 75, and the C-terminal side peptide is composed of the CH (CH1, hinge, CH2 and CH3) of IgG4 PER409K comprising the amino acid sequence shown in SEQ ID NO: 77; and a bispecific antibody comprising the following linker and the following C-terminal side peptide, the linker is composed of the CH of IgG4 PER409K comprising the amino acid sequence shown in SEQ ID NO: 77, and the C-terminal side peptide is composed of the CH1 of IgG4 comprising the amino acid sequence shown in SEQ ID NO: 75.

[0206] In the bispecific antibody of the present invention, Figure 1 As shown in (A), an antibody composed of two heavy chains and four light chains containing the amino acid sequence of VH1, CH1, VH2, CH1, hinge, CH2 and CH3 in order from the N-terminal side is called an N-terminal type bispecific antibody. Figure 1 As shown in (B), an antibody composed of two heavy chains and four light chains containing the amino acid sequence of VH1, CH1, hinge, CH2, CH3, VH2, and CH1 in this order from the N-terminal side is called a C-terminal bispecific antibody.

[0207] Bispecific antibodies or bispecific antibody fragments thereof that have one or more amino acid residues deleted, added, substituted, or inserted into the amino acid sequence constituting the bispecific antibodies or bispecific antibody fragments of the present invention and that have the same activity as the above-mentioned bispecific antibodies or bispecific antibody fragments are also included in the bispecific antibodies or bispecific antibody fragments of the present invention.

[0208] The number of amino acids to be deleted, substituted, inserted and / or added is one or more, and the number is not particularly limited. It is the number that can be deleted, substituted, inserted or added by known techniques such as the site-specific mutation introduction method described in Molecular Cloning, The Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Willy & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985), etc. For example, the number is usually 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5.

[0209] The deletion, substitution, insertion, or addition of one or more amino acid residues in the amino acid sequence of the bispecific antibody of the present invention described above indicates the following: It means that one or more amino acid residues are deleted, substituted, inserted, or added in any one or more amino acid sequences within the same sequence. Furthermore, deletion, substitution, insertion, or addition may occur simultaneously, and the substituted, inserted, or added amino acid residues may be either naturally occurring or non-naturally occurring.

[0210] Examples of naturally occurring amino acid residues include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and L-cysteine.

[0211] Preferred examples of amino acid residues that can be substituted for each other are shown below: Amino acid residues included in the same group can be substituted for each other.

[0212] Group A: Leucine, Isoleucine, Norleucine, Valine, Norvaline, Alanine, 2-Aminobutyric Acid, Methionine, O-Methylserine, Tert-Butylglycine, Tert-Butylalanine, Cyclohexylalanine

[0213] Group B: Aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid

[0214] Group C: Asparagine, Glutamine

[0215] Group D: lysine, arginine, ornithine, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid

[0216] Group E: Proline, 3-hydroxyproline, 4-hydroxyproline

[0217] Group F: serine, threonine, homoserine

[0218] Group G: Phenylalanine, Tyrosine

[0219] The bispecific antibodies or bispecific antibody fragments of the present invention also include antibodies containing any amino acid residues that have been post-translationally modified. Examples of post-translational modifications include deletion of lysine residues at the C-terminus of the H chain (lysine clipping) and substitution of glutamine residues at the N-terminus of the polypeptide with pyroglutamine (pyroGlu) [Beck et al., Analytical Chemistry, 85, 715-736 (2013)].

[0220] Specific examples of the bispecific antibody of the present invention include any one of the bispecific antibodies selected from the following (1) to (3).

[0221] (1) a bispecific antibody comprising the V region of an anti-CD40 antibody (referring to the V region derived from the anti-CD40 antibody) and the V region of an anti-EpCAM antibody (referring to the V region derived from the anti-EpCAM antibody),

[0222] (2) a bispecific antibody comprising CDR1 to 3 of VH and CDR1 to 3 of VL of an anti-CD40 antibody, and CDR1 to 3 of VH and CDR1 to 3 of VL of an anti-EpCAM antibody, and

[0223] (3) A bispecific antibody comprising VH and VL of an anti-CD40 antibody and VH and VL of an anti-EpCAM antibody.

[0224] In the bispecific antibody described in (2) above, CDRs 1 to 3 of the VL of the anti-CD40 antibody and CDRs 1 to 3 of the VL of the anti-EpCAM antibody may be the same or different, but are preferably the same.

[0225] In the bispecific antibody described in (3) above, the VL of the anti-CD40 antibody and the VL of the anti-EpCAM antibody may be the same or different, but are preferably the same.

[0226] The anti-CD40 antibodies of the present invention may or may not have CD40 agonist activity, preferably anti-CD40 antibodies without CD40 agonist activity. Furthermore, the CD40 antibodies of the present invention may or may not have CD40 antagonist activity, preferably anti-CD40 antibodies without CD40 antagonist activity.

[0227] An example of the anti-CD40 antibody of the present invention includes an anti-CD40 antibody comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 23, 24, and 25, respectively, and a VH selected from any one of the following (1a) to (1d).

[0228] (1a) VH, VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH21, VH22, VH33, VH34, VH35, VH36, VH37, VH48, VH59, VH60, VH71, VH72, VH83, VH94, VH105, VH110, V

[0229] (1b) VH, VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH21, VH22, VH33, VH34, VH35, VH36, VH37, VH48, VH59, VH60, VH71, VH72, VH83, VH94, VH105, VH110, V

[0230] (1c) VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 38, 39, and 40, respectively, and

[0231] (1d) VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 43, 44, and 45, respectively.

[0232] The anti-CD40 antibodies of the present invention also include anti-CD40 antibodies comprising VH and VL CDR1-3 amino acid sequences that are at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequences of VH CDR1-3 set forth in any one of (1a) to (1d) above and the amino acid sequences of VL CDR1-3 set forth in SEQ ID NOs: 23 to 25, respectively.

[0233] The anti-CD40 antibodies of the present invention also include the antibodies described in (i) or (ii) below.

[0234] (i) An antibody that binds to CD40 in competition with an anti-CD40 antibody having a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 23, 24, and 25, respectively, and a VH shown in any one of (1a) to (1d) above.

[0235] (ii) An antibody that binds to the same epitope as an anti-CD40 antibody having a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 23, 24, and 25, respectively, and a VH shown in any one of (1a) to (1d) above.

[0236] Another example of the anti-CD40 antibody of the present invention includes an anti-CD40 antibody comprising a VL comprising the amino acid sequence shown in SEQ ID NO: 22 and a VH comprising the amino acid sequence shown in SEQ ID NO: 27, 32, 37, or 42.

[0237] An example of the anti-EpCAM antibody of the present invention includes an anti-EpCAM antibody having a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 23, 24, and 25, respectively, and a VH selected from any one of the following (2a) to (2d).

[0238] (2a) VH, VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH21, VH22, VH33, VH44, VH55, VH56, VH57, VH58, VH60, VH71, VH72, VH83, VH94, VH95, VH106, VH110, V

[0239] (2b) VH, VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH21, VH22, VH33, VH44, VH55, VH56, VH57, VH58, VH59, VH60, VH71, VH72, VH73, VH74, VH85, VH96, VH9

[0240] (2c) VH, VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH21, VH22, VH33, VH44, VH55, VH66, VH77, VH88, VH99, VH110, VH120, VH130, VH140, VH150, VH160, VH

[0241] (2d) VH comprising CDR1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NOs: 67, 68 and 69, respectively.

[0242] The anti-EpCAM antibodies of the present invention also include: anti-EpCAM antibodies comprising amino acid sequences of VH and VL CDR1-3 that are at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequences of CDR1-3 of VH shown in any one of (2a) to (2d) above and the amino acid sequences of CDR1-3 of VL shown in SEQ ID NOs. 23 to 25, respectively.

[0243] The anti-EpCAM antibodies of the present invention also include the antibodies described in (i) or (ii) below.

[0244] (i) An antibody that binds to EpCAM in competition with an anti-EpCAM antibody having a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 23, 24, and 25, respectively, and a VH shown in any one of (2a) to (2d) above.

[0245] (ii) An antibody that binds to the same epitope as an anti-EpCAM antibody having a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 23, 24, and 25, respectively, and a VH shown in any one of (2a) to (2d) above.

[0246] Another example of the anti-EpCAM antibody of the present invention is a bispecific antibody comprising a VL comprising the amino acid sequence shown in SEQ ID NO: 22 and a VH comprising the amino acid sequence shown in SEQ ID NO: 51, 56, 61, or 66.

[0247] More specifically, the bispecific antibodies of the present invention include those comprising VL comprising CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 23, 24, and 25, respectively, and VH1 and VH2 shown in (i) or (ii) below.

[0248] (i) H1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 38, 39, and 40; or SEQ ID NOs: 43, 44, and 45, respectively, that bind to CD40;

[0249] and a VH2 that binds to EpCAM and comprises CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 52, 53, and 54; SEQ ID NOs. 57, 58, and 59; SEQ ID NOs. 62, 63, and 64; or SEQ ID NOs. 67, 68, and 69, respectively.

[0250] (ii) a VH1, a VH2, a VH3, a VH4, a VH5, a VH6, a VH7, a VH8, a VH9, a VH10, a VH11, a VH12, a VH13, a VH14, a VH15, a VH16, a VH17, a VH18, a VH19, a VH20, a VH21, a VH22, a VH23, a VH24, a VH25, a VH26, a VH27, a VH28, a VH29, a VH30, a VH31, a VH32, a VH33, a VH34, a VH35, a VH36, a VH37, a VH38, a VH39, a VH40, a VH41, a VH42, a VH43, a VH44, a VH45, a VH50, a VH51, a VH52, a VH53, a VH54, a VH55, a VH56, a VH57, a VH58, a VH59, a VH60, a VH61, a VH62, a VH63, a VH64, a VH65

[0251] and a CD40-binding VH2 comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 28, 29, and 30; SEQ ID NOs. 33, 34, and 35; SEQ ID NOs. 38, 39, and 40; or SEQ ID NOs. 43, 44, and 45, respectively.

[0252] Another specific example of the bispecific antibody of the present invention includes a bispecific antibody comprising VL comprising the amino acid sequence shown in SEQ ID NO: 22, and VH1 and VH2 shown in (i) or (ii) below.

[0253] (i) VH1 that binds to CD40 and comprises the amino acid sequence shown in SEQ ID NO: 27, 32, 37 or 42,

[0254] And VH2 that binds to EpCAM and comprises the amino acid sequence shown in SEQ ID NO: 51, 56, 61 or 66.

[0255] (ii) VH1 that binds to EpCAM and comprises the amino acid sequence shown in SEQ ID NO: 51, 56, 61 or 66,

[0256] And a VH2 that binds to CD40 and comprises the amino acid sequence shown in SEQ ID NO: 27, 32, 37 or 42.

[0257] One embodiment of the present invention includes a bispecific antibody comprising a heavy chain composed of a polypeptide represented by the formula VH1-X-VH2-Y from the N-terminus {wherein, VH1 represents the VH of a first antibody (referring to the VH derived from the first antibody), VH2 represents the VH of a second antibody (referring to the VH derived from the second antibody), and X and Y each represent a polypeptide (wherein, at least one of X and Y further comprises an antibody hinge region); hereinafter referred to as Formula (I)}, wherein either the first antibody or the second antibody is an anti-CD40 antibody, and the other is an anti-EpCAM antibody.

[0258] Examples of X and Y in the above formula (I) include polypeptides comprising any one of CH1, CH2, CH3, and hinge of each antibody (wherein at least one of X and Y further comprises the hinge region of the antibody).

[0259] As one embodiment of the present invention, examples include: wherein one of X and Y in the above-mentioned formula (I) is CH1, and the other comprises a polypeptide comprising CH1, CH2, CH3, and a hinge. Specifically, examples include: X is a polypeptide comprising the CH1, hinge, CH2, and CH3 of human IgG4 or human IgG1, and Y is a polypeptide comprising the CH1 of human IgG4 or human IgG1; X is a polypeptide comprising a constant region of human IgG4 in which position 228 is Pro, position 235 is Glu, and position 409 is Lys, and Y is a polypeptide comprising the CH1 of human IgG4 or human IgG1; X is a polypeptide comprising the CH1 of human IgG4 or human IgG1, and Y is a polypeptide comprising the CH1 of human IgG4 or human IgG1. 1; X is a polypeptide comprising CH1 of human IgG4 or human IgG1, and Y is a polypeptide comprising a constant region in which position 228 is Pro, position 235 is Glu, and position 409 is Lys in the constant region of human IgG4; X is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 75, and Y is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 77; X is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 77, and Y is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 75.

[0260] As one embodiment of the present invention, a bispecific antibody comprising two identical heavy chains composed of a polypeptide represented by formula (I) is more preferred, a bispecific antibody comprising two identical heavy chains composed of a polypeptide represented by formula (I) and four identical light chains or two of each of two light chains is even more preferred, and a bispecific antibody comprising two identical heavy chains composed of a polypeptide represented by formula (I) and four identical light chains is further preferred.

[0261] In the preferred embodiment described above, two identical heavy chains are preferably disulfide-bonded via cysteine ​​residues in the hinge region to form a polymer. Alternatively, one heavy chain is preferably disulfide-bonded to a cysteine ​​residue in the constant region of a light chain via a cysteine ​​residue in CH1 to form a polymer. Therefore, the bispecific antibody of the present invention preferably has a heterohexameric structure composed of two heavy chains and four light chains.

[0262] As a preferred embodiment of the present invention, the following bispecific antibody can be mentioned: it contains: two heavy chains, each composed of a polypeptide represented by formula (I) {VH1-X-VH2-Y from the N-terminus {wherein, VH1 represents the VH of the first antibody, VH2 represents the VH of the second antibody, X represents a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 75, and Y represents a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 77}; and four light chains, each having a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 23, 24, and 25, respectively, wherein VH1 and VH2 are VH represented by (i) or (ii) below.

[0263] (i) VH1 is a VH of an anti-human CD40 antibody comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 38, 39, and 40; or SEQ ID NOs: 43, 44, and 45, respectively;

[0264] VH2 is an anti-human EpCAM antibody VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 52, 53, and 54; SEQ ID NOs: 57, 58, and 59; SEQ ID NOs: 62, 63, and 64; or SEQ ID NOs: 67, 68, and 69, respectively.

[0265] (ii) VH1 is a VH of an anti-human EpCAM antibody comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 52, 53, and 54; SEQ ID NOs: 57, 58, and 59; SEQ ID NOs: 62, 63, and 64; or SEQ ID NOs: 67, 68, and 69, respectively;

[0266] VH2 is an anti-human CD40 antibody VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 38, 39, and 40; or SEQ ID NOs: 43, 44, and 45, respectively.

[0267] The light chain may be a λ chain or a κ chain, and is preferably a κ chain.

[0268] In addition, an example of a bispecific antibody in which VH1 is VH of an anti-human CD40 antibody comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 33, 34, and 35, respectively, and VH2 is VH of an anti-human EpCAM antibody comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 57, 58, and 59, respectively, is R1090S55A-Ep203.

[0269] As another preferred embodiment of the present invention, the following bispecific antibody can be cited, which comprises: two heavy chains composed of a polypeptide represented by formula (I) {VH1-X-VH2-Y from the N-terminus {wherein, VH1 represents the VH of the first antibody, VH2 represents the VH of the second antibody, X represents a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 75, and Y represents a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 77}; and four light chains each having a VL comprising the amino acid sequence shown in SEQ ID NO: 22, wherein VH1 and VH2 are VH represented by (i) or (ii) below.

[0270] (i) VH1 is VH of an anti-human CD40 antibody comprising the amino acid sequence shown in SEQ ID NO: 27, 32, 37, or 42,

[0271] VH2 is VH of an anti-human EpCAM antibody comprising the amino acid sequence shown in SEQ ID NO: 51, 56, 61, or 66.

[0272] (ii) VH1 is a VH of an anti-human EpCAM antibody comprising the amino acid sequence shown in SEQ ID NO: 51, 56, 61 or 66,

[0273] VH2 is VH of an anti-human CD40 antibody comprising the amino acid sequence shown in SEQ ID NO: 27, 32, 37, or 42.

[0274] The light chain may be a λ chain or a κ chain, and is preferably a κ chain.

[0275] Among them, R1090S55A-Ep203 is an example of a bispecific antibody in which VH1 is VH of an anti-human CD40 antibody comprising the amino acid sequence shown in SEQ ID NO: 32, and VH2 is VH of an anti-human EpCAM antibody comprising the amino acid sequence shown in SEQ ID NO: 56.

[0276] As a preferred embodiment of the present invention, the following bispecific antibody can be cited, which comprises: two heavy chains composed of a polypeptide represented by formula (I) {VH1-X-VH2-Y from the N-terminus {wherein, VH1 represents the VH of the first antibody, VH2 represents the VH of the second antibody, X represents a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 77, and Y represents a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 75}; and four light chains each having a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 23, 24, and 25, respectively, wherein VH1 and VH2 are VH represented by (i) or (ii) below.

[0277] (i) VH1 is a VH of an anti-human CD40 antibody comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 38, 39, and 40; or SEQ ID NOs: 43, 44, and 45, respectively;

[0278] VH2 is an anti-human EpCAM antibody VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 52, 53, and 54; SEQ ID NOs: 57, 58, and 59; SEQ ID NOs: 62, 63, and 64; or SEQ ID NOs: 67, 68, and 69, respectively.

[0279] (ii) VH1 is a VH of an anti-human EpCAM antibody comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 52, 53, and 54; SEQ ID NOs: 57, 58, and 59; SEQ ID NOs: 62, 63, and 64; or SEQ ID NOs: 67, 68, and 69, respectively;

[0280] VH2 is an anti-human CD40 antibody VH comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 38, 39, and 40; or SEQ ID NOs: 43, 44, and 45, respectively.

[0281] The light chain may be either a λ chain or a κ chain, and is preferably a κ chain.

[0282] In addition, an example of a bispecific antibody in which VH1 is VH of an anti-human CD40 antibody comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 33, 34, and 35, respectively, and VH2 is VH of an anti-human EpCAM antibody comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 57, 58, and 59, respectively, is Ct R1090S55A-Ep203.

[0283] In addition, as an example of a bispecific antibody in which VH1 is VH of an anti-human EpCAM antibody comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 67, 68, and 69, respectively, and VH2 is VH of an anti-human CD40 antibody comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively, Ct Epc112-R1066 can be cited.

[0284] As another preferred embodiment of the present invention, the following bispecific antibody can be cited, which contains: two heavy chains, each of which is composed of a polypeptide represented by formula (I) {VH1-X-VH2-Y from the N-terminus {wherein, VH1 represents the VH of the first antibody, VH2 represents the VH of the second antibody, X represents a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 77, and Y represents a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 75}; and four light chains, each of which has a VL comprising the amino acid sequence shown in SEQ ID NO: 22, wherein VH1 and VH2 are VH represented by the following (i) or (ii).

[0285] (i) VH1 is VH of an anti-human CD40 antibody comprising the amino acid sequence shown in SEQ ID NO: 27, 32, 37, or 42,

[0286] VH2 is VH of an anti-human EpCAM antibody comprising the amino acid sequence shown in SEQ ID NO: 51, 56, 61, or 66.

[0287] (ii) VH1 is a VH of an anti-human EpCAM antibody comprising the amino acid sequence shown in SEQ ID NO: 51, 56, 61 or 66,

[0288] VH2 is VH of an anti-human CD40 antibody comprising the amino acid sequence shown in SEQ ID NO: 27, 32, 37, or 42.

[0289] The light chain may be a λ chain or a κ chain, and is preferably a κ chain.

[0290] Among them, an example of a bispecific antibody in which VH1 is VH of an anti-human CD40 antibody comprising the amino acid sequence shown in SEQ ID NO: 32, and VH2 is VH of an anti-human EpCAM antibody comprising the amino acid sequence shown in SEQ ID NO: 56 is Ct R1090S55A-Ep203.

[0291] Furthermore, an example of a bispecific antibody in which VH1 is VH of an anti-human EpCAM antibody comprising the amino acid sequence of SEQ ID NO: 66, and VH2 is VH of an anti-human CD40 antibody comprising the amino acid sequence of SEQ ID NO: 27 is Ct Epc112-R1066.

[0292] The bispecific antibodies or bispecific antibody fragments of the present invention also include antibodies or bispecific antibody fragments having effector activity.

[0293] Effector activity refers to antibody-dependent cellular cytotoxic activity induced via the Fc region of an antibody, and examples thereof include antibody-dependent cellular cytotoxicity activity (ADCC activity), complement-dependent cytotoxicity activity (CDC activity), antibody-dependent cellular phagocytosis activity (ADCP activity) of phagocytes such as macrophages and dendritic cells, and opsonization.

[0294] In the present invention, ADCC activity and CDC activity can be measured by a known measurement method [Cancer Immunol. Immunother., 36, 373 (1993)].

[0295] ADCC activity refers to the activity of an antibody that has bound to an antigen on a target cell to bind to an Fc receptor on an immune cell via the Fc region of the antibody, thereby activating immune cells (such as natural killer cells) and damaging the target cell.

[0296] Fc receptors (FcRs) are receptors that bind to the Fc region of antibodies and induce various effector activities through antibody binding. Each FcR corresponds to an antibody subclass, with IgG, IgE, IgA, and IgM specifically binding to FcγR, FcεR, FcαR, and FcμR, respectively. Furthermore, FcγRs have subtypes: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). Within each subtype, there are isotypes: FcγRIA, FcγRIB, FcγRIC, FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA, and FcγRIIIB. These different FcγRs are present on different cells [Annu. Rev. Immunol. 9:457-492 (1991)]. In humans, FcγRIIIB is specifically expressed on neutrophils, while FcγRIIIA is expressed on monocytes, natural killer cells (NK cells), macrophages, and some T cells. Antibody binding to FcγRIIIA induces NK cell-dependent ADCC activity.

[0297] CDC activity refers to the ability of antibodies bound to antigens on target cells to kill target cells by initiating a series of cascade reactions (complement activation pathway) consisting of complement-related proteins in the blood. Furthermore, protein fragments generated by complement activation induce chemotaxis and activation of immune cells. The CDC activity cascade begins with C1q binding to the Fc region, followed by binding to two serine proteases, C1r and C1s, to form the C1 complex.

[0298] The CDC activity or ADCC activity of the bispecific antibody of the present invention or the bispecific antibody fragment against antigen-expressing cells can be evaluated by a known measurement method [Cancer Immunol. Immunother., 36, 373 (1993)].

[0299] Known methods for controlling the effector activity of the bispecific antibody of the present invention include methods for controlling the amount of fucose (also called core fucose) bound via an α-1,6 linkage to N-acetylglucosamine (GlcNAc) present at the reducing terminus of the N-linked complex sugar chain bound to asparagine (Asn) at position 297 of the antibody Fc region (constant region composed of the CH2 and CH3 domains) (International Publication Nos. 2005 / 035586, 2002 / 31140, and 00 / 61739), and methods for controlling the effector activity by modifying amino acid residues in the antibody Fc region (International Publication No. 00 / 42072).

[0300] By controlling the amount of fucose added to a bispecific antibody, the antibody's ADCC activity can be enhanced or reduced. For example, as a method for reducing the amount of fucose bound to the N-linked complex sugar chains bound to the Fc region of the antibody, bispecific antibodies with high ADCC activity can be obtained by expressing them in host cells deficient in the α1,6-fucosyltransferase gene. On the other hand, as a method for increasing the amount of fucose bound to the N-linked complex sugar chains bound to the Fc region of the bispecific antibody, bispecific antibodies with low ADCC activity can be obtained by expressing them in host cells into which the α1,6-fucosyltransferase gene has been introduced.

[0301] In addition, by changing the amino acid residues in the Fc region of the bispecific antibody, ADCC activity or CDC activity can be increased or decreased. For example, by using the amino acid sequence of the Fc region described in U.S. Patent Application Publication No. 2007 / 0148165, the CDC activity of the bispecific antibody can be increased. In addition, by performing amino acid changes described in U.S. Patent No. 6,737,056, U.S. Patent No. 7,297,775 or U.S. Patent No. 7,317,091, etc., ADCC activity or CDC activity can also be increased or decreased.

[0302] Furthermore, by combining the above-mentioned methods, bispecific antibodies with controlled effector activity can also be obtained.

[0303] The stability of the bispecific antibodies of the present invention can be evaluated by measuring the amount of aggregates (oligomers) formed in a sample during the purification process or stored under certain conditions. That is, a decrease in the amount of aggregates under the same conditions is evaluated as an improvement in antibody stability. The amount of aggregates can be measured by separating aggregated antibodies from non-aggregated antibodies using an appropriate chromatographic method, including gel filtration chromatography.

[0304] The productivity of the bispecific antibodies of the present invention can be evaluated by measuring the amount of antibody produced in the culture medium by antibody-producing cells. More specifically, it can be evaluated by measuring the amount of antibody contained in the culture supernatant after removing the producing cells from the culture medium using an appropriate method such as HPLC or ELISA.

[0305] In the present invention, an antibody fragment is a protein that contains an antigen-binding domain and has binding activity against the antigen. Examples of antibody fragments include Fab, Fab', F(ab')2, scFv, diabody, dsFv, and peptides containing CDRs.

[0306] Fab is an antibody fragment obtained by treating an IgG antibody with the protease papain (in which amino acid residue 224 of the H chain is cleaved). Approximately half of the N-terminal H chain is bound to the entire L chain via a disulfide bond (SS bond). It has a molecular weight of approximately 50,000 and exhibits antigen-binding activity.

[0307] F(ab')2 is an antibody fragment obtained by treating IgG with the protease pepsin (amino acid residue 234 of the H chain is cleaved). It is slightly larger than the Fab fragment bound via the S-S bond of the hinge region and has a molecular weight of approximately 100,000 and antigen-binding activity.

[0308] Fab' is an antibody fragment having antigen-binding activity and a molecular weight of approximately 50,000, obtained by cleaving the S-S bond of the hinge region of the aforementioned F(ab')2.

[0309] scFv is an antibody fragment having antigen-binding activity, which is a VH-P-VL or VL-P-VH polypeptide obtained by linking one VH and one VL using an appropriate peptide linker (P) of 12 residues or longer.

[0310] Diabodies are antibody fragments formed by dimers of scFvs with the same or different antigen-binding specificities, and have bivalent antigen-binding activity against the same antigen, or have antigen-binding activity specific for different antigens.

[0311] dsFv is a fragment in which one amino acid residue each in VH and VL is substituted with a cysteine ​​residue, and the polypeptides are linked via an SS bond between the cysteine ​​residues.

[0312] The peptide comprising CDR comprises at least one region of the CDR of VH or VL and is constituted. The peptide comprising multiple CDRs can be produced by combining the CDRs directly or via an appropriate peptide linker. The peptide comprising CDR can be produced by constructing a DNA encoding the CDRs of the VH and VL of the bispecific antibody of the present invention, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and importing the expression vector into a prokaryotic or eukaryotic organism to express and manufacture the peptide. In addition, the peptide comprising CDR can also be produced by chemical synthesis methods such as the Fmoc method or the tBoc method.

[0313] The bispecific antibody fragment of the present invention is essentially a protein composed of a portion of the bispecific antibody structure, comprising two antigen-binding domains with different antigen-binding specificities of the bispecific antibody, and having binding activity to both antigens.

[0314] Examples of the bispecific antibody fragments of the present invention include Figure 12 Fab-type bispecific antibody fragment shown in (B), Figure 12 (C) shows the F(ab')2 type bispecific antibody fragment.

[0315] Fc regions containing amino acid residue modifications for the purpose of enhancing or abolishing antibody effector activity, stabilizing antibodies, and controlling blood half-life can also be used in the bispecific antibodies of the present invention.

[0316] The bispecific antibodies or bispecific antibody fragments of the present invention include antibody derivatives obtained by chemically or genetically engineering the bispecific antibodies or bispecific antibody fragments thereof by binding radioactive isotopes, low molecular weight drugs, high molecular weight drugs, proteins, or antibody drugs.

[0317] The antibody derivatives of the present invention can be produced by chemical methods [Introduction to Antibody Engineering, Diren Shuguan (1994)] by binding radioactive isotopes, low-molecular-weight drugs, high-molecular-weight drugs, immunostimulants, proteins, or antibody drugs to the N-terminal or C-terminal side of the H chain or L chain of the bispecific antibody of the present invention or its bispecific antibody fragment, to appropriate substituents or side chains in the bispecific antibody or its bispecific antibody fragment, or to sugar chains in the bispecific antibody or its bispecific antibody fragment.

[0318] Furthermore, the antibody derivatives of the present invention can be produced by genetic engineering methods by ligating DNA encoding the bispecific antibody or bispecific antibody fragment of the present invention to DNA encoding a desired protein or antibody pharmaceutical, inserting the DNA into an expression vector, and introducing the expression vector into an appropriate host cell for expression.

[0319] Examples of radioisotopes include 111 In, 131 I. 125 I. 90 Y. 64 Cu, 99 Tc, 77 Lu or 211 At, etc. Radioactive isotopes can be directly bound to antibodies using the chloramine T method, etc. Alternatively, a substance that chelates radioactive isotopes can be bound to the antibody. Examples of chelating agents include 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA).

[0320] Examples of low-molecular-weight drugs include anticancer agents such as alkylating agents, nitrosoureas, metabolic antagonists, antibiotics, plant alkaloids, topoisomerase inhibitors, hormone therapy agents, hormone antagonists, aromatase inhibitors, P-glycoprotein inhibitors, platinum coordination compound inducers, M phase inhibitors, or kinase inhibitors [Clinical Oncology, Cancer and Chemotherapy Society (1996)], steroid agents such as hydrocortisone and prednisone, non-steroidal agents such as aspirin and indomethacin, immunomodulators such as gold thiomalate and penicillamine, immunosuppressants such as cyclophosphamide and azathioprine, and anti-inflammatory agents such as antihistamines such as chlorpheniramine maleate and clemastine [Inflammation and Anti-Inflammation Therapy, Ichiyaku Publishing Co., Ltd. (1982)].

[0321] Examples of anticancer agents include amifostine (ETHYOL), cisplatin, dacarbazine (DTIC), actinomycin, dichloromethane (nitrogen mustard,Nitrogen Mustard), streptozotocin, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (Adriamycin), epirubicin, gemcitabine (Gemzar), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, 5-fluorouracil, fluorouracil, vinblastine, vincristine, bleomycin, daunorubicin, peplomycin, estramustine, paclitaxel (Taxol), docetaxel (Taxotere), aldesleukin, asparaginase, busulfan, carboplatin, oxaliplatin, nedaplatin, cladribine, camptothecin, 10-hydroxy-7-ethyl-camptothecin (SN38), floxuridine, fludarabine, hydroxyurea, idarubicin, mesna, irinotecan (CP T-11), nogitecan, mitoxantrone, topotecan, leuprolide, megestrol acetate, melphalan, mercaptopurine, hydroxyurea, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, tamoxifen, goserelin, leuprolide, flutamide, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, hydrocortisone, prednisolone, methylprednisolone, vindesine, nimustine, semustine, capecitabine, touide, azacytidine, UFT, oxaliplatin, gefitinib (Iressa), imatinib (STI571), erlotinib, FMS-like tyrosine kinase 3 (Flt3) inhibitors, vascular endothelial growth factor receptor (vascular endothelial growth factor receptor (VEGFR) inhibitors, fibroblast growth factor receptor (FGFR) inhibitors, epidermal growth factor receptor (EGFR) inhibitors such as Tarceva, radicicol, 17-allylamino-17-demethoxygeldanamycin, rapamycin, amsacrine, all-trans retinoic acid, thalidomide, lenalidomide, anastrozole, faldrozole, letrozole, exemestane, bucillamine, mizoribine, cyclosporine, hydrocortisone, bexarotene (tatagliptin), dexamethasone, progestins, estrogen class, anastrozole (Arimidex), leupirin, aspirin, indomethacin, celecoxib, azathioprine, penicillamine, gold thiomalate, chlorpheniramine maleate, chlorpheniramine, clemastine, tretinoin, bexarotene, arsenic, bortezomib, allopurinol, calicheamicin, ibritumomab tiuxetan, tatagliptin, ozogamicin, clarithromycin, folinic acid, ketoconazole, aminoglutethimide, suramin, methotrexate or maytansinol or its derivatives, etc.

[0322] Examples of methods for binding a low-molecular-weight drug to the bispecific antibody of the present invention include a method of binding the drug to an amino group of the antibody via glutaraldehyde, and a method of binding the amino group of the drug to a carboxyl group of the antibody via a water-soluble carbodiimide.

[0323] Examples of high molecular weight drugs include polyethylene glycol (PEG), albumin, dextran, polyethylene oxide, styrene-maleic acid copolymers, polyvinyl pyrrolidone, pyran copolymers, and hydroxypropyl methacrylamide. By binding these high molecular weight compounds to the bispecific antibodies or bispecific antibody fragments of the present invention, effects such as (1) improved stability against various chemical, physical, or biological factors, (2) significant extension of blood half-life, and (3) elimination of immunogenicity or suppression of antibody production can be expected [Bioconjugate Pharmaceuticals, Hirokawa Shoten (1993)].

[0324] For example, as a method for conjugating PEG to the bispecific antibody of the present invention, a method of reacting with a PEGylation modification agent can be mentioned [Bioconjugate Pharmaceuticals, Hirokawa Shoten (1993)]. Examples of PEGylation modification agents include modifiers for the ε-amino group of lysine (Japanese Patent Application Laid-Open No. 61-178926), modifiers for the carboxyl groups of aspartic acid and glutamic acid (Japanese Patent Application Laid-Open No. 56-23587), and modifiers for the guanidinium group of arginine (Japanese Patent Application Laid-Open No. 2-117920).

[0325] The immunostimulant may be a natural product known as an immunoadjuvant. Specific examples include immunostimulating agents such as β(1→3) glucan (e.g., lentinan or schizophyllan) or α-galactosylceramide (KRN7000).

[0326] Examples of the protein include cytokines or growth factors that activate immunocompetent cells such as NK cells, macrophages, and neutrophils, and toxin proteins.

[0327] Examples of cytokines or growth factors include interferon (hereinafter referred to as IFN)-α, IFN-β, IFN-γ, interleukin (hereinafter referred to as IL)-2, IL-12, IL-15, IL-18, IL-21, IL-23, granulocyte colony-stimulating factor (G-CSF), granulocyte / macrophage colony-stimulating factor (GM-CSF), and macrophage colony-stimulating factor (M-CSF).

[0328] Examples of toxin proteins include ricin, diphtheria toxin, and ONTAK, and also include protein toxins in which mutations have been introduced to adjust toxicity.

[0329] Fusion antibodies with proteins or antibody drugs can be produced by ligating a cDNA encoding the protein to a cDNA encoding the bispecific antibody or bispecific antibody fragment of the present invention to construct a DNA encoding the fusion antibody, inserting the DNA into an expression vector for prokaryotes or eukaryotes, and introducing the expression vector into the prokaryote or eukaryote to express the antibody.

[0330] When using derivatives of the above antibodies as detection methods, quantitative methods, detection reagents, quantitative reagents or diagnostic drugs, the agents that bind to the bispecific antibodies of the present invention or their bispecific antibody fragments include conventional immunological detection or assay labels. As labels, for example, enzymes such as alkaline phosphatase, peroxidase or luciferase, luminescent substances such as acridinium esters or rofenine, or fluorescent substances such as fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (RITC), Alexa (registered trademark) Fluor 488, R-phycoerythrin (R-PE) and the like can be cited.

[0331] The present invention includes bispecific antibodies and bispecific antibody fragments thereof having cytotoxic activity such as CDC activity or ADCC activity. The CDC activity or ADCC activity of the bispecific antibodies or bispecific antibody fragments of the present invention against antigen-expressing cells can be evaluated by known assay methods [Cancer Immunol. Immunother., 36, 373 (1993)].

[0332] The present invention also relates to a composition comprising a bispecific antibody or a bispecific antibody fragment thereof that specifically recognizes and binds to CD40 and EpCAM, or a therapeutic agent for a disease associated with at least one of CD40 and EpCAM, preferably a disease associated with cells expressing CD40 and EpCAM, comprising the bispecific antibody or the bispecific antibody fragment as an active ingredient.

[0333] The disease related to at least one of CD40 and EpCAM may be any disease, for example, malignant tumors and cancer.

[0334] In the present invention, examples of malignant tumors and cancers include colon cancer, colorectal cancer, lung cancer, breast cancer, glioma, malignant melanoma (melanoma), thyroid cancer, renal cell carcinoma, leukemia, lymphoma, T-cell lymphoma, gastric cancer, pancreatic cancer, cervical cancer, endometrial cancer, ovarian cancer, bile duct cancer, esophageal cancer, liver cancer, head and neck cancer, skin cancer, urethral cancer, bladder cancer, prostate cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer, mesothelioma, pleural tumor, male embryonal tumor, endometrial hyperplasia, endometriosis, embryonal tumor, fibrosarcoma, Kaposi's sarcoma, hemangioma, cavernous hemangioma, hemangioblastoma, retinoblastoma, astrocytoma, neurofibroma, oligodendroglioma, medulloblastoma, neuroblastoma, glioma, rhabdomyosarcoma, glioblastoma, osteosarcoma, leiomyosarcoma, and Wilms' tumor.

[0335] Therapeutic drugs comprising the bispecific antibodies or bispecific antibody fragments of the present invention, or derivatives thereof, may contain only the antibodies or bispecific antibody fragments or derivatives thereof as active ingredients, but are generally preferably provided as pharmaceutical preparations prepared by any method known in the art of pharmaceutical preparations, mixed with one or more pharmacologically acceptable carriers.

[0336] The route of administration is preferably the most effective route for treatment, and examples thereof include oral administration, or parenteral administration such as oral, intrarespiratory, rectal, subcutaneous, intramuscular, or intravenous administration. Among them, intravenous administration is preferred.

[0337] Examples of the administration form include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, and patches.

[0338] The dosage or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc., but is generally 10 μg / kg to 10 mg / kg per day for adults.

[0339] Furthermore, the present invention relates to a reagent for immunologically detecting or measuring at least one of CD40 and EpCAM, comprising the bispecific antibody of the present invention or a bispecific antibody fragment thereof, or a diagnostic agent for a disease associated with at least one of CD40 and EpCAM, preferably a disease associated with cells expressing CD40 and EpCAM. Furthermore, the present invention relates to a method for immunologically detecting or measuring at least one of CD40 and EpCAM, using the bispecific antibody of the present invention or a bispecific antibody fragment thereof, a method for treating a disease associated with at least one of CD40 and EpCAM, preferably a disease associated with cells expressing CD40 and EpCAM, or a method for diagnosing a disease associated with at least one of CD40 and EpCAM, preferably a disease associated with cells expressing CD40 and EpCAM.

[0340] In the present invention, any known method can be used as a method for detecting or measuring the amount of at least one of CD40 and EpCAM, for example, immunological detection or measurement methods.

[0341] Immunological detection or determination methods refer to methods for detecting or determining the amount of antibodies or antigens using labeled antigens or antibodies. Examples of immunological detection or determination methods include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, Western blotting, and physicochemical methods.

[0342] By detecting or measuring cells expressing at least one of CD40 and EpCAM using the bispecific antibody of the present invention or the bispecific antibody fragment thereof, diseases associated with at least one of CD40 and EpCAM, preferably diseases associated with cells expressing CD40 and EpCAM, can be diagnosed.

[0343] Known immunological detection methods can be used to detect cells expressing at least one of CD40 and EpCAM, and examples thereof include immunoprecipitation, immunocytostaining, immunohistostaining, and fluorescent antibody staining. Furthermore, examples thereof include fluorescent antibody staining methods such as the FMAT8100 HTS system (manufactured by Applied Biosystems).

[0344] In the present invention, biological samples that are the subject of detection or measurement of at least one of CD40 and EpCAM include, for example, tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium, and are not particularly limited as long as they are samples that are likely to contain cells expressing at least one of CD40 and EpCAM.

[0345] Diagnostic drugs containing the bispecific antibodies of the present invention, or bispecific antibody fragments thereof, or derivatives thereof, may contain reagents for antigen-antibody reactions and reagents for detecting such reactions, depending on the intended diagnostic method. Examples of reagents for antigen-antibody reactions include buffers and salts.

[0346] Examples of detection reagents include labeled secondary antibodies capable of binding to the aforementioned bispecific antibodies, bispecific antibody fragments, or derivatives thereof, and substrates corresponding to the labels, which are reagents used in conventional immunological detection or measurement methods.

[0347] The following specifically describes methods for producing the bispecific antibodies of the present invention, methods for evaluating the activity of the bispecific antibodies or bispecific antibody fragments, and methods for treating and diagnosing diseases using the bispecific antibodies or bispecific antibody fragments.

[0348] 1. Method for producing monoclonal antibodies

[0349] The method for producing a monoclonal antibody of the present invention includes the following steps: (1) purifying at least one of an antigen used as an immunogen and producing cells that overexpress the antigen on their cell surfaces; (2) after immunizing an animal with the antigen, collecting blood, testing its antibody titer, determining the time for spleen removal, and preparing antibody-producing cells; (3) preparing myeloma cells; (4) fusing the antibody-producing cells with myeloma cells; (5) screening a hybridoma population that produces the target antibody; (6) isolating monoclonal cells (clones) from the hybridoma population; (7) culturing hybridomas for mass production of monoclonal antibodies, or raising animals transplanted with hybridomas, as appropriate; and (8) studying the physiological activity and antigen-binding specificity of the monoclonal antibodies thus produced, or examining their properties as labeling agents.

[0350] The following describes in detail the methods for producing monoclonal antibodies that bind to CD40 and monoclonal antibodies that bind to EpCAM, used in the bispecific antibodies that bind to CD40 and EpCAM of the present invention, according to the aforementioned steps. The methods for producing these antibodies are not limited thereto; for example, antibody-producing cells other than spleen cells and myeloma cells may also be used.

[0351] (1) Antigen purification

[0352] Cells expressing at least one of CD40 and EpCAM can be obtained by introducing an expression vector containing a cDNA encoding the full length or partial length of at least one of CD40 and EpCAM into Escherichia coli, yeast, insect cells, or animal cells. Alternatively, at least one of CD40 and EpCAM can be purified from various human tumor cultured cells or human tissues that express at least one of CD40 and EpCAM in large quantities and used as an antigen. Alternatively, the tumor cultured cells or tissues can be used directly as an antigen. Furthermore, synthetic peptides containing a partial sequence of at least one of CD40 and EpCAM can be prepared by chemical synthesis methods such as the Fmoc method or the tBoc method and used as antigens.

[0353] At least one of CD40 and EpCAM used in the present invention can be produced by expressing DNA encoding at least one of CD40 and EpCAM in host cells using the methods described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) or Current Protocols In Molecular Biology, John Wiley & Sons (1987-1997), for example, by the following method.

[0354] A recombinant vector is prepared by inserting a full-length cDNA containing a portion encoding at least one of CD40 and EpCAM downstream of a promoter in an appropriate expression vector. Alternatively, a DNA fragment of appropriate length containing a portion encoding a polypeptide, prepared from the full-length cDNA, can be used instead. The resulting recombinant vector is then introduced into a host cell suitable for the expression vector, thereby obtaining a transformant that produces at least one of CD40 and EpCAM.

[0355] Any expression vector can be used as long as it can autonomously replicate or integrate into the chromosome in the host cell used and contains an appropriate promoter at a position capable of transcribing DNA encoding at least one of CD40 and EpCAM.

[0356] As the host cell, any cell can be used as long as it is a microorganism belonging to the genus Escherichia such as Escherichia coli, yeast, insect cells, or animal cells that can express the target gene.

[0357] When using a prokaryotic organism such as Escherichia coli as a host cell, the recombinant vector is preferably capable of autonomous replication in the prokaryotic organism and comprises a promoter, a ribosome binding sequence, a DNA encoding at least one of CD40 and EpCAM, and a transcription termination sequence. While the transcription termination sequence is not necessarily present in the recombinant vector, it is preferably located immediately below the structural gene. Furthermore, the recombinant vector may also comprise a gene that controls the promoter.

[0358] As the recombinant vector, a plasmid in which the distance between the Shine Dalgarno sequence, which is a ribosome binding sequence, and the start codon is adjusted to an appropriate distance (for example, 6 to 18 bases) is preferably used.

[0359] Furthermore, the base sequence of the DNA encoding at least one of CD40 and EpCAM can be substituted with codons that are optimal for expression in a host, thereby improving the productivity of at least one of the target CD40 and EpCAM.

[0360] As the expression vector, any vector can be used as long as it can function in the host cell used, and examples thereof include pBTrp2, pBTac1, pBTac2 (all manufactured by Roche Diagnostics), pKK233-2 (manufactured by Pharmacia), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by Qiagen), pKYP10 (Japanese Patent Application Laid-Open No. 58-110600), pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], and pBluescript II. SK(-) (manufactured by Stratagene), pTrs30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrs32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pGHA2 [prepared from Escherichia coli IGHA2 (FERM BP-400), JP-A-60-221091], pGKA2 [prepared from Escherichia coli IGKA2 (FERM BP-6798), Japanese Patent Application Laid-Open No. 60-221091], pTerm2 (U.S. Patent No. 4,686,191, U.S. Patent No. 4,939,094, U.S. Patent No. 5,160,735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (manufactured by Pharmacia), pET system (manufactured by Novagen), or pME18SFL3 (manufactured by Toyobo Co., Ltd.), etc.

[0361] As a promoter, any promoter may be used as long as it can function in the host cell used. For example, promoters derived from Escherichia coli or bacteriophages such as the trp promoter (Ptrp), the lac promoter, the PL promoter, the PR promoter, or the T7 promoter may be mentioned. In addition, for example, promoters artificially designed and modified such as a tandem promoter formed by connecting two Ptrp promoters in series, the tac promoter, the lacT7 promoter, or the let I promoter may be mentioned.

[0362] Examples of host cells include Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No. 49, Escherichia coli W3110, Escherichia coli NY49, and Escherichia coli DH5α.

[0363] As a method for introducing a recombinant vector into a host cell, any method can be used as long as it is a method for introducing DNA into the host cell to be used, and examples thereof include methods using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)].

[0364] When animal cells are used as hosts, any expression vector can be used as long as it can function in animal cells. Examples thereof include pcDNAI (manufactured by Invitrogen), pcDM8 (manufactured by Funakoshi Co., Ltd.), pAGE107 [Japanese Patent Application Publication No. 3-22979; Cytotechnology, 3, 133 (1990)], pAS3-3 (Japanese Patent Application Publication No. 2-227075), pCDM8 [Nature, 329, 840 (1987)], pcDNAI / Amp (manufactured by Invitrogen), pcDNA3.1 (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 [J. Biochemistry, 101, 1307 (1987)], pAGE210, pME18SFL3, and pKANTEX93 (International Publication No. 97 / 10354).

[0365] As a promoter, any promoter can be used as long as it can function in animal cells. Examples include the promoter of the immediate early (IE) gene of cytomegalovirus (CMV), the early promoter of SV40, a retroviral promoter, a metallothionein promoter, a heat shock promoter, the SRα promoter, or a promoter or enhancer of Moloney murine leukemia virus. In addition, the enhancer of the IE gene of human CMV can also be used together with the promoter.

[0366] Examples of host cells include human Burkitt's lymphoma Namalwa cells, African green monkey kidney-derived COS cells, Chinese hamster ovary-derived CHO cells, and human leukemia cells HBT5637 (Japanese Patent Application Laid-Open No. 63-000299).

[0367] As a method for introducing a recombinant vector into a host cell, any method can be used as long as it is a method for introducing DNA into animal cells, for example, electroporation [Cytotechnology, 3, 133 (1990)], the calcium phosphate method (Japanese Patent Application Publication No. 2-227075), or lipofection [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] and the like can be mentioned.

[0368] At least one of CD40 and EpCAM can be produced by culturing a transformant derived from a microorganism or animal cell, etc., which harbors a recombinant vector introduced with a DNA encoding at least one of CD40 and EpCAM, in a culture medium, producing and accumulating the at least one of CD40 and EpCAM in the culture medium, and collecting the transformed cells from the culture medium. Culturing the transformant in a culture medium can be carried out according to conventional methods used for culturing hosts.

[0369] When expressed in cells derived from eukaryotic organisms, at least one of CD40 and EpCAM to which sugars or sugar chains are added can be obtained.

[0370] When culturing microorganisms transformed with a recombinant vector using an inducible promoter, an inducer can be added to the culture medium as needed. For example, when culturing microorganisms transformed with a recombinant vector using the lac promoter, isopropyl-β-D-thiogalactoside can be added to the culture medium, and when culturing microorganisms transformed with a recombinant vector using the trp promoter, indoleacrylic acid can be added to the culture medium.

[0371] Examples of culture media for culturing transformants obtained using animal cells as hosts include commonly used RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)], 199 medium [Proc. Soc. Exp. Biol. Med., 73, 1 (1950)], Iscove's Modified Dulbecco's Medium (IMDM) medium, or these mediums supplemented with fetal bovine serum (FBS). Culture is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40°C, and in the presence of 5% CO2. Furthermore, antibiotics such as kanamycin and penicillin may be added to the culture medium as needed during the culture.

[0372] As methods for expressing genes encoding at least one of CD40 and EpCAM, in addition to direct expression, methods such as secretory production or fusion protein expression can be used [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)]. Examples of methods for producing at least one of CD40 and EpCAM include production within host cells, secretion outside the host cells, and production on the outer membrane of the host cells. The appropriate method can be selected by modifying the host cells used or the structure of at least one of CD40 and EpCAM produced.

[0373] For example, antigen fusion proteins can be produced by preparing DNA encoding the amino acid sequence of the extracellular region, linking DNA encoding the Fc region of an antibody, DNA encoding glutathione S-transferase (GST), DNA encoding a FLAG tag, or DNA encoding a Histidine tag, and expressing and purifying the DNA. Specific examples include an Fc fusion protein in which the extracellular region of at least one of CD40 and EpCAM is bound to the Fc region of human IgG, and a fusion protein of at least one of the extracellular region of CD40 and EpCAM with glutathione S-transferase (GST).

[0374] When at least one of CD40 and EpCAM is produced within host cells or on the outer membrane of host cells, at least one of CD40 and EpCAM can be actively secreted outside the host cells by using the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], the method of Law et al. [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], Japanese Patent Application Publication No. 05-336963, or International Publication No. 94 / 23021. Furthermore, the production of at least one of CD40 and EpCAM can be increased by using a gene amplification system using the dihydrofolate reductase gene (Japanese Patent Application Publication No. 2-227075).

[0375] At least one of the produced CD40 and EpCAM can be isolated and purified, for example, as follows.

[0376] When at least one of CD40 and EpCAM is expressed in a soluble state in the cells, after the culture is completed, the cells are recovered by centrifugation, suspended in an aqueous buffer, and then disrupted using an ultrasonic disruptor, French press, Menton Gaulin homogenizer, or bead mill (DYNO-MILL) to obtain a cell-free extract. From the supernatant obtained by centrifugation of the cell-free extract, a purified protein can be obtained by using, alone or in combination, conventional protein isolation and purification methods, namely, solvent extraction, salting-out using ammonium sulfate or the like, desalting, precipitation using an organic solvent, anion exchange chromatography using a resin such as diethylaminoethyl (DEAE)-Sepharose or DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), cation exchange chromatography using a resin such as S-Sepharose FF (manufactured by Pharmacia), hydrophobic chromatography using a resin such as butyl sepharose or phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, or electrophoresis such as isoelectric point electrophoresis.

[0377] When at least one of CD40 and EpCAM is expressed as an insoluble form within the cells, the cells are recovered and disrupted, followed by centrifugation, as described above, to recover the insoluble form of at least one of CD40 and EpCAM as a precipitate. The recovered insoluble form of at least one of CD40 and EpCAM is solubilized using a protein denaturant. The solubilized solution is diluted or dialyzed to restore the normal three-dimensional structure of at least one of CD40 and EpCAM. Purified polypeptide protein can then be obtained using the same separation and purification methods as described above.

[0378] When at least one of CD40 and EpCAM, or derivatives thereof such as glycomodified forms, is secreted extracellularly by cells, the at least one of CD40 and EpCAM, or derivatives thereof such as glycomodified forms, can be recovered in the culture supernatant. The culture supernatant can be treated by centrifugation or other methods as described above to obtain a soluble fraction, and the purified protein can be obtained from the soluble fraction using the same separation and purification methods as described above.

[0379] Furthermore, at least one of CD40 and EpCAM used in the present invention can also be produced by chemical synthesis methods such as the Fmoc method or the tBoc method. Specifically, chemical synthesis can be performed using peptide synthesizers such as those manufactured by Devanst Chemtech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, Perceptive, or Shimadzu Corporation.

[0380] (2) Preparation process of antibody-producing cells

[0381] An animal such as a mouse, rat, or hamster aged 3 to 20 weeks is immunized with the antigen obtained in (1), and antibody-producing cells are collected from the spleen, lymph nodes, or peripheral blood of the animal. Examples of the animal to be immunized include transgenic mice producing human-derived antibodies as described in Tomizuka et al. [Proc Natl Acad Sci USA, 97, 722, (2000)], and conditional knockout mice for CD40 or EpCAM to enhance immunogenicity.

[0382] Immunization is performed by administering the antigen together with a suitable adjuvant such as Freund's complete adjuvant, aluminum hydroxide gel, or pertussis vaccine. Immunogen administration for mouse immunization can be any method such as subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, intramuscular injection, or plantar injection, with intraperitoneal injection, plantar injection, or intravenous injection being preferred. When the antigen is a partial peptide, a complex with a carrier protein such as BSA (bovine serum albumin) or KLH (Keyhole Limpet hemocyanin) is prepared and used as the immunogen.

[0383] After the first administration, the antigen is administered 5 to 10 times at intervals of 1 to 2 weeks. Blood is collected from the venous plexus of the retina 3 to 7 days after each administration, and the antibody titer of the serum is measured using enzyme immunoassay [Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. If animals whose serum exhibits sufficient antibody titers against the antigen used in immunization are used as a source of cells producing antibodies for fusion, the effectiveness of subsequent operations can be improved.

[0384] Three to seven days after the final administration of the antigen, tissues containing antibody-producing cells, such as the spleen, are removed from the immunized animal to collect the antibody-producing cells. Antibody-producing cells are plasma cells and lymphocytes, which are their precursors. These cells can be obtained from any part of the individual, typically from the spleen, lymph nodes, bone marrow, tonsils, peripheral blood, or an appropriate combination thereof. Spleen cells are most commonly used. When using spleen cells, the spleen is minced and centrifuged to remove red blood cells, thereby obtaining antibody-producing cells for fusion.

[0385] (3) Myeloma preparation process

[0386] As myeloma cells, cells without the ability to produce self-antibodies can be used that are derived from mammals such as mice, rats, guinea pigs, hamsters, rabbits, or humans. In general, cell lines derived from mice are used, for example, the 8-azaguanine-resistant mouse (BALB / c-derived) myeloma cell line P3-X63Ag8-U1 (P3-U1) [Current Topics in Microbiology and Immunology, 18, 1 (1978)], P3-NS1 / 1-Ag41 (NS-1) [European J. Immunology, 6, 511 (1976)], SP2 / 0-Ag14 (SP-2) [Nature, 276, 269 (1978)], P3-X63-Ag86 53 (653) [J. Immunology, 123, 1548 (1979)] or P3-X63-Ag8 (X63) [Nature, 256, 495 (1975)], etc. The cell line is subcultured in an appropriate medium, such as 8-azaguanine medium [RPMI-1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, FCS, and 8-azaguanine], Iscove's Modified Dulbecco's Medium (hereinafter referred to as "IMDM"), or Dulbecco's Modified Eagle Medium (hereinafter referred to as "DMEM"). 3 to 4 days before cell fusion, the above cell line is subcultured in a normal medium (for example, DMEM medium containing 10% FCS), and on the day of fusion, 2×10 7 More than the number of cells.

[0387] (4) Cell fusion

[0388] The antibody-producing cells for fusion obtained in (2) and the myeloma cells obtained in (3) were thoroughly washed with Minimum Essential Medium (MEM) or PBS (1.83 g disodium phosphate, 0.21 g monopotassium phosphate, 7.65 g sodium chloride, 1 liter distilled water, pH 7.2), mixed at a ratio of 5:1 to 10:1 for antibody-producing cells for fusion: myeloma cells, and centrifuged. The supernatant was removed. After the precipitated cell aggregates were thoroughly dispersed, a mixture of polyethylene glycol-1000 (PEG-1000), MEM medium, and dimethyl sulfoxide was added while stirring at 37°C. 1 to 2 mL of MEM medium was then added every 1 to 2 minutes. After this was repeated several times, MEM medium was added to a total volume of 50 mL. After centrifugation, the supernatant was removed, the precipitated cell aggregates were gradually dispersed, and the cells were slowly suspended in HAT medium (normal medium supplemented with hypoxanthine, thymidine, and aminopterin). The suspension was cultured in a 5% CO 2 incubator at 37° C. for 7 to 14 days.

[0389] Alternatively, cell fusion can be performed using the following method. Spleen cells and myeloma cells are thoroughly washed with serum-free medium (e.g., DMEM) or phosphate-buffered saline (hereinafter referred to as "phosphate buffer"), mixed to a spleen cell to myeloma cell ratio of approximately 5:1 to 10:1, and centrifuged. The supernatant is removed, and after the precipitated cell aggregates are thoroughly dispersed, 1 mL of serum-free medium containing 50% (w / v) polyethylene glycol (molecular weight 1000-4000) is added dropwise while stirring. Subsequently, 10 mL of serum-free medium is slowly added, followed by centrifugation. The supernatant is again discarded, and the precipitated cells are suspended in an appropriate amount of normal culture medium containing hypoxanthine, aminopterin, and thymidine (HAT) solution and human interleukin-2 (IL-2) (hereinafter referred to as HAT medium). The cells are dispensed into each well of a culture dish (hereinafter referred to as a culture plate) and cultured at 37°C in the presence of 5% carbon dioxide for approximately two weeks. HAT medium is replenished as appropriate during the process.

[0390] (5) Selection of hybridoma groups

[0391] In the case where the myeloma cell used in fusion is 8-azaguanine resistant strain, that is, in the case of hypoxanthine guanine phosphoribosyltransferase (HGPRT) defective strain, the myeloma cell that does not have fusion and the fusion cell of myeloma cell each other can not survive in HAT culture medium.On the other hand, the fusion cell of antibody-producing cell each other and the hybridoma of antibody-producing cell and myeloma cell can survive in HAT culture medium, but the fusion cell of antibody-producing cell each other will not just reach lifespan.Therefore, by continuing the cultivation in HAT culture medium, only the hybridoma of antibody-producing cell and myeloma cell will be left to survive, and as a result, hybridoma can be obtained.

[0392] About the hybridoma that grows into colony shape, HAT culture medium is replaced with the culture medium (hereinafter referred to as HT culture medium) that has removed aminopterin.Then, gather a part for culture supernatant, use the antibody titer determination method described later, can select the hybridoma that produces antibody.As the assay method of antibody titer, for example, can enumerate various known technologies such as radioisotope immunoassay (RIA method), solid phase enzyme immunoassay (ELISA method), fluorescent antibody method and passive hemagglutination reaction method, from the viewpoint of the possibility etc. of detection sensitivity, rapidity, accuracy and operation automation, preferably RIA method or ELISA method.

[0393] Hybridomas that have been determined to produce the desired antibody by measuring the antibody titer are transferred to another culture plate for cloning. Examples of such cloning methods include the limiting dilution method, which involves diluting and culturing the cells so that each well of the culture plate contains a single cell; the soft agar method, which involves culturing the cells in a soft agar medium and recovering colonies; methods for isolating a single cell using micromanipulation; and methods for isolating a single cell using a cell sorter.

[0394] For wells where antibody titers were confirmed, cloning by the limiting dilution method was repeated 2 to 4 times, and wells where antibody titers were stably confirmed were selected as hybridoma lines producing monoclonal antibodies against human CD40 or EpCAM.

[0395] (6) Preparation of monoclonal antibodies

[0396] The monoclonal antibody obtained in (5) was intraperitoneally injected into 8-10 week old mice or nude mice treated with pristane [0.5 mL of 2,6,10,14-tetramethylpentadecane (Pristane) was intraperitoneally administered and maintained for 2 weeks] to produce hybridomas. After 10-21 days, the hybridomas developed ascites cancer. Ascites was collected from the mice, centrifuged to remove the solid components, and then salted out with 40-50% ammonium sulfate. The ascites was purified by octanoic acid precipitation, DEAE-agarose gel column, protein A column or gel filtration column, and the IgG or IgM fraction was collected as purified monoclonal antibodies. In addition, by allowing the hybridoma to grow in the peritoneal cavity of mice (e.g., BALB / c) or Nu / Nu mice of the system, rats, guinea pigs, hamsters or rabbits, ascites containing a large amount of monoclonal antibodies that bind to CD40 or EpCAM can be obtained.

[0397] The monoclonal antibody-producing hybridoma obtained in (5) is cultured in RPMI1640 medium supplemented with 10% FBS, then centrifuged, the supernatant removed, and suspended in GIT medium or Hybridoma SFM medium supplemented with 5% Daigo GF21, and cultured for 3 to 7 days by flask culture, rotary culture, or inverted culture. The resulting cell suspension is centrifuged, and the resulting supernatant is purified using a protein A column or protein G column to collect the IgG fraction, thereby obtaining a purified monoclonal antibody. As a simple purification method, a commercially available monoclonal antibody purification kit (e.g., MAbTrap GII Kit; manufactured by Amersham Pharmacia Biotech) can also be used.

[0398] The subclass of the antibody was determined by enzyme immunoassay using a subclass typing kit. Protein quantity was quantified by the Lowry method and absorbance at 280 nm [1.4 (OD 280 )=immunoglobulin 1 mg / mL] was calculated using the method.

[0399] (7) Monoclonal antibody binding test to CD40 or EpCAM

[0400] The binding activity of monoclonal antibodies to CD40 or EpCAM can be measured using binding assay systems such as immunodiffusion (Ouchterlony), ELISA, RIA, flow cytometry (FCM), or surface plasmon resonance (SPR).

[0401] The immunodiffusion method is simple, but requires concentration when the antibody concentration is low. On the other hand, when using the ELISA method or RIA method, the culture supernatant can be directly reacted with the antigen-adsorbed solid phase, and antibodies corresponding to various immunoglobulin isotypes and subclasses can be used as secondary antibodies to identify the antibody isotype and subclass, and the antibody binding activity can be measured.

[0402] As a specific example of the procedure, at least one of purified or partially purified recombinant CD40 and EpCAM is adsorbed onto the solid surface of a 96-well ELISA plate, etc. The solid surface free of antigen is then blocked with an antigen-independent protein, such as bovine serum albumin (BSA). The ELISA plate is then washed with phosphate-buffered saline (PBS) and PBS containing 0.05% Tween 20 (Tween-PBS). The plate is then reacted with a first antibody (e.g., mouse serum, culture supernatant, etc.) diluted in stages to allow the antibody to bind to the antigen immobilized on the plate. An anti-immunoglobulin antibody labeled with biotin, an enzyme (horseradish peroxidase; HRP, alkaline phosphatase; ALP, etc.), a chemiluminescent substance, or a radioactive compound is then dispensed as a second antibody, allowing the second antibody to react with the first antibody bound to the plate. After thorough washing with Tween-PBS, a reaction occurs corresponding to the second antibody's labeling substance, allowing selection of a monoclonal antibody specifically reactive with the target antigen.

[0403] The binding activity of antibodies to antigen-expressing cells can be measured by FCM [Cancer Immunol. Immunother., 36, 373 (1993)]. The binding of an antibody to a membrane protein antigen expressed on the cell membrane means that the antibody recognizes the three-dimensional structure of the naturally occurring antigen and binds thereto.

[0404] As an SPR method, kinetic analysis by Biacore can be cited. For example, using Biacore T100, the kinetics of binding between the antigen and the test substance are measured, and the results are analyzed using the analysis software included with the instrument. As a specific example of the operating steps, after the anti-mouse IgG antibody is fixed to the sensor chip CM5 by the amine coupling method, the test substance such as hybridoma culture supernatant or purified monoclonal antibody is flowed to bind an appropriate amount, and then the antigen with multiple concentrations of known concentrations is flowed to measure the binding and dissociation. Then, the obtained data is subjected to kinetic analysis using a 1:1 binding model using the software included with the instrument to obtain various parameters. Alternatively, after at least one of CD40 and EpCAM is fixed to the sensor chip, for example, using the amine coupling method, the purified monoclonal antibody with multiple concentrations of known concentrations is flowed to measure the binding and dissociation. The obtained data is subjected to kinetic analysis using a bivalent binding model using the software included with the instrument to obtain various parameters.

[0405] Furthermore, in the present invention, antibodies that compete with antibodies against CD40 or EpCAM for binding to CD40 or EpCAM can be selected by reacting the test antibody in the presence of the test antibody in the aforementioned binding assay system. Specifically, by screening for antibodies that inhibit antigen binding when the test antibody is added, antibodies that compete with the antibodies obtained above for binding to CD40 or EpCAM can be obtained.

[0406] (8) Identification of epitopes of monoclonal antibodies against CD40 or EpCAM

[0407] In the present invention, identification of the epitope recognized and bound by an antibody can be performed as follows.

[0408] For example, a partial deletion of an antigen, a variant having different amino acid residues between species, or a variant having a specific structural domain is produced. If the reactivity of an antibody to the deletion or variant is reduced, the defect or amino acid change site is known to be the epitope of the antibody. Such partial deletions and variants of an antigen can be obtained as secretory proteins using appropriate host cells such as Escherichia coli, yeast, plant cells, or mammalian cells, or can be expressed on the cell membrane of a host cell and prepared as antigen-expressing cells. In the case of a membrane-type antigen, in order to express while maintaining the three-dimensional structure of the antigen, it is preferably expressed on the membrane of the host cell. In addition, synthetic peptides that mimic the primary structure or three-dimensional structure of the antigen can also be produced to confirm the reactivity of the antibody. Regarding synthetic peptides, methods for producing various partial peptides of the molecule using known peptide synthesis techniques can be cited.

[0409] For example, with respect to the extracellular region of CD40 or EpCAM of humans and mice, a chimeric protein that appropriately combines the domains constituting each region can be prepared, and the reactivity of the antibody to the protein is confirmed, thereby identifying the epitope of the antibody. Then, further subdivided, various oligopeptides of the corresponding parts or variants of the peptide are synthesized using oligopeptide synthesis techniques well known to those skilled in the art, and the reactivity of the antibody to the peptide is confirmed, thereby determining the epitope. As a simple method for obtaining a variety of oligopeptides, commercially available kits [e.g., SPOTs kit (manufactured by Genosys Biotechnologies), a series of multicenter peptide synthesis kits (manufactured by Chiron) using a multicenter synthesis method, etc.] can also be utilized.

[0410] Antibodies that bind to the same epitope as that bound by antibodies that bind to CD40 or EpCAM can be obtained by identifying the epitope of the antibody obtained by the above-mentioned binding test system, preparing a synthetic peptide that is a portion of the epitope, a synthetic peptide that mimics the three-dimensional structure of the epitope, or a recombinant of the epitope, and then immunizing with the resulting peptide.

[0411] For example, if the epitope is a membrane protein, the entire extracellular region or a portion of the extracellular domain can be linked to an appropriate tag such as a FLAG tag, a Histidine tag, a GST protein, or an antibody Fc region to produce a recombinant fusion protein, and the recombinant protein can be used for immunization, thereby more efficiently producing antibodies specific for the epitope.

[0412] 2. Production of recombinant antibodies

[0413] As examples of the preparation of genetically recombinant antibodies, there are general descriptions in PJ Delves., ANTIBODY PRODUCTION ESSENTIALTECHNIQUES., 1997 WILEY, P. Shepherd and C. Dean. Monoclonal Antibodies., 2000 OXFORD UNIVERSITY PRESS and JW Goding., Monoclonal Antibodies: principles and practice., 1993 ACADEMIC PRESS, etc., and the following shows methods for preparing chimeric antibodies, humanized antibodies and human antibodies. In addition, genetically recombinant mouse, rat, hamster and rabbit antibodies can also be prepared using the same method.

[0414] (1) Obtaining cDNA encoding the V region of a monoclonal antibody from a hybridoma

[0415] cDNAs encoding VH and VL of a monoclonal antibody can be obtained, for example, as follows.

[0416] First, mRNA is extracted from a hybridoma producing a monoclonal antibody and cDNA is synthesized. The synthesized cDNA is then cloned into a vector such as a phage or plasmid to create a cDNA library. From this library, DNA encoding the C region or V region of the antibody is used as a probe to isolate recombinant phage or recombinant plasmids harboring cDNA encoding the VH or VL, respectively. The complete base sequence of the VH or VL in the isolated recombinant phage or recombinant plasmid is determined, and the complete amino acid sequence of the VH or VL is inferred from this base sequence.

[0417] As non-human animals used for hybridoma production, mice, rats, hamsters, rabbits, etc. are used, but any animals can be used as long as hybridomas can be produced.

[0418] In order to prepare total RNA from hybridomas, a kit such as the guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol., 154, 3 (1987)] or RNA easy kit (manufactured by Qiagen) is used.

[0419] When preparing mRNA from total RNA, the oligo(dT)-immobilized cellulose column method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)] or a kit such as Oligo-dT30<Super> mRNA Purification Kit (manufactured by Takara Bio) is used. Alternatively, mRNA can be prepared using a kit such as Fast Track mRNA Isolation Kit (manufactured by Invitrogen) or QuickPrep mRNA Purification Kit (manufactured by Pharmacia).

[0420] For cDNA synthesis and cDNA library preparation, known methods [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)] or kits such as SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (manufactured by Invitrogen) or ZAP-cDNA Synthesis Kit (manufactured by Stratagene) are used.

[0421] When preparing a cDNA library, any vector can be used as long as it is a vector recombined with cDNA synthesized using mRNA extracted from a hybridoma as a template.

[0422] For example, ZAP Express [Strategies, 5, 58 (1992)], pBluescript IISK(+) [Nucleic Acids Research, 17, 9494 (1989)], λZAPII (manufactured by Stratagene), λgt10, λgt11 [DNA Cloning: A Practical Approach, 1, 49 (1985)], LambdaBlueMid (manufactured by Clontech), λExCell, pT7T3-18U (manufactured by Pharmacia), pcD2 [Mol. Cell. Biol., 3, 280 (1983)], or pUC18 [Gene, 33, 103 (1985)] can be used.

[0423] As the E. coli for introducing a cDNA library constructed using a phage or plasmid vector, any E. coli capable of introducing, expressing, and maintaining the cDNA library can be used, for example, XL1-Blue MRF' [Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science, 222, 778 (1983)], NM522 [J. Mol. Biol., 166, 1 (1983)], K802 [J. Mol. Biol., 16, 118 (1966)], or JM105 [Gene, 38, 275 (1985)].

[0424] When selecting cDNA clones encoding VH or VL of non-human antibodies from a cDNA library, colony hybridization or plaque hybridization using isotope- or fluorescently labeled probes [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)] is used.

[0425] Alternatively, cDNA encoding VH or VL can be prepared by preparing primers and performing the polymerase chain reaction method [hereinafter referred to as PCR method, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)] using cDNA synthesized from mRNA or a cDNA library as a template.

[0426] The selected cDNA is cleaved with an appropriate endonuclease or the like and cloned onto a plasmid such as pBluescript SK(-) (manufactured by Stratagene), and the base sequence of the cDNA is determined by a commonly used base sequence analysis method. For example, the base sequence can be analyzed using an automatic base sequence analyzer such as the ALF DNA sequencer (manufactured by Pharmacia) after performing a reaction such as the dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)].

[0427] The complete amino acid sequences of VH and VL were estimated from the determined complete base sequences and compared with the complete amino acid sequences of VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] to confirm whether the obtained cDNAs encode the complete amino acid sequences of VH and VL of the antibody, including the secretion signal sequence.

[0428] The complete amino acid sequences of VH and VL of antibodies containing secretory signal sequences can be compared with the complete amino acid sequences of VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] to infer the length of the secretory signal sequence and the N-terminal amino acid sequence, thereby identifying the subgroup to which they belong.

[0429] In addition, the amino acid sequence of each CDR of VH and VL can be estimated by comparison with the amino acid sequences of VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)].

[0430] Furthermore, for the obtained complete amino acid sequences of VH and VL, a homology search can be performed using any database such as SWISS-PROT or PIR-Protein using the BLAST method [J. Mol. Biol., 215, 403 (1990)] to confirm whether the complete amino acid sequences of VH and VL are novel sequences.

[0431] (2) Construction of vectors for expressing recombinant antibodies

[0432] A vector for expressing a genetically recombinant antibody can be constructed by cloning a DNA encoding at least one of CH and CL of a human antibody into an expression vector for animal cells.

[0433] As the human antibody C region, any human antibody CH and CL can be used, for example, human antibody γ1 subclass CH and κ class CL can be used. As DNA encoding human antibody CH and CL, cDNA is used, but chromosomal DNA containing exons and introns can also be used.

[0434] As an expression vector for animal cells, any vector can be used as long as it can be recombined with a gene encoding the C region of a human antibody and expressed. For example, pAGE107 [Cytotechnol., 3, 133 (1990)], pAGE103 [J. Biochem., 101, 1307 (1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc. Natl. Acad. Sci. USA, 78, 1527 (1981)], pSG1bd2-4 [Cytotechnol., 4, 173 (1990)], or pSE1UK1Sed1-3 [Cytotechnol., 13, 79 (1993)], INPEP4 (manufactured by Biogen-IDEC), N5KG1val (U.S. Patent No. 6,001,358), N5KG4PE R409K (described in International Publication No. 2006 / 033386), N5KG2 vector (described in International Publication No. 2003 / 033538), transposon vector (International Publication No. 2010 / 143698), etc.

[0435] As promoters and enhancers for expression vectors for animal cells, the SV40 early promoter [J. Biochem., 101, 1307 (1987)], Moloney murine leukemia virus LTR [Biochem. Biophys. Res. Commun., 149, 960 (1987)], CMV promoter (U.S. Patent No. 5,168,062), or the immunoglobulin H chain promoter [Cell, 41, 479 (1985)] and enhancer [Cell, 33, 717 (1983)] can be used.

[0436] In the expression of genetically recombinant antibodies, from the viewpoints of ease of vector construction, ease of introduction into animal cells, and balanced expression levels of the antibody H and L chains in cells, vectors carrying both antibody H and L chain genes (tandem vectors) are used [J. Immunol. Methods, 167, 271 (1994)]. However, a combination of multiple vectors (separate vectors) each carrying a gene for the antibody H and L chains may also be used.

[0437] As tandem gene recombinant antibody expression vectors, pKANTEX93 (International Publication No. 97 / 10354), pEE18 [Hybridoma, 17,559 (1998)], N5KG1val (U.S. Patent No. 6,001,358), N5KG4PE R409K (described in International Publication No. 2006 / 033386), N5KG2 vector (described in International Publication No. 2003 / 033538), Tol2 transposon vector (International Publication No. 2010 / 143698), etc. are used.

[0438] (3) Construction of chimeric antibody expression vector

[0439] A chimeric antibody expression vector can be constructed by cloning the cDNA encoding the VH or VL of the non-human antibody obtained in (1) upstream of the gene encoding the CH or CL of the human antibody in the vector for expressing the genetically recombinant antibody obtained in (2).

[0440] First, in order to link the 3' end of the cDNA encoding VH or VL of a non-human antibody with the 5' end of the CH or CL of a human antibody, VH and VL cDNAs are designed so that the base sequence of the linking portion encodes appropriate amino acids and serves as an appropriate endonuclease recognition sequence. Next, the prepared VH and VL cDNAs are cloned upstream of the gene encoding the CH or CL gene of the human antibody in the recombinant antibody expression vector obtained in (2) so that they are expressed in an appropriate form, thereby constructing a chimeric antibody expression vector.

[0441] Alternatively, a chimeric antibody expression vector may be constructed by using synthetic DNA having recognition sequences for appropriate endonucleases at both ends to amplify cDNA encoding VH or VL of a non-human antibody by PCR and cloning the cDNA into the recombinant antibody expression vector obtained in (2).

[0442] (4) Preparation of cDNA encoding the V region of a humanized antibody

[0443] The cDNA encoding VH or VL of a humanized antibody can be prepared as follows: First, the amino acid sequence of the framework region (hereinafter referred to as FR) of VH or VL of a human antibody to be transplanted with the amino acid sequence of the CDR of VH or VL of the non-human antibody obtained in (1) is selected.

[0444] Any sequence can be used as long as it is derived from a human antibody in the selected FR amino acid sequence. For example, the amino acid sequence of human antibody FRs registered in databases such as the Protein Data Bank or the common amino acid sequence of each subgroup of human antibody FRs [Sequences of Proteins of Immunological Interest, US Patent No. 2002, Health and Human Services (1991)] can be used. To suppress the reduction of antibody binding activity, human FR amino acid sequences with the highest possible homology (60% or more) to the amino acid sequence of the VH or VL FRs of the original non-human antibody are selected.

[0445] Then, the amino acid sequence of the CDR of the original non-human antibody is transplanted into the amino acid sequence of the FR of the selected human antibody VH or VL, respectively, to design the amino acid sequence of the VH or VL of the humanized antibody. Taking into account the codon usage frequency that can be regarded as the base sequence of the antibody gene [Sequences of Proteins of Immunological Interest, US Pat. Health and Human Services (1991)], the designed amino acid sequence is converted into a DNA sequence, thereby designing the cDNA sequence of the VH or VL of the humanized antibody.

[0446] Based on the designed cDNA sequence, several synthetic DNA strands of approximately 100 to 150 bases are synthesized and used for PCR reactions. In this case, from the perspectives of PCR reaction efficiency and the length of DNA that can be synthesized, it is preferred to design 4 to 6 synthetic DNA strands for each of the H and L chains. Alternatively, synthetic DNAs spanning the entire length of the variable region can be synthesized for use.

[0447] Furthermore, by introducing a recognition sequence for an appropriate endonuclease into the 5' end of the synthetic DNA at both ends, the cDNA encoding the VH or VL of the humanized antibody can be easily cloned into the recombinant antibody expression vector obtained in (2). After the PCR reaction, the amplified products are cloned into a plasmid such as pBluescript SK(-) (manufactured by Stratagene), and the base sequence is determined by the same method as described in (1) to obtain a plasmid having a DNA sequence encoding the amino acid sequence of the VH or VL of the desired humanized antibody.

[0448] (5) Modification of the amino acid sequence of the V region of the humanized antibody

[0449] If only the CDRs of VH and VL of a non-human antibody are grafted onto the FRs of VH and VL of a human antibody, the antigen-binding activity of the humanized antibody is reduced compared to that of the original non-human antibody [BIO / TECHNOLOGY, 9, 266 (1991)]. Therefore, by identifying the amino acid residues in the amino acid sequences of the FRs of VH and VL of human antibodies that are directly involved in antigen binding, that interact with the amino acid residues of the CDRs, and that indirectly participate in antigen binding by maintaining the steric structure of the antibody, and by substituting these amino acid residues with amino acid residues of the original non-human antibody, the reduced antigen-binding activity of the humanized antibody can be increased.

[0450] In order to identify amino acid residues in FRs that are associated with antigen-binding activity, the three-dimensional structure of the antibody can be constructed and analyzed using X-ray crystallography [J. Mol. Biol., 112, 535 (1977)] or computer modeling [Protein Engineering, 7, 1501 (1994)]. In addition, by repeatedly studying the correlation between each variant produced by each antibody and its antigen-binding activity and conducting trial and error, a modified humanized antibody having the desired antigen-binding activity can be obtained.

[0451] The amino acid residues of the FRs of VH and VL of human antibodies can be modified by performing the PCR reaction described in (4) using synthetic DNA for modification. The base sequence of the amplified product after the PCR reaction is determined by the method described in (1) to confirm that the target modification has been carried out.

[0452] (6) Construction of humanized antibody expression vector

[0453] A humanized antibody expression vector can be constructed by cloning cDNA encoding VH or VL of a humanized antibody upstream of the gene encoding CH or CL of the gene recombinant antibody expression vector obtained in (2).

[0454] For example, by introducing recognition sequences for appropriate endonucleases into the 5' ends of the synthetic DNAs used to construct VH or VL of humanized antibodies obtained in (4) and (5), they can be cloned upstream of the genes encoding CH or CL of human antibodies in the recombinant antibody expression vector obtained in (2) so that they are expressed in an appropriate form.

[0455] (7) Construction of human antibody expression vector

[0456] When a human antibody-producing animal is used as an immunized animal to establish a monoclonal antibody-producing hybridoma, the amino acid sequences and cDNA sequences of the VH and VL of the human antibody can be obtained in (1). Therefore, by cloning the gene encoding the VH or VL of the human antibody obtained in (1) upstream of the gene encoding the CH or CL of the human antibody in the vector for expressing the genetically recombinant antibody obtained in (2), a human antibody expression vector can be constructed.

[0457] (8) Transient expression of recombinant antibodies

[0458] By using the recombinant antibody expression vectors obtained in (3), (6) and (7), or their modified expression vectors, the recombinant antibodies can be transiently expressed, and the antigen binding activity of the obtained multiple recombinant antibodies can be effectively evaluated.

[0459] Any host cell capable of expressing a recombinant antibody can be used as the host cell into which the expression vector is introduced, for example, COS-7 cells [American Type Culture Collection (ATCC) code: CRL1651]. The expression vector can be introduced into COS-7 cells using the DEAE-dextran method [Methods in Nucleic Acids Res., CRC press (1991)] or the lipofectamine transfection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].

[0460] After the expression vector is introduced, the expression level and antigen-binding activity of the recombinant antibody in the culture supernatant are measured using enzyme immunoassay [Monoclonal Antibodies - Principles and Practice, Third Edition, Academic Press (1996), Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Monoclonal Antibodies Laboratory Manual, Kodansha Scientific (1987)].

[0461] (9) Obtaining a Stable Expression Strain of Genetically Recombinant Antibodies and Preparation of Genetically Recombinant Antibodies

[0462] By introducing the recombinant antibody expression vectors obtained in (3), (6) and (7) into appropriate host cells, transformants stably expressing the recombinant antibody can be obtained.

[0463] Examples of methods for introducing expression vectors into host cells include electroporation [Japanese Patent Application Laid-Open No. 2-257891, Cytotechnology, 3, 133 (1990)], the calcium ion method, electroporation, the spheroplast method, the lithium acetate method, the calcium phosphate method, and the lipofection method. Furthermore, examples of methods for introducing genes into animals described below include microinjection, methods for introducing genes into ES cells using electroporation or lipofection, and nuclear transfer.

[0464] As host cells for introducing a recombinant antibody expression vector, any host cell capable of expressing a recombinant antibody can be used, for example, mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), Chinese hamster CHO-K1 cells (ATCC CCL-61), DUKXB11 (ATCC CCL-9096), Pro-5 cells (ATCC CCL-1781), CHO-S cells (Life Technologies, Cat No. 11619), CHO cells deficient in the dihydrofolate reductase gene (dhfr) (CHO / DG44 cells) [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)], Lec13 cells acquired lectin resistance [Somatic Cell and Molecular Biology, 1996], and the like. genetics, 12, 55 (1986)], α1,6-fucosyltransferase gene-deficient CHO cells (International Publication No. 2005 / 035586, International Publication No. 02 / 31140), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC No.: CRL1662), etc.

[0465] Alternatively, host cells in which the activity of proteins such as enzymes involved in the synthesis of the intracellular sugar nucleotide GDP-fucose, proteins such as enzymes involved in the modification of the sugar chain at the 6-position of N-acetylglucosamine in which fucose is α-bound at the 1-position to the reducing end of the N-glycoside-bound complex sugar chain, or proteins involved in the transport of the intracellular sugar nucleotide GDP-fucose to the Golgi apparatus may be reduced or deleted, for example, CHO cells deficient in the α1,6-fucosyltransferase gene (International Publication Nos. 2005 / 035586 and 02 / 31140).

[0466] After the expression vector is introduced, transformants stably expressing the recombinant antibody are selected by culturing them in an animal cell culture medium containing a drug such as G418 sulfate (hereinafter referred to as G418) (Japanese Patent Application Laid-Open No. 2-257891).

[0467] Zoocell culture medium uses RPMI1640 medium (Invitrogen company system), GIT medium (Nippon Pharmaceutical Co., Ltd. system), EX-CELL301 medium (JRH company system), EX-CELL302 medium (JRH company system), EX-CELL325 medium (JRH company system), IMDM medium (Invitrogen company system) or Hybridoma-SFM medium (Invitrogen company system) or in these mediums, has added the medium of various additives such as FBS etc..By cultivating the resulting transformant in medium, make the recombinant antibody express, accumulate in culture supernatant.The expression level of the recombinant antibody in culture supernatant and antigen-binding activity can be measured by ELISA method etc. In addition, also can utilize DHFR amplification system (Japanese Patent Laid-Open No. 2-257891 communique) etc., the expression level of the recombinant antibody that transformant produces rises.

[0468] Recombinant antibodies can be purified from the culture supernatant of the transformant using a protein A column [Monoclonal Antibodies - Principles and Practice, Third Edition, Academic Press (1996), Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. Alternatively, purification can be performed by combining methods commonly used for protein purification, such as gel filtration, ion exchange chromatography, and ultrafiltration.

[0469] The molecular weight of the H chain, L chain, or entire antibody molecule of the purified recombinant antibody can be measured using polyacrylamide gel electrophoresis [Nature, 227, 680 (1970)] or Western blotting [Monoclonal Antibodies - Principles and Practice, Third Edition, Academic Press (1996), Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].

[0470] 3. Preparation of Bispecific Antibodies or Bispecific Antibody Fragments

[0471] The bispecific antibodies of the present invention can be produced by, for example, first obtaining a plurality of monoclonal antibodies that bind to different epitopes using the method described in 1. above, then determining the VH and VL cDNA sequences of each antibody using the method described in 2. above, and designing a bispecific antibody or bispecific antibody fragment in which the antigen-binding sites of each antibody are linked.

[0472] Specifically, the bispecific antibody or bispecific antibody fragment can be produced by synthesizing DNA that appropriately combines the antigen-binding site of each antibody with a linker, and then recombining it into the gene recombinant antibody expression vector described in 2. (2) above to express the bispecific antibody or bispecific antibody fragment. More specifically, the bispecific antibody or bispecific antibody fragment can be produced by synthesizing DNA that encodes a polypeptide formed by linking the VH of each antibody and synthesizing DNA that encodes the VL of each antibody, and then recombining these DNAs into the gene recombinant antibody expression vector described in 2. (2) above to express the bispecific antibody or bispecific antibody fragment. Alternatively, instead of the gene encoding CH or CL in 2. (2), an expression vector for animal cells into which a gene encoding an arbitrary polypeptide chain is introduced can be used to produce a bispecific antibody or bispecific antibody fragment having an arbitrary polypeptide chain (C-terminal side polypeptide) on the C-terminal side of the antigen-binding site closest to the C-terminus. The gene encoding any polypeptide chain can be combined with the DNA encoding the polypeptide formed by linking the VH of each antibody via a linker and then recombined into an expression vector for animal cells. Alternatively, the gene encoding the polypeptide chain can be separately recombined with the DNA encoding the polypeptide formed by linking the VH of each antibody via a linker and then recombined into an expression vector for animal cells.

[0473] In addition to the hybridoma method described in 1. above, antigen-binding sites can also be isolated and obtained using techniques such as the Phage Display method and the Yeast Display method [Emmanuelle Laffy et al., Human Antibodies 14, 33-55, (2005)].

[0474] Examples of the linker and C-terminal polypeptide include polypeptide chains. Specifically, for example, polypeptides formed by binding multiple antigen-binding domains. Examples include immunoglobulin domains composed of CH1-hinge-CH2-CH3 arranged sequentially from the N-terminus to the C-terminus, immunoglobulin domains composed of CH1-hinge-CH2, immunoglobulin domains composed of CH1-hinge, immunoglobulin domains composed of CH1, N-terminal fragments of CH1, CH1 fragments composed of 14 amino acid residues with Cys at amino acid residue position 14, and CH1 fragments composed of amino acid residues 1 to 14 from the N-terminus of CH1, as well as fragments in which one or more amino acid residues are altered in the amino acid sequence of these immunoglobulin domain fragments.

[0475] More specifically, as the above-mentioned linker and C-terminal side polypeptide, for example, there can be cited a polypeptide composed of 14 amino acid residues at positions 1 to 14 of the N-terminus of CH1 of IgG4 shown in sequence number 75, a polypeptide composed of CH1 of IgG4 shown in sequence number 75, and a polypeptide composed of CH (CH1, hinge, CH2 and CH3) of IgG4PE R409K shown in sequence number 77.

[0476] Furthermore, when producing bispecific antibodies composed of multiple VHs and a single VL or such bispecific antibody fragments, screening using phage display methods or the like is performed so that each antigen-binding site contained in the bispecific antibody reacts with each specific antigen, and each VH that is most suitable for a single VL is selected.

[0477] Specifically, first, using the method described in 1. above, an animal is immunized with a first antigen, hybridomas are prepared from the spleen, and a DNA sequence encoding the first antigen-binding site is cloned. Next, an animal is immunized with a second antigen, a cDNA library is prepared from the spleen, and DNA encoding the VH amino acid sequence is obtained from this library by PCR.

[0478] Next, a phage library expressing a scFv consisting of the VH obtained by immunization with the second antigen linked to the VL of the first antigen-binding site is prepared. Panning using this phage library is used to select phage presenting scFvs that specifically bind to the second antigen. From the selected phage, a DNA sequence encoding the amino acid sequence of the VH of the second antigen-binding site is cloned.

[0479] Furthermore, a DNA sequence encoding the amino acid sequence of a polypeptide composed of the VH of the first antigen-binding site and the VH of the second antigen-binding site linked via the aforementioned linker is designed, and this DNA sequence and a DNA sequence encoding the amino acid sequence of a single VL are inserted into, for example, the genetic recombinant antibody expression vector described in 2.(2) above, thereby constructing an expression vector for the bispecific antibody or bispecific antibody fragment of the present invention. Alternatively, a bispecific antibody or bispecific antibody fragment having an arbitrary polypeptide chain (C-terminal polypeptide) at the C-terminal side of the VH of the second antigen-binding site can be produced by using an expression vector for animal cells into which a gene encoding an arbitrary polypeptide chain has been introduced, instead of the gene encoding the CH of 2.(2).

[0480] 4. Activity Evaluation of the Bispecific Antibody or Bispecific Antibody Fragment of the Present Invention

[0481] The activity of the purified bispecific antibody or bispecific antibody fragment can be evaluated as follows.

[0482] The binding activity of the bispecific antibody or the bispecific antibody fragment of the present invention toward a cell line expressing at least one of CD40 and EpCAM can be measured using the binding assay system described in 1.(7) above.

[0483] The CDC activity or ADCC activity on cells expressing at least one of CD40 and EpCAM can be measured by a known measurement method [Cancer Immunol. Immunother., 36, 373 (1993)].

[0484] The cell death-inducing activity of the bispecific antibodies or bispecific antibody fragments of the present invention can be measured by the following method: For example, cells are seeded in a 96-well plate, and after adding the antibody and culturing for a certain period of time, they are reacted with WST-8 reagent (manufactured by Dojindo Co., Ltd.), and the absorbance at 450 nm is measured using a plate reader to determine the cell survival rate.

[0485] 5. Methods for treating diseases using the bispecific antibodies or bispecific antibody fragments of the present invention

[0486] The bispecific antibodies or bispecific antibody fragments of the present invention can be used to treat diseases associated with at least one of CD40 and EpCAM, preferably diseases associated with cells expressing CD40 and EpCAM. Examples of diseases associated with at least one of CD40 and EpCAM include malignant tumors and cancers.

[0487] Examples of malignant tumors and cancers include colon cancer, colorectal cancer, lung cancer, breast cancer, glioma, malignant melanoma, thyroid cancer, renal cell carcinoma, leukemia, lymphoma, T-cell lymphoma, gastric cancer, pancreatic cancer, cervical cancer, endometrial cancer, ovarian cancer, bile duct cancer, esophageal cancer, liver cancer, squamous cell carcinoma of the head and neck, skin cancer, urinary tract cancer, bladder cancer, prostate cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer, pleural tumor, male embryonal tumor, endometrial hyperplasia, endometriosis, embryonal tumor, fibrosarcoma, Kaposi's sarcoma, hemangioma, cavernous hemangioma, hemangioblastoma, retinoblastoma, astrocytoma, neurofibroma, oligodendroglioma, medulloblastoma, neuroblastoma, glioma, rhabdomyosarcoma, glioblastoma, osteosarcoma, leiomyosarcoma, and Wilms' tumor.

[0488] Therapeutic drugs comprising the bispecific antibodies or bispecific antibody fragments of the present invention, or derivatives thereof, may contain only the antibodies or bispecific antibody fragments or derivatives thereof as active ingredients, or may be provided as pharmaceutical preparations prepared by methods known in the art of pharmaceutical preparations by mixing with one or more generally pharmacologically acceptable carriers.

[0489] As the route of administration, for example, oral administration, or parenteral administration such as oral, intratracheal, rectal, subcutaneous, intramuscular or intravenous administration can be mentioned. As the form of administration, for example, sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments or patches can be mentioned. Various preparations can be manufactured by conventional methods using commonly used excipients, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, dissolution aids, preservatives, colorants, flavoring agents and stabilizers.

[0490] Examples of excipients include lactose, fructose, glucose, corn starch, sorbitol, crystalline cellulose, sterile water, ethanol, glycerol, physiological saline, and buffer solutions. Examples of disintegrants include starch, sodium alginate, gelatin, calcium carbonate, calcium citrate, dextrin, magnesium carbonate, and synthetic magnesium silicate.

[0491] Examples of binders include methylcellulose or its salts, ethylcellulose, gum arabic, gelatin, hydroxypropylcellulose, and polyvinylpyrrolidone, etc. Examples of lubricants include talc, magnesium stearate, polyethylene glycol, and hydrogenated vegetable oils.

[0492] Examples of the stabilizer include amino acids such as arginine, histidine, lysine, and methionine, human serum albumin, gelatin, dextran 40, methylcellulose, sodium sulfite, and sodium metabisulfite.

[0493] Examples of other additives include syrup, vaseline, glycerin, ethanol, propylene glycol, citric acid, sodium chloride, sodium nitrite, and sodium phosphate.

[0494] Examples of preparations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, and granules.

[0495] Liquid preparations such as emulsions and syrups can be produced using water, sugars such as sucrose, sorbitol, or fructose, glycols such as polyethylene glycol or propylene glycol, oils such as sesame oil, olive oil, or soybean oil, preservatives such as parabens, or flavorings such as strawberry flavor or mint as additives.

[0496] Capsules, tablets, powders or granules are manufactured using excipients such as lactose, glucose, sucrose or mannitol, disintegrants such as starch or sodium alginate, lubricants such as magnesium stearate or talc, binders such as polyvinyl alcohol, hydroxypropyl cellulose or gelatin, surfactants such as fatty acid esters, or plasticizers such as glycerol as additives.

[0497] Examples of preparations suitable for parenteral administration include injections, suppositories, and sprays. Injections are prepared using a carrier containing a saline solution, a glucose solution, or a mixture thereof.

[0498] Suppositories are manufactured using carriers such as cocoa butter, hydrogenated fats, or carboxylic acids. Sprays are manufactured using carriers that do not irritate the oral and respiratory mucosa of the recipient and disperse the bispecific antibody or bispecific antibody fragment of the present invention into fine particles for easy absorption. Examples of carriers include lactose and glycerol. Alternatively, they can be manufactured as aerosols or dry powders. Furthermore, the aforementioned parenteral formulations may contain ingredients listed as additives in formulations suitable for oral administration.

[0499] The effective dose of the bispecific antibody of the present invention administered in combination with an appropriate diluent and a pharmacologically acceptable carrier is 0.0001 mg to 100 mg per kg of body weight, and is repeated 2 days later and at intervals of 8 weeks.

[0500] 6. Methods for diagnosing diseases using the bispecific antibodies or bispecific antibody fragments of the present invention

[0501] By detecting or measuring cells expressing at least one of CD40 and EpCAM using the bispecific antibody or bispecific antibody fragment of the present invention, diseases associated with at least one of CD40 and EpCAM, preferably diseases associated with cells expressing CD40 and EpCAM, can be diagnosed.

[0502] Diagnosis of a malignant tumor or cancer, which is a disease associated with at least one of CD40 and EpCAM, can be performed, for example, by detecting or measuring at least one of CD40 and EpCAM as follows.

[0503] First, biological samples collected from multiple healthy subjects are subjected to detection or measurement of at least one of CD40 and EpCAM using the bispecific antibodies of the present invention, bispecific antibody fragments, or derivatives thereof, using the immunological methods described below, to investigate the amount of at least one of CD40 and EpCAM present in the biological samples from the healthy subjects.

[0504] Next, the amount of at least one of CD40 and EpCAM in the biological sample of the subject is similarly measured and compared with that of a healthy person. If the amount of at least one of CD40 and EpCAM in the subject is increased compared to that of a healthy person, the subject is diagnosed with cancer. The same method can be used to diagnose other diseases related to at least one of CD40 and EpCAM.

[0505] Immunological methods refer to methods for detecting or measuring the amount of antibodies or antigens using labeled antigens or antibodies. Examples include radioactive substance-labeled immunoantibody methods, enzyme immunoassays, fluorescent immunoassays, luminescent immunoassays, immunoblotting, and physicochemical methods.

[0506] Examples of radioactive substance-labeled immunoantibody methods include reacting the bispecific antibody of the present invention or a bispecific antibody fragment with an antigen or a cell expressing the antigen, and then reacting with a radiolabeled anti-immunoglobulin antibody or binding fragment, followed by measurement using a scintillation counter or the like.

[0507] Examples of enzyme immunoassays include methods in which the bispecific antibody or bispecific antibody fragment of the present invention is reacted with an antigen or cells expressing the antigen, and then reacted with a labeled anti-immunoglobulin antibody or binding fragment, followed by measuring the color pigment using an absorptiometry. Examples include sandwich ELISA.

[0508] As the labeling substance used in the enzyme immunoassay, a known enzyme label [Enzyme Immunoassay, Medical College (1987)] can be used, for example, an alkaline phosphatase label, a peroxidase label, a luciferase label, or a biotin label.

[0509] The sandwich ELISA method is to capture the antigen as the detection or measurement object after the solid phase is combined with the antibody, and the method for reacting the second antibody with the captured antigen. In this ELISA method, two different antibodies with different antigen binding sites are prepared, as antibodies or antibody fragments that are combined with the antigen to be detected or measured, wherein the first antibody or antibody fragment is adsorbed on a culture plate (for example, a 96-well plate) in advance, and then the second antibody or antibody fragment is labeled with fluorescent substances such as FITC, enzymes such as peroxidase, or biotin. After the culture plate having adsorbed the above-mentioned antibody reacts with cells separated from a living body or their crushed liquid, tissue or their crushed liquid, cell culture supernatant, serum, pleural effusion, ascites, or eye fluid, it is reacted with the antibody or antibody fragment after the labeling, and the detection reaction corresponding to the labeling substance is carried out. The antigen with a known concentration is diluted in stages to make a standard curve, and the antigen concentration in the sample to be tested is calculated according to the standard curve.

[0510] As the antibodies used in the sandwich ELISA method, either polyclonal antibodies or monoclonal antibodies can be used, and antibody fragments such as Fab, Fab', or F(ab)2 can also be used. As the combination of two antibodies used in the sandwich ELISA method, it can be a combination of monoclonal antibodies or antibody fragments that bind to different epitopes, or a combination of a polyclonal antibody and a monoclonal antibody or an antibody fragment thereof.

[0511] Fluorescence immunoassays can be performed, for example, by methods described in the literature [Monoclonal Antibodies - Principles and Practice, Third Edition, Academic Press (1996), Monoclonal Antibody Laboratory Manual, Kodansha Scientific (1987)]. Labels used in fluorescence immunoassays can be well-known fluorescent labels [Fluorescent Antibody Method, Soft Science (1983)]. For example, FITC or RITC can be used.

[0512] Luminescence immunoassays can be performed using methods described in, for example, [Bioluminescence and Chemiluminescence Clinical Examination 42, Hirokawa Shoten (1998)]. Labels used in luminescence immunoassays include known luminescent labels, such as acridinium esters and lofenac.

[0513] As immunoblotting, after utilizing SDS (sodium dodecyl sulfate)-PAGE [Antibodies-A Laboratory Manual Cold Spring Harbor Laboratory (1988)] with antigen or the cell that has expressed antigen etc. to be fractionated, this gel is blotted on polyvinylidene fluoride (PVDF) film or nitrocellulose membrane, this film is reacted with the antibody or antibody fragment that binds to the antigen, and then after reacting the anti-IgG antibody or its antibody fragment of the enzyme labeling such as fluorescent substance, peroxidase etc. or biotin labeling etc. that has implemented FITC etc., by making this mark visual to measure. An example is shown below.

[0514] First, cells or tissues expressing a polypeptide with a desired amino acid sequence are dissolved and electrophoresed by SDS-PAGE under reducing conditions with a protein amount of 0.1 to 30 μg per band. Then, the protein after electrophoresis is transferred to a PVDF membrane and blocked in PBS containing 1 to 10% BSA (hereinafter referred to as BSA-PBS) at room temperature for 30 minutes. The bispecific antibody of the present invention is reacted, washed with PBS containing 0.05 to 0.1% Tween-20 (Tween-PBS), and reacted with peroxidase-labeled goat anti-mouse IgG at room temperature for 2 hours. Washed with Tween-PBS, the band bound to the antibody is detected using ECL Western Blotting Detection Reagents (made by Amersham), thereby detecting the antigen. As the antibody used in the detection by Western blotting, an antibody that can bind to a polypeptide that does not maintain its native three-dimensional structure can be used.

[0515] As a physicochemical method, for example, at least one of the antigens CD40 and EpCAM is bound to the bispecific antibody of the present invention or a bispecific antibody fragment thereof to form aggregates, and the aggregates are detected. Alternatively, capillary methods, one-dimensional immunodiffusion, immunoturbidimetry, or latex immunoturbidimetry [Summary of Clinical Laboratory Methods, Kanehara Publishing (1998)] may be used.

[0516] In latex immunoturbidimetry, a carrier such as polystyrene latex, sensitized with an antibody or antigen, is used. The antigen-antibody reaction is induced by the corresponding antigen or antibody. This results in an increase in scattered light and a decrease in transmitted light in the reaction solution. This change is detected as absorbance or integrating sphere turbidimetry, allowing the determination of antigen concentrations in the sample.

[0517] On the other hand, cells expressing at least one of CD40 and EpCAM can be detected or measured by known immunological detection methods, preferably immunoprecipitation, immunocytostaining, immunohistostaining, or fluorescent antibody staining.

[0518] In the immunoprecipitation method, cells expressing at least one of CD40 and EpCAM are reacted with the bispecific antibody of the present invention or a bispecific antibody fragment thereof, and then a carrier having specific binding ability to immunoglobulins, such as Protein G Sepharose, is added to precipitate the antigen-antibody complex.

[0519] Alternatively, the following method can be used. First, the bispecific antibody of the present invention or a bispecific antibody fragment thereof is immobilized on a 96-well ELISA plate and blocked with BSA-PBS. The BSA-PBS is then discarded, the plate is thoroughly washed with PBS, and then reacted with a lysate of cells or tissues expressing at least one of CD40 and EpCAM. The immunoprecipitate from the thoroughly washed plate is extracted with sample buffer using SDS-PAGE and detected by the aforementioned Western blotting method.

[0520] Immunocytological staining or immunohistochemical staining involves treating antigen-expressing cells or tissues with a surfactant or methanol, as appropriate, to improve antibody permeability, reacting them with the bispecific antibody of the present invention, and then reacting them with an anti-immunoglobulin antibody or binding fragment thereof labeled with a fluorescent marker such as FITC, an enzyme such as peroxidase, or biotin. The marker is then visualized and observed under a microscope. Alternatively, fluorescently labeled antibodies are reacted with cells, and detection is performed using a fluorescent antibody staining method analyzed by flow cytometry [Monoclonal Antibodies - Principles and Practice, Third Edition, Academic Press (1996), Monoclonal Antibodies: A Laboratory Manual, Kodansha Scientific (1987)]. In particular, the bispecific antibodies of the present invention or their bispecific antibody fragments can detect at least one of CD40 and EpCAM expressed on the cell membrane using fluorescent antibody staining.

[0521] Furthermore, when the fluorescent antibody staining method is performed using the FMAT8100HTS system (manufactured by Applied Biosystems), the amount of antigen or antibody can be measured without separating the formed antibody-antigen complex from the free antibody or antigen that does not participate in the formation of the antibody-antigen complex. Example

[0522] Hereinafter, the present invention will be specifically described using examples, but the present invention is not limited to the following examples.

[0523] [Example 1] Preparation of soluble human and monkey CD40 antigens and soluble human, monkey and mouse EpCAM antigens

[0524] 1. Preparation of Soluble Antigens of Human CD40 and Monkey CD40

[0525] Human and monkey CD40 extracellular domain proteins with FLAG-Fc added to the C-terminus were prepared using the methods described below. The base sequence encoding the human CD40 extracellular domain is shown as SEQ ID NO: 1, and the amino acid sequence deduced from this base sequence is shown as SEQ ID NO: 2. The base sequence encoding the monkey CD40 extracellular domain is shown as SEQ ID NO: 3, and the amino acid sequence deduced from this base sequence is shown as SEQ ID NO: 4.

[0526] (1) Preparation of human and monkey CD40-FLAG-Fc vectors

[0527] According to the human CD40 gene sequence (Genbank Accession Number: NM ＿ 001250, SEQ ID NO: 5; the amino acid sequence encoded by this gene is represented by SEQ ID NO: 6), and a gene fragment of the extracellular domain of human CD40 consisting of the base sequence shown in SEQ ID NO: 1 was prepared.

[0528] The INPEP4 vector (Biogen-IDEC) containing the FLAG-tag and the Fc region of human IgG was digested with the endonucleases KpnI and XbaI, and a gene fragment of the extracellular domain to which the human CD40 signal sequence encoding region consisting of base sequence from bases 1 to 60 of the sequence shown in SEQ ID NO: 1 was added was inserted into an appropriate site to produce a human CD40-FLAG-Fc expression vector.

[0529] In the same manner, a monkey CD40-FLAG-Fc expression vector containing a gene fragment of the monkey CD40 extracellular domain consisting of the base sequence shown in SEQ ID NO: 3 was prepared based on the monkey CD40 gene sequence cloned from monkey peripheral blood mononuclear cells (PBMC) (SEQ ID NO: 7; the amino acid sequence encoded by this gene is shown as SEQ ID NO: 8).

[0530] (2) Preparation of human and monkey CD40-FLAG-Fc proteins

[0531] The human CD40-FLAG-Fc expression vector prepared in 1-(1) was introduced into HEK293 cells using the FreeStyle (trademark) 293 Expression System (manufactured by Thermo Fisher Scientific), and the cells were cultured to express the protein using a transient expression system. Five days after vector introduction, the culture supernatant was collected and filtered through a membrane filter with a pore size of 0.22 μm (manufactured by Millipore).

[0532] The culture supernatant was affinity purified using Protein A resin (MabSelect, manufactured by GE Healthcare). Antibodies adsorbed to Protein A were washed with Dulbecco's phosphate-buffered saline (D-PBS(-) solution without Ca and Mg; hereinafter referred to as D-PBS(-). Manufactured by Nakarai Tesque Co., Ltd.), eluted with 20 mM sodium citrate, 50 mM NaCl buffer (pH 3.4), and recovered in a test tube containing 1 M sodium phosphate buffer (pH 7.0).

[0533] Then, human CD40-FLAG-Fc protein was produced by ultrafiltration using VIVASPIN (manufactured by Sartrius Stealin Co., Ltd.), replacement with D-PBS(-), and filtration sterilization using a 0.22 μm pore size membrane filter, Millex-Gv (manufactured by Millipore Co., Ltd.). Monkey CD40-FLAG-Fc protein was produced by the same method using the monkey CD40-FLAG-Fc expression vector prepared in 1-(1). The concentration of the obtained protein was calculated by measuring absorbance at a wavelength of 280 nm using the absorption coefficient estimated from the amino acid sequence of each protein.

[0534] (3) Preparation of human and monkey CD40-GST vectors

[0535] The N5 vector (manufactured by Biogen-IDEC) containing the GST region was digested with the endonucleases BglII and KpnI, and the gene fragment of the extracellular domain of human CD40 consisting of the base sequence shown in SEQ ID NO: 1 described in 1-(1) was inserted into the appropriate site to produce a human CD40-GST expression vector. A monkey CD40-GST expression vector containing the gene fragment of the extracellular domain consisting of the base sequence shown in SEQ ID NO: 3 was produced using the same method.

[0536] (4) Preparation of human and monkey CD40-GST proteins

[0537] The human CD40-GST vector prepared in 1-(3) was introduced into HEK293 cells using the same method as in 1-(2). After culturing the cells, the culture supernatant was filtered through a membrane filter. The culture supernatant was reacted with Glutathione Sepharose 4B (GE Healthcare), washed with D-PBS(-), and affinity purified using 50 mM Tris-HCl (pH 8.0) containing 10 mM glutathione as an elution buffer.

[0538] The eluted fusion protein solution was sterilized by ultrafiltration and membrane filtration using the same method as in step 1-(2) to obtain human CD40-GST protein. Separately, monkey CD40-GST protein was obtained using the same method using a monkey CD40-GST vector. The concentration of the obtained protein was calculated by measuring absorbance at a wavelength of 280 nm using the absorption coefficient estimated from the amino acid sequence of each protein.

[0539] 2. Preparation of Soluble Antigens of Human, Monkey, and Mouse EpCAM

[0540] The extracellular domain proteins of human, monkey, or mouse EpCAM with FLAG-Fc or GST added to the C-terminus were prepared by the methods described below. The base sequence encoding the human EpCAM extracellular domain is shown as SEQ ID NO: 9, and the amino acid sequence deduced from this base sequence is shown as SEQ ID NO: 10. The base sequence encoding the monkey EpCAM extracellular domain is shown as SEQ ID NO: 11, and the amino acid sequence deduced from this base sequence is shown as SEQ ID NO: 12. The base sequence encoding the mouse EpCAM extracellular domain is shown as SEQ ID NO: 13, and the amino acid sequence deduced from this base sequence is shown as SEQ ID NO: 14.

[0541] According to the human EpCAM gene shown in sequence number 15 (Genbank Accession Number: NM_002354, the amino acid sequence encoded by this gene is represented as sequence number 16), the monkey EpCAM gene shown in sequence number 17 (Genbank Accession Number: XM_015433685, the amino acid sequence encoded by this gene is represented as sequence number 18), and the mouse EpCAM gene shown in sequence number 19 (Genbank Accession Number: NM_008532, the amino acid sequence encoded by this gene is represented as sequence number 20), the same operation as the method described in 1-(1) and (2) was performed to obtain human, monkey and mouse EpCAM-FLAG-Fc proteins, respectively.

[0542] In addition, a base sequence consisting of bases 1 to 63 of the base sequence shown in SEQ ID NO: 15 was incorporated into the EpCAM-FLAG-Fc expression vector as a signal sequence coding region. Human, monkey, and mouse EpCAM-GST proteins were obtained in the same manner as described in 1-(3) and (4). The concentration of the obtained protein was calculated by measuring absorbance at a wavelength of 280 nm using the extinction coefficient estimated from the amino acid sequence of each protein.

[0543] [Example 2] Preparation of membrane-expressing human EpCAM-expressing HEK293 cells

[0544] The human EpCAM gene shown in SEQ ID NO: 15 was TOPO cloned using the pEF6 / V5-His TOPO TA Expression Kit (Invitrogen), and clones with the human EpCAM gene inserted in the forward direction were selected to obtain the human EpCAM expression vector pEF6-humanEpCAM full for membrane expression.

[0545] The obtained expression vector pEF6-human EpCAM full was introduced into HEK293 cells using the FreeStyle (trademark) 293 Expression System (manufactured by Thermo Fisher Scientific) and cultured to express the protein using a transient expression system. After gene introduction, the cells were shaken and cultured for 24 hours, followed by centrifugation to obtain human EpCAM / HEK293 cells.

[0546] [Example 3] Acquisition of anti-CD40 antibodies

[0547] 1. Preparation of CD40 immune human antibody M13 phage library

[0548] Human antibody-producing mice [Ishida & Lonberg, IBC's 11th Antibody Engineering, Abstract 2000; Ishida, I. et al., Cloning & Stem Cells 4, 85-96 (2002) and Ishida Isao (2002) Experimental Medicine 20, 6, 846-851] were intraperitoneally administered with human CD40-FLAG-Fc prepared in Example 1 as an immunogen a total of four times. Alum gel (2 mg / mouse) and pertussis vaccine (1×10 9 pieces / pieces).

[0549] The second immunization was performed 2 weeks after the first immunization, the third immunization was performed 1 week after the first immunization, and the final immunization was performed 10 days after the third immunization. Four days after the final immunization, the spleen was surgically removed. The removed spleen was placed on a cell strainer (manufactured by Falcon) and gently crushed with a silicon rod while transferring the cells to a test tube. The cells were centrifuged to precipitate the cells and then reacted with an erythrocyte removal reagent (manufactured by Sigma Aldrich) on ice for 3 minutes, followed by further centrifugation.

[0550] RNA was extracted from the resulting spleen cells using the RNeasy Mini kit (QIAGEN), cDNA was amplified using the SMARTer RACE cDNA Amplification Kit (Clontech), and the VH gene fragment was further amplified by PCR. This VH gene fragment and a VL gene fragment containing the L6 sequence consisting of the base sequence shown in SEQ ID NO: 21, which is a human antibody germ-line sequence, were inserted into the phagemid pCANTAB 5E (Amersham Pharmacia), and Escherichia coli TG1 ...

Claims

1. A bispecific antibody, wherein: The bispecific antibody comprises an antigen binding domain that binds to CD40 and an antigen binding domain that binds to epithelial cell adhesion molecule (EpCAM). wherein the bispecific antibody binds to CD40 and EpCAM in a divalent manner; The antigen binding domain that binds to CD40 includes the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody that specifically binds to CD40 (anti-CD40 antibody); the antigen binding domain that binds to EpCAM includes the VH and VL of an antibody that specifically binds to EpCAM (anti-EpCAM antibody); The bispecific antibody comprises two identical heavy chains containing a polypeptide represented by the formula VH1-X-VH2-Y in order from the N-terminus, and four light chains containing the same VL, In the VH1-X-VH2-Y formula, The VH1 represents the VH of the first antibody, The VH2 represents the VH of the second antibody, X is a polypeptide consisting of CH1, hinge region, CH2 and CH3 of human IgG, and Y is a polypeptide consisting of CH1 of human IgG, Either one of the first antibody and the second antibody is an anti-CD40 antibody, and the other is an anti-EpCAM antibody; wherein the VL comprises complementarity determining regions (CDRs) 1 to 3, and the complementarity determining regions (CDRs) 1 to 3 are respectively composed of the amino acid sequences shown in sequence numbers 23 to 25; and Wherein, the anti-CD40 antibody comprises: a VH comprising CDR1-3 consisting of the amino acid sequences shown in sequence numbers 28 to 30, respectively; and the anti-EpCAM antibody comprises: a VH comprising CDR1-3 consisting of the amino acid sequences shown in sequence numbers 57 to 59, respectively, or a VH comprising CDR1-3 consisting of the amino acid sequences shown in sequence numbers 67 to 69, respectively; or The anti-CD40 antibody comprises as a first antibody: a VH comprising CDR1-3 consisting of the amino acid sequences shown in SEQ ID NOs: 28-30, respectively; and the anti-EpCAM antibody comprises as a second antibody: a VH comprising CDR1-3 consisting of the amino acid sequences shown in SEQ ID NOs: 52-54, respectively, or a VH comprising CDR1-3 consisting of the amino acid sequences shown in SEQ ID NOs: 62-64, respectively; or The anti-CD40 antibody comprises as a first antibody: a VH comprising CDR1 to 3 consisting of the amino acid sequences shown in sequence numbers 33 to 35, respectively; and the anti-EpCAM antibody comprises as a second antibody: a VH comprising CDR1 to 3 consisting of the amino acid sequences shown in sequence numbers 52 to 54, respectively, or a VH comprising CDR1 to 3 consisting of the amino acid sequences shown in sequence numbers 57 to 59, respectively.

2. The bispecific antibody according to claim 1, wherein The first antibody is an anti-CD40 antibody, and the second antibody is an anti-EpCAM antibody; The VL consists of the amino acid sequence shown in SEQ ID NO: 22; The anti-CD40 antibody comprises VH, wherein VH consists of the amino acid sequence shown in SEQ ID NO: 32; The anti-EpCAM antibody contains VH, and the VH consists of the amino acid sequence shown in SEQ ID NO:

56.

3. The bispecific antibody according to claim 2, wherein: The light chain is a kappa chain.

4. The bispecific antibody according to claim 3, wherein In the VH1-X-VH2-Y formula, X is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 77, and Y is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

75.

5. The bispecific antibody according to claim 1, wherein The light chain is a kappa chain.

6. The bispecific antibody according to claim 5, wherein In the VH1-X-VH2-Y formula, X is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 77, and Y is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

75.

7. The bispecific antibody according to claim 5, wherein The VL consists of the amino acid sequence shown in sequence number 22.

8. The bispecific antibody according to claim 6, wherein The VL consists of the amino acid sequence shown in sequence number 22.

9. The bispecific antibody according to any one of claims 1 to 8, wherein The bispecific antibody has CD40 agonist activity.

10. The bispecific antibody according to any one of claims 1 to 8, wherein In the absence of cells expressing EpCAM, CD40 agonist activity was not exhibited, and CD40 agonist activity was exhibited only in the presence of cells expressing EpCAM.

11. The bispecific antibody according to any one of claims 1 and 5 to 8, wherein The first antibody is an anti-CD40 antibody, and the second antibody is an anti-EpCAM antibody.

12. The bispecific antibody according to any one of claims 1 and 5 to 8, wherein The first antibody is an anti-EpCAM antibody, and the second antibody is an anti-CD40 antibody.

13. The bispecific antibody according to any one of claims 1 and 5 to 8, wherein The anti-CD40 antibody contains VH, and the VH consists of the amino acid sequence shown in any one of SEQ ID NOs. 27 and 32.

14. The bispecific antibody according to any one of claims 1 and 5 to 8, wherein The anti-EpCAM antibody contains VH, and the VH consists of an amino acid sequence represented by any one of SEQ ID NOs. 51, 56, 61 and 66. 15 . A DNA encoding the bispecific antibody according to claim 1 .

16. A recombinant vector comprising the DNA according to claim 15.

17. A transformant obtained by introducing the recombinant vector according to claim 16 into a non-plant-derived host cell.

18. The method for producing a bispecific antibody according to any one of claims 1 to 14, wherein The transformant according to claim 17 is cultured in a culture medium to produce and accumulate the bispecific antibody according to any one of claims 1 to 14 in the culture, and the bispecific antibody or the bispecific antibody fragment is collected from the culture.

Images