Cytotoxicity-inducing therapeutic agent

Abstract

To provide multispecific antigen-binding molecules comprising a first antigen-binding domain having RNF43-binding activity and a second antigen-binding domain having T cell receptor complex-binding activity, and to provide uses of such multispecific antigen-binding molecules, and the like.SOLUTION: The present invention relates to novel multispecific antigen-binding molecules comprising a first antigen-binding domain with RNF43-binding activity and a second antigen-binding domain with T-cell receptor complex-binding activity and having excellent cytotoxicity and high stability. Since the molecules of the present invention exhibit strong cytotoxicity against cells and tissues expressing RNF43, they are possible to produce novel pharmaceutical compositions containing multispecific antigen-binding molecules for treating or preventing various cancers.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a multispecific antigen-binding molecule comprising a first antigen-binding domain having RNF43 binding activity and a second antigen-binding domain having T cell receptor complex binding activity, and to the use of the same. [Background technology]

[0002] Cancer is one of the leading causes of death worldwide. With the exception of certain types of cancer, tumors are often inoperable at the time of diagnosis. Conventional cancer treatments include radiation therapy, chemotherapy, and immunotherapy. These treatments are often ineffective, ultimately leading to cancer recurrence or metastasis after treatment. Lack of tumor specificity is one factor limiting maximum effectiveness; therefore, more tumor-specific molecular targeted therapies are becoming a viable option in cancer treatment.

[0003] Antibodies are attracting attention as pharmaceuticals due to their high stability in plasma and low side effects. Among the various therapeutic antibodies, some types of antibodies require effector cells to produce an antitumor response. Antibody-dependent cell-mediated cytotoxicity (ADCC) is cytotoxicity exhibited by effector cells against cells to which an antibody is bound, when the Fc region of the antibody binds to Fc receptors present on NK cells and macrophages. To date, several therapeutic antibodies capable of inducing ADCC and exerting antitumor effects have been developed as pharmaceuticals for treating cancer (Non-Patent Literature 1). While therapies that target tumor-specific antigens using conventional therapeutic antibodies show excellent antitumor activity, the administration of such antibodies does not always yield satisfactory results.

[0004] In addition to antibodies that employ ADCC by recruiting NK cells or macrophages as effector cells, T-cell recruiting antibodies (TR antibodies) that employ cytotoxicity by recruiting T cells as effector cells have been known since the 1980s (Non-Patent Literature 2-4). TR antibodies are bispecific antibodies that recognize and bind to one of the subunits that form the T-cell receptor complex on T cells, particularly the CD3ε chain, and antigens on cancer cells. Several TR antibodies are currently under development. Catumakisomab, a TR antibody against EpCAM, has been approved in the EU for the treatment of malignant ascites. Furthermore, a type of TR antibody called a "bispecific T-cell engager (BiTE)" has recently been found to exhibit potent antitumor activity (Non-Patent Literature 5 and 6). Blinatumomab, a BiTE molecule against CD19, was the first to receive FDA approval in 2014. Blinatumomab was found to exhibit far more potent cytotoxic activity against CD19 / CD20-positive cancer cells in vitro compared to rituximab, which induces antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (Non-Patent Literature 7).

[0005] However, trifunctional antibodies are known to bind simultaneously to both T cells and cells such as NK cells or macrophages, independently of cancer antigens. As a result, receptors expressed on these cells are cross-linked, inducing the expression of various cytokines independently of cancer antigens. Systemic administration of trifunctional antibodies is thought to cause cytokine storm-like side effects as a result of this induction of cytokine expression. In fact, in a phase I clinical trial, it was reported that the maximum tolerated dose of catumaxomab for systemic administration to non-small cell lung cancer patients was an extremely low 5 μg / body, and that various serious side effects occurred with doses higher than this (Non-Patent Literature 8). With such low doses, catumaxomab cannot possibly reach its effective blood concentration. In other words, the expected antitumor effect cannot be obtained with such low doses of catumaxomab.

[0006] On the other hand, unlike catumaxomab, BiTE lacks an Fcγ receptor binding site and therefore does not cross-link receptors expressed on cells such as T cells, NK cells, and macrophages in a cancer antigen-dependent manner. Therefore, BiTE has been shown not to induce cancer antigen-independent cytokines, as is observed when catumaxomab is administered. However, because BiTE is a low-molecular-weight modified antibody molecule lacking an Fc region, it has the problem of having a significantly shorter half-life in the blood after administration to a patient compared to conventionally used IgG antibodies as therapeutic antibodies. In fact, the half-life of BiTE administered to a living organism has been reported to be only a few hours (Non-patent documents 9 and 10). In clinical trials of blinatumomab, administration is performed by continuous intravenous infusion using a minipump. This method of administration is not only extremely inconvenient for patients but also carries the potential risk of medical errors due to equipment malfunctions, etc. Therefore, this method of administration is not desirable.

[0007] Ubiquitin E3 ligase ring finger protein 43 (RNF43) is a single-pass type 1 transmembrane protein. RNF43 has been suggested as a negative feedback regulator of the Wnt signaling pathway (Non-Patent Literature 11). Several reports exist regarding the controversial role of RNF43 in tumorigenesis. Some reports consider RNF43 an oncogene based on the fact that it is one of the genes upregulated in colorectal tumors, is frequently overexpressed at both mRNA and protein levels in hepatocellular carcinoma, but is not significantly expressed in normal tissues (Non-Patent Literature 12). It has also been demonstrated that knockdown of RNF43 inhibits the proliferation of cancer cell lines (Non-Patent Literature 12 and 13). On the other hand, some other reports consider RNF43 a tumor suppressor based on the fact that RNF43 expression is downregulated at the protein level in tumor tissues such as pancreatic and gastric cancer, and that overexpression of RNF43 suppresses the proliferation of cancer cell lines (Non-Patent Literature 14 and 15). RNF43 is one of the genes that frequently mutates in pancreatic cancer, and it is also known that decreased RNF43 expression is associated with the presence of such mutations, which implies a tumor suppressor function of RNF43 (Non-Patent Literature 16). As a result, the ability of RNF43 as a cancer therapeutic target has not yet been evaluated.

[0008] Peptide vaccine therapy using RNF43-derived epitope peptides has been clinically evaluated in patients with advanced or recurrent colorectal cancer. While the vaccine therapy is well-tolerated, it has been found to have limited efficacy (Non-Patent Literature 17). An anti-RNF43 antibody-drug conjugate (ADC) has been constructed, which has shown cytotoxic activity against human RNF43-overexpressing HEK293T cells in vitro (Patent Literature 1), but its efficacy against RNF43-positive tumor cells and its potential as a treatment for RNF43-positive tumors remain unclear. [Prior art documents] [Patent Documents]

[0009]

Patent Document 1

Non-licensed literature

[0010] [Non-licensed document 1] Clin Cancer Res. 2010 Jan 1;16(1):11-20 [Non-licensed document 2] Nature. 1985 Apr 18-24;314(6012):628-31 [Non-licensed document 3] Int J Cancer. 1988 Apr 15;41(4):609-15

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

[0011] An anti - RNF43 antibody - drug conjugate (ADC) has been constructed, but its efficacy against RNF43 - positive tumor cells and its treatment potential for treating RNF43 - positive tumors have not yet been elucidated. Those skilled in the art will know that ADCs are not sufficiently effective when the abundance of the antigen in the tumor is low, or when the internalization rate of the ADC - antigen complex is low, and the conjugated drug is affected by the activity of drug transporters. The present invention was achieved by providing an effective therapeutic method that targets RNF43, based on the analysis of tumors with high RNF43 levels. An object of the present invention is to provide a multispecific antigen-binding molecule that enables the treatment of cancer by bringing T cells into close proximity to RNF43-expressing cells. An object of the present invention is to provide a method for producing a multispecific antigen-binding molecule using the cytotoxicity of T cells against RNF43-expressing cancer cells, and a therapeutic agent for inducing cytotoxicity, comprising such a multispecific antigen-binding molecule as an active ingredient. Another object of the present invention is to provide a pharmaceutical composition for treating or preventing various cancers, comprising the above-mentioned therapeutic agent for inducing cytotoxicity as an active ingredient, and a therapeutic method using the pharmaceutical composition. [Means for solving the problem]

[0012] The inventors have discovered that a multispecific antigen-binding molecule containing a first antigen-binding domain having RNF43-binding activity and a second antigen-binding domain having T-cell receptor complex-binding activity can damage RNF43-expressing cells and exhibit excellent antitumor activity. Furthermore, the inventors have discovered a pharmaceutical composition containing this antigen-binding molecule as an active ingredient that can treat various cancers, particularly RNF43-positive tumors. More specifically, the present invention provides the following: [1] A multispecific antigen-binding molecule comprising a first antigen-binding domain having RNF43 binding activity and a second antigen-binding domain having T cell receptor complex binding activity. [2] A cytotoxic, multispecific antigen-binding molecule as described in [1]. [3] A multispecific antigen-binding molecule as described in [1] or [2], wherein the cytotoxicity is T cell-dependent. [4] A multispecific antigen-binding molecule according to any one of [2] to [3], which has cytotoxic effects on cells expressing RNF43 on its surface. [5] RNF43-expressing cells are cancer cells, and the multispecific antigen-binding molecule described in [4]. [6] A multispecific antigen-binding molecule as described in any of [1] to [5], wherein the T cell receptor complex binding activity is binding activity to the T cell receptor. [7] A multispecific antigen-binding molecule described in any of [1] to [5], wherein the T cell receptor complex binding activity is binding activity to the CD3ε chain. [8] A multispecific antigen-binding molecule described in any of [1] to [7], wherein the RNF43 binding activity is binding activity to human RNF43. [9] A multispecific antigen-binding molecule as described in any of [1] to [7], wherein the RNF43 binding activity is the binding activity to RNF43 on the surface of eukaryotic cells.

[10] A multispecific antigen-binding molecule as described in any of [1] to [9], wherein the RNF43 binding activity is the binding activity to human RNF43 on the surface of eukaryotic cells.

[11] A multispecific antigen-binding molecule according to any one of [1] to

[10] , wherein the first antigen-binding domain is a domain containing the variable regions of the heavy and light chains of the antibody, and / or the second antigen-binding domain is a domain containing the variable regions of the heavy and light chains of the antibody.

[12] A multispecific antigen-binding molecule according to any one of [1] to

[11] , wherein the first antigen-binding domain is a domain containing an antibody-variable fragment, and / or the second antigen-binding domain is a domain containing an antibody-variable fragment.

[13] A multispecific antigen-binding molecule according to any one of [1] to

[12] , wherein the first antigen-binding domain is a domain containing a Fab structure and / or the second antigen-binding domain is a domain containing a Fab structure.

[14] A multispecific antigen-binding molecule according to any of [1] to

[13] , wherein the first antigen-binding domain contains one of the following antibody-variable fragments: (a) Antibody variable fragments comprising an antibody heavy chain variable region including HVR-H1 containing the amino acid sequence of SEQ ID NO: 28, HVR-H2 containing the amino acid sequence of SEQ ID NO: 48, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 68, and an antibody light chain variable region including HVR-L1 containing the amino acid sequence of SEQ ID NO: 38, HVR-L2 containing the amino acid sequence of SEQ ID NO: 58, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 78; (b) Antibody variable fragments comprising an antibody heavy chain variable region including HVR-H1 containing the amino acid sequence of SEQ ID NO: 31, HVR-H2 containing the amino acid sequence of SEQ ID NO: 51, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 71, and an antibody light chain variable region including HVR-L1 containing the amino acid sequence of SEQ ID NO: 41, HVR-L2 containing the amino acid sequence of SEQ ID NO: 61, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 81; (c) Antibody variable fragments comprising an antibody heavy chain variable region including HVR-H1 containing the amino acid sequence of SEQ ID NO: 33, HVR-H2 containing the amino acid sequence of SEQ ID NO: 53, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 73, and an antibody light chain variable region including HVR-L1 containing the amino acid sequence of SEQ ID NO: 43, HVR-L2 containing the amino acid sequence of SEQ ID NO: 63, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 83; (d) Antibody variable fragments comprising an antibody heavy chain variable region including HVR-H1 containing the amino acid sequence of SEQ ID NO: 34, HVR-H2 containing the amino acid sequence of SEQ ID NO: 54, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 74, and an antibody light chain variable region including HVR-L1 containing the amino acid sequence of SEQ ID NO: 44, HVR-L2 containing the amino acid sequence of SEQ ID NO: 64, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 84; (e) Antibody variable fragments comprising an antibody heavy chain variable region containing HVR-H1 containing the amino acid sequence of SEQ ID NO: 35, HVR-H2 containing the amino acid sequence of SEQ ID NO: 55, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 75, and an antibody light chain variable region containing HVR-L1 containing the amino acid sequence of SEQ ID NO: 45, HVR-L2 containing the amino acid sequence of SEQ ID NO: 65, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 85; (f) an antibody variable fragment that competes with any one of the antibody variable fragments of (a) to (e) for binding to human RNF43; and (g) An antibody variable fragment that binds to the same epitope on human RNF43 as any one of the antibody variable fragments (a) to (e).

[15] A multispecific antigen-binding molecule according to any one of [1] to

[14] , further comprising a domain containing an Fc region with reduced Fcγ receptor binding activity.

[16] The multispecific antigen-binding molecule according to

[15] , wherein the Fc region of the multispecific antigen-binding molecule has reduced Fcγ receptor binding activity compared to the Fc domain of an IgG1, IgG2, IgG3, or IgG4 antibody.

[17] A multispecific antigen-binding molecule according to

[15] or

[16] , wherein the Fc region is an Fc region having an amino acid mutation in any of the Fc region constituent amino acids of SEQ ID NOs: 122-125 (IgG1-IgG4).

[18] The Fc region is located at the following amino acid positions, as specified by EU numbering: 220th, 226th, 229th, 231st, 232nd, 233rd, 234th, 235th, 236th, 237th, 238th, 239th, 240th, 264th, 265th, 266th, 2 67th, 269th, 270th, 295th, 296th, 297th, 298th, 299th, 300th, 325th, 327th, 328th, 329th, 330th, 331st, and 332nd A multispecific antigen-binding molecule as described in

[17] , which is an Fc region having a mutation in at least one more selected amino acid.

[19] A bispecific antibody comprising a first antibody variable fragment having RNF43 binding activity, a second antibody variable fragment having CD3ε chain binding activity, and an Fc region with reduced Fcγ receptor binding activity, as described in any of [1] to

[18] .

[20] A bispecific antibody, which is a multispecific antigen-binding molecule as described in any of [1] to

[19] . A nucleic acid encoding a multispecific antigen-binding molecule as described in any of

[21] , [1], to

[20] . A vector into which the nucleic acids described in

[22] and

[21] have been introduced. Cells containing the nucleic acid described in

[23] or

[21] or the vector described in

[22] . A method for producing a multispecific antigen-binding molecule described in any of [1] to

[20] by culturing the cells described in

[24] and

[23] . A multispecific antigen-binding molecule produced by the method described in

[25] and

[24] . A pharmaceutical composition comprising a multispecific antigen-binding molecule as described in any of

[26] [1] to

[20] . A pharmaceutical composition for use in inducing cytotoxicity, comprising a multispecific antigen-binding molecule as described in any of

[27] [1] to

[20] . A pharmaceutical composition for use in the treatment or prevention of cancer, comprising a multispecific antigen-binding molecule as described in any of

[28] [1] to

[20] .

[29] The pharmaceutical composition according to

[28] , wherein the cancer is colorectal cancer or gastric cancer. A method for treating or preventing cancer, wherein a multispecific antigen-binding molecule described in any of

[30] [1] to

[20] is administered to a patient in need thereof.

[31] The method according to

[30] , wherein the cancer is colorectal cancer or gastric cancer. [Effects of the Invention]

[0013] This invention provides a multispecific antigen-binding molecule that enables cancer treatment by bringing T cells into close proximity to RNF43-expressing cells and utilizing the cytotoxicity of T cells against RNF43-expressing cancer cells, a method for producing the multispecific antigen-binding molecule, and a therapeutic agent for inducing cytotoxicity containing such a multispecific antigen-binding molecule as an active ingredient. The multispecific antigen-binding molecule of this invention has potent antitumor activity, induces cytotoxicity, and can target and damage RNF43-expressing cells, thus enabling the treatment and prevention of various cancers. Furthermore, in certain embodiments, the multispecific antigen-binding molecule of this invention has a long blood half-life and excellent safety characteristics, such as not inducing cancer antigen-independent cytokine storms. This enables desirable treatment with high safety and convenience, and reduces the physical burden on patients. [Brief explanation of the drawing]

[0014] [Figure 1] Box plots of human RNF43 mRNA expression profiles in normal and tumor tissues, constructed using data downloaded from TCGA. [Figure 2] Binding of anti-RNF43 monospecific antibody to Ba / F3 E12 transfectant (a) and NUGC-4 cancer cell line (b), as determined by FACS analysis. [Figure 3] Antibody binding ability (ABC) of RNF43 on the surface of cancer cells. [Figure 4] T cell-dependent cytotoxicity (TDCC) of anti-RNF43 / CD3 bispecific antibodies against RNF43-expressing cell lines (a: NUGC-4 cell line; b: SW48 cell line). [Figure 5] In vivo antitumor activity of anti-RNF43 / CD3 bispecific antibodies. [Figure 6] Inhibition of binding between monospecific anti-RNF43 antibodies. [Modes for carrying out the invention]

[0015] Description of the mannerThe techniques and procedures described or referenced herein are generally well understood, and refer to, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press;Animal Cell Culture (RI Freshney), ed., 1987);Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons;Handbook of Experimental Immunology (DM Weir and CCBlackwell, eds.);Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987);PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology (JE Coligan et al., eds., 1991);Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (CA Janeway and P. Travers, 1997);Antibodies (P. Finch, 1997);Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989);Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using Antibodies: A Laboratory Manual (E. This methodology is commonly used by those skilled in the art, employing conventional methodologies such as those described in Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0016] antigen binding molecule As used herein, the term "antigen-binding molecule" refers to any molecule containing an antigen-binding domain, and may further refer to molecules such as peptides or proteins having a length of approximately 5 amino acids or more. Peptides and proteins are not limited to those of biological origin, but may also be polypeptides produced from artificially designed sequences, for example. They may also be natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc.

[0017] A preferred example of the antigen-binding molecule of the present invention is an antigen-binding molecule comprising multiple antigen-binding domains. In a particular embodiment, the antigen-binding molecule of the present invention is an antigen-binding molecule comprising two antigen-binding domains with different antigen-binding specificities. In a particular embodiment, the antigen-binding molecule of the present invention is an antigen-binding molecule comprising two antigen-binding molecules comprising two antigen-binding domains with different antigen-binding specificities and an FcRn-binding domain contained in the Fc region of an antibody. A well-known method for extending the blood half-life of a protein administered to a living organism is to attach the FcRn-binding domain of an antibody to the target protein and utilize the recycling function via FcRn.

[0018] antigen-binding domain As used herein, the term “antigen-binding domain” refers to a portion of an antibody that contains a region that specifically binds to and is complementary to all or part of an antigen. When the molecular weight of an antigen is large, the antibody may bind only to a specific portion of the antigen. This specific portion is referred to as an “epitope.” An antigen-binding domain may be provided from one or more antibody variable domains. Preferably, the antigen-binding domain includes both an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Such preferred antigen-binding domains include, for example, “single-chain Fv (scFv),” “single-chain antibody,” “Fv,” “single-chain Fv2 (scFv2),” “Fab,” and “F(ab')2.”

[0019] The antigen-binding domain of the antigen-binding molecule of the present invention may bind to the same epitope. The epitope may be present in a protein containing the amino acid sequence of SEQ ID NO: 94 or 102. Alternatively, the antigen-binding domain of the polypeptide complex of the present invention may bind to individually different epitopes. The epitopes may be present in a protein containing the amino acid sequence of SEQ ID NO: 94 or 102.

[0020] The antigen-binding domain of the antigen-binding molecule of the present invention "has RNF43 binding activity or T cell receptor complex binding activity." That is, RNF43 and the T cell receptor complex are the preferred antigens of interest. As used herein, "has binding activity" means that an antigen-binding domain, antibody, antigen-binding molecule, antibody variable fragment, etc. (hereinafter referred to as "antigen-binding domain, etc.") has the activity to bind to the antigen of interest at a level of specific binding higher than the level of nonspecific binding or background binding. In other words, such an antigen-binding domain, etc. "has specific / significant binding activity" to the antigen of interest. Specificity can be measured by any method for detecting affinity or binding activity that is mentioned herein or known in the art. The level of specific binding may be high enough to be recognized as significant by a person skilled in the art. For example, an antigen-binding domain, etc. can be said to have "specific / significant binding activity" to the antigen of interest if a person skilled in the art can detect or observe any significant or relatively strong signal or value of binding between the antigen-binding domain, etc. and the antigen of interest in a suitable binding assay. Alternatively, "having specific / significant binding activity" can be rephrased as "binding specifically / significantly (to the target antigen)." In some cases, the phrase "having binding activity" has substantially the same meaning as the phrase "having specific / significant binding activity" in this art.

[0021] RNF43 As used herein, the term “RNF43” refers to any native RNF43 (ring finger protein 43) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. This term encompasses both “full-length” unprocessed RNF43 and any form of RNF43 resulting from in-cellular processing. The term also encompasses naturally occurring variants of RNF43, such as splice variants and allele variants. An exemplary human RNF43 amino acid sequence is shown in SEQ ID NO: 89.

[0022] Ring finger protein 43 (RNF43; also known as E3 ubiquitin protein ligase RNF43 or RNF124) is a single-pass type 1 transmembrane protein that functions as an important feedback regulator of WNT signaling. Representative RNF43 protein orthologues include, but are not limited to, humans (NP_060233, SEQ ID NO: 89), chimpanzees (XP_001172611, SEQ ID NO: 90), rhesus monkeys (XP_001106574, SEQ ID NO: 91), rats (NP_001129393, SEQ ID NO: 92), and mice (NP_766036, SEQ ID NO: 93). In humans, the RNF43 gene consists of 10 exons extending approximately 63.9 kBp on chromosome 17 at cytogenetic location 17q22. Transcription of the human RNF43 locus yields a spliced ​​4.6 kBp mature mRNA transcript (NM_017763) encoding a 783-amino acid protein precursor (NP_060233, SEQ ID NO: 89). Processing of the RNF43 protein precursor is predicted to involve the removal of the first 23 amino acids, including the secretory signal peptide. The mature RNF43 protein is predicted to contain a 174-amino acid extracellular domain (amino acids 24-197 of SEQ ID NO: 89), a 21-amino acid helical transmembrane domain (amino acids 198-218 of SEQ ID NO: 89), and a 565-amino acid cytoplasmic domain (amino acids 219-783 of SEQ ID NO: 89), with a portion of the cytoplasmic domain containing the atypical RING domain zinc finger (amino acids 272-313 of SEQ ID NO: 89), from which the protein is named. The RING domain is a clearly defined domain associated with the formation of zinc finger structures that mediate protein-protein interactions, and is commonly found in proteins involved in protein ubiquitination.

[0023] Affinity "Affinity" refers to the strength of the combined non-covalent interactions between one binding site of a molecule (e.g., an antigen-binding molecule or antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antigen-binding molecule and an antigen, or an antibody and an antigen). The affinity of molecule X to its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific examples and exemplary embodiments for measuring binding affinity are described below.

[0024] How to determine affinity In certain embodiments, the antigen-binding domain of the antigen-binding molecule or antibody provided herein is capable of binding to its antigen in the following ranges: ≤1 μM, ≤120 nM, ≤100 nM, ≤80 nM, ≤70 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤10 nM, ≤2 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (for example, 10 -8 M or less, 10 -8 M~10 -13 M, 10 -9 M~10 -13 It has a dissociation constant (Kd) of M. In a particular embodiment, the Kd value of the first antigen-binding domain of the antibody / antigen-binding molecule for RNF43 falls within the range of 1-40, 1-50, 1-70, 1-80, 30-50, 30-70, 30-80, 40-70, 40-80, or 60-80 nM.

[0025] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using the Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of Fab to the antigen is measured in the presence of a gradual increase in the concentration of the unlabeled antigen. 125I) Fab is equilibrated with a labeled antigen, and then the bound antigen is captured by a plate coated with anti-Fab antibody. (See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the measurement conditions, a MICROTITER® multiwell plate (Thermo Scientific) is coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ 125 Mix the [I]-antigen with serial dilutions of the Fab of interest (e.g., as in the evaluation of anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). Then incubate the Fab of interest overnight, although this incubation may be extended for a longer period (e.g., about 65 hours) to ensure equilibrium is achieved. Subsequently, transfer the mixture to a capture plate for incubation at room temperature (e.g., 1 hour). Then remove the solution and wash the plate eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, add 150 μl / well of scintillant (MICROSCINT-20®, Packard) and count the plate for 10 minutes on a TOPCOUNT® gamma counter (Packard). Select concentrations of each Fab that give less than 20% of maximum binding for use in competitive binding assays.

[0026] According to another aspect, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, a measurement method using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) is carried out at 25° C using a CM5 chip with approximately 10 response units (RU) of antigen immobilized thereon. In one aspect, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated using N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) using 10 mM sodium acetate, pH 4.8 before being injected at a flow rate of 5 μl / min to achieve binding of approximately 10 response units (RU) of protein. After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold dilutions (0.78 nM to 500 nM) of Fab in PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20™) surfactant are injected at 25° C and a flow rate of approximately 25 μl / min. The association rate (k on ) and dissociation rate (k off ) are calculated by simultaneously fitting the binding and dissociation sensorgrams using a simple 1:1 Langmuir binding model (BIACORE® evaluation software version 3.2). The equilibrium dissociation constant (Kd) is calculated as the ratio of k off / k on . See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). The association rate by the above surface plasmon resonance assay is 10 6 M -1 s -1If it exceeds this, the ON rate can be determined by measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) at 25°C in the presence of gradually increasing concentrations of antigen using a spectrometer (e.g., a stop-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO® spectrophotometer (ThermoSpectronic) using a stirred cuvette).

[0027] A method for measuring the affinity of an antibody's antigen-binding domain has been described above, and those skilled in the art can perform affinity measurements of other antigen-binding domains.

[0028] antibody In this specification, the term “antibody” is used in its broadest sense and encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0029] Antibody class The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0030] framework The "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of HVR and FR usually appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0031] Human Consensus Framework The "Human Consensus Framework" is a framework that shows the most commonly occurring amino acid residues in selected human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Typically, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI by Kabat et al. As described above. In another embodiment, for VH, the subgroup is subgroup III by Kabat et al. As described above.

[0032] HVR As used herein, the term “hypervariable region” or “HVR” refers to each region of the variable domain of an antibody that is hypervariable in sequence (a “complementarity determining region” or “CDR”), and / or forms a structurally defined loop (a “hypervariable loop”), and / or contains an antigen contact residue (a “antigen contact”). Typically, an antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Illustrative HVRs as used herein include: (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and, (d) A combination of (a), (b), and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein in accordance with Kabat et al.

[0033] Variable region The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening complementary libraries of VL or VH domains, respectively, using the VH or VL domains from antibodies that bind to that antigen. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0034] Chimeric antibodies The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remaining heavy chain and / or light chain originates from a different source or species. Similarly, the term "chimeric antibody variable domain" refers to an antibody variable region in which a portion of the heavy chain and / or light chain variable region originates from a specific source or species, while the remaining heavy chain and / or light chain variable region originates from a different source or species.

[0035] Humanized antibodies A “humanized” antibody is a chimeric antibody that contains amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization. The “humanized antibody variable region” refers to the variable region of a humanized antibody.

[0036] Human antibodies A "human antibody" is an antibody that possesses an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an antibody derived from a non-human source that uses the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues. The "variable region of a human antibody" refers to the variable region of a human antibody.

[0037] Method for producing an antibody having a desired binding activity. Methods for producing antibodies with desired binding activity are known to those skilled in the art. The following is an example of a method for producing an antibody that binds to the ring finger protein 43 (hereinafter also referred to as RNF43) (anti-RNF43 antibody). Antibodies that bind to T cell receptor complexes, etc., can also be produced according to the example described below.

[0038] Anti-RNF43 antibodies can be obtained as polyclonal or monoclonal antibodies using known methods. Preferably, anti-RNF43 antibodies are mammalian monoclonal antibodies. Such mammalian monoclonal antibodies include antibodies produced by hybridomas or antibodies produced by host cells transformed with expression vectors containing the antibody gene using genetic engineering techniques.

[0039] Monoclonal antibody-producing hybridomas can be manufactured using known techniques, for example, as described below. Specifically, mammals are immunized using conventional immunization methods with the RNF43 protein as a sensitizing antigen. The resulting immune cells are fused with known parent cells using conventional cell fusion methods. Then, hybridomas that produce anti-RNF43 antibodies can be selected by screening monoclonal antibody-producing cells using conventional screening methods.

[0040] Specifically, monoclonal antibodies are prepared as follows. First, the RNF43 gene, whose nucleotide sequence is disclosed in RefSeq registry number NM_017763.5, can be expressed to produce the RNF43 protein shown in RefSeq registry number NP_060233.3 (SEQ ID NO: 89), which is then used as a sensitizing antigen for antibody preparation. Alternatively, the nucleotide encoding the extracellular domain (ECD) of RNF43 can be expressed to produce the RNF43 ECD-containing protein whose amino acid sequence is described in SEQ ID NO: 94. That is, the gene sequence encoding full-length RNF43 or RNF43 ECD is inserted into a known expression vector, and appropriate host cells are transformed with this vector. The desired human full-length RNF43 or RNF43 ECD protein is purified from the host cells or their culture supernatant by known methods. Alternatively, purified native RNF43 protein can be used as a sensitizing antigen.

[0041] Purified full-length RNF43 or RNF43 ECD protein can be used as a sensitizing antigen for use in mammalian immunization. Partial peptides of full-length RNF43 or RNF43 ECD can also be used as sensitizing antigens. In this case, the partial peptide may be obtained by chemical synthesis from the human RNF43 amino acid sequence. Furthermore, the partial peptide may be obtained by incorporating a portion of the RNF43 gene into an expression vector and expressing it. In addition, the partial peptide may be obtained by degrading the RNF43 protein using a protease, but the region and size of the RNF43 peptide used as a partial peptide are not particularly limited to any specific embodiment. Preferred regions may be any sequence from the amino acid sequence corresponding to amino acids 1-197 in the amino acid sequence of SEQ ID NO: 89. The number of amino acids constituting the peptide used as a sensitizing antigen is at least 5, preferably 6 or 7. More specifically, peptides consisting of 8-50 residues, or preferably 10-30 residues, can be used as sensitizing antigens.

[0042] Alternatively, a fusion protein prepared by fusing a desired partial polypeptide or peptide of full-length RNF43 or RNF43 ECD protein with a different polypeptide can be used as a sensitizing antigen. For example, an antibody Fc fragment and a peptide tag are preferably used to generate a fusion protein to be used as a sensitizing antigen. A vector for expressing such a fusion protein can be constructed by fusing genes encoding two or more desired polypeptide fragments in-frame and inserting the fusion gene into an expression vector as described above. A method for generating a fusion protein is described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab. Press). A method for preparing RNF43 to be used as a sensitizing antigen and an immunization method using RNF43 will be described later in the examples of this specification.

[0043] There are no particular limitations on the mammals immunized with the sensitizing antigen. However, it is preferable to select mammals considering their compatibility with the parent cells used for cell fusion. Generally, rodents such as mice, rats, hamsters, rabbits, and monkeys are preferred.

[0044] The animals described above are immunized with sensitizing antigens by known methods. For example, commonly performed immunization methods include intraperitoneal or subcutaneous injection of sensitizing antigens into mammals. Specifically, the sensitizing antigen is appropriately diluted with PBS (phosphate-buffered saline), saline, etc. If desired, a conventional adjuvant, such as Freund's complete adjuvant, is mixed with the antigen, and the mixture is emulsified. The sensitizing antigen is then administered to mammals several times every 4 to 21 days. A suitable carrier may be used in immunization with sensitizing antigens. In particular, when a low molecular weight partial peptide is used as the sensitizing antigen, it may be desirable to conjugate the sensitizing antigen peptide to a carrier protein such as albumin or keyhole limpet hemocyanin for immunization.

[0045] Alternatively, hybridomas that produce the desired antibody can be prepared using DNA immunization as follows. DNA immunization is an immunization method in which immune stimulation is provided by administering vector DNA, which is constructed so that the gene encoding the antigen protein can be expressed in the animal, thereby causing the sensitized antigen to be expressed in the immunized animal. Compared to conventional immunization methods in which protein antigens are administered to immunized animals, DNA immunization is expected to have the following advantages: -Immunostimulation can be delivered while maintaining the structure of membrane proteins such as RNF43; and - There is no need to purify antigens for immunization.

[0046] To prepare the monoclonal antibody of the present invention using DNA immunization, first, DNA expressing the RNF43 protein is administered to an immunized animal. The DNA encoding RNF43 can be synthesized by known methods such as PCR. The obtained DNA is inserted into a suitable expression vector, and then this vector is administered to an immunized animal. A commercially available expression vector such as pcDNA3.1 is a preferred example. The vector can be administered to the organism using conventional methods. For example, DNA immunization is performed by introducing gold particles coated with the expression vector into cells in the body of an immunized animal using a gene gun. Furthermore, antibodies that recognize RNF43 can also be produced by the method described in WO2003 / 104453.

[0047] As described above, after immunization of mammals, an increase in the titer of RNF43-conjugated antibodies is confirmed in the serum. Subsequently, immune cells are collected from the mammals and then subjected to cell fusion. Splenocytes, in particular, are preferably used as immune cells.

[0048] Mammalian myeloma cells are used as the cells fused with the above-mentioned immune cells. It is preferable that the myeloma cells possess appropriate selection markers for screening. These selection markers provide cells with characteristics that allow them to survive (or die) under specific culture conditions. Known selection markers include hypoxanthine-guanine phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). Cells lacking HGPRT or TK are hypoxanthine-aminopterin-thymidine sensitive (hereinafter abbreviated as HAT sensitive). HAT-sensitive cells die because they cannot synthesize DNA in HAT-selective medium. However, when these cells fuse with normal cells, they can continue DNA synthesis using the normal cell's salvage pathway, and thus can proliferate even in HAT-selective medium.

[0049] HGPRT-deficient and TK-deficient cells can be selected in media containing 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells dies because they incorporate these pyrimidine analogs into their DNA. On the other hand, cells lacking these enzymes can survive in the selective medium because they cannot incorporate these pyrimidine analogs. Furthermore, a selection marker called G418 resistance, mediated by the neomycin resistance gene, confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs). Various myeloma cells suitable for cell fusion are known.

[0050] For example, myeloma cells including the following cells may be suitably used: P3 (P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519), MPC-11 (Cell (1976) 8 (3), 405-415), SP2 / 0 (Nature (1978) 276 (5685), 269-270), FO (J. Immunol. Methods (1980) 35 (1-2), 1-21), S194 / 5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323), R210 (Nature (1979) 277 (5692), 131-133), etc.

[0051] Basically, cell fusion between immune cells and myeloma cells is performed using known methods, such as the method of Kohler and Milstein et al. (Methods Enzymol. (1981) 73: 3-46). More specifically, cell fusion can be performed in conventional culture media, for example, in the presence of a cell fusion promoter. Examples of fusion promoters include polyethylene glycol (PEG) and Sendai virus (HVJ). If necessary, auxiliary substances such as dimethyl sulfoxide may also be added to enhance fusion efficiency.

[0052] The ratio of immune cells to myeloma cells can be set arbitrarily; for example, 1 to 10 immune cells to 1 myeloma cell is preferred. Examples of culture media used for cell fusion include media suitable for the proliferation of myeloma cell lines, such as RPMI1640 medium and MEM medium, as well as other conventional media used for this type of cell culture. Furthermore, serum solutions such as fetal bovine serum (FCS) can be suitably added to the culture medium.

[0053] For cell fusion, a predetermined amount of the above-mentioned immune cells and myeloma cells is thoroughly mixed in the above-mentioned culture medium. Then, a PEG solution (for example, with an average molecular weight of about 1000 to 6000) that has been preheated to about 37°C is added to it at a concentration of usually 30% to 60% (w / v). When this is gently mixed, the desired fused cells (hybridomas) are generated. Next, the above-mentioned appropriate culture medium is sequentially added to the cells, and the mixture is repeatedly centrifuged to remove the supernatant. In this way, cell fusion agents and other substances unfavorable to hybridoma growth can be removed.

[0054] The hybridomas obtained in this manner can be selected by culturing them in a conventional selective medium, such as HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). Cells other than the desired hybridoma (non-fused cells) can be killed by continuing to culture them in the HAT medium for a sufficient period of time. This period is usually several days to several weeks. Then, hybridomas that produce the desired antibody are screened and single-cloned using a conventional limiting dilution method.

[0055] The hybridomas obtained in this way can be selected using a selective medium based on the selection markers present in the myeloma used for cell fusion. For example, HGPRT-deficient cells or TK-deficient cells can be selected by culturing them in HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that successfully fuse with normal cells can be selectively proliferated in HAT medium. Cells other than the desired hybridoma (non-fused cells) can be killed by continuing to culture them in the HAT medium for a sufficient period of time. Specifically, the desired hybridoma can generally be selected by culturing for several days to several weeks. Then, hybridomas that produce the desired antibody are screened and single-cloned using the conventional limiting dilution method.

[0056] Desired antibodies can be suitably selected and single-cloned by known antigen / antibody reaction-based screening methods. For example, a monoclonal antibody that binds to RNF43 can bind to RNF43 expressed on the cell surface. Such monoclonal antibodies can be screened by fluorescence-activated cell sorting (FACS). FACS is a system that evaluates antibody binding to the cell surface by analyzing cells contacted with a fluorescent antibody using laser light and measuring the fluorescence emitted by individual cells.

[0057] To screen hybridomas that produce the monoclonal antibody of the present invention by FACS, cells expressing RNF43 are first prepared. Mammalian cells that overexpress RNF43 are preferably used for screening. As a control, untransformed mammalian cells can be used as host cells to selectively detect the antibody binding activity to RNF43 on the cell surface. That is, hybridomas that produce antibodies that bind to RNF43-overexpressing cells but not to host cells can be isolated.

[0058] Alternatively, the binding activity of antibodies against immobilized RNF43-expressing cells can be evaluated based on the principles of ELISA. For example, RNF43-expressing cells are immobilized in the wells of an ELISA plate. The culture supernatant of hybridomas is brought into contact with the immobilized cells in the wells, and antibodies that bind to the immobilized cells are detected. If the monoclonal antibody is derived from a mouse, the antibody bound to the cells can be detected using an anti-mouse immunoglobulin antibody. Hybridomas that produce the desired antibody with antigen-binding ability are selected by the above screening, and these can be cloned by methods such as limiting dilution.

[0059] The monoclonal antibody-producing hybridomas prepared in this manner can be subcultured in conventional culture media and stored for extended periods in liquid nitrogen.

[0060] The above-mentioned hybridomas can be cultured using conventional methods, and the desired monoclonal antibody can be prepared from the culture supernatant. Alternatively, the hybridomas can be administered to a compatible mammal to grow, and the monoclonal antibody can be prepared from its ascites fluid. The former method is suitable for preparing high-purity antibodies.

[0061] Antibodies encoded by antibody genes cloned from antibody-producing cells such as the hybridomas mentioned above can also be suitably utilized. The cloned antibody gene is inserted into a suitable vector, which is then introduced into a host to express the antibody encoded by that gene. Methods for isolating antibody genes, inserting them into vectors, and transforming host cells have already been established, for example, by Vandamme et al. (Eur.J. Biochem. (1990) 192(3), 767-775). Methods for producing recombinant antibodies are also known, as described below.

[0062] Preferably, the present invention provides nucleic acids encoding the multispecific antigen-binding molecules of the present invention. The present invention also provides vectors into which the nucleic acids encoding the multispecific antigen-binding molecules are introduced, i.e., vectors containing the nucleic acids. Furthermore, the present invention provides cells containing the nucleic acids or the vectors. The present invention also provides a method for producing the multispecific antigen-binding molecules by culturing the cells. The present invention further provides multispecific antigen-binding molecules produced by the method.

[0063] For example, cDNA encoding the variable region (V region) of an anti-RNF43 antibody can be prepared from hybridoma cells expressing an anti-RNF43 antibody. To do this, total RNA is first extracted from the hybridoma. Methods used to extract mRNA from cells include, for example, the following: -Guanidine ultracentrifugation (Biochemistry (1979) 18(24), 5294-5299), and - AGPC method (Anal. Biochem. (1987) 162(1), 156-159)

[0064] The extracted mRNA can be purified using an mRNA Purification Kit (GE Healthcare Bioscience), or similar kits for directly extracting total mRNA from cells, such as the QuickPrep mRNA Purification Kit (GE Healthcare Bioscience). mRNA can be prepared from hybridomas using such kits. From the prepared mRNA, cDNA encoding the antibody V region can be synthesized using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation), or similar kits. Additionally, the SMART RACE cDNA amplification kit (Clontech) and the PCR-based 5'-RACE method (Proc. Natl. Acad. Sci. USA (1988) 85(23), 8998-9002, Nucleic Acids Res. (1989) 17(8), 2919-2932) may be used as appropriate for cDNA synthesis and amplification. During this cDNA synthesis process, appropriate restriction enzyme sites, as described later, can be introduced at both ends of the cDNA.

[0065] The desired cDNA fragment is purified from the obtained PCR product and then ligated to vector DNA. A recombinant vector is thus constructed and introduced into Escherichia coli (E. coli), etc. After colony selection, the desired recombinant vector can be prepared from the E. coli that formed the colony. Then, whether the recombinant vector has the desired cDNA nucleotide sequence is tested by a known method, such as dideoxynucleotide chain intermination.

[0066] To isolate genes encoding variable regions, the 5'-RACE method, which uses primers to amplify variable region genes, is a convenient method. First, a 5'-RACE cDNA library is constructed by synthesizing cDNA using RNA extracted from hybridoma cells as a template. Commercially available kits, such as the SMART RACE cDNA amplification kit, are used as appropriate for synthesizing the 5'-RACE cDNA library.

[0067] The antibody gene is amplified by PCR using a prepared 5'-RACE cDNA library as a template. Based on known antibody gene sequences, primers for mouse antibody gene amplification can be designed. The nucleotide sequences of these primers differ for each immunoglobulin subclass. Therefore, it is preferable to pre-determine the subclass using a commercially available kit such as the Iso Strip mouse monoclonal antibody isotyping kit (Roche Diagnostics).

[0068] Specifically, for example, to isolate the gene encoding mouse IgG, primers capable of amplifying the genes encoding γ1, γ2a, γ2b, and γ3 heavy chains, as well as the κ and λ light chains, are used. To amplify the variable region genes of IgG, a primer that anneals to a constant region site close to the variable region is generally used as the 3' primer. On the other hand, the primers included with the 5' RACE cDNA library construction kit are used as the 5' primer.

[0069] The amplified PCR product is then used to reconstitute an immunoglobulin consisting of a combination of heavy and light chains. The RNF43 binding activity of the reconstituted immunoglobulin can be used as an indicator to select the desired antibody. For example, when the goal is to isolate an antibody against RNF43, the specificity of the antibody's binding to RNF43 is even more preferable. Antibodies that bind to RNF43 can be screened, for example, by the following steps: (1) A step of contacting RNF43-expressing cells with an antibody containing a V region encoded by cDNA isolated from a hybridoma, (2) A step to detect the binding of RNF43-expressing cells to an antibody, and (3) A step of selecting an antibody that binds to RNF43-expressing cells.

[0070] Methods for detecting the binding of antibodies to RNF43-expressing cells are known. Specifically, the binding of antibodies to RNF43-expressing cells can be detected using methods such as FACS, as mentioned earlier. Fixed samples of RNF43-expressing cells are used as appropriate to evaluate the binding activity of the antibodies.

[0071] A preferred antibody screening method that uses binding activity as an indicator is the panning method using phage vectors. When antibody genes are isolated from a library of heavy and light chain subclasses from a polyclonal antibody-expressing cell population, screening using phage vectors is advantageous. Genes encoding the variable regions of the heavy and light chains can be linked with a suitable linker sequence to form a single-chain Fv (scFv). By inserting the gene encoding scFv into a phage vector, a phage that displays scFv on its surface can be generated. This phage is brought into contact with the target antigen. Subsequently, by recovering the phage bound to the antigen, DNA encoding scFv with the desired binding activity can be isolated. By repeating this process as needed, scFv with the desired binding activity can be enriched.

[0072] After the cDNA encoding the V region of the target anti-RNF43 antibody is isolated, the cDNA is digested by a restriction enzyme that recognizes restriction enzyme sites introduced at both ends of the cDNA. A preferred restriction enzyme recognizes and cleaves nucleotide sequences that appear infrequently in the nucleotide sequence of the antibody gene. Furthermore, in order to insert one copy of the digested fragment in the correct orientation, it is preferable to introduce the restriction enzyme site of the enzyme that provides the adherent ends into the vector. By digesting the cDNA encoding the V region of the anti-RNF43 antibody as described above and inserting it into a suitable expression vector, an antibody expression vector is constructed. At this time, if the gene encoding the antibody constant region (C region) and the gene encoding the V region are fused in frame, a chimeric antibody is obtained. Here, "chimeric antibody" means that the origin of the constant region is different from the origin of the variable region. Therefore, in addition to mouse / human heterologous chimeric antibodies, human / human allologous chimeric antibodies are also included in the chimeric antibodies of the present invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector that already has a constant region. Specifically, for example, a recognition sequence for a restriction enzyme that excises the V region gene can be appropriately placed on the 5' end of an expression vector containing DNA encoding the desired antibody constant region (C region). A chimeric antibody expression vector is constructed by in-frame fusion of both genes digested with the same combination of restriction enzymes.

[0073] To produce an anti-RNF43 monoclonal antibody, the antibody gene is inserted into an expression vector so that it is expressed under the control of an expression regulatory region. This regulatory region for antibody expression includes, for example, an enhancer and a promoter. Furthermore, an appropriate signal sequence may be added to the amino terminus so that the expressed antibody is secreted extracellularly. In the examples described later, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 103) is used as the signal sequence. Alternatively, other suitable signal sequences may be added. The expressed polypeptide is cleaved at the carboxyl terminus of the above sequence, and the resulting polypeptide is secreted extracellularly as a mature polypeptide. Then, a suitable host cell is transformed using this expression vector to obtain recombinant cells expressing DNA encoding the anti-RNF43 antibody.

[0074] For antibody gene expression, the DNA encoding the antibody heavy chain (H chain) and light chain (L chain) is inserted separately into different expression vectors. By co-transfecting the same host cell with vectors containing both the H chain and L chain genes, antibody molecules with both H and L chains can be expressed. Alternatively, host cells can be transformed by a single expression vector containing both H and L chain encoding DNA (see WO94 / 11523).

[0075] Various host cell / expression vector combinations are known for preparing antibodies by introducing isolated antibody genes into suitable hosts. These expression systems can all be applied to isolate the domain containing the antibody variable region of the present invention. Suitable eukaryotic cells to be used as host cells include animal cells, plant cells, and fungal cells. Specifically, examples of animal cells include the following: (1) Mammalian cells: CHO, COS, myeloma, baby hamster kidney cells (BHK), HeLa, Vero, etc. (2) Amphibian cells: such as African clawed frog oocytes; and (3) Insect cells: sf9, sf21, Tn5, etc.

[0076] Furthermore, antibody gene expression systems using cells derived from the Nicotiana genus, such as Nicotiana tabacum, are known. Callus-cultured cells can be used as appropriate for plant cell transformation.

[0077] Furthermore, the following types of fungal cells can be used: Yeast: Saccharomyces genus, such as Saccharomyces cerevisiae, and Pichia genus, such as Pichia pastoris; and Filamentous fungi: Aspergillus genus, such as Aspergillus niger.

[0078] Furthermore, antibody gene expression systems utilizing prokaryotic cells are also known. For example, when using bacterial cells, Escherichia coli cells, Bacillus subtilis cells, etc., can be appropriately used in this invention. An expression vector containing the target antibody gene is introduced into these cells by transfection. The transfected cells are cultured in vitro, and the desired antibody can be prepared from the culture of the transformed cells.

[0079] In addition to the host cells mentioned above, transgenic animals can also be used to produce recombinant antibodies. That is, the antibody can be obtained from an animal into which the gene encoding the antibody of interest has been introduced. For example, the antibody gene can be constructed as a fusion gene by inserting it in-frame into a gene encoding a protein that is specifically produced in milk. For example, goat β-casein can be used as the protein secreted in milk. The DNA fragment containing the fusion gene into which the antibody gene is inserted is injected into a goat embryo, and then this embryo is introduced into a female goat. The desired antibody can be obtained as a fusion protein with the milk protein from the milk produced by the transgenic goat (or its offspring) born from the goat that received the embryo. Furthermore, hormones can be administered to the transgenic goat as needed to increase the amount of milk containing the desired antibody produced by the transgenic goat (Ebert, KM et al., Bio / Technology (1994) 12 (7), 699-702).

[0080] Method for producing humanized antibodies When the antigen-binding molecules described herein are administered to humans, the domain containing the antibody variable region of the antigen-binding molecule may, as appropriate, be a domain derived from a recombinant antibody that has been artificially modified for purposes such as reducing heterologous antigenicity to humans. Such recombinant antibodies include, for example, humanized antibodies. These modified antibodies can be appropriately manufactured by known methods. Furthermore, generally, the binding specificity of one antibody can be introduced into another antibody by transplantation of a CDR.

[0081] Specifically, humanized antibodies prepared by transplanting CDRs of non-human animal antibodies, such as mouse antibodies, into human antibodies are known. General genetic engineering techniques for obtaining humanized antibodies are also known. Specifically, overlap extension PCR is a known method for transplanting mouse antibody CDRs into human FRs. In overlap extension PCR, the nucleotide sequence encoding the mouse antibody CDR to be transplanted is added to the primer for synthesizing the human antibody FR. Primers are prepared for each of the four FRs. Generally, when transplanting mouse CDRs into human FRs, it is considered advantageous to select human FRs with high identity to the mouse FRs in order to maintain the function of the CDRs. That is, it is generally preferable to use human FRs that contain amino acid sequences with high identity to the amino acid sequences of the FRs adjacent to the mouse CDR to be transplanted.

[0082] The nucleotide sequences to be ligated are designed to be linked in-frame. Human FR is synthesized individually using each primer. This results in a product in which the DNA encoding mouse CDR is attached to each FR-encoding DNA. The nucleotide sequences encoding mouse CDR in each product are designed to overlap. Subsequently, a complementary chain synthesis reaction is performed, annealing the overlapping CDR regions of the product synthesized using the human antibody gene as a template. This reaction ligates the human FR via the mouse CDR sequence.

[0083] The final full-length V-region gene, consisting of three CDRs and four FRs, is amplified using primers that anneal to its 5' or 3' end, to which an appropriate restriction enzyme recognition sequence is added. By inserting the DNA obtained as described above and the DNA encoding the human antibody C-region into an expression vector in a frame-linked manner, an expression vector for a humanized antibody can be generated. After transfecting a host with the recombinant vector to establish recombinant cells, the recombinant cells are cultured and the DNA encoding the humanized antibody is expressed, thereby producing the humanized antibody in the cell culture (see European Patent Application Publication EP 239400 and International Publication WO1996 / 002576).

[0084] By qualitatively or quantitatively measuring and evaluating the antigen-binding activity of the humanized antibody produced as described above, a human antibody FR capable of forming a good antigen-binding site when linked to a CDR can be suitably selected. If necessary, amino acid residues of the FR can be substituted so that the reconstituted human antibody CDR forms an appropriate antigen-binding site. For example, amino acid sequence mutations can be introduced into the FR by applying the PCR method used for transplanting mouse CDRs into human FRs. More specifically, partial nucleotide sequence mutations can be introduced into primers that anneal to the FR. Nucleotide sequence mutations are introduced into the FR synthesized using such primers. By measuring and evaluating the antigen-binding activity of the mutant antibody with substituted amino acids using the method described above, a mutant FR sequence with desired properties can be selected (Sato, K. et al., Cancer Res. (1993) 53: 851-856).

[0085] Method for producing human antibodies Alternatively, desired human antibodies can be obtained by immunizing transgenic animals possessing the entire repertoire of human antibody genes (see WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, WO1996 / 033735) via DNA immunization.

[0086] Furthermore, techniques for preparing human antibodies by panning using human antibody libraries are also known. For example, the V region of a human antibody is expressed as a single-chain antibody (scFv) on the surface of a phage by phage display. A phage expressing an scFv that binds to an antigen can be selected. By analyzing the genes of the selected phage, the DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined. The DNA sequence of the scFv that binds to the antigen is determined. This V region sequence is fused in-frame with the C region sequence of the desired human antibody and inserted into a suitable expression vector to prepare an expression vector. This expression vector is introduced into cells suitable for expression, such as the cells described above. The human antibody is produced by expressing the gene encoding the human antibody in these cells. These methods are already publicly known (see WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, WO1995 / 015388).

[0087] vector As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Some vectors can result in the expression of the nucleic acid to which they are operationally ligated. Such vectors are also referred to herein as "expression vectors."

[0088] host cell The terms “host cell,” “host cell line,” and “host cell culture” refer to cells (including their offspring) that are interchangeably used and into which foreign nucleic acids have been introduced. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring do not have to be completely identical to the parent cells in terms of nucleic acid content and may contain mutations. Mutant offspring that have the same function or biological activity as those used when the original transformed cells were screened or selected are also included herein.

[0089] Epitope An "epitope" refers to an antigenic determinant in an antigen, and is the site on an antigen to which the antigen-binding domain of an antigen-binding molecule or antibody disclosed herein binds. Therefore, for example, an epitope may be defined by its structure. Alternatively, an epitope may be defined by the antigen-binding activity of an antigen-binding molecule or antibody that recognizes it. If the antigen is a peptide or polypeptide, the epitope can also be identified by the amino acid residues that form the epitope. Alternatively, if the epitope is a glycan, the epitope can also be identified by a specific glycan structure.

[0090] A linear epitope is an epitope that contains an epitope whose amino acid primary sequence is recognized. Such linear epitopes typically contain at least three, and most commonly at least five, amino acids, for example, about 8–10 or 6–20, in their intrinsic sequence.

[0091] A "structural epitope," in contrast to a linear epitope, is an epitope in which the amino acid primary sequence containing the epitope is not the sole determinant of the recognized epitope (for example, the amino acid primary sequence of a structural epitope is not necessarily recognized by the antibody that defines the epitope). A structural epitope may contain a larger number of amino acids compared to a linear epitope. The antigen-binding domain that recognizes a structural epitope recognizes the three-dimensional structure of the peptide or protein. For example, when a protein molecule folds to form a three-dimensional structure, the amino acids and / or polypeptide backbone that form the structural epitope are parallel, and the epitope becomes recognizable by the antigen-binding domain. Methods for determining the three-dimensional structure of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance, site-specific spin labeling, and electron paramagnetic resonance. For example, see Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0092] An example of a method for evaluating epitope binding by a test antigen-binding molecule or antibody containing an anti-RNF43 antigen-binding domain is described below. Methods for evaluating epitope binding by a test antigen-binding molecule or antibody containing antigen-binding domains for antigens other than RNF43 can also be appropriately carried out according to the following examples.

[0093] For example, whether a test antigen-binding molecule or antibody containing an anti-RNF43 antigen-binding domain recognizes a linear epitope in the RNF43 molecule can be confirmed, for example, as follows: For the above purpose, a linear peptide containing an amino acid sequence that forms the extracellular domain of RNF43 is synthesized. This peptide can be synthesized chemically or obtained by genetic engineering using a region in RNF43 cDNA that encodes an amino acid sequence corresponding to the extracellular domain. The test antigen-binding molecule or antibody containing the anti-RNF43 antigen-binding domain is then evaluated for its binding activity to the linear peptide containing the amino acid sequence that forms the extracellular domain. For example, ELISA can be used to evaluate the binding activity of a polypeptide complex to an immobilized linear peptide as an antigen. Alternatively, the binding activity to the linear peptide can be evaluated based on the level to which the linear peptide inhibits the binding of the antigen-binding molecule or antibody to RNF43-expressing cells. These tests can demonstrate the binding activity of the antigen-binding molecule or antibody to the linear peptide.

[0094] Whether a test antigen-binding molecule or antibody containing an anti-RNF43 antigen-binding domain recognizes a structural epitope can be evaluated as follows: For the above purpose, RNF43-expressing cells are prepared. If a test antigen-binding molecule or antibody containing an anti-RNF43 antigen-binding domain binds strongly to RNF43-expressing cells upon contact, but substantially does not bind to an immobilized linear peptide containing the amino acid sequence that forms the extracellular domain of RNF43, it can be determined that it recognizes a structural epitope. In this specification, "substantially not binding" means that the binding activity is 80% or less, generally 50% or less, preferably 30% or less, and particularly preferably 15% or less, compared to the binding activity to RNF43-expressing cells.

[0095] Methods for assaying the binding activity of a test antigen-binding molecule or antibody containing an anti-RNF43 antigen-binding domain to RNF43-expressing cells include, for example, the method described in Antibodies: A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). Specifically, the evaluation can be performed using RNF43-expressing cells as the antigen, based on the principles of ELISA or fluorescence-activated cell sorting (FACS).

[0096] In the ELISA format, the binding activity of a test antigen-binding molecule or antibody containing an anti-RNF43 antigen-binding domain to RNF43-expressing cells can be quantitatively evaluated by comparing the level of signal produced by the enzymatic reaction. Specifically, the test polypeptide complex is added to an ELISA plate immobilized with RNF43-expressing cells. Then, the test antigen-binding molecule or antibody bound to the cells is detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule or antibody. Alternatively, if FACS is used, a dilution series of the test antigen-binding molecule or antibody can be prepared, and the antibody binding titer to RNF43-expressing cells can be determined to compare the binding activity of the test antigen-binding molecule or antibody to RNF43-expressing cells.

[0097] The binding of a test antigen-binding molecule or antibody to an antigen expressed on the surface of cells suspended in a buffer can be detected using a flow cytometer. Known flow cytometers include, for example, the following devices: FACSCanto(TM) II FACSAria(trademark) FACSArray (trademark) FACSVantage(TM) SE FACSCalibur (trademark) (all are product names of BD Biosciences) EPICS ALTRA HyPerSort Cytomics FC 500 EPICS XL-MCL ADC EPICS XL ADC Cell Lab Quanta / Cell Lab Quanta SC (both are product names from Beckman Coulter)

[0098] Preferred methods for assaying the binding activity of a test antigen-binding molecule or antibody containing an anti-RNF43 antigen-binding domain to an antigen include, for example, the following: First, RNF43-expressing cells are reacted with the test antigen-binding molecule or antibody, and then stained with a FITC-labeled secondary antibody that recognizes the antigen-binding molecule or antibody. The test antigen-binding molecule or antibody is then appropriately diluted with a suitable buffer to prepare the antigen-binding molecule or antibody at the desired concentration. For example, the antigen-binding molecule or antibody can be used at concentrations in the range of 10 μg / ml to 10 ng / ml. Next, the fluorescence intensity and cell number are determined using FACSCalibur (BD). The fluorescence intensity, i.e., the geometric mean, obtained by analysis using CELL QUEST Software (BD) reflects the amount of antibody bound to the cells. That is, by measuring the geometric mean, the binding activity of the test antigen-binding molecule or antibody, expressed by the amount of bound test antigen-binding molecule or antibody, can be determined.

[0099] Whether a test antigen-binding molecule or antibody containing an anti-RNF43 antigen-binding domain shares a common epitope with another antigen-binding molecule or antibody can be evaluated based on competition between the two antigen-binding molecules or antibodies for the same epitope. Competition between antigen-binding molecules or antibodies can be detected by cross-blocking assays, for example. A competitive ELISA assay is a preferred cross-blocking assay.

[0100] Specifically, in a cross-blocking assay, RNF43 protein immobilized in wells of a microtiter plate is pre-incubated in the presence or absence of candidate competitor antigen-binding molecules or antibodies, and then the test antigen-binding molecule or antibody is added. The amount of the test antigen-binding molecule or antibody bound to the RNF43 protein in the well is indirectly correlated with the binding ability of the candidate competitor antigen-binding molecules or antibodies that compete for binding to the same epitope. That is, the higher the affinity of the competitor antigen-binding molecule or antibody to the same epitope, the lower the binding activity of the test antigen-binding molecule or antibody to the RNF43 protein-coated well.

[0101] The amount of the test antigen-binding molecule or antibody bound to the well via the RNF43 protein can be easily determined by pre-labeling the antigen-binding molecule or antibody. For example, biotin-labeled antigen-binding molecules or antibodies are measured using an avidin / peroxidase conjugate and a suitable substrate. In particular, cross-blocking assays using enzymatic labeling such as peroxidase are called "competitive ELISA assays." Antigen-binding molecules or antibodies can also be labeled with other labeling substances that enable detection or measurement. Specifically, radiolabeling, fluorescent labeling, etc., are well known.

[0102] If a candidate competitor antigen-binding molecule or antibody can block binding by the test antigen-binding molecule or antibody containing an anti-RNF43 antigen-binding domain by at least 20%, preferably at least 20-50%, and more preferably at least 50%, compared to the binding activity in a control experiment performed in the absence of the competitor antigen-binding molecule or antibody, then the test antigen-binding molecule or antibody is determined to substantially bind to the same epitope to which the competitor antigen-binding molecule or antibody binds, or to compete for binding to the same epitope.

[0103] If the structure of the epitope to which the test antigen-binding molecule or antibody, which contains an anti-RNF43 antigen-binding domain, binds has already been identified, whether the test antigen-binding molecule or antibody and the control antigen-binding molecule or antibody share a common epitope can be evaluated by comparing the binding activity of both antigen-binding molecules or antibodies to a peptide prepared by introducing amino acid mutations into the peptide that forms the epitope.

[0104] Alternatively, epitope binning can be performed to identify the epitopes of various antigen-binding molecules, such as anti-RNF43 antibodies, as follows: The variable region DNA is amplified by PCR and recombined with the DNA encoding the rabbit heavy and light chain constant regions. The cloned antibody is expressed in cells and purified from the culture supernatant. The antibody is biotinylated, and then free biotin is removed, for example, by dialysis.

[0105] For example, the EC50 concentration of each antibody bound to RNF43 ECD with an Fc region (RNF43-Fc) is determined by an ELISA assay using a biotinylated antibody. For example, a plate is coated with RNF43-Fc, and a biotinylated antibody is added and incubated. After washing, for example, StAv-HRP (PIERCE) is added and incubated. After washing, for example, ABTS peroxidase substrate (SeraCare Life Sciences) is added and the signal intensity is measured. The EC50 concentration of the anti-RNF43 monospecific antibody bound to RNF43-Fc is calculated, for example, using a nonlinear regression 4-parameter fit. The normalized absorbance at 405 nm / 570 nm measured when the EC50 concentration of the anti-RNF43 antibody is fitted is A OThis is expressed as follows. To evaluate binding competition between anti-RNF43 monospecific antibodies, an ELISA assay can be performed similarly. For example, a plate is coated with RNF43-Fc and incubated with a non-biotinized primary antibody (test antibody) at a 10-fold concentration of each EC50. Without washing, a biotinized secondary antibody (reference antibody) is added at its EC50 concentration and incubated. After washing, a peroxidase substrate is added and the signal intensity is measured. The normalized absorbance at 405 nm / 570 nm is expressed as A. The binding inhibition (%) is calculated using the following formula: TIFF2023123726000001.tif15128 Binning can be determined using a 20% binding inhibition cutoff value. If the binding inhibition between antibodies is less than 20%, they are classified into different bins. In other words, if the test antibody Ab1 shows more than 20% binding inhibition when another antibody Ab2 is used as the reference antibody, and antibody Ab2 also shows more than 20% binding inhibition when Ab2 is used as the test antibody and Ab1 is used as the reference antibody, then antibodies Ab1 and Ab2 are classified into the same bin. Antibodies in the same bin compete with each other and can be said to bind to the same (or at least closely located) epitopes.

[0106] To measure the binding activity described above, for example, the binding activity of a test antigen-binding molecule or antibody against a mutated linear peptide is compared with that of a control antigen-binding molecule or antibody in the ELISA format described above. In addition to the ELISA method, the binding activity to the mutated peptide bound to the column can also be determined by passing the test antigen-binding molecule or antibody and the control antigen-binding molecule or antibody through a column, and then quantifying the antigen-binding molecule or antibody eluted into the eluate. For example, methods for adsorbing the mutated peptide onto a column in the form of a GST fusion peptide are known.

[0107] Alternatively, if the identified epitope is a structural epitope, whether the test antigen-binding molecule or antibody and the control antigen-binding molecule or antibody share a common epitope can be evaluated by the following method: First, prepare RNF43-expressing cells and cells expressing RNF43 with a mutation introduced into the epitope. Add the test antigen-binding molecule or antibody and the control antigen-binding molecule or antibody to the cell suspension prepared by suspending these cells in a suitable buffer such as PBS. Next, wash the cell suspension with buffer as appropriate and add the FITC-labeled antibody that recognizes the test antigen-binding molecule or antibody and the control antigen-binding molecule or antibody. Determine the fluorescence intensity and number of cells stained with the labeled antibody using FACSCalibur (BD). The test antigen-binding molecule or antibody and the control antigen-binding molecule or antibody are diluted as appropriate with a suitable buffer and used at the desired concentration. For example, they can be used at concentrations in the range of 10 μg / ml to 10 ng / ml. The fluorescence intensity, or geometric mean, determined by analysis using CELL QUEST Software (BD), reflects the amount of labeled antibody bound to the cell. In other words, by measuring the geometric mean, the binding activity of the test antigen-binding molecule or antibody and the control antigen-binding molecule or antibody, expressed by the amount of bound labeled antibody, can be determined.

[0108] In the above method, whether the antigen-binding molecule or antibody "substantially does not bind to cells expressing mutant RNF43" can be evaluated, for example, by the following method. First, the test antigen-binding molecule or antibody and the control antigen-binding molecule or antibody bound to cells expressing mutant RNF43 are stained with a labeled antibody. Next, the fluorescence intensity of the cells is determined. If FACSCalibur is used for fluorescence detection by flow cytometry, the determined fluorescence intensity can be analyzed using CELL QUEST Software. From the geometric mean values ​​in the presence and absence of the antigen-binding molecule or antibody, a comparative value (ΔGeo-Mean) can be calculated according to the following formula to determine the rate of increase in fluorescence intensity as a result of binding by the antigen-binding molecule or antibody.

[0109] ΔGeo-Mean = Geo-Mean (in the presence of antigen-binding molecules or antibodies) / Geo-Mean (in the absence of antigen-binding molecules or antibodies)

[0110] The geometric mean comparison value (ΔGeo-Mean value for the mutant RNF43 molecule) determined by the above analysis, which reflects the amount of the test antigen-binding molecule or antibody bound to cells expressing the mutant RNF43, is compared with the ΔGeo-Mean comparison value that reflects the amount of the test antigen-binding molecule or antibody bound to RNF43-expressing cells. In this case, it is particularly preferable that the concentrations of the test antigen-binding molecule or antibody used to determine the ΔGeo-Mean comparison values ​​for RNF43-expressing cells and cells expressing the mutant RNF43 are adjusted to be equal or substantially equal. An antigen-binding molecule or antibody that has been confirmed to recognize the epitope in RNF43 is used as the control antigen-binding molecule or antibody.

[0111] If the ΔGeo-Mean comparison value of the test antigen-binding molecule or antibody against cells expressing mutant RNF43 is at least 80%, preferably 50%, more preferably 30%, and particularly preferably 15% smaller than the ΔGeo-Mean comparison value of the test antigen-binding molecule or antibody against RNF43-expressing cells, then the test antigen-binding molecule or antibody is considered "substantially unbinding to cells expressing mutant RNF43." The formula for determining the Geo-Mean (geometric mean) value is described in the CELL QUEST Software User's Guide (BD biosciences). If the comparison shows that the comparison values ​​are substantially equal, the epitopes of the test antigen-binding molecule or antibody and the control antigen-binding molecule or antibody can be determined to be the same.

[0112] specificity "Specific" means that a molecule that specifically binds to one or more binding partners does not show any significant binding to molecules other than those partners. Furthermore, "specific" is also used when an antigen-binding domain is specific to a particular epitope among several epitopes contained in an antigen. If the epitope to which the antigen-binding domain binds is contained in multiple different antigens, the antigen-binding molecule containing the antigen-binding domain can bind to various antigens that have that epitope.

[0113] Single-specific antigen-binding molecule The term "monospecific antigen-binding molecule" is used to refer to an antigen-binding molecule that specifically binds to only one type of antigen. A preferred example of a monospecific antigen-binding molecule is an antigen-binding molecule containing a single type of antigen-binding domain. A monospecific antigen-binding molecule may contain a single antigen-binding domain or multiple antigen-binding domains of the same type. A preferred example of a monospecific antigen-binding molecule is a monospecific antibody. If the monospecific antigen-binding molecule is an IgG-type monospecific antibody, the monospecific antibody contains two antibody variable fragments having the same antigen-binding specificity.

[0114] antibody fragment An "antibody fragment" refers to a molecule other than the complete antibody, containing a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments, but not limited to these, include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0115] The terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a native antibody, or having a heavy chain containing an Fc region as defined herein.

[0116] Variable fragment (Fv) In this specification, the term "variable fragment (Fv)" refers to the smallest unit of an antibody-derived antigen-binding domain, consisting of a pair of the antibody's light chain variable region (VL) and its heavy chain variable region (VH). In 1988, Skerra and Pluckthun discovered that homogeneous and active antibodies could be prepared from the periplasmic fraction of E. coli by inserting an antibody gene downstream of a bacterial signal sequence and inducing the expression of that gene in E. coli (Science (1988) 240 (4855), 1038-1041). In Fv prepared from the periplasmic fraction, VH and VL associate in a manner that binds to the antigen.

[0117] scFv, single-chain antibody, and sc(Fv)2 In this specification, the terms “scFv,” “monoclonal antibody,” and “sc(Fv)2” all refer to antibody fragments of a single polypeptide chain that include variable regions derived from the heavy and light chains but do not include a constant region. Generally, monoclonal antibodies further include a polypeptide linker between the VH and VL domains, which enables the formation of a desired structure that is expected to allow antigen binding. Monoclonal antibodies are discussed in detail by Pluckthun in “The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, 269-315 (1994).” See also International Publication WO1988 / 001649, U.S. Patents 4,946,778 and 5,260,203. In certain embodiments, monoclonal antibodies may be bispecific and / or humanized.

[0118] scFv is an antigen-binding domain in which the VH and VL that form Fv are linked by a peptide linker (Proc. Natl. Acad. Sci. USA (1988) 85 (16), 5879-5883). This peptide linker allows the VH and VL to be kept in close proximity.

[0119] sc(Fv)2 is a single-chain antibody in which four variable regions, two VLs and two VHs, are linked by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231 (1-2), 177-189). These two VHs and two VLs may be derived from different monoclonal antibodies. A preferred example of such sc(Fv)2 is a bispecific sc(Fv)2 that recognizes two epitopes present in a single antigen, as disclosed in Journal of Immunology (1994) 152 (11), 5368-5374. sc(Fv)2 can be produced by methods known to those skilled in the art. For example, sc(Fv)2 can be produced by linking scFv with a linker such as a peptide linker.

[0120] In this specification, the antigen-binding domain that forms sc(Fv)2 is defined as an antibody in which two VH units and two VL units are arranged in the order VH, VL, VH, VL ([VH]-linker-[VL]-linker-[VH]-linker-[VL]) starting from the N-terminus of a single-chain polypeptide. The order of the two VH units and two VL units is not limited to the above configuration and may be arranged in any order. Examples of configurations are listed below. [VL]-Linker-[VH]-Linker-[VH]-Linker-[VL] [VH]-Linker-[VL]-Linker-[VL]-Linker-[VH] [VH]-Linker-[VH]-Linker-[VL]-Linker-[VL] [VL]-Linker-[VL]-Linker-[VH]-Linker-[VH] [VL]-Linker-[VH]-Linker-[VL]-Linker-[VH]

[0121] The molecular morphology of sc(Fv)2 is also described in detail in WO2006 / 132352. Those skilled in the art can prepare the desired sc(Fv)2 to produce the polypeptide complexes disclosed herein by following these descriptions.

[0122] Furthermore, the antigen-binding molecule or antibody of the present invention may be conjugated with a carrier polymer such as PEG or an organic compound such as an anticancer agent. Alternatively, a glycosylation sequence may be suitably inserted into the antigen-binding molecule or antibody so that the glycosylation produces the desired effect.

[0123] Linkers used for linking the variable region of an antibody include any peptide linker that can be introduced by genetic engineering, synthetic linkers, and linkers disclosed, for example, in Protein Engineering, 9 (3), 299-305, 1996. However, peptide linkers are preferred in the present invention. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose. The length is preferably 5 amino acids or more (not particularly limited, but the upper limit is usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. If sc(Fv)2 contains three peptide linkers, their lengths may all be the same or different.

[0124] For example, such peptide linkers include the following: Ser Gly Ser Gly Gly Ser Ser Gly Gly Gly Gly Gly Ser (Sequence ID: 104) Ser Gly Gly Gly (Sequence No.: 105) Gly Gly Gly Gly Ser (Sequence No.: 106) Ser Gly Gly Gly Gly (Sequence No.: 107) Gly Gly Gly Gly Gly Ser (Sequence No.: 108) Ser Gly Gly Gly Gly Gly (Sequence code: 109) Gly Gly Gly Gly Gly Gly Ser (Line number: 110) Ser Gly Gly Gly Gly Gly Gly (Line code: 111) (Gly Gly Gly Gly Ser (Sequence No.: 106))n (Ser Gly Gly Gly Gly (Sequence No.: 107))n Here, n is an integer greater than or equal to 1. The length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0125] Synthetic linkers (chemical crosslinking agents) are commonly used for crosslinking peptides. Examples include N-hydroxysuccinimide (NHS), disuccinimidylsverate (DSS), bis(sulfosuccinimidyl)sverate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethylene glycol bis(succinimidylsuccinate) (EGS), ethylene glycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimideoxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimideoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). These crosslinking agents are commercially available.

[0126] To link four antibody variable regions, typically three linkers are required. The linkers used may be of the same type or different types.

[0127] Fab, F(ab')2, and Fab' "Fab" consists of one light chain and one heavy chain with a CH1 domain and a variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with other heavy chain molecules.

[0128] "F(ab')2" or "Fab" refers to an antibody fragment produced by treating immunoglobulin (monoclonal antibody) with proteases such as pepsin and papain, and by digesting the immunoglobulin (monoclonal antibody) near the disulfide bond located between the hinge regions of each of the two H chains. For example, papain cleaves IgG upstream of the disulfide bond located between the hinge regions of each of the two H chains, producing two homologous antibody fragments in which the L chain containing VL (variable L chain region) and CL (constant L chain region) is linked by a disulfide bond at the C-terminal region to an H chain fragment containing VH (variable H chain region) and CHγ1 (γ1 region in the constant H chain region). These two homologous antibody fragments are each called Fab'.

[0129] "F(ab')2" consists of two light chains and two heavy chains containing constant regions of the CH1 domain and a portion of the CH2 domain such that a disulfide bond is formed between the two heavy chains. The F(ab')2 disclosed herein can be suitably prepared as follows: A monoclonal whole antibody containing the desired antigen-binding domain is partially digested with a protease such as pepsin, and the Fc fragment is removed by adsorption onto a protein A column. The protease is not particularly limited as long as it can selectively cleave the whole antibody to produce F(ab')2 under appropriate setting enzymatic reaction conditions such as pH. For example, such proteases include pepsin and ficin.

[0130] Fc area In this specification, the terms “Fc region” or “Fc domain” are used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes both the native sequence Fc region and mutant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, provided that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region is present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0131] Fc receptor The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is the native human FcR. In some embodiments, the FcR is one that binds to an IgG antibody (gamma receptor) and includes the FcγRI, FcγRII, and FcγRIII subclass receptors, including allelic variants and alternative splicing forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997).) FcRs have been reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med 126:330-41 (1995). Other FcRs, including those to be identified in the future, are also included in the term “FcR” as used herein.

[0132] The term “Fc receptor” or “FcR” also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are publicly known (see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO2004 / 92219 (Hinton et al.)).

[0133] The in vivo binding to human FcRn and the plasma half-life of human FcRn high-affinity binding polypeptides can be measured, for example, in transgenic mice expressing human FcRn or transfected human cell lines, or in primates administered polypeptides with mutant Fc regions. WO2000 / 42072 (Presta) describes antibody variants with increased or decreased binding to FcR. See also, for example, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0134] Fcγ receptor Fcγ receptors are receptors that can bind to the Fc domain of IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies, and this includes all members of the family of proteins substantially encoded by the Fcγ receptor gene. In humans, this family includes FcγRI (CD64), which contains isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which contains isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which contains isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2); as well as all unidentified human Fcγ receptors, Fcγ receptor isoforms, and their allotypes. However, Fcγ receptors are not limited to these examples. Fcγ receptors include those derived from humans, mice, rats, rabbits, and monkeys, but are not limited to these. Fcγ receptors may originate from any organism. Mouse Fcγ receptors include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as all unidentified mouse Fcγ receptors, Fcγ receptor isoforms, and their allotypes. Such preferred Fcγ receptors include, for example, human FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16), and / or FcγRIIIB (CD16).The polynucleotide and amino acid sequences of FcγRI are shown in SEQ ID NOs: 112 (NM_000566.3) and 113 (NP_000557.1), respectively; the polynucleotide and amino acid sequences of FcγRIIA are shown in SEQ ID NOs: 114 (BC020823.1) and 115 (AAH20823.1), respectively; the polynucleotide and amino acid sequences of FcγRIIB are shown in SEQ ID NOs: 116 (BC146678.1) and 117 (AAI46679.1), respectively; the polynucleotide and amino acid sequences of FcγRIIIA are shown in SEQ ID NOs: 118 (BC033678.1) and 119 (AAH33678.1), respectively; and the polynucleotide and amino acid sequences of FcγRIIIB are shown in SEQ ID NOs: 120 (BC128562.1), respectively. And as shown in 121 (AAI28563.1) (RefSeq registry numbers are shown in parentheses). Whether the Fcγ receptor has binding activity to the Fc domain of IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies can be evaluated by ALPHA screen (amplified luminescence proximity homogeneous assay), surface plasmon resonance (SPR) based BIACORE method, and others, in addition to the FACS and ELISA formats described above (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0135] On the other hand, "Fc ligand" or "effector ligand" refers to a molecule, preferably a polypeptide, that binds to the antibody Fc domain to form an Fc / Fc ligand complex. The molecule may originate from any organism. Binding of an Fc ligand to Fc preferably induces one or more effector functions. Such Fc ligands include, but are not limited to, Fc receptors, Fcγ receptors, Fcα receptors, Fcβ receptors, FcRn, C1q, and C3, mannan-binding lectins, mannose receptors, Staphylococcus protein A, Staphylococcus protein G, and viral Fcγ receptors. Fc ligands also include Fc receptor homologs (FcRH) (Davis et al., (2002) Immunological Reviews 190, 123-136), which are a family of Fc receptors homologous to the Fcγ receptor. Fc ligands also include unidentified molecules that bind to Fc.

[0136] Fcγ receptor binding activity Impaired binding activity of the Fc domain to any of the Fcγ receptors FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and / or FcγRIIIB can be assessed using the FACS and ELISA formats described above, as well as the ALPHA screen (amplified luminescence proximity homogeneous assay) and surface plasmon resonance (SPR)-based BIACORE method (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0137] The ALPHA screen is performed using the ALPHA technology, which is based on the following principle and uses two types of beads: donor beads and acceptor beads. A luminescence signal is detected only when the molecule linked to the donor bead biologically interacts with the molecule linked to the acceptor bead, and the two beads are located in close proximity. The photosensitizer in the donor bead is excited by laser light, converting the oxygen around the bead into excited singlet oxygen. When the singlet oxygen diffuses around the donor bead and reaches the nearby acceptor bead, a chemiluminescent reaction is induced in the acceptor bead. This reaction ultimately emits light. If the molecule linked to the donor bead does not interact with the molecule linked to the acceptor bead, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and the chemiluminescent reaction does not occur.

[0138] For example, a biotin-labeled antigen-binding molecule or antibody is immobilized on donor beads, and a glutathione S-transferase (GST)-tagged Fcγ receptor is immobilized on acceptor beads. In the absence of antigen-binding molecules or antibodies containing competitive mutant Fc domains, the Fcγ receptor interacts with antigen-binding molecules or antibodies containing wild-type Fc domains, resulting in a signal at 520–620 nm. Antigen-binding molecules or antibodies with untagged mutant Fc domains compete with antigen-binding molecules or antibodies containing wild-type Fc domains for interaction with the Fcγ receptor. Relative binding affinity can be determined by quantifying the decrease in fluorescence resulting from the competition. Methods for biotinylating antigen-binding molecules or antibodies, such as antibodies, using sulfo-NHS-biotin are known. Appropriate methods for attaching a GST tag to the Fcγ receptor include in-frame fusion of a polypeptide encoding the Fcγ receptor and a polypeptide encoding GST, expression of the fusion gene using cells into which a vector containing the fusion gene has been introduced, and subsequent purification using a glutathione column. The induced signal can preferably be analyzed by fitting it to a one-site competition model based on nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad; San Diego).

[0139] One of the substances to be observed for interaction is immobilized as a ligand on a gold thin film of the sensor chip. When light is shone from the back of the sensor chip so that total internal reflection occurs at the interface between the gold thin film and the glass, the intensity of the reflected light partially decreases at a specific site (SPR signal). The other substance to be observed for interaction is injected as an analyte onto the surface of the sensor chip. When the analyte binds to the ligand, the mass of the immobilized ligand molecule increases. This changes the refractive index of the solvent on the surface of the sensor chip. The change in refractive index causes a shift in the position of the SPR signal (conversely, when it dissociates, the signal shifts back to its original position). In the Biacore system, the amount of the above shift (i.e., the change in mass on the surface of the sensor chip) is plotted on the vertical axis, and the change in mass over time is displayed as measurement data (sensorgram). Dynamic parameters (binding rate constant (ka) and dissociation rate constant (kd)) are determined from the sensorgram curve, and the affinity (KD) is determined from the ratio of these two constants. In the BIACORE method, inhibition assays are preferably used. Examples of such inhibitory assays are described in Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010.

[0140] Fc region with reduced Fcγ receptor binding activity In this specification, "decreased Fcγ receptor binding activity" means, for example, that based on the analysis method described above, the competitive activity of the test antigen-binding molecule or antibody is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, or 15% or less, and particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, of the competitive activity of the control antigen-binding molecule or antibody.

[0141] Antigen-binding molecules or antibodies containing the Fc domain of IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies can be used as appropriate as control antigen-binding molecules or antibodies. The Fc domain structures are shown in SEQ ID NOs: 122 (with an A added to the N-terminus of RefSeq registry number AAC82527.1), 123 (with an A added to the N-terminus of RefSeq registry number AAB59393.1), 124 (with an A added to the N-terminus of RefSeq registry number CAA27268.1), and 125 (with an A added to the N-terminus of RefSeq registry number AAB59394.1). Furthermore, when an antigen-binding molecule or antibody containing an Fc domain variant of a specific isotype of antibody is used as the test substance, the effect of the variant mutation on Fcγ receptor binding activity should be evaluated using an antigen-binding molecule or antibody containing the same isotype of Fc domain as a control. As described above, antigen-binding molecules or antibodies containing Fc domain variants that are judged to have reduced Fcγ receptor binding activity are prepared as appropriate.

[0142] Such known variants include, for example, the variant lacking amino acids 231A-238S (EU numbering) (WO2009 / 011941), as well as the variants C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34, 11); C226S and C229S (Hum. Antibod. Hybridomas (1990) 1(1), 47-54); C226S, C229S, E233P, L234V, and L235A (Blood (2007) 109, 1185-1192).

[0143] Specifically, preferred antigen-binding molecules or antibodies include those containing an Fc domain having a mutation (such as a substitution) of at least one amino acid selected from the following amino acid positions in the amino acids forming the Fc domain of a particular isotype of antibody: positions 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331, or 332 (EU numbering). The antibody isotype from which the Fc domain originates is not particularly limited, and a suitable Fc domain derived from IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies can be used. It is preferable to use an Fc domain derived from an IgG1 antibody.

[0144] Preferred antigen-binding molecules or antibodies include, for example, an Fc domain in which the amino acids forming the Fc domain of an IgG1 antibody have one of the substitutions shown below (each number represents the position of the amino acid residue in EU numbering; the single-letter amino acid symbol before the number represents the amino acid residue before substitution, and the single-letter amino acid symbol after the number represents the amino acid residue after substitution), the position of which is identified according to EU numbering: (a) L234F, L235E, P331S; (b) C226S, C229S, P238S; (c) C226S, C229S; or (d) C226S, C229S, E233P, L234V, L235A; Furthermore, this includes those that have an Fc domain with a deletion in the amino acid sequence between positions 231 and 238.

[0145] Furthermore, preferred antigen-binding molecules or antibodies also include those containing an Fc domain in the amino acids forming the Fc domain of an IgG2 antibody, the Fc domain having one of the substitutions shown below, the position of which is identified according to EU numbering: (e) H268Q, V309L, A330S, and P331S; (f) V234A; (g) G237A; (h) V234A and G237A; (i) A235E and G237A; or (j) V234A, A235E, and G237A. Each number represents the position of the amino acid residue in EU numbering; the single-letter amino acid symbol before the number represents the amino acid residue before substitution, and the single-letter amino acid symbol after the number represents the amino acid residue after substitution.

[0146] Furthermore, preferred antigen-binding molecules or antibodies also include those containing an Fc domain in the amino acids forming the Fc domain of an IgG3 antibody, the Fc domain having one of the substitutions shown below, the substitution whose position is identified according to EU numbering: (k) F241A; (l) D265A; or (m) V264A. Each number represents the position of the amino acid residue in EU numbering; the single-letter amino acid symbol before the number represents the amino acid residue before substitution, and the single-letter amino acid symbol after the number represents the amino acid residue after substitution.

[0147] Furthermore, preferred antigen-binding molecules or antibodies also include those containing an Fc domain in the amino acids forming the Fc domain of an IgG4 antibody, the Fc domain having one of the substitutions shown below, the position of which is identified according to EU numbering: (n) L235A, G237A, and E318A; (o) L235E; or (p) F234A and L235A. Each number represents the position of the amino acid residue in EU numbering; the single-letter amino acid symbol before the number represents the amino acid residue before substitution, and the single-letter amino acid symbol after the number represents the amino acid residue after substitution.

[0148] Other preferred antigen-binding molecules or antibodies include, for example, those containing an Fc domain in which any of the amino acids forming the Fc domain of an IgG1 antibody at positions 233, 234, 235, 236, 237, 327, 330, or 331 (EU numbering) is substituted with the amino acid at the corresponding EU numbering position in the corresponding IgG2 or IgG4.

[0149] Preferred antigen-binding molecules or antibodies also include those containing an Fc domain in which one or more of the amino acids at positions 234, 235, and 297 (EU numbering) of the amino acids forming the Fc domain of an IgG1 antibody are substituted with other amino acids. The type of substituted amino acid is not particularly limited; however, antigen-binding molecules or antibodies containing an Fc domain in which one or more of the amino acids at positions 234, 235, and 297 are substituted with alanine are particularly preferred.

[0150] Preferred antigen-binding molecules or antibodies also include, for example, those containing an Fc domain in which the amino acid at position 265 (EU numbering) of the amino acids forming the Fc domain of an IgG1 antibody is substituted with another amino acid. The type of substituted amino acid is not particularly limited; however, antigen-binding molecules or antibodies containing an Fc domain in which the amino acid at position 265 is substituted with alanine are particularly preferred.

[0151] Antigen-binding domain with RNF43 binding activity As used herein, the terms "antigen-binding domain having RNF43-binding activity" or "anti-RNF43 antigen-binding domain" refer to an antigen-binding domain that specifically binds to the RNF43 protein or a partial peptide of the RNF43 protein, in whole or in part.

[0152] In certain embodiments, the antigen-binding domain having RNF43 binding activity is a domain containing the variable regions (VL and VH) of the antibody's light and heavy chains. Suitable examples of such domains containing the variable regions of the antibody's light and heavy chains include "single-chain Fv (scFv)", "single-chain antibody", "Fv", "single-chain Fv 2 (scFv2)", "Fab", "F(ab')2", etc. In specific embodiments, the antigen-binding domain having RNF43 binding activity is a domain containing an antibody variable fragment. A domain containing an antibody variable fragment may be provided from the variable domains of one or more antibodies.

[0153] In certain embodiments, the antigen-binding domain having RNF43 binding activity includes the heavy chain variable region and the light chain variable region of the anti-RNF43 antibody. In certain embodiments, the antigen-binding domain having RNF43 binding activity is a domain comprising a Fab structure.

[0154] Preferably, the anti-RNF43 antibody comprises an H chain containing one amino acid sequence (variable H chain region) from SEQ ID NOs: 5 to 14 and an L chain containing one amino acid sequence (variable L chain region) from SEQ ID NOs: 15 to 24.

[0155] In some embodiments, the antigen-binding domain having RNF43 binding activity specifically binds to the extracellular domain of RNF43 (amino acids 24-194 of SEQ ID NO: 94 and SEQ ID NO: 89). In some embodiments, the antigen-binding domain having RNF43 binding activity specifically binds to an epitope within the extracellular domain of RNF43 (amino acids 24-194 of SEQ ID NO: 94 and SEQ ID NO: 89). In some embodiments, the antigen-binding domain having RNF43 binding activity binds to RNF43 protein expressed on the surface of eukaryotic cells. In some embodiments, the antigen-binding domain having RNF43 binding activity binds to RNF43 protein expressed on the surface of cancer cells.

[0156] In a specific embodiment, the antigen-binding domain having RNF43 binding activity includes one of the antibody variable fragments shown in Table 1 below.

[0157] [Table 1]

[0158] In a specific embodiment, the antigen-binding domain having RNF43 binding activity is a domain containing an antibody variable fragment that competes with any one of the antibody variable fragments shown in Table 1 for binding to human RNF43. In a specific embodiment, the antigen-binding domain having RNF43 binding activity is a domain containing an antibody variable fragment that binds to the same human RNF43 epitope as any one of the antibody variable fragments shown in Table 1.

[0159] Alternatively, the antigen-binding domain having RNF43 binding activity includes an antibody-variable fragment that competes with any one of the above antibody-variable fragments for binding to human RNF43. Alternatively, the antigen-binding domain having RNF43 binding activity includes an antibody-variable fragment that binds to the same epitope on human RNF43 as any one of the above antibody-variable fragments binds to.

[0160] Antigen-binding domain with T cell receptor complex binding activity As used herein, the terms "antigen-binding domain having T cell receptor complex binding activity" or "anti-T cell receptor complex antigen-binding domain" refer to an antigen-binding domain that specifically binds to all or part of a partial peptide of a T cell receptor complex. The T cell receptor complex may be the T cell receptor itself, or it may be an adapter molecule that, together with the T cell receptor, constitutes the T cell receptor complex. CD3 is suitable as an adapter molecule.

[0161] In certain embodiments, the antigen-binding domain having T cell receptor complex binding activity is a domain containing the variable regions (VL and VH) of the antibody's light and heavy chains. Suitable examples of such domains containing the variable regions of the antibody's light and heavy chains include "single-chain Fv (scFv)", "single-chain antibody", "Fv", "single-chain Fv 2 (scFv2)", "Fab", "F(ab')2", etc. In specific embodiments, the antigen-binding domain having T cell receptor complex binding activity is a domain containing an antibody variable fragment. A domain containing an antibody variable fragment may be provided from the variable domains of one or more antibodies.

[0162] In certain embodiments, the antigen-binding domain having T cell receptor complex binding activity includes the heavy chain variable region and the light chain variable region of the anti-T cell receptor complex antibody. In certain embodiments, the antigen-binding domain having T cell receptor complex binding activity is a domain containing a Fab structure.

[0163] Antigen-binding domain with T cell receptor binding activity As used herein, the terms “antigen-binding domain having T cell receptor binding activity” or “anti-T cell receptor antigen-binding domain” refer to an antigen-binding domain that specifically binds to all or part of a partial peptide of a T cell receptor. The portion of the T cell receptor to which the antigen-binding domain binds may be the variable region or the constant region of the T cell receptor; however, an epitope located in the constant region is preferred. Examples of constant region sequences include the T cell receptor α chain (SEQ ID NO: 95) of RefSeq registry number CAA26636.1, the T cell receptor β chain (SEQ ID NO: 96) of RefSeq registry number C25777, the T cell receptor γ1 chain (SEQ ID NO: 97) of RefSeq registry number A26659, the T cell receptor γ2 chain (SEQ ID NO: 98) of RefSeq registry number AAB63312.1, and the T cell receptor δ chain (SEQ ID NO: 99) of RefSeq registry number AAA61033.1.

[0164] In certain embodiments, the antigen-binding domain having T cell receptor binding activity is a domain containing the variable regions (VL and VH) of the antibody's light and heavy chains. Suitable examples of such domains containing the variable regions of the antibody's light and heavy chains include "single-chain Fv (scFv)", "single-chain antibody", "Fv", "single-chain Fv 2 (scFv2)", "Fab", "F(ab')2", etc. In specific embodiments, the antigen-binding domain having T cell receptor binding activity is a domain containing an antibody variable fragment. A domain containing an antibody variable fragment may be provided from the variable domains of one or more antibodies.

[0165] In certain embodiments, the antigen-binding domain having T cell receptor binding activity includes the heavy chain variable region and the light chain variable region of an anti-T cell receptor antibody. In certain embodiments, the antigen-binding domain having T cell receptor binding activity is a domain containing a Fab structure.

[0166] Antigen-binding domain with CD3 binding activity As used herein, the terms “antigen-binding domain having CD3 binding activity” or “anti-CD3 antigen-binding domain” refer to an antigen-binding domain that specifically binds to all or part of a partial peptide of CD3. An antigen-binding domain having CD3 binding activity may be any epitope-binding domain, as long as the epitope is present in the γ, δ, or ε chain sequence constituting human CD3. Regarding the structures of the γ, δ, or ε chains constituting CD3, their polynucleotide sequences are disclosed in RefSeq registry numbers NM_000073.2, NM_000732.4, and NM_000733.3, and their polypeptide sequences are shown in SEQ ID NOs: 100 (NP_000064.1), 101 (NP_000723.1), and 102 (NP_000724.1), where the RefSeq registry number is indicated in parentheses.

[0167] In certain embodiments, the antigen-binding domain having CD3 binding activity is a domain containing the variable regions (VL and VH) of the antibody's light and heavy chains. Suitable examples of such domains containing the variable regions of the antibody's light and heavy chains include "single-chain Fv (scFv)", "single-chain antibody", "Fv", "single-chain Fv 2 (scFv2)", "Fab", "F(ab')2", etc. In specific embodiments, the antigen-binding domain having CD3 binding activity is a domain containing an antibody variable fragment. A domain containing an antibody variable fragment may be provided from the variable domains of one or more antibodies.

[0168] In certain embodiments, the antigen-binding domain having CD3 binding activity includes the heavy chain variable region and the light chain variable region of the anti-CD3 antibody. In certain embodiments, the antigen-binding domain having CD3 binding activity is a domain containing a Fab structure.

[0169] The antigen-binding domain having CD3 binding activity of the present invention can bind to any epitope, as long as the epitope is located within the γ, δ, or ε chain sequence that forms human CD3. In the present invention, preferred antigen-binding domains having CD3 binding activity include those comprising a CD3 antibody light chain variable region (VL) and a CD3 antibody heavy chain variable region (VH) that bind to an epitope in the extracellular domain of the ε chain of the human CD3 complex. Such preferred antigen-binding domains having CD3-binding activity include those containing the CD3 antibody light chain variable region (VL) and CD3 antibody heavy chain variable region (VH) of various known CD3 antibodies, such as the OKT3 antibody (Proc. Natl. Acad. Sci. USA (1980) 77, 4914-4917) or the antibody having the light chain variable region (VL) of NCBI registry number AAB24132 and the heavy chain variable region (VH) of NCBI registry number AAB24133 (Int. J. Cancer Suppl. 7, 45-50 (1992)). Furthermore, such suitable antigen-binding domains having CD3-binding activity include those derived from CD3 antibodies with desired characteristics obtained by immunizing the desired animal with the γ, δ, or ε chains that form human CD3 by the method described above. Suitable anti-CD3 antibodies from which antigen-binding domains having CD3-binding activity originate include human antibodies and, as appropriate, humanized antibodies as described above.

[0170] Multispecific antigen binding molecules A "multispecific antigen-binding molecule" refers to an antigen-binding molecule that specifically binds to two or more antigens. In a preferred embodiment, the multispecific antigen-binding molecule of the present invention comprises two or more antigen-binding domains, where different antigen-binding domains specifically bind to different antigens.

[0171] The multispecific antigen-binding molecule of the present invention comprises a first antigen-binding domain having RNF43 binding activity and a second antigen-binding domain having T cell receptor complex binding activity. A combination of an antigen-binding domain having RNF43 binding activity, selected from those described in the above section "Antigen-binding domain having RNF43 binding activity," and an antigen-binding domain having T cell receptor complex binding activity, selected from those described in the above section "Antigen-binding domain having T cell receptor complex binding activity" to "Antigen-binding domain having CD3 binding activity," can be used.

[0172] For example, the first antigen-binding domain is a domain containing the variable regions of the antibody's heavy and light chains, and / or the second antigen-binding domain is a domain containing the variable regions of the antibody's heavy and light chains. Alternatively, the first antigen-binding domain is a domain containing an antibody variable fragment, and / or the second antigen-binding domain is a domain containing an antibody variable fragment. Alternatively, the first antigen-binding domain is a domain containing a Fab structure, and / or the second antigen-binding domain is a domain containing a Fab structure.

[0173] In a particular embodiment, the present invention provides a multispecific antigen-binding molecule comprising a first antigen-binding domain containing an antibody-variable fragment and having RNF43-binding activity, and a second antigen-binding domain containing an antibody-variable fragment and having T cell receptor complex-binding activity. In a particular embodiment, the present invention provides a bispecific antigen-binding molecule comprising a first antigen-binding domain having RNF43-binding activity, a second antigen-binding domain having T cell receptor complex-binding activity, and a domain comprising an Fc region with reduced Fcγ receptor-binding activity. The Fc region may have reduced Fcγ receptor-binding activity compared to the Fc domain of an IgG1, IgG2, IgG3, or IgG4 antibody. In one embodiment, the Fc region is an Fc region having an amino acid mutation in any of the Fc region constituent amino acids of SEQ ID NOs: 122-125 (IgG1-IgG4).

[0174] In a particular embodiment, the present invention provides a bispecific antibody comprising a first antibody variable fragment having human RNF43 binding activity and a second antibody variable fragment having CD3 binding activity. In a particular embodiment, the present invention provides a bispecific antibody comprising a first antibody variable fragment having human RNF43 binding activity, a second antibody variable fragment having CD3 binding activity, and an Fc region with reduced Fcγ receptor binding activity. In a particular embodiment, the present invention provides a bispecific antibody comprising a first antibody variable fragment having human RNF43 binding activity, a second antibody variable fragment having CD3ε chain binding activity, and an Fc region having reduced Fcγ receptor binding activity compared to the natural IgG Fc region.

[0175] A preferred embodiment of the "multispecific antigen-binding molecule" of the present invention is a multispecific antibody. When using an Fc region with reduced Fcγ receptor binding activity as the Fc region of the multispecific antibody, an Fc region derived from the multispecific antibody may be used as appropriate. A bispecific antibody is particularly preferred as the multispecific antibody of the present invention. Here, a bispecific antibody is an antibody having two different specificities. An IgG-type bispecific antibody can be secreted from a hybrid hybridoma (quadroma) produced by fusing two hybridomas that produce IgG antibodies (Milstein et al., Nature (1983) 305, 537-540).

[0176] Furthermore, IgG-type bispecific antibodies are secreted by introducing the genes for the light and heavy chains (L and H chains) of the two target IgGs—a total of four genes—into cells and co-expressing them. However, theoretically, there are only 10 possible combinations of IgG H and L chains that can be produced by these methods. Therefore, it is difficult to purify IgG containing the desired combination of H and L chains from 10 different types of IgG. Moreover, the amount of IgG secreted with the desired combination is theoretically significantly reduced, requiring large-scale culture, which further increases manufacturing costs.

[0177] Therefore, techniques for promoting the association of H chains and L chains with desired combinations can be applied to the multispecific antigen-binding molecules of the present invention. For example, to suppress undesirable H chain association, a technique can be applied to the association of multispecific antibodies by introducing electrostatic repulsion at the interface of the second or third constant region (CH2 or CH3) of the antibody H chain (WO2006 / 106905).

[0178] In a technique for suppressing unintended H chain association by introducing electrostatic repulsion at the CH2 or CH3 interface, examples of amino acid residues in contact at the interface of the other constant region of the H chain include regions corresponding to the residues at EU numbering positions 356, 439, 357, 370, 399, and 409 in the CH3 region.

[0179] More specifically, an example of an antibody containing two types of H chain CH3 regions is one in which 1 to 3 pairs of amino acid residues selected from the following pairs of amino acid residues in the first H chain CH3 region have the same charge: (1) amino acid residues at EU numbering positions 356 and 439 in the H chain CH3 region, (2) amino acid residues at EU numbering positions 357 and 370 in the H chain CH3 region, and (3) amino acid residues at EU numbering positions 399 and 409 in the H chain CH3 region.

[0180] Furthermore, the antibody may be one in which a pair of amino acid residues in a second H chain CH3 region, which is different from the first H chain CH3 region, is selected from the pairs of amino acid residues (1) to (3), and 1 to 3 pairs of amino acid residues corresponding to the pairs of amino acid residues (1) to (3) that have the same charge in the first H chain CH3 region have the opposite charge to the corresponding amino acid residues in the first H chain CH3 region.

[0181] The amino acid residues shown in (1) to (3) above are close to each other when they associate. A person skilled in the art can find the positions corresponding to the amino acid residues in (1) to (3) above in a desired H chain CH3 region or H chain constant region by homology modeling using commercially available software, and can modify the amino acid residues at these positions as appropriate.

[0182] In the above antibody, the "charged amino acid residue" is preferably selected from, for example, an amino acid residue belonging to one of the following groups: (a) Glutamic acid (E) and aspartic acid (D), and (b) Lysine (K), arginine (R), and histidine (H).

[0183] In the above antibodies, the phrase "having the same charge" means, for example, that any of the two or more amino acid residues are selected from amino acid residues belonging to either group (a) or (b) above. The phrase "having opposite charges" means, for example, that if at least one of the two or more amino acid residues is selected from amino acid residues belonging to either group (a) or (b) above, then the remaining amino acid residues are selected from amino acid residues belonging to the other group.

[0184] In a preferred embodiment, the antibody may have a first H chain CH3 region and a second H chain CH3 region crosslinked by a disulfide bond.

[0185] The amino acid residues to be modified in this invention are not limited to the amino acid residues in the antibody variable region or antibody constant region described above. Those skilled in the art can identify the amino acid residues that form interfaces in mutant polypeptides or heterologous polymers using homology modeling with commercially available software, and then modify the amino acid residues at these positions to control their association.

[0186] Furthermore, other known techniques can also be used for the association of the multispecific antibodies of the present invention. By substituting an amino acid side chain in the Fc region of one H chain of the antibody with a larger side chain (knob; projection), and substituting an amino acid side chain in the opposing Fc region of the other H chain with a smaller side chain (hole; void), the projection can be positioned within the void, thereby enabling efficient association of Fc region-containing polypeptides containing different amino acids (WO1996 / 027011, Ridgway JB et al., Protein Engineering (1996) 9, 617-621, Merchant AM et al. Nature Biotechnology (1998) 16, 677-681, and US20130336973).

[0187] In addition, other known techniques can also be used to form the multispecific antibodies of the present invention. By replacing a portion of the CH3 of one H chain of an antibody with a corresponding IgA-derived sequence and introducing the corresponding IgA-derived sequence into the complementary portion of the CH3 of the other H chain, a strand-exchange engineered domain CH3 is generated. This allows for the efficient induction of association of polypeptides having different sequences through complementary association of CH3 (Protein Engineering Design & Selection, 23; 195-202, 2010). This known technique can also be used to efficiently form the desired multispecific antibodies.

[0188] In addition, the formation of multispecific antibodies involves antibody production techniques utilizing the association of CH1 and CL and VH and VL in antibodies, as described in WO2011 / 028952, WO2014 / 018572, and Nat Biotechnol. 2014 Feb;32(2):191-8; techniques for producing bispecific antibodies using a combination of separately prepared monoclonal antibodies (Fab Arm Exchange), as described in WO2008 / 119353 and WO2011 / 131746; techniques for controlling the association between CH3 groups of antibody heavy chains, as described in WO2012 / 058768 and WO2013 / 063702; techniques for producing bispecific antibodies composed of two types of light chains and one type of heavy chain, as described in WO2012 / 023053; and Christoph et al. (Nature Biotechnology Vol. 31, p 753-758) Techniques for producing bispecific antibodies using two bacterial cell lines that each express one of the antibody chains, one of which contains one heavy chain and one light chain, as described in (2013), may also be used.

[0189] Alternatively, even if it is not possible to efficiently form the desired multispecific antibody, the multispecific antibody of the present invention can be obtained by separating and purifying the desired multispecific antibody from the produced antibody. For example, a method has been reported that allows for the purification of two homozygous antibodies and the desired heteroantibody by ion exchange chromatography by introducing amino acid substitutions into the variable regions of two types of H chains to create a difference in isoelectric points (WO2007114325). As a method for purifying heteroantibodies, a method has been reported to purify heterodimerized antibodies containing the H chain of mouse IgG2a that binds to protein A and the H chain of rat IgG2b that does not bind to protein A, using protein A (WO98050431 and WO95033844). Furthermore, by using heavy chains in which the amino acid residues at EU numbering positions 435 and 436, which are the binding sites between IgG and protein A, are substituted with amino acids such as Tyr and His, which result in different protein A affinitys, or by using heavy chains with different protein A affinitys, the interaction between each heavy chain and protein A is altered, and then by using a protein A column, heterodimerized antibodies can be efficiently purified.

[0190] Alternatively, a common light chain capable of conferring binding ability to multiple different heavy chains can be obtained and used as the common light chain for multispecific antibodies. By introducing the genes for such a common light chain and multiple different heavy chains into cells and expressing IgG, efficient expression of multispecific IgG becomes possible (Nature Biotechnology (1998) 16, 677-681). When selecting a common heavy chain, a method can also be used to select a common light chain that exhibits high binding ability to any different heavy chain (WO2004 / 065611).

[0191] Furthermore, as the Fc region of the present invention, an Fc region in which the heterogeneity of the C-terminus of the Fc region is improved may be used as appropriate. More specifically, the present invention provides an Fc region generated by deleting glycine at position 446 and lysine at position 447, which are identified by EU numbering, from the amino acid sequences of two polypeptides constituting an Fc region derived from IgG1, IgG2, IgG3, or IgG4.

[0192] Multiple of these techniques can be used in combination, for example, two or more. Furthermore, these techniques can be applied separately to two H chains to be associated, as appropriate. Moreover, these techniques can be used in combination with the Fc region having reduced binding activity to the Fcγ receptor as described above. Furthermore, the antigen-binding molecule of the present invention may be a molecule separately manufactured based on the antigen-binding molecule subjected to the above modification, having the same amino acid sequence.

[0193] Preferably, the antigen-binding molecule of the present invention may comprise a first antigen-binding domain having RNF43-binding activity and a second antigen-binding domain having T cell receptor complex-binding activity. In one embodiment, T cell receptor complex-binding activity is binding activity to the T cell receptor. In another embodiment, T cell receptor complex-binding activity is binding activity to the CD3ε chain. In one embodiment, RNF43-binding activity is binding activity to human RNF43. In a further embodiment, RNF43-binding activity is binding activity to RNF43 on the surface of eukaryotic cells. In one embodiment, RNF43-binding activity is binding activity to human RNF43 on the surface of eukaryotic cells.

[0194] Preferably, the antigen-binding molecule of the present invention may have cellular cytotoxicity (also referred to as "cytotoxicity"). In one embodiment, the cytotoxicity is T cell-dependent cytotoxicity (TDCC). In another embodiment, the cytotoxicity is cytotoxicity to cells expressing RNF43 on their surface. RNF43-expressing cells may be cancer cells.

[0195] In a preferred scenario, the antibody (or antigen-binding molecule) of the present invention exhibits cytotoxicity (or cellular cytotoxicity), or preferably T-cell-dependent cytotoxicity (TDCC), against RNF43-expressing cells such as cancer cells. RNF43 may be expressed on the surface of such cells. The cytotoxicity or TDCC of the antibody (or antigen-binding molecule) of the present invention can be evaluated by any suitable method known in the art. For example, the method described in Example 6.2.2 can be used to measure TDCC. In this case, cytotoxic activity is evaluated by the percentage of cell growth inhibition by the antibody (or antigen-binding molecule) of the present invention. Cell growth is measured using a suitable analyzer, such as an xCELLigence real-time cell analyzer. Cancer cells are used as target cells, and they are subjected to appropriate cell concentrations (e.g., about 10 cells). 4 Seeds are seeded on a multi-well plate at a concentration of (1 cell / well). The following day, the test antibody, prepared at an appropriate concentration (e.g., 0.01-10 nM), is added to the plate. After a 15-minute reaction, a solution containing T cells (e.g., PBMCs) is added in an appropriate effector (PBMC) / target (cancer cells) ratio, such as 10. The reaction is carried out using carbon dioxide gas. After adding the T cells, the CGI rate (%) is determined using the formula: Cell Growth Inhibition (CGI) rate (%) = (AB) × 100 / (A-1) (wherein A represents the average Cell Index value of the wells without antibody (or antigen-binding molecule), i.e., containing only target cells and T cells; B represents the average Cell Index value of the wells with antibody (or antigen-binding molecule)). The Cell Index values ​​used in the calculation are normalized values, i.e., the Cell Index value immediately before antibody addition is defined as 1. If the CGI rate of an antibody (or antigen-binding molecule) is high, i.e., has a significantly positive value, then that antibody (or antigen-binding molecule) has TDCC activity, which is more preferable in the present invention.

[0196] cancer The terms “cancer” and “malignant” refer to or describe a physiological condition in mammals typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas (e.g., Hodgkin and non-Hodgkin lymphomas), blastomas, sarcomas, and leukemias. More detailed examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, leukemia and other lymphoproliferative disorders, as well as various types of head and neck cancers.

[0197] tumor The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as used herein.

[0198] Colorectal tumors The term “colorectal tumor” or “colorectal cancer” refers to any tumor or cancer of the large intestine, including the colon (from the cecum to the rectum) and the rectum, including, for example, adenocarcinoma, and less common forms such as lymphoma and squamous cell carcinoma.

[0199] gastric tumor The term “gastric tumor” or “gastric cancer” or “stomach tumor” or “stomach cancer” refers to any tumor or cancer of the stomach, including, for example, adenocarcinoma (e.g., diffuse and intestinal types) as well as less common forms such as lymphoma, leiomyosarcoma, and squamous cell carcinoma.

[0200] Pharmaceutical preparations The term "pharmaceutical preparation" refers to a preparation in a form such that the biological activity of the active ingredient contained therein can exert an effect, and the preparation does not contain additional elements that are toxic to an unacceptable degree to the subject to whom the preparation is administered.

[0201] Pharmacologically acceptable carriers "Pharmaceutically acceptable carrier" refers to a component other than the active ingredient in a pharmaceutical preparation that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0202] treatment As used herein, "treatment" (and its grammatical derivatives, such as "treat", "treating", etc.) means a clinical intervention intended to modify the natural course of the individual being treated and can be carried out for prevention as well as during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of the occurrence or recurrence of a disease, alleviation of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction of the rate of progression of the disease, restoration or remission of the disease state, and improved prognosis or remission. In some embodiments, the antibodies of the present invention are used to delay the onset or slow the progression of a disease.

[0203] In one aspect, the present invention is in part based on a multispecific antigen-binding molecule comprising a first antigen-binding domain having RNF43-binding activity and a second antigen-binding domain having T-cell receptor complex-binding activity, and its use. The antigen-binding molecules and antibodies of the present invention are useful, for example, for the diagnosis or treatment of tumors, particularly colorectal tumors and gastric tumors.

[0204] Pharmaceutical composition The pharmaceutical compositions of the present invention, the therapeutic agents for inducing cytotoxicity, the cell growth inhibitors, or the anticancer agents of the present invention can be formulated together with different types of multispecific antigen-binding molecules as needed. For example, a cocktail of multiple multispecific antigen-binding molecules of the present invention can enhance the cytotoxic effect on cells expressing a particular antigen.

[0205] If necessary, the multispecific antigen-binding molecules of the present invention may be encapsulated in microcapsules (microcapsules made from hydroxymethylcellulose, gelatin, poly[methylmethacrylate], etc.) and used as components of colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) (see, for example, "Remington's Pharmaceutical Science 16th edition", Oslo Ed. (1980)). Furthermore, methods for preparing drugs as sustained-release agents are also known and can be applied to the multispecific antigen-binding molecules of the present invention (J. Biomed. Mater. Res. (1981) 15, 267-277; Chemtech. (1982) 12, 98-105; U.S. Patent No. 3,773,719; European Patent Applications (EP) Nos. EP58481 and EP133988; Biopolymers (1983) 22, 547-556).

[0206] The pharmaceutical compositions, cell growth inhibitors, or anticancer agents of the present invention may be administered to patients either orally or parenterally. Parenteral administration is preferred. Specifically, such administration methods include injection, nasal administration, pulmonary administration, and transdermal administration. Injections include, for example, intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. For example, the pharmaceutical compositions, therapeutic agents for inducing cell damage, cell growth inhibitors, or anticancer agents of the present invention can be administered locally or systemically by injection. Furthermore, an appropriate administration method can be selected according to the patient's age and symptoms. Dosages may be selected, for example, from a range of 0.0001 mg to 1,000 mg per kg of body weight per dose. Alternatively, doses may be selected, for example, from a range of 0.001 mg / body to 100,000 mg / body per patient. However, the doses of the pharmaceutical compositions of the present invention are not limited to these doses.

[0207] The pharmaceutical compositions of the present invention can be formulated according to conventional methods (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA) and may contain pharmaceutically acceptable carriers or additives. Examples, but not limited to, include surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, flow enhancers, and flavoring agents, and other commonly used carriers may be used as appropriate. Specific examples of carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts, and the like.

[0208] Preferably, the pharmaceutical composition of the present invention comprises the multispecific antigen-binding molecule of the present invention. In one embodiment, the composition is a pharmaceutical composition for use in inducing cytotoxicity. In another embodiment, the composition is a pharmaceutical composition for use in the treatment or prevention of cancer. Preferably, the cancer is colorectal cancer or gastric cancer. The pharmaceutical composition of the present invention can be used to treat or prevent cancer. Accordingly, the present invention provides a method for treating or preventing cancer in which the multispecific antigen-binding molecule of the present invention is administered to a patient in need thereof.

[0209] The present invention also provides a method for damaging or inhibiting the cell growth of RNF43-expressing cells by contacting RNF43-expressing cells with the multispecific antigen-binding molecules of the present invention that bind to RNF43. Monoclonal antibodies that bind to RNF43 are described above as the multispecific antigen-binding molecules of the present invention, and are included in the therapeutic agents for inducing cell damage, cell growth inhibitors, and anticancer agents of the present invention. The cells to which the multispecific antigen-binding molecules of the present invention bind are not particularly limited, as long as they express RNF43. Specifically, preferred cancer antigen-expressing cells in the present invention include ovarian cancer cells, prostate cancer cells, breast cancer cells, uterine cancer cells, liver cancer cells, lung cancer cells, pancreatic cancer cells, gastric cancer cells, bladder cancer cells, and colon cancer cells.

[0210] In the present invention, "contact" can be achieved, for example, by adding the multispecific antigen-binding molecule of the present invention to a culture medium of RNF43-expressing cells cultured in vitro. In this case, the multispecific antigen-binding molecule to be added can be used in a suitable form, such as a solution or a solid prepared by lyophilization or the like. When the multispecific antigen-binding molecule of the present invention is added as an aqueous solution, the solution may be a pure aqueous solution containing the multispecific antigen-binding molecule alone, or a solution containing, for example, the above-mentioned surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, flow enhancers, and flavoring agents. The concentration added is not particularly limited; however, the final concentration in the culture medium is preferably in the range of 1 pg / ml to 1 g / ml, more preferably 1 ng / ml to 1 mg / ml, and even more preferably 1 μg / ml to 1 mg / ml.

[0211] In another embodiment of the present invention, "contact" may also be carried out by administering RNF43-expressing cells in vivo to a non-human animal or to an animal having cancer cells that endogenously express RNF43. The method of administration may be oral or parenteral. Parenteral administration is particularly preferred. Specifically, parenteral administration methods include injection, nasal administration, pulmonary administration, and transdermal administration. Injections include, for example, intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. For example, the pharmaceutical compositions of the present invention, therapeutic agents for inducing cytotoxicity, cell growth inhibitors, or anticancer agents may be administered locally or systemically by injection. Furthermore, an appropriate method of administration may be selected according to the age and condition of the animal subject. When the multispecific antigen-binding molecule is administered as an aqueous solution, the solution may be a pure aqueous solution containing the multispecific antigen-binding molecule alone, or a solution containing, for example, the surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, flow enhancers, and flavoring agents. The dosage may be selected, for example, from a range of 0.0001 to 1,000 mg per kg of body weight per administration. Alternatively, the dose may be selected, for example, from a range of 0.001 to 100,000 mg / body weight per patient. However, the dosage of the multispecific antigen-binding molecule of the present invention is not limited to these examples.

[0212] The following method is preferred as a method for evaluating or determining cytotoxicity caused by contacting the multispecific antigen-binding molecule of the present invention with RNF43-expressing cells to which the antigen-binding domain forming the multispecific antigen-binding molecule of the present invention binds. Methods for evaluating or determining cytotoxic activity in vitro include methods for determining the activity of cytotoxic T cells, etc. Whether the multispecific antigen-binding molecule of the present invention has the activity to induce T cell-mediated cytotoxicity can be determined by known methods (see, for example, Current protocols in Immunology, Chapter 7. Immunologic studies in humans, Editor, John E, Coligan et al., John Wiley & Sons, Inc., (1993)). In the cytotoxicity assay, a multispecific antigen-binding molecule whose antigen-binding domain binds to an antigen different from RNF43 and not expressed in the cells is used as a control multispecific antigen-binding molecule. The control multispecific antigen-binding molecule is assayed in the same manner. Then, the activity is evaluated by testing whether the multispecific antigen-binding molecule of the present invention exhibits stronger cytotoxic activity than that of the control multispecific antigen-binding molecule.

[0213] On the other hand, in vivo cytotoxic activity can be evaluated or determined, for example, by the following procedure: Cells expressing an antigen to which the antigen-binding domain forming the multispecific antigen-binding molecule of the present invention binds are transplanted intradermally or subcutaneously into a non-human animal subject. The test multispecific antigen-binding molecule is then administered intravenously or intraperitoneally daily or at intervals of several days, starting on the day of transplantation or thereafter. Tumor size is measured over time. The difference in the change in tumor size can be defined as cytotoxic activity. A control multispecific antigen-binding molecule is administered, similar to the in vitro assay. If the tumor size is smaller in the group administered with the multispecific antigen-binding molecule of the present invention than in the group administered with the control multispecific antigen-binding molecule, the multispecific antigen-binding molecule of the present invention can be determined to have cytotoxic activity.

[0214] To evaluate or determine the effect of contacting the multispecific antigen-binding molecule of the present invention with cells expressing an antigen to which the antigen-binding domain forming the multispecific antigen-binding molecule binds, the MTT method and measurement of the uptake of isotope-labeled thymidine into cells are preferably used. On the other hand, to evaluate or determine the activity of inhibiting cell growth in vivo, the same method as the above-described method for evaluating or determining in vivo cytotoxic activity may be preferably used.

[0215] The present invention also provides a kit for use in the method of the present invention, comprising the multispecific antigen-binding molecule of the present invention or a multispecific antigen-binding molecule produced by the method of the present invention. The kit may be packaged with additional pharmaceutically acceptable carriers or media, or with instructions for use describing the kit.

[0216] In addition, the present invention relates to a multispecific antigen-binding molecule of the present invention or a multispecific antigen-binding molecule produced by the present invention, for use in the method of the present invention.

[0217] All references cited herein are incorporated herein by reference. [Examples]

[0218] The following are examples of the methods and compositions of the present invention. In light of the general description above, it will be understood that various other embodiments may be implemented.

[0219] Example 1. RNF43 expression in tumor / normal tissue Figure 1 shows the RNF43 mRNA expression profile based on data created by the TCGA Research Network: http: / / cancergenome.nih.gov / . The human RNF43 mRNA expression profiles in normal and tumor tissues, analyzed using data downloaded from TCGA, are shown as box plots. The data consists of minimum, maximum, and three quartiles. The boxes represent the interquartile range. The lines inside the boxes represent the median. The lines and points extending outside the boxes represent the minimum and maximum values. The results indicate that RNF43 mRNA expression is upregulated in multiple cancer types, particularly in gastrointestinal tumor tissues.

[0220] Example 2. Expression and purification of the extracellular domain (ECD) of human RNF43. A synthetic polypeptide (SEQ ID NO: 1) containing amino acids 1-190 of human RNF43 ECD with a Flag tag at its C-terminus was transiently expressed in the FreeStyle293F cell line (Thermo Fisher). The conditioned medium expressing the synthetic polypeptide was applied to a column packed with anti-Flag M2 affinity resin (Sigma), and eluted with Flag peptide (Sigma). The fraction containing the synthetic polypeptide was collected and subsequently subjected to a Superdex 200 gel filtration column (GE Healthcare) equilibrated with 1×D-PBS. The fraction containing the synthetic polypeptide was then pooled and stored at -80°C (°F). Human RNF43 ECD (named RNF43-Fc, SEQ ID NO: 128), in which an Fc region is fused to the C-terminus, was transiently expressed using the FreeStyle293F cell line (Thermo Fisher). The conditioned medium expressing RNF43-Fc was purified using a HiTrap MabSelect SuRe column (GE healthcare). The fraction containing RNF43-Fc was collected and subsequently subjected to Superdex 200 gel filtration column (GE healthcare) equilibrated with 1×D-PBS. The fraction containing RNF43-Fc was then pooled and stored at -80°C.

[0221] Example 3. Establishment of a Ba / F3 cell line expressing cleaved human RNF43. Inserted a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 2 consisting of truncated human RNF43 with a C-terminal FLAG tag into the pCXND3 expression vector (WO / 2008 / 156083).

[0222] Introduced 400 ng of linearized truncated human RNF43-pCXND3 into the mouse IL-3-dependent pro-B cell-derived cell line Ba / F3 by electroporation (LONZA, 4D-Nucleofector X).

[0223] After introduction, added geneticin and cultured the cells to obtain a cell line resistant to geneticin. Plated the transfected cell line in a 96-well plate by limiting dilution and expanded it. Named the established cell line Ba / F3 E12 (truncated human RNF43).

[0224] Example 4: Preparation and screening of monospecific anti-RNF43 antibodies Prepared, selected, and assayed an anti-RNF43 monoclonal antibody as follows.

[0225] 12-16 week old NZW rabbits were intradermally immunized with human RNF43 (50-100 μg / dose / rabbit) prepared as described in Example 2. This dose was repeated three times over a period of one month. One week after the final immunization, spleens and blood were collected from the immunized rabbits. Antigen-specific B cells were stained with labeled antigens and sorted using an FCM cell sorter (FACS aria III, BD). These cells were plated in 96-well plates at a rate of 1 cell / well with 25,000 EL4 cells (European Collection of Cell Cultures) per well and 20-fold diluted activated rabbit T cell conditioned medium. The cells were cultured for 7-12 days. The EL4 cells were pre-treated with mitomycin C (Sigma, catalog no. M4287) for 2 hours and washed three times. Activated rabbit T cell conditioned medium was prepared by culturing rabbit thymocytes in RPMI-1640 containing phytohemagglutinin M (Roche, catalog no. 1 1082132-001), phorbol 12-myristate 13-acetate (Sigma, catalog no. P1585), and 2% FBS. After culturing, the B cell culture supernatant was collected for further analysis, and the pellet was cryopreserved.

[0226] The specificity of antibodies in B cell culture supernatant was tested using FCM analysis. RNF43-expressing Ba / F3 cells (Ba / F3 E12 established in Example 3) or parental Ba / F3 cells (1 × 10⁶ cells) were used. 5The cells were dispensed into a V-bottom 96-well plate (BD Falcon 353263) and centrifuged at 500 xg for 2 minutes. The supernatant was aspirated, and 30 μL of B cell culture supernatant was added to resuspend the cells. The cells were incubated on ice for 30 minutes and centrifuged at 500 xg for 2 minutes. The supernatant was aspirated, and the cells were washed with 150 μL of HEPES-buffered saline (HEPES-BSA) containing 0.02 M HEPES, 5 mM KCl, 4 mM NaHCO3, 138 mM NaCl, 2 mM CaCl2, 5 mM glucose, 0.4 mM KH2PO4, 0.34 mM Na2HPO4, and 0.1% BSA. After washing, 100 μL of mouse anti-rabbit IgG PE conjugate (SouthernBiotech, 4090-09, 100-fold dilution in HEPES-BSA) was added, and the cells were resuspended. The cells were incubated on ice for 30 minutes and then washed. The cells were resuspended in 100 μL of HEPES-BSA, and the binding of rabbit antibodies was analyzed by FACS Verse (BD).

[0227] A total of 8,670 B cell lines were screened for binding to human RNF43. 470 cell lines were selected as RNF43-specific conjugates that bind to the Ba / F3 E12 cell line but not to the parental Ba / F3 cell line, and these were named RNN0184-0653. Their RNA was purified from cryopreserved cell pellets using the ZR-96 Quick-RNA kit (ZYMO RESEARCH, catalog number R1053). The DNA of the antibody heavy chain variable region was amplified by reverse transcription PCR and recombined with the DNA encoding the BS03aHis (SEQ ID NO: 3) heavy chain constant region. The DNA of the antibody light chain variable region was amplified by reverse transcription PCR and recombined with the DNA encoding the hk0MC light chain constant region (SEQ ID NO: 4). The cloned antibodies were expressed in FreeStyle® 293-F cells (Invitrogen), purified from the culture supernatant, and their functional activity was evaluated. The specific binding of antibodies to RNF43 was evaluated by FCM analysis. Several anti-RNF43 monospecific antibodies were selected for further analysis and are listed in Table 2 (SEQ ID NOs: 5-24 and 27-86).

[0228] [Table 2]

[0229] Example 5. Characterization of an anti-RNF43 monospecific antibody Example 5.1 Analysis of antibody binding to membrane RNF43 Figures 2a and 2b show the binding of the anti-RNF43 antibody to the Ba / F3 E12 transfectant and the NUGC-4 cancer cell line, as determined by FACS analysis.

[0230] Anti-RNF43 monospecific antibodies were incubated with each cell line at room temperature for 30 minutes and washed with FACS buffer (2% FBS in PBS, 2 mM EDTA). Then, goat F(ab')2 anti-human IgG and mouse ads-PE (Southern Biotech, catalog no. 2043-09) were added, incubated at 4°C for 20 minutes, and washed with FACS buffer. Data was collected using FACS Verse (Becton Dickinson) and subsequently analyzed using FlowJo software (Tree Star) and GraphPad Prism software (GraphPad).

[0231] Figures 2a and 2b show that all anti-RNF43 monospecific antibodies produced in Example 4, namely RNN0187jj, RNN0191kk, RNN0192nn, RNN0193jj, RNN0198oo, RNN0207ii, RNN0242nn, RNN0246jj, RNN0275kk, and RNN0276oo, bind to the target antigen, RNF43. In these figures, the mean fluorescence intensity (MFI) obtained by the antibodies was normalized against the negative control, keyhole limpet hemocyanin (KLH) antibody. The data are expressed as dMFI values.

[0232] Example 5.2 Affinity measurement of anti-RNF43 monospecific antibody The affinity of anti-RNF43 monospecific antibodies to human RNF43 at pH 7.4 was determined at 25°C using a Biacore T200 instrument (GE Healthcare). Anti-human Fc (GE Healthcare) was immobilized on all flow cells of the CM4 sensor tip using an amine coupling kit (GE Healthcare). All antibodies and analytes were prepared in pH 7.4 ACES containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, and 0.005% NaN3. Each antibody was captured on the sensor surface by anti-human Fc. The antibody capture level was aimed at 312 resonance units (RU). Recombinant human RNF43 was injected at 200 nM, 50 nM, and 12.5 nM, prepared by 4-fold serial dilutions, and then dissociated. The sensor surface was regenerated with 3 M MgCl2 after each cycle. The binding affinity was determined by processing the data using Biacore T200 Evaluation software, version 2.0 (GE Healthcare), and fitting it to a one-to-one binding model.

[0233] Table 3 shows the affinity of anti-RNF43 monospecific antibodies against human RNF43. [Table 3]

[0234] Example 6. Functional evaluation of an anti-RNF43 / CD3 bispecific antibody. Example 6.1 Absolute quantification of RNF43 on the surface of cancer cells The antibody binding ability (ABC) of RNF43 on the cell surface of cultured cancer cell lines (SW48, LS1034, and LS513 were purchased from ATCC; PC-10 from IBL; and NUGC4 from HSRRB) was evaluated using QIFIKIT (DAKO) flow cytometry.

[0235] Cancer cells (1 cell x 10⁻⁶ 5 ~5×10 5Cells were washed with 0.5% BSA-supplemented CellWASH (BD Bioscience) (hereinafter referred to as FACS / PBS). The variable region of RNN0246jj was ligated to mouse Fc (SEQ ID NOs: 87 and 88) to create bivalent RNN0246-mFc. RNN0246-mFc or control antibody was added to 50 μL of solution at a final concentration of 20 μg / mL. These were allowed to stand at 4°C for 30-60 minutes. After washing the cells with FACS / PBS, FITC-labeled goat anti-mouse IgG antibody diluted 50-fold in FACS / PBS was added to the cells. These were allowed to stand at 4°C for 30-60 minutes. The cells were washed with FACS / PBS and analyzed by flow cytometry.

[0236] The ABC of RNF43 on the surface of cancer cells was calculated using QIFI KIT (Figure 3).

[0237] Example 6.2 Functional Characterization of Anti-RNF43 / CD3 Bispecific Antibody Example 6.2.1 Preparation of human peripheral blood mononuclear cells (PBMC solution) The primary human PBMC solution was newly isolated from healthy volunteers or purchased in frozen form, where indicated (STEMCELL).

[0238] For fresh PBMC solutions, 50 mL of peripheral blood was collected from each healthy volunteer (individual adult) using a syringe pre-filled with 100 μL of 1,000 units / mL heparin solution (Novo Nordisk for injection, 5,000 units). This peripheral blood was diluted 2-fold with PBS (-), divided into four equal parts, and added to Leucosep tubes (catalog number 227290, Greiner Bio-One) for lymphocyte separation, which had been pre-filled with 15 mL of Ficoll-Paque PLUS and subjected to centrifugation. These separation tubes were centrifuged (2,150 rpm at room temperature for 10 minutes) to collect the mononuclear cell fraction. Cells in the mononuclear cell fraction were washed once with Dulbecco's modified Eagle medium (SIGMA) containing 10% FBS, and the cell density was reduced to 4 × 10⁶ using 10% FBS / D-MEM. 6The cell suspension was prepared to a concentration of cells / mL. This cell suspension was used as the human PBMC solution in the following experiments.

[0239] For frozen PBMCs, the frozen cells were thawed by placing the cryopreservation vials in a 37°C water bath. The cells were then dispensed into 15 mL Falcon tubes containing 9 mL of culture medium for target cells. The cell suspension was then centrifuged at 1,200 rpm at room temperature for 5 minutes. The supernatant was gently aspirated, and fresh warmed medium was added for resuspension. The cell suspension was used as the human PBMC solution in the following experiments.

[0240] Example 6.2.2 Measurement of T cell-dependent cytotoxicity of anti-RNF43 / CD3 bispecific antibody Using the anti-RNF43 monospecific antibody and anti-CD3 antibody (SEQ ID NOs: 25 and 26) listed in Table 2, anti-RNF43 / CD3 bispecific antibodies were prepared using conventional methods published elsewhere. The CDR sequences of the RNF43-binding arms in the anti-RNF43 / CD3 bispecific antibodies are shown in Table 4.

[0241] [Table 4]

[0242] The resulting bispecific antibodies contain silent Fc, which has reduced affinity for the Fcγ receptor.

[0243] Figure 4 shows the T cell-dependent cytotoxicity (TDCC) of the anti-RNF43 / CD3 bispecific antibody. Cytotoxic activity was evaluated by the ratio of cell growth inhibition using the xCELLigence real-time cell analyzer (Roche Diagnostics). The NUGC-4 human cancer cell line was used as the target cell. The target cells were detached from the dish and the cells were divided into 1 × 10⁶ cells. 4By adjusting the cells / well, they were plated in 100 μL / well fractions onto an E-plate 96 (Roche Diagnostics), and cell growth was measured using an xCELLigence real-time cell analyzer. After 24 hours, the plate was removed, and 50 μL of each antibody prepared at each concentration (0.016, 0.08, 0.4, 2, or 10 nM) was added to the plate. After a 15-minute reaction at room temperature, 50 μL of fresh human PBMC solution prepared in Example 6.2.1 was added at an effector (PBMC) / target (NUGC-4) ratio of 10 (i.e., 1 × 10 cells). 5 The antibody was added (in cells / well), and cell growth was resumed using the xCELLigence real-time cell analyzer. The reaction was carried out at 37°C under 5% carbon dioxide gas conditions. 72 hours after the addition of PBMCs, the cell growth inhibition (CGI) rate (%) was determined using the following formula. The Cell Index value obtained from the xCELLigence real-time cell analyzer used in the calculation was a normalized value, in which case the Cell Index value immediately before antibody addition was defined as 1.

[0244] Cell growth inhibition rate (%) = (AB) × 100 / (A - 1) A represents the average Cell Index value in the well without antibody (containing only target cells and human PBMCs), and B represents the average Cell Index value in the target well. The experiment was performed in sets of three.

[0245] All antibodies from Example 5 were subjected to a TDCC assay using a NUGC-4 cell line with moderate RNF43 expression. Bispecific antibodies 191, 193, 198, 242, 246, and 275 showed the strongest TDCC activity at a 10 nM concentration (Figure 4a). In particular, 242 and 246 showed the strongest T cell-dependent cytotoxicity. Similarly, these two antibodies also showed potent T cell-dependent cytotoxicity in SW48, a cell line with high RNF43 surface expression (Figure 4b).

[0246] Example 7. Evaluation of in vivo drug efficacy We evaluated the in vivo efficacy of some of the antibodies mentioned above using a tumor-bearing model.

[0247] In vivo efficacy was evaluated using anti-human RNF43 / CD3 bispecific antibodies (242 and 246) that were confirmed to have cytotoxic activity in the in vitro assay described in Example 6. Cell lines were transplanted into NOD scid mice, and NOD scid mice in which tumor formation was confirmed were subjected to transplantation of T cells proliferated by in vitro culture of human PBMCs. Mice (referred to as the T cell injection model) were treated with administration of anti-human RNF43 / CD3 bispecific antibodies.

[0248] More specifically, in the efficacy study of an anti-human RNF43 / CD3 bispecific antibody using an SCC152 (ATCC) transplantation and T-cell injection model, the following tests were performed: T cells were cultured in large quantities using purchased PBMCs and a T-cell activation / expansion kit for humans (MACS Miltenyi biotec). Human cancer cell line SCC152 (cells 1 × 10⁶) 7 T cells were mixed with Matrigel® basement membrane matrix (BD) and transplanted into the subcutaneous region of the inguinal region of NOD scid mice (CLEA Japan, female, 6W-8W). The day of transplantation was defined as day 0. On the day before transplantation (day 0), the mice were intraperitoneally administered 0.2 mg / mice of anti-asialoGM1 antibody (Wako Pure Chemical Industries). On day 17 after transplantation, the mice were divided into groups according to their body weight and tumor size, and the mice were again intraperitoneally administered 0.2 mg / mice of anti-asialoGM1 antibody. The following day, the T cells obtained by the above-mentioned expansion culture were cultured to 3 × 10⁶ cells. 7 Individual cells were transplanted into the peritoneal cavity. Four hours after T cell transplantation, an anti-human RNF43 / CD3 bispecific antibody was administered intravenously via the tail vein at a dose of 10 mg / kg. The anti-human RNF43 / CD3 bispecific antibody was administered only once.

[0249] As a result, antitumor activity was observed in the group administered with the anti-human RNF43 / CD3 bispecific antibody compared to the solvent-administered control group (Figure 5a).

[0250] The efficacy studies of the anti-human RNF43 / CD3 bispecific antibody in the SW48 (ATCC) transplantation and T-cell injection model were conducted using a similar method. The anti-human RNF43 / CD3 bispecific antibody was administered intravenously twice via the tail vein at doses of 10 mg / kg and 7 mg / kg on days 7 and 14, respectively.

[0251] As a result, antitumor activity was observed in the group administered with the anti-human RNF43 / CD3 bispecific antibody compared to the solvent-administered control group (Figure 5b).

[0252] Example 8. Epitope binning of anti-RNF43 monospecific antibody 8.1 Preparation of an anti-RNF43 monospecific antibody containing a rabbit constant region The plasmid prepared in Example 4 was used as a template for PCR amplification of the variable region and was recombinant with DNA encoding the rabbit heavy chain constant region (SEQ ID NO: 126) and the rabbit light chain constant region (SEQ ID NO: 127). The cloned antibody was expressed in FreeStyle® 293-F cells (Invitrogen) and purified from the culture supernatant. The antibody was biotinylated by incubating 50 micrograms (μg) of purified antibody with 2 μg of NHS-PEG2-biotin (PIERCE) on ice for 2 hours. Subsequently, free biotin was removed by dialysis using an Easy Sep chamber (TOMY) in PBS.

[0253] 8.2 Binding competition of anti-RNF43 monospecific antibodies First, the EC50 concentration of each anti-RNF43 monospecific antibody bound to RNF43-Fc (described in Example 2) was determined by ELISA assay using biotinylated antibodies. Briefly, 5 μg / mL or 1 μg / mL of RNF43-Fc was coated onto Maxisorp plates (NUNC) overnight at 4°C. The coated plates were then washed with PBS-T and subsequently blocked with Blocking One solution (Nacalai Tesque) at room temperature for 2 hours. Next, serially diluted biotinylated anti-RNF43 monospecific antibodies were added and incubated at room temperature for 1 hour. After washing with PBS-T, StAv-HRP (PIERCE) was added and incubated at room temperature for 1 hour. After washing with PBS-T, ABTS peroxidase substrate (SeraCare Life Sciences) was added, and the signal intensity was measured using a Multiskan® GO microplate spectrophotometer. The EC50 concentration of an anti-RNF43 monospecific antibody bound to RNF43-Fc was calculated using a nonlinear regression 4-parameter fit. The normalized absorbance at 405 nm / 570 nm measured when the EC50 concentration of the anti-RNF43 antibody was fitted was defined as A. O It is expressed as follows. To evaluate binding competition between monospecific anti-RNF43 antibodies, an ELISA assay was performed using a similar setup. RNF43-Fc was first coated onto a Maxisorp plate overnight. The coated plate was blocked with Blocking One solution and then incubated for 15 minutes with a 10-fold concentration of the unbiotinized primary antibody (test antibody) at each EC50. Without washing, the biotinylated secondary antibody (reference antibody) was added at its EC50 concentration and incubated at room temperature for 1 hour. After washing with PBS-T, ABTS peroxidase substrate was added, and the signal intensity was measured using a Multiskan® GO microplate spectrophotometer. Normalized absorbance at 405 nm / 570 nm is denoted as A. The binding inhibition (%) was calculated using the following formula: Figure 6 shows the binding inhibition between anti-RNF43 monospecific antibodies. Binning was determined using a 20% binding inhibition cutoff value, meaning that antibodies with less than 20% binding inhibition were classified into different bins. In other words, if the test antibody Ab1 shows binding inhibition of more than 20% when another antibody Ab2 is used as the reference antibody, and antibody Ab2 also shows binding inhibition of more than 20% when Ab2 is used as the test antibody and Ab1 is used as the reference antibody, then antibodies Ab1 and Ab2 are classified into the same bin. The antibodies were classified into four bins as follows: RNN0207ii in bin A; RNN0187jj and RNN0192nn in bin B; RNN0193jj in bin C; RNN0242nn and RNN0246jj in bin D.

[0254] The invention described herein has been described in detail with examples and illustrations for the purpose of aiding clear understanding, but the descriptions and illustrations herein should not be construed as limiting the scope of the invention. All disclosures of patent and scientific documents cited herein are expressly incorporated herein by reference throughout.

[0255] Industrial applicability This invention provides a novel multispecific antigen-binding molecule with potent antitumor activity, excellent safety characteristics such as not inducing cancer antigen-independent cytokine storms, and a long blood half-life. A cytotoxicity inducer containing the antigen-binding molecule of this invention as an active ingredient can target RNF43-expressing cells and tumor tissues containing these cells, and induce cell damage. Administering the multispecific antigen-binding molecule of this invention to patients enables a desirable treatment that is not only highly safe but also reduces physical burden and offers greater convenience.

Claims

1. A pharmaceutical composition for use in treating cancer, comprising a multispecific antigen-binding molecule comprising a first antigen-binding domain having RNF43-binding activity and a second antigen-binding domain having T cell receptor complex-binding activity.

2. A pharmaceutical composition for use according to claim 1, wherein the multispecific antigen-binding molecule has cytotoxicity.

3. 3. The pharmaceutical composition for use according to claim 2, wherein the cytotoxicity is T-cell dependent cytotoxicity.

4. The pharmaceutical composition for use according to any one of claims 1 to 3, wherein the T cell receptor complex binding activity is binding activity to a T cell receptor.

5. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the T cell receptor complex binding activity is binding activity to the CD3ε chain.

6. A pharmaceutical composition for use according to any one of claims 1 to 5, wherein the RNF43 binding activity is binding activity to human RNF43 on the surface of a eukaryotic cell.

7. 7. The pharmaceutical composition for use according to any one of claims 1 to 6, wherein the first antigen-binding domain is a domain comprising an antibody variable fragment and / or the second antigen-binding domain is a domain comprising an antibody variable fragment.

8. 8. The pharmaceutical composition for use according to any one of claims 1 to 7, wherein the first antigen-binding domain is a domain comprising a Fab structure and / or the second antigen-binding domain is a domain comprising a Fab structure.

9. 9. The pharmaceutical composition for use according to any one of claims 1 to 8, wherein the first antigen-binding domain comprises any one of the following antibody variable fragments: (a) an antibody variable fragment comprising an antibody heavy chain variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 28, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 48, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 68, and an antibody light chain variable region comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 58, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 78; (b) an antibody variable fragment comprising an antibody heavy chain variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 51, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 71, and an antibody light chain variable region comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 41, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 81; (c) an antibody variable fragment comprising an antibody heavy chain variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 53, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 73, and an antibody light chain variable region comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 63, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 83; (d) an antibody variable fragment comprising an antibody heavy chain variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 34, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 54, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 74, and an antibody light chain variable region comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 44, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 64, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 84; (e) an antibody variable fragment comprising an antibody heavy chain variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 55, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75, and an antibody light chain variable region comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 45, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 65, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 85; (f) an antibody variable fragment that competes with any one of the antibody variable fragments (a) to (e) for binding to human RNF43; and (g) An antibody variable fragment that binds to the same epitope on human RNF43 as any one of the antibody variable fragments (a) to (e).

10. The pharmaceutical composition for use according to any one of claims 1 to 9, wherein the first antigen-binding domain is one of the following (a) to (e): (a) an antigen-binding domain comprising a VH comprising SEQ ID NO: 6 and a VL comprising SEQ ID NO: 16; (b) an antigen-binding domain comprising a VH comprising SEQ ID NO: 9 and a VL comprising SEQ ID NO: 19; (c) an antigen-binding domain comprising a VH comprising SEQ ID NO: 11 and a VL comprising SEQ ID NO: 21; (d) an antigen-binding domain comprising a VH comprising SEQ ID NO: 12 and a VL comprising SEQ ID NO: 22; (e) An antigen-binding domain comprising a VH comprising SEQ ID NO: 13 and a VL comprising SEQ ID NO:

23.

11. The pharmaceutical composition for use according to any one of claims 1 to 10, wherein the multispecific antigen-binding molecule further comprises a domain comprising an Fc region having reduced Fcγ receptor binding activity compared to a native human Fc region.

12. The pharmaceutical composition for use according to any one of claims 1 to 11, wherein the multispecific antigen-binding molecule is a bispecific antibody comprising a first antibody variable fragment having RNF43-binding activity, a second antibody variable fragment having CD3ε chain-binding activity, and an Fc region having reduced Fcγ receptor-binding activity compared to a native human Fc region.

13. 13. The pharmaceutical composition for use according to any one of claims 1 to 12, wherein the cancer is colorectal cancer or gastric cancer.

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