HER2 S310F-specific antigen-binding molecule

Abstract

An object of the present invention is to provide an antigen-binding molecule that specifically binds to a HER2 mutant. The present invention also provides an antigen-binding molecule that specifically binds to a HER2 mutant and can regulate and / or activate an immune response, a composition comprising the antigen-binding molecule, and methods of using the same. TIFF2022505144000015.tif150170

Description

[Technical field]

[0001] The present invention relates to an antigen-binding molecule that specifically binds to HER2 (human epidermal growth factor receptor 2) having an S310F mutation (hereinafter also referred to as HER2 S310F); and a multispecific antigen-binding molecule comprising a first domain comprising a first antibody variable region that specifically binds to HER2 S310F, a second domain comprising a second antibody variable region that binds to a T cell receptor complex, and a third domain comprising an Fc region; and uses thereof. [Background technology]

[0002] Cancer is one of the leading causes of death worldwide. Except for certain carcinomas, tumors are often difficult to effectively cure. Traditional cancer treatments include radiation therapy, chemotherapy, and immunotherapy. These treatments are often not effective enough, and eventually cancer recurrence or metastasis occurs after treatment. Lack of tumor specificity is one of the factors that limit maximum efficacy; therefore, more tumor-specific molecular targeted therapy has become an additional viable option in cancer treatment.

[0003] Antibodies are attracting attention as pharmaceuticals because they are highly stable in plasma and have few side effects. Among multiple therapeutic antibodies, some types of antibodies require effector cells to exert antitumor responses. Antibody-dependent cell-mediated cytotoxicity (ADCC) is a cytotoxic activity exhibited by effector cells against antibody-bound cells through binding of the Fc region of the antibody to Fc receptors present on NK cells and macrophages. To date, multiple therapeutic antibodies capable of inducing ADCC and exerting antitumor efficacy have been developed as pharmaceuticals for treating cancer (Non-Patent Document 1). Therapies using conventional therapeutic antibodies targeting tumor-specific antigens show excellent antitumor activity, but administration of such antibodies does not always result in satisfactory treatment outcomes.

[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 cytotoxic activity by recruiting T cells as effector cells have been known since the 1980s (Non-Patent Documents 2-4). TR antibodies are bispecific antibodies that recognize and bind to any one of the subunits that form the T cell receptor complex on T cells, in particular the CD3ε chain, and an antigen on a cancer cell. Several TR antibodies are currently being developed. Catumaxomab, a TR antibody against EpCAM, has been approved in Europe for the treatment of malignant ascites. Furthermore, a type of TR antibody called "bispecific T-cell engager (BiTE)" has recently been found to exhibit strong antitumor activity (Non-Patent Documents 5 and 6). Blinatumomab, a BiTE molecule against CD19, was the first to receive FDA approval in 2014. Blinatumomab has been shown to exhibit much stronger 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 Document 7).

[0005] However, it is known that trifunctional antibodies simultaneously bind to both T cells and cells such as NK cells or macrophages in a cancer antigen-independent manner, resulting in cross-linking of receptors expressed on cells and induction of expression of various cytokines in a cancer antigen-independent manner. Systemic administration of trifunctional antibodies is thought to cause cytokine storm-like side effects as a result of induction of such cytokine expression. In fact, it has been reported that a very low dose of 5 μg / body was the maximum tolerated dose for systemic administration of catumaxomab to patients with non-small cell lung cancer in a phase I clinical trial, and that administration of higher doses causes various severe side effects (Non-Patent Document 8). When administered at such a low dose, catumaxomab can never reach an effective blood level. That is, the expected antitumor effect cannot be achieved by administering catumaxomab at such a low dose.

[0006] On the other hand, unlike catumaxomab, BiTE does not have an Fcγ receptor binding site and therefore does not crosslink receptors expressed on T cells and cells such as NK cells and macrophages in a cancer antigen-independent manner. Therefore, it has been demonstrated that BiTE does not cause the cancer antigen-independent cytokine induction observed when catumaxomab is administered. However, since BiTE is a modified low molecular weight antibody molecule that does not have an Fc region, the problem is that its blood half-life after administration to patients is significantly shorter than that of IgG type antibodies that have traditionally been used as therapeutic antibodies. In fact, it has been reported that the blood half-life of BiTE administered in vivo is about several hours (Non-Patent Documents 9 and 10). In clinical trials of blinatumomab, it is administered by continuous intravenous infusion using a minipump. This administration method is not only extremely inconvenient for patients, but also has a potential risk of medical accidents due to device malfunctions, etc. Therefore, it cannot be said that such an administration method is desirable.

[0007] HER2 (ErbB2 or ErbB-2, also known as human epidermal growth factor receptor 2, CD340) is a single-pass type 1 transmembrane protein and a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases that activate signaling pathways that control cell proliferation and survival. HER2 overexpression and HER2 gene alterations, such as HER2 gene amplification, HER2 mutations (point mutations, insertion mutations, and deletion mutations), have been identified as oncogenic drivers and potential therapeutic targets in cancer (Non-Patent Documents 11-13). HER2 targeted therapy has dramatically improved clinical outcomes for breast and gastric cancer patients with HER2 overexpression or HER2 amplification. However, targeted therapy for cancer patients with HER2 mutations remains an unmet need in the clinical setting.

[0008] HER2 mutations have been identified in various cancers. One example of HER2 mutation is the HER2 S310F mutation observed in breast cancer, non-small cell lung cancer, bladder cancer, colorectal cancer, cervical cancer, endometrial cancer, and gastroesophageal cancer (Non-Patent Document 14).

[0009] To date, there have been no reports of an antibody that recognizes HER2 S310F but does not recognize wild-type HER2. Therefore, an object of the present invention is to provide a monoclonal antibody that binds to HER2 S310F but does not bind to wild-type HER2. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Clin Cancer Res. 2010 Jan 1;16(1):11-20. [Non-Patent Document 2] Nature. 1985 Apr 18-24;314(6012):628-31. [Non-Patent Document 3] Int J Cancer. 1988 Apr 15;41(4):609-15. [Non-Patent Document 4] Proc Natl Acad Sci US A. 1986 Mar;83(5):1453-7. [Non-Patent Document 5] Proc Natl Acad Sci US A. 1995 Jul 18;92(15):7021-5. [Non-Patent Document 6] Drug Discov Today. 2005 Sep 15;10(18):1237-44. [Non-Patent Document 7] Int J Cancer. 2002 Aug 20;100(6):690-7. [Non-Patent Document 8] Cancer Immunol Immunother (2007) 56 (10), 1637-44. [Non-Patent Document 9] Cancer Immunol Immunother. (2006) 55 (5), 503-14. [Non-Patent Document 10] Cancer Immunol Immunother. (2009) 58 (1), 95-109. [Non-Patent Document 11] Nat Rev Cancer. 2009 Jul;9(7):463-75. [Non-Patent Document 12] Cancer Discov. 2013 Feb;3(2):224-37. [Non-Patent Document 13] Cancer Discov. 2015 Aug;5(8):832-41. [Non-Patent Document 14] Nature. 2018 Feb 8;554(7691):189-194. Summary of the Invention [Problem to be solved by the invention]

[0011] The object of the present invention is to provide an antigen binding molecule that binds to HER2 variants, particularly HER2 S310F, with high specificity; and its multispecific antigen binding molecule that enables cancer treatment by recruiting T cells near HER2 S310F-expressing cells and induces the cytotoxic activity of T cells against HER2 S310F-expressing cancer cells; a method for producing the antigen binding molecule; and a therapeutic agent that contains such an antigen binding molecule as an active ingredient for inducing cellular cytotoxic activity. Another object of the present invention is to provide a pharmaceutical composition for use in the treatment or prevention of various cancers, which contains one of the above-mentioned antigen binding molecules as an active ingredient, and a therapeutic method using the pharmaceutical composition. [Means for solving the problem]

[0012] The inventors of the present invention have succeeded in generating an antigen-binding molecule that exhibits high specificity and binding activity to HER2 S310F and has low or no cross-reactivity to wild-type HER2. To the best of the inventors' knowledge, to date, there have been no reports of antibodies that specifically recognize HER2 with a single amino acid residue mutation (e.g., S310F) but do not recognize wild-type HER2. Such mutant-specific anti-HER2 antigen-binding molecules can demonstrate excellent specificity because they only target tumor cells that express tumor-specific antigens (i.e., HER2 S310F); whereas conventional anti-HER2 antibodies target / bind to wild-type HER2, which is also expressed in normal tissues, and thus may result in on-target toxicity and healthy organ damage. Due to the lack of expression of HER2 S310F in non-tumor tissues, the HER2 S310F mutant-specific antigen-binding molecule of the present invention (e.g., anti-HER2 S310F antibody) is expected to be a superior therapeutic agent in terms of high accuracy and specificity of tumor targeting, as well as its safety (reduced toxicity), compared with conventional anti-HER2 antibodies. In another aspect, the present inventors have generated a multispecific antigen-binding molecule comprising a first domain comprising a first antibody variable region that specifically binds to HER2 S310F and a second domain comprising a second antibody variable region that binds to a T cell receptor complex (e.g., CD3), which can effectively kill cells expressing HER2 S130F and exert excellent cytotoxicity / antitumor activity. The multispecific antigen-binding molecule and pharmaceutical composition comprising the antigen-binding molecule as an active ingredient can treat various cancers, particularly those associated with HER2 S130F mutation, such as HER2 S130F-positive tumors. The multispecific antigen-binding molecule of the present invention has a strong antitumor activity that induces cellular cytotoxicity activity, and can specifically target and damage HER2 S130F-expressing cells (but not HER2-expressing cells), thus enabling highly accurate and specific treatment and prevention of HER2 S310F-positive cancer. Furthermore, the multispecific antigen-binding molecules of the present invention have a long half-life in blood and excellent safety profile, which does not induce any cancer antigen-independent cytokine storm, etc. This enables desirable treatments that are highly safe and convenient, and reduces the physical burden on patients.

[0013] More specifically, the present invention provides the following: [1] An antigen-binding molecule comprising a first antigen-binding domain that specifically binds to human HER2 having an S310F mutation (HER2 S310F) and has weaker binding activity or no binding activity to wild-type human HER2. [2] An antigen-binding molecule or the antigen-binding molecule of [1] that specifically binds to an epitope containing a phenylalanine residue at position 310 of human HER2. [3] Preferably, by surface plasmon resonance (SPR), under the following conditions: 37°C, pH 7.4, 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3; antigen-binding molecules are immobilized on a CM4 sensor chip, and the HER2 S310F antigen serves as the analyte. An antigen-binding molecule or any one of the antigen-binding molecules of [1] to [2] that binds to human HER2 S310F with a KD of less than 100 nanomolar (nM) when measured by. [4] Preferably, the SPR satisfies the following conditions: 37°C, pH 7.4, 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3; antigen-binding molecules are immobilized on a CM4 sensor chip, and antigen serves as the analyte a KD ratio (i.e., KD value for binding to human HER2 S310F vs. KD value for binding to human wild-type HER2) that is at least 10-fold, 100-fold, 1,000-fold, or 10,000-fold, or 100,000-fold smaller when measured by HER2 S310F / KD 野生型HER2 or any one of the antigen-binding molecules of [1] to [3]. [5] An antigen-binding molecule selected from [1] to [4] that does not bind to wild-type human HER2. [6] An antigen-binding molecule or any one of the antigen-binding molecules [1] to [5] that inhibits ligand-independent dimerization of human HER2 S130F. [6A] An antigen-binding molecule selected from any one of [1] to [5], which is a monoclonal antibody. [6B] An antigen-binding molecule according to any one of [1] to [5], which is a human antibody, a humanized antibody, or a chimeric antibody. [6C] An antigen-binding molecule of any one of [1] to [5], which is human IgG1. [7] An antigen-binding molecule according to any one of [1] to [6], further comprising a second antigen-binding domain that specifically binds to a T cell receptor complex molecule. [8] The antigen-binding molecule of [7], wherein the T cell receptor complex molecule is CD3, preferably a human CD3ε chain. [9] The antigen-binding molecule of any one of [7] to [8], which is a multispecific antigen-binding molecule, preferably a bispecific antibody. [9A] An antigen-binding molecule according to any one of [1] to [9], further comprising an Fc domain.

[10] The antigen-binding molecule of [9A], wherein the Fc domain is an Fc domain having reduced Fcγ receptor-binding activity compared to a wild-type Fc domain of IgG1, preferably human IgG1. [10A] The Fc region comprises the following amino acid positions as identified 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 The antigen-binding molecule of

[10] , wherein the Fc region has at least one amino acid mutation selected from the group consisting of:

[11] An antigen-binding molecule according to any one of [1] to [10A], which has cytotoxic activity.

[12] The antigen-binding molecule of

[11] , which comprises a second antigen-binding domain that specifically binds to a T cell receptor complex molecule, and the cytotoxic activity is T cell-dependent cytotoxicity (TDCC).

[13] The antigen-binding molecule of any one of [1] to

[12] , wherein the first antigen-binding domain and / or the second antigen-binding domain comprises a Fab, Fv, scFv, or VHH structure.

[14] The antigen-binding molecule of any one of [1] to

[13] , wherein the first antigen-binding domain and / or the second antigen-binding domain comprises a Fab structure.

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

[14] , comprising an F(ab')2 structure.

[16] Any one of the antigen-binding molecules of [1] to

[15] , wherein the first antigen-binding domain comprises any one of the following antibody variable regions: (a1) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 29, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 41, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 53, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 65, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 77, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 89; (a2) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 30, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 42, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 54, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 66, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 78, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 90; (a3) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 31, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 43, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 55, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 67, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 79, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 91; (a4) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 32, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 44, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 56, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 68, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 80, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 92; (a5) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 33, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 45, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 57, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 69, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 81, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 93; (a6) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 34, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 46, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 58, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 70, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 82, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 94; (a7) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 35, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 47, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 59, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 71, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 83, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 95; (a8) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 36, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 48, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 60, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 72, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 84, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 96; (a9) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 37, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 49, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 61, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 73, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 85, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 97; (a10) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 38, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 50, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 62, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 74, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 86, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 98; (a11) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 39, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 51, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 63, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 75, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 87, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 99; (a12) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 40, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 52, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 64, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 76, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 88, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 100; (b1) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 29, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 41, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 53, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 65, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 77, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 89; (b2) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 30, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 42, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 54, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 66, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 78, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 90; (b3) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 31, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 43, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 67, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 79, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 91; (b4) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 44, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 56, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 68, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 80, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 92; (b5) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 33, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 45, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 57, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 69, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 81, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 93; (b6) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 46, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 58, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 70, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 82, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 94; (b7) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 35, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 47, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 59, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 71, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 83, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 95; (b8) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 36, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 60, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 72, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 84, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 96; (b9) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 37, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 49, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 61, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 73, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 85, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 97; (b10) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 38, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 50, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 62, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 74, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 86, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 98; (b11) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 39, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 51, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 63, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 75, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 87, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 99; (b12) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 40, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 52, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 64, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 76, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 100; (c1) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO:5, and a light chain variable domain (VL) of SEQ ID NO:17; (c2) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO:6, and a light chain variable domain (VL) of SEQ ID NO:18; (c3) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 7, and a light chain variable domain (VL) of SEQ ID NO: 19; (c4) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 8, and a light chain variable domain (VL) of SEQ ID NO: 20; (c5) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 9, and a light chain variable domain (VL) of SEQ ID NO: 21; (c6) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 10, and a light chain variable domain (VL) of SEQ ID NO: 22; (c7) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 11, and a light chain variable domain (VL) of SEQ ID NO: 23; (c8) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 12, and a light chain variable domain (VL) of SEQ ID NO: 24; (c9) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 13, and a light chain variable domain (VL) of SEQ ID NO: 25; (c10) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 14, and a light chain variable domain (VL) of SEQ ID NO: 26; (c11) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 15, and a light chain variable domain (VL) of SEQ ID NO: 27; (c12) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 16, and a light chain variable domain (VL) of SEQ ID NO: 28; (d) an antibody variable region that competes with any one of the antibody variable regions of (a1) to (c12) for binding to HER2; (e) An antibody variable region that binds to the same epitope on HER2 as any one of the antibody variable regions of (a1) to (c12).

[17] Any one of the antigen-binding molecules of [1] to

[16] , wherein the second antigen-binding domain comprises any one of the following antibody variable regions: (a) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 103, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 104, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 105, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 106, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 107, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 108; (b) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO:101, and a light chain variable domain (VL) of SEQ ID NO:102.

[18] A pharmaceutical composition for use in inducing cellular cytotoxicity, comprising any one of the antigen-binding molecules of [1] to

[17] and a pharma- ceutical acceptable excipient.

[19] A pharmaceutical composition for use in treating cancer, comprising any of the antigen-binding molecules of [1] to

[17] and a pharma- ceutical acceptable excipient.

[20] A diagnostic kit comprising any one of the antigen-binding molecules described in [1] to

[17] .

[21] An immunoconjugate comprising any one of the antigen-binding molecules according to [1] to

[17] and a cytotoxic agent.

[22] An antigen-binding molecule according to any one of [1] to

[17] for use as a medicine.

[23] An antigen-binding molecule of any one of [1] to

[17] for use in the treatment, prevention, or diagnosis of cancer.

[24] Use of any one of the antigen-binding molecules [1] to

[17] in the manufacture of a medicine for the treatment of cancer.

[25] A method for diagnosing cancer in an individual or predicting cancer treatment response in an individual, the method comprising determining the presence of human HER2 S310F using an antigen-binding molecule according to any one of [1] to

[17] .

[26] A method for detecting the presence of human HER2 S310F in an isolated sample, comprising the steps of incubating the sample with an antigen-binding molecule according to any one of [1] to

[17] , and detecting the presence of a complex formed between the antigen-binding molecule and human HER2 S310F.

[27] Any of the pharmaceutical compositions

[18] -

[19] , the diagnostic kit

[20] , any of the antigen-binding molecules for use in

[22] -

[23] , the use of

[24] , or the method of

[25] , wherein the cancer is characterized by expression of human HER2 S310F.

[28] The cancer is bladder cancer, cervical cancer, colorectal cancer (CRC), non-small cell lung cancer (NSCLC), esophageal cancer, head and neck cancer, skin cancer (melanoma), bile duct cancer, kidney cancer, gastric cancer, small intestine cancer, liver cancer, uterine cancer, duodenal cancer, breast cancer, gallbladder cancer, or ovarian cancer. Any of the pharmaceutical compositions

[18] -

[19] , the diagnostic kits

[20] , any of the antigen-binding molecules for use in

[22] -

[23] , the use of

[24] , or the method of

[25] .

[29] A nucleic acid encoding any one of the antigen-binding molecules of [1] to

[17] , optionally in a vector.

[30]

[29] A cell containing nucleic acid.

[31] A method for preparing the antigen-binding molecule of any one of [1] to

[17] , comprising the step of expressing a nucleic acid encoding the antigen-binding molecule in a cell. The present invention also provides the following. [1] An antigen-binding molecule comprising a first antigen-binding domain that specifically binds to human HER2 having an S310F mutation (HER2 S310F) but does not bind to wild-type human HER2. [2] The antigen-binding molecule of [1], which specifically binds to an epitope containing a phenylalanine residue at position 310 of human HER2. [3] The antigen-binding molecule of [1] or [2], which inhibits ligand-independent dimerization of human HER2 S310F. [4] The antigen-binding molecule of any one of [1] to [3], further comprising a second antigen-binding domain that specifically binds to a T cell receptor complex molecule. [5] The antigen-binding molecule of [4], wherein the T cell receptor complex molecule is CD3, preferably a human CD3ε chain. [6] The antigen-binding molecule of [4] or [5], which is a multispecific antigen-binding molecule, preferably a bispecific antibody. [7] The antigen-binding molecule of any one of [4] to [6], further comprising an Fc domain having reduced Fcγ receptor-binding activity compared to a wild-type Fc domain of IgG1. [8] The antigen-binding molecule of any one of [4] to [7], which has T cell-dependent cytotoxicity (TDCC). [9] The antigen-binding molecule of any one of [1] to [8], wherein the first antigen-binding domain and / or the second antigen-binding domain comprises a Fab, Fv, scFv, or VHH structure.

[10] The antigen-binding molecule of any one of [1] to [9], wherein the first antigen-binding domain and / or the second antigen-binding domain comprises a Fab structure.

[11] The antigen-binding molecule of any one of [1] to

[10] , wherein the first antigen-binding domain comprises any one of the following antibody variable regions: (a1) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 29, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 41, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 53, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 65, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 77, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 89; (a2) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 30, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 42, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 54, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 66, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 78, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 90; (a3) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 31, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 43, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 67, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 79, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 91; (a4) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 44, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 56, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 68, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 80, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 92; (a5) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 33, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 45, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 57, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 69, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 81, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 93; (a6) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 46, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 58, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 70, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 82, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 94; (a7) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 35, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 47, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 59, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 71, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 83, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 95; (a8) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 36, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 60, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 72, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 84, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 96; (a9) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 37, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 49, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 61, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 73, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 85, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 97; (a10) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 38, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 50, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 62, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 74, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 86, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 98; (a11) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 39, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 51, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 63, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 75, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 87, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 99; (a12) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 40, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 52, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 64, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 76, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 100; (b1) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO:5, and a light chain variable domain (VL) of SEQ ID NO:17; (b2) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO:6, and a light chain variable domain (VL) of SEQ ID NO:18; (b3) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 7, and a light chain variable domain (VL) of SEQ ID NO: 19; (b4) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 8, and a light chain variable domain (VL) of SEQ ID NO: 20; (b5) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 9, and a light chain variable domain (VL) of SEQ ID NO: 21; (b6) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 10, and a light chain variable domain (VL) of SEQ ID NO: 22; (b7) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 11, and a light chain variable domain (VL) of SEQ ID NO: 23; (b8) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 12, and a light chain variable domain (VL) of SEQ ID NO: 24; (b9) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 13, and a light chain variable domain (VL) of SEQ ID NO: 25; (b10) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 14, and a light chain variable domain (VL) of SEQ ID NO: 26; (b11) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 15, and a light chain variable domain (VL) of SEQ ID NO: 27; (b12) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 16, and a light chain variable domain (VL) of SEQ ID NO: 28; (c) an antibody variable region that competes with any one of the antibody variable regions (a1) to (b12) for binding to HER2; (d) An antibody variable region that binds to the same epitope on HER2 as any one of the antibody variable regions of (a1) to (b12).

[12] The antigen-binding molecule of any one of [1] to

[11] for use in the treatment, prevention, or diagnosis of cancer.

[13] The antigen-binding molecule for use according to

[12] , wherein the cancer is characterized by expression of HER2 S310F.

[14] The antigen-binding molecule for use according to

[12] or

[13] , wherein the cancer is bladder cancer, cervical cancer, colorectal cancer (CRC), non-small cell lung cancer (NSCLC), esophageal cancer, head and neck cancer, skin cancer (melanoma), bile duct cancer, kidney cancer, gastric cancer, small intestine cancer, liver cancer, uterine cancer, duodenal cancer, breast cancer, or gallbladder cancer.

[15] A method for preparing the antigen-binding molecule according to any one of [1] to

[14] , comprising the step of expressing a nucleic acid encoding the antigen-binding molecule in a cell.

[0014] In one embodiment, the antigen-binding molecule of the present invention comprises a first antigen-binding domain that specifically binds to human HER2 with S310F mutation (HER2 S310F) and has weaker or no binding activity to wild-type human HER2. In another embodiment, the antigen-binding molecule of the present invention comprises a first antigen-binding domain that specifically binds to an epitope containing a phenylalanine residue at position 310 of human HER2. In yet another embodiment, the antigen-binding molecule of the present invention preferably comprises a first antigen-binding domain that binds to human HER2 S310F with a KD of less than 100 nanomolar (nM) when measured by surface plasmon resonance (SPR) under the following conditions: 37° C., pH 7.4, 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3; the antigen-binding molecule is immobilized on a CM4 sensor chip, and the HER2 S310F antigen serves as the analyte. In one embodiment, the antigen-binding molecule of the present invention that is specific for the HER2 S310F mutant has a KD value for binding to human wild-type HER2 (KD 野生型HER2 ) for binding to human HER2 S310F (KD HER2 S310FIn a preferred embodiment, the antigen-binding molecule of the present invention preferably comprises a first antigen-binding domain having a KD value for binding to human HER2 S310F that is at least 10-fold, 100-fold, 1,000-fold, or 10,000-fold, or 100,000-fold lower (i.e., 1 / 10, 1 / 100, 1 / 1000, 1 / 10000, or 1 / 100000) compared to the KD value for binding to human wild-type HER2, when measured by SPR under the following conditions: 37° C., pH 7.4, 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3; the antigen-binding molecule is immobilized on a CM4 sensor chip, and the antigen serves as the analyte. In a preferred embodiment, the antigen-binding molecule of the present invention comprises a first antigen-binding domain that specifically binds to human HER2 having an S310F mutation (HER2 S310F) but does not bind to wild-type human HER2. In another preferred embodiment, the antigen-binding molecule of the present invention comprises a first antigen-binding domain that prevents ligand-independent dimerization of human HER2 S310F.

[0015] In a preferred embodiment, the antigen-binding molecule of the present invention comprises [A] A first antigen-binding domain comprising any one of the following antibody variable regions: (a1) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 29, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 41, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 53, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 65, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 77, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 89; (a2) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 30, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 42, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 54, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 66, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 78, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 90; (a3) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 31, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 43, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 55, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 67, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 79, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 91; (a4) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 32, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 44, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 56, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 68, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 80, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 92; (a5) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 33, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 45, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 57, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 69, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 81, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 93; (a6) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 34, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 46, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 58, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 70, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 82, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 94; (a7) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 35, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 47, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 59, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 71, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 83, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 95; (a8) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 36, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 48, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 60, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 72, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 84, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 96; (a9) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 37, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 49, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 61, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 73, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 85, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 97; (a10) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 38, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 50, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 62, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 74, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 86, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 98; (a11) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 39, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 51, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 63, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 75, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 87, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 99; (a12) an antibody variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 40, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 52, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 64, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 76, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 88, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 100; (b1) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 29, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 41, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 53, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 65, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 77, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 89; (b2) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 30, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 42, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 54, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 66, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 78, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 90; (b3) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 31, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 43, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 67, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 79, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 91; (b4) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 44, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 56, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 68, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 80, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 92; (b5) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 33, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 45, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 57, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 69, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 81, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 93; (b6) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 46, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 58, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 70, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 82, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 94; (b7) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 35, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 47, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 59, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 71, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 83, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 95; (b8) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 36, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 60, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 72, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 84, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 96; (b9) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 37, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 49, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 61, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 73, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 85, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 97; (b10) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 38, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 50, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 62, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 74, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 86, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 98; (b11) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 39, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 51, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 63, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 75, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 87, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 99; (b12) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 40, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 52, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 64, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 76, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 100; (c1) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO:5, and a light chain variable domain (VL) of SEQ ID NO:17; (c2) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO:6, and a light chain variable domain (VL) of SEQ ID NO:18; (c3) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 7, and a light chain variable domain (VL) of SEQ ID NO: 19; (c4) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 8, and a light chain variable domain (VL) of SEQ ID NO: 20; (c5) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 9, and a light chain variable domain (VL) of SEQ ID NO: 21; (c6) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 10, and a light chain variable domain (VL) of SEQ ID NO: 22; (c7) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 11, and a light chain variable domain (VL) of SEQ ID NO: 23; (c8) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 12, and a light chain variable domain (VL) of SEQ ID NO: 24; (c9) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 13, and a light chain variable domain (VL) of SEQ ID NO: 25; (c10) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 14, and a light chain variable domain (VL) of SEQ ID NO: 26; (c11) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 15, and a light chain variable domain (VL) of SEQ ID NO: 27; (c12) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 16, and a light chain variable domain (VL) of SEQ ID NO: 28; (d) an antibody variable region that competes with any one of the antibody variable regions of (a1) to (c12) for binding to HER2; (e) an antibody variable region that binds to the same epitope on HER2 as any one of the antibody variable regions of (a1) to (c12); and preferably further comprising: [B] A second antigen-binding domain comprising any one of the following antibody variable regions: (a) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 103, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 104, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 105, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 106, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 107, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 108; (b) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 101, and a light chain variable domain (VL) of SEQ ID NO: 102. Includes.

[0016] In a preferred embodiment, the antigen-binding molecule of the present invention is a bispecific antibody that specifically binds to human HER2 S310 and CD3.

[0017] In one embodiment, the antigen-binding molecule of the present invention comprises a first antigen-binding domain having a binding activity to human HER2 S310F that is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 1000-fold, 10000-fold stronger than any of the reference antibodies as described in Table 2 or 3. Preferably, the binding activity is measured by SPR under the following conditions: 37°C, pH 7.4, 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3; the antibody is immobilized on a CM4 sensor chip and the antigen serves as the analyte. In one embodiment, the antigen-binding molecule of the present invention specific for the HER2 S310F mutant has a KD ratio (i.e., KD value for binding to human HER2 S310F to KD value for binding to human wild-type HER2) that is smaller than that of a reference antibody, preferably a reference antibody as described in Table 2 or 3. HER2 S310F / KD 野生型HER2 In a preferred embodiment, the antigen-binding molecule of the present invention comprises a first antigen-binding domain having a KD ratio (i.e., KD value for binding to human HER2 S310F to the KD value for binding to human wild-type HER2) that is at least 10-fold, 100-fold, 1,000-fold, or 10,000-fold, or 100,000-fold smaller (i.e., 1 / 10, 1 / 100, 1 / 1000, 1 / 10000, or 1 / 100000) compared to any of the reference antibodies as described in Tables 2 or 3. HER2 S310F / KD 野生型HER2 Preferably, the binding activity is measured by SPR under the following conditions: 37° C., pH 7.4, 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3; the antigen-binding molecule is immobilized on a CM4 sensor chip, and the antigen serves as the analyte.

[0018] In one aspect, the present invention provides a human T cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR). In some embodiments, the CAR of the present invention comprises a first antigen binding domain of the present invention, preferably one of the antigen binding domains as described in Table 2 or 3. In further embodiments, the CAR of the present invention comprises a first antigen binding domain and a transmembrane domain, and an intracellular domain and a signaling domain of a costimulatory molecule. In some embodiments, the intracellular domain may comprise a CD3ζ signaling domain. In some embodiments, the antigen binding fragment is an scFv. In some embodiments, the T cell comprises a vector comprising the nucleic acid sequence. For example, the vector is a lentiviral vector. In another aspect, the present invention provides a pharmaceutical composition comprising the human T cell as described above. [Brief description of the drawings]

[0019] [Figure 1] Binding analysis of anti-HER2 S310F monospecific antibody to transfectants. [Diagram 2] Binding analysis of anti-HER2 S310F monospecific antibody to cancer cell lines. [Diagram 3] T cell activation activity of anti-HER2 S310F / CD3 bispecific antibodies in the presence of transfectants expressing HER2 S310F or wild-type HER2. [Figure 4] T cell-dependent cytotoxicity of anti-HER2 S310F / CD3 bispecific antibodies against transfectants expressing HER2 S310F or wild-type HER2. [Diagram 5] T cell-dependent cytotoxicity of anti-HER2 S310F / CD3 bispecific antibody against cancer cell lines. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Description of the Aspects The techniques and procedures described or referenced herein are generally well understood and may be found in, 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)); Methods in Enzymology (Academic Press, Inc.) series: 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. 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), using conventional methodologies commonly employed by those skilled in the art.

[0021] The following definitions and detailed descriptions are provided to facilitate the understanding of the invention as exemplified herein.

[0022] amino acid Amino acids are referred to herein by single or three letter codes or both, e.g., Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, or Val / V.

[0023] Amino acid modification For amino acid modification in the amino acid sequence of an antigen-binding molecule, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR may be used as appropriate. In addition, some known methods may also be used as amino acid modification methods for substitution with unnatural amino acids (Annu Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, it is suitable to use a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA having an unnatural amino acid bound to a complementary amber suppressor tRNA of the UAG codon (amber codon), which is one of the stop codons.

[0024] As used herein, the meaning of the term "and / or" when describing the site of amino acid modification includes any suitable combination of "and" and "or." Specifically, for example, "amino acids at positions 33, 55, and / or 96 are substituted" includes the following variations of amino acid modification: (a) amino acids at positions 33, (b) 55, (c) 96, (d) 33 and 55, (e) 33 and 96, (f) 55 and 96, and (g) 33, 55, and 96.

[0025] Furthermore, in the present specification, as an expression indicating an amino acid modification, an expression indicating the one-letter or three-letter code of the amino acid before and after the modification may be appropriately used before and after the number indicating a specific position. For example, the modification N100bL or Asn100bLeu used when replacing an amino acid contained in an antibody variable region indicates the replacement of Asn at position 100b (according to Kabat numbering) with Leu. That is, the number indicates the position of the amino acid according to Kabat numbering, the one-letter or three-letter code written before the number indicates the amino acid before the replacement, and the one-letter or three-letter code written after the number indicates the amino acid after the replacement. Similarly, the modification P238D or Pro238Asp used when replacing an amino acid in the Fc region contained in an antibody constant region indicates the replacement of Pro at position 238 (according to EU numbering) with Asp. That is, the number indicates the position of the amino acid according to EU numbering, the one-letter or three-letter amino acid code written before the number indicates the amino acid before substitution, and the one-letter or three-letter amino acid code written after the number indicates the amino acid after substitution.

[0026] antigen binding molecule The term "antigen-binding molecule" as used herein refers to any molecule that contains an antigen-binding domain or has binding activity to an antigen, and may further refer to molecules such as peptides or proteins having a length of about 5 amino acids or more.Peptides and proteins are not limited to those derived from organisms, and may be, for example, polypeptides made from artificially designed sequences.They may also be naturally occurring polypeptides, synthetic polypeptides, recombinant polypeptides, etc.

[0027] A convenient example of the antigen-binding molecule of the present invention is an antigen-binding molecule that comprises multiple antigen-binding domains.In a specific embodiment, the antigen-binding molecule of the present invention is an antigen-binding molecule that comprises two antigen-binding domains with different antigen-binding specificities.In a specific embodiment, the antigen-binding molecule of the present invention is an antigen-binding molecule that comprises two antigen-binding domains with different antigen-binding specificities and an FcRn-binding domain contained in an antibody Fc region.As a method for extending the blood half-life of a protein administered to a living body, a method of adding an antibody FcRn-binding domain to a protein of interest and utilizing the function of FcRn-mediated recycling is well known.

[0028] Antigen-binding domain The term "antigen-binding domain" as used herein refers to an antibody portion that contains a region that specifically binds to and is complementary to all or a portion of an antigen. If the molecular weight of the antigen is large, the antibody can only bind to a specific portion of the antigen. The specific portion is called an "epitope". An antigen-binding domain can be provided by one or more antibody variable domains. Preferably, the antigen-binding domain contains an antibody variable region, including 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", "F(ab')2", and VHH (Variable domain of Heavy chain of Heavy chain [antibody])) structures.

[0029] The antigen-binding domain of the antigen-binding molecule of the present invention "specifically binds to HER2 S310F or a T cell receptor complex molecule." That is, HER2 S310F and a T cell receptor complex molecule are each preferred antigens of interest, and the antigen-binding domain has binding activity to the antigen of interest, but does not substantially recognize or bind to other molecules in a sample containing a mixed population of antigen molecules. As used herein, the phrase "has binding activity" refers to the activity of an antigen-binding domain, antibody, antigen-binding molecule, antibody variable fragment, etc. (hereinafter, "antigen-binding domain, etc.") to bind to an antigen of interest at a level of specific binding higher than the level of non-specific binding or background binding. In other words, such an antigen-binding domain, etc. "has specific / significant binding activity" to an antigen of interest. Specificity can be measured by any method for detecting affinity or binding activity, as described herein or known in the art. The level of specific binding described above will be high enough that a person skilled in the art would recognize it as significant. For example, an antigen-binding domain or the like can be said to have "specific / significant binding activity" for an antigen of interest when 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 or the like and the antigen of interest in a suitable binding assay. Alternatively, "having specific / significant binding activity" can be rephrased as "specifically / significantly binding" (to an antigen of interest). Sometimes, the phrase "having binding activity" has substantially the same meaning in the art as the phrase "having specific / significant binding activity". As used herein, an antigen-binding molecule or antibody that "specifically binds" to an antigen is one that has a binding activity greater than or equal to 100% of the antigen of interest as measured by a surface plasmon resonance assay or a cell binding assay. -7 M or less, preferably 10 -8 M or less, and even more preferably 10 -9 M or less, and most preferably 10 -8 M~10 -10This refers to an antigen-binding molecule or antibody that binds to its antigen and substantially identical antigens with high affinity, meaning that it has a KD of less than or equal to M, but does not bind with high affinity to unrelated antigens.

[0030] In some embodiments, the binding activity or affinity of the antigen-binding domain of the present invention (e.g., anti-HER2 S310F / CD3 bispecific antibody) to an antigen of interest (e.g., human HER2 S310F) is evaluated at 37°C, for example, using a Biacore T200 instrument (GE Healthcare) or a Biacore 8K instrument (GE Healthcare). Anti-human Fc (e.g., GE Healthcare) is immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (e.g., GE Healthcare). The antigen-binding molecule or antigen-binding domain is captured on the anti-Fc sensor surface, and then the antigen (e.g., HER2 S310F or CD3) is injected across the flow cells. The capture level of the molecule / domain may aim for 200 resonance units (RU). Recombinant human HER2 S310F or wild-type HER2 may be injected at 400 to 25 nM prepared by two-fold serial dilution, followed by dissociation. All antigen-binding domains and analytes are prepared in ACES pH 7.4 containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3. The sensor surface is regenerated with 3M MgCl2 every cycle. The binding affinity is determined by processing the data and fitting to a 1:1 binding model, for example, using Biacore T200 Evaluation software, version 2.0 (GE Healthcare) or Biacore 8K Evaluation software (GE Healthcare). To evaluate the specific binding activity or affinity of the antigen-binding domain of the present invention, the KD values ​​for HER2 S310F and wild-type HER2 are determined and the KD ratio (i.e., KD HER2 S310F / KD 野生型HER2 ) to calculate

[0031] HER2 and HER2 S310F As used herein, the term "HER2" in the context of "wild type HER2" (also known as proto-oncogene Neu, ErbB-2, tyrosine kinase cell surface receptor HER2, CD340) is a member of the HER2 family of proteins identified in RefSeq Accession Nos. NP_001005862.1 (receptor tyrosine protein kinase erbB-2 isoform b; SEQ ID NO: 162), NP_001276865.1 (receptor tyrosine protein kinase erbB-2 isoform c; SEQ ID NO: 163), and NP_001276865.1 (receptor tyrosine protein kinase erbB-2 isoform c; SEQ ID NO: 164). No.: 163), NP_001276866.1 (receptor tyrosine protein kinase erbB-2 isoform d; SEQ ID NO: 164), NP_001276867.1 (receptor tyrosine protein kinase erbB-2 isoform e; SEQ ID NO: 165), NP_004439.2 (receptor tyrosine protein kinase erbB-2 isoform a; SEQ ID NO: 166). The HER2 S310F protein can dimerize in a ligand-independent manner to transmit a signal to a downstream signaling pathway. In some embodiments, the present invention provides an antigen-binding molecule or an antigen-binding molecule that blocks the ligand-independent dimerization of the human HER2 S310F mutant. As used herein, the term "HER2 S310F" refers to any HER2 mutant having a serine to phenylalanine mutation at the amino acid residue corresponding to position 310 of NP_004439.2 (receptor tyrosine protein kinase erbB-2 isoform a; SEQ ID NO: 166). TIFF0007625516000001.tif244164TIFF0007625516000002.tif134166

[0032] Affinity / Avidity "Affinity" or "binding activity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antigen-binding molecule or an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity / activity" refers to the inherent binding affinity that reflects a 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 a molecule X for its partner Y can generally be represented by a dissociation constant (KD). Affinity can be measured by common methods known in the art, including the methods described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below. As used herein, "no binding activity" (or "no specific binding activity") refers to the activity of an antibody to bind to a non-target antigen at a level of binding that includes non-specific binding or background binding but does not include specific binding. In other words, such an antibody "does not bind" (or "does not specifically bind") to a non-target antigen. Specificity can be measured by any assay method described herein or known in the art by determining the KD ratio between the target antigen and the non-target antigen. The level of non-specific binding or background binding described above may be zero, or may be close to zero but not zero, or may be very low enough to be technically ignored by a person skilled in the art. For example, an antibody can be said to have "no (specific) binding activity" or "does not (specifically) bind" to a non-target antigen when a person skilled in the art cannot detect or observe any significant (or relatively strong) signal for binding between the antibody and the non-target antigen in a suitable binding assay. On the other hand, "weaker binding activity" refers to the activity of an antibody to bind to a target antigen at a level lower than the level of binding of a certain reference (or control) antibody to the antigen. The degree of "weakness" may be measured by any assay method described herein or known in the art, for example, by determining the difference in KD values ​​or ratios between the antibody of interest and a reference / control antibody.

[0033] How to determine affinity In certain embodiments, the antigen-binding domain of an antigen-binding molecule or antibody provided herein has an affinity for its antigen of 1 μM or less, 120 nM or less, 100 nM or less, 80 nM or less, 70 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 2 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, 10 -8 M~10 -13 M, 10 -9 M~10 -13In certain embodiments, the KD value of the first antigen-binding domain of the antibody / antigen-binding molecule for HER2 S130F falls within the range of 1 to 40, 1 to 50, 1 to 70, 1 to 80, 30 to 50, 30 to 70, 30 to 80, 40 to 70, 40 to 80, or 60 to 80 nM.

[0034] In one embodiment, KD is measured by radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is determined by measuring the binding affinity of the Fab to the antigen at the lowest concentration ( 125 I) Fab is equilibrated with labeled antigen, and then bound antigen is captured by a plate coated with anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)). To establish measurement conditions, MICROTITER® multi-well plates (Thermo Scientific) are 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 non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., as in the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although the incubation can be continued for longer (e.g., about 65 hours) to ensure that equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™; Packard) is added and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in the competitive binding assay.

[0035] In another embodiment, KD is measured using a BIACORE® surface plasmon resonance assay. For example, measurements using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25°C using a CM5 chip with approximately 10 response units (RU) of antigen immobilized. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with 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) with 10 mM sodium acetate, pH 4.8, before being injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of protein binding. After injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. with a flow rate of approximately 25 μl / min. The binding rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the association and dissociation sensorgrams with a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). The equilibrium dissociation constant (KD) is k off / k on The on-rate is calculated as a ratio of 10 to 10 by the surface plasmon resonance assay described above. 6 M -1 s -1If the on-rate exceeds , the on-rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, band pass 16 nm) of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 at 25°C in the presence of increasing concentrations of antigen, as measured in a spectrometer (e.g., a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) using a stirred cuvette).

[0036] Methods for measuring the affinity of an antigen-binding domain of an antibody are described above and those skilled in the art can perform affinity measurements for other antigen-binding domains.

[0037] antibody The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0038] Antibody classes The "class" of an antibody refers to the type of constant domain or constant region present in its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0039] Framework "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of the HVRs and FRs generally appear in the following sequence in the VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0040] Human Consensus Framework A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the human immunoglobulin VL or VH sequence is selected from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup 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 kappa I in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III in Kabat et al., supra.

[0041] HVR The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain residues that contact the antigen ("antigen contacts"). Generally, antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include: (a) the hypervariable loops located 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 located 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 sites present 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) combinations 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 according to Kabat et al., supra. HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 are also referred to as "HCDR1," "HCDR2," "HCDR3," "LCDR1," "LCDR2," and "LCDR3," respectively.

[0042] Variable region The term "variable region" or "variable domain" refers to the domain of 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 a natural antibody (VH and VL, respectively) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al. Kuby Immunology, 6 th ed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Moreover, antibodies that bind to a specific antigen may be isolated by screening a library of complementary VL or VH domains, respectively, with a VH or VL domain from an antibody that binds to the antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0043] Identity (sequence identity) "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps if necessary, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0044] The ALIGN-2 sequence comparison computer program is the property of Genentech, Inc., and its source code has been submitted with user documentation to the US Copyright Office, Washington DC, 20559, and is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, it can be phrased as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y. where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program, as described in the immediately preceding paragraph.

[0045] Chimeric antibodies The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived 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 and / or light chain variable region is derived from a particular source or species, while the remainder of the heavy and / or light chain variable region is derived from a different source or species.

[0046] Humanized antibodies A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. A "humanized antibody variable region" refers to the variable region of a humanized antibody.

[0047] Human antibodies A "human antibody" is an antibody with an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues. "Human antibody variable region" refers to the variable region of a human antibody.

[0048] T cell recruitment antibody "T cell recruiting (TR) antibodies" have cytotoxic activity by recruiting T cells as effector cells. Examples of TR antibodies include bispecific antibodies that recognize and bind to (i) any one of the subunits forming the T cell receptor complex on T cells (i.e., "T cell receptor complex molecules"), such as CD3, particularly the human CD3ε chain, and (ii) an antigen on cancer cells. TR antibodies recruit T cells and promote the killing of cancer cells by binding to both the T cell receptor complex molecule (e.g., CD3 or human CD3ε chain) and the cancer antigen. In the present context, preferably, the antigen is human HER2 S310F mutant.

[0049] Methods for producing antibodies with desired binding activity The method for producing an antibody with desired binding activity is known to those skilled in the art.The following is an example that describes the method for producing an antibody that binds to HER2 S310F (anti-HER2 S310F antibody).Antibody that binds to T cell receptor complex, etc. can also be produced according to the following example.

[0050] Anti-HER2 S310F antibody can be obtained as polyclonal antibody or monoclonal antibody by known methods.Preferably prepared anti-HER2 S310F antibody is a monoclonal antibody derived from a mammal.Such mammalian monoclonal antibody includes the antibody produced by hybridoma or by host cell transformed with an expression vector carrying antibody gene by genetic engineering techniques.

[0051] Monoclonal antibody-producing hybridomas can be prepared, for example, by using known methods such as those described below. Specifically, a mammal is immunized by a conventional immunization method using HER2 S310F protein as a sensitizing antigen. The resulting immune cells are fused with known parent cells by a conventional cell fusion method. Then, a hybridoma that produces anti-HER2 S310F antibody can be selected by screening monoclonal antibody-producing cells using a conventional screening method.

[0052] Specifically, monoclonal antibody is prepared as described below. First, a polypeptide containing HER2 S310F can be synthesized or expressed, and used as a sensitizing antigen or immunogen for antibody preparation. Alternatively, nucleotides encoding the full-length or preferably extracellular domain (ECD) of HER2 S310F can be expressed to produce HER2 S310F-containing protein. That is, the gene sequence encoding full-length HER2 S310F or HER2 S310F ECD is inserted into a known expression vector, and a suitable host cell is transformed with this vector. The desired human full-length HER2 S310F or HER2 S310F ECD protein is purified from host cell or its culture supernatant by known methods.

[0053] Purified full-length HER2 S310F or HER2 S310F ECD protein can be used as sensitizing antigen or immunogen for use in mammalian immunization.Full-length HER2 S310F or partial peptide of HER2 S310F ECD can also be used as sensitizing antigen.In this case, partial peptide can also be obtained by chemical synthesis from human HER2 amino acid sequence with S310F substitution.Furthermore, they can also be obtained by incorporating a part of HER2 gene with S310F mutation into an expression vector and expressing it.

[0054] Alternatively, for the sensitizing antigen, it is possible to use a fusion protein, which is prepared by fusing the desired partial polypeptide or peptide of full-length HER2 S310F or HER2 S310F ECD protein with a different polypeptide.For example, antibody Fc fragment and peptide tag are preferably used to prepare a fusion protein used as a sensitizing antigen.The vector for expressing such a fusion protein can be constructed by fusing the genes encoding two or more desired polypeptide fragments in frame and inserting the fusion gene into an expression vector as described above.The method for preparing 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).The method for preparing HER2 S310F used as a sensitizing antigen and the immunization method using HER2 S310F are also described later in the examples of this specification.

[0055] In one embodiment, a synthetic polypeptide comprising amino acids 23-646 of human HER2 S310F (i.e., the extracellular domain or ECD) with a rabbit Fc tag at its C-terminus (SEQ ID NO:1 shown below) was transiently expressed using the Expi293 cell line (Thermo Fisher), purified, and used for immunization. TIFF0007625516000003.tif124166

[0056] As shown above, SEQ ID NO:1 comprises amino acid sequence 23-646 of human HER2 S310F ECD (the F amino acid residue corresponding to position 310 of HER2 is in bold and underlined), including a signal sequence (italics, first 19 amino acid residues), with a linker (bold) at its C-terminus followed by a rabbit Fc tag (underlined). The signal sequence is not part of the mature polypeptide chain.

[0057] There is no particular limitation on the mammalian animal to be immunized with the sensitizing antigen. However, it is preferable to select the mammalian animal by considering the compatibility with the parent cell used in cell fusion. Generally, rodents such as mice, rats, and hamsters, rabbits, and monkeys are preferably used.

[0058] The above animals are immunized with the sensitizing antigen by a known method. Commonly performed immunization methods include, for example, intraperitoneal or subcutaneous injection of the sensitizing antigen into the mammal. Specifically, the sensitizing antigen is appropriately diluted with PBS (phosphate buffered saline), physiological saline, or the like. If desired, a conventional adjuvant such as Freund's complete adjuvant is mixed with the antigen to emulsify the mixture. The sensitizing antigen is then administered to the mammal several times at intervals of 4 to 21 days. A suitable carrier may be used in immunization together with the sensitizing antigen. In particular, when a low molecular weight partial peptide is used as the sensitizing antigen, it may be desirable to couple the sensitizing antigen peptide to a carrier protein such as albumin or keyhole limpet hemocyanin for immunization.

[0059] Alternatively, hybridomas producing the desired antibodies can be prepared using DNA immunization as described below. DNA immunization is an immunization method that stimulates the immune system by expressing a sensitizing antigen in an immunized animal as a result of administering a vector DNA constructed to enable expression of an antigen protein-encoding gene in the animal. Compared with conventional immunization methods in which a protein antigen is administered to an immunized animal, DNA immunization is expected to be superior in the following respects: - immune stimulation can be provided while preserving the structure of membrane proteins such as HER2 S310F; and - There is no need to purify the antigen for immunization.

[0060] To prepare the monoclonal antibody of the present invention by DNA immunization, first, DNA expressing HER2 S310F protein is administered to the animal to be immunized. HER2 S310F-encoding DNA can be synthesized by known methods such as PCR. The obtained DNA is inserted into a suitable expression vector, which is then administered to the animal to be immunized. The expression vector preferably used includes, for example, commercially available expression vectors such as pcDNA3.1. The vector can be administered to the organism using conventional methods. For example, DNA immunization is performed by using a gene gun to introduce gold particles coated with the expression vector into the cells of the body of the animal to be immunized. The antibody that recognizes HER2 S310F can also be produced by the method described in WO 2003 / 104453.

[0061] After immunizing mammal as described above, confirm the increase in the titer of HER2 S310F binding antibody in serum.Then, collect immune cells from mammal and then carry out cell fusion.In particular, splenocytes are preferably used as immune cells.

[0062] Mammalian myeloma cells are used as cells to be fused with the above-mentioned immune cells. Myeloma cells preferably contain a selection marker suitable for screening. The selection marker gives the cell a characteristic for its survival (or death) under a specific culture condition. Hypoxanthine-guanine phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency) are known as selection markers. Cells with HGPRT deficiency or TK deficiency have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviated as HAT sensitivity). HAT-sensitive cells cannot synthesize DNA in HAT selection medium, and therefore die. However, when cells are fused with normal cells, they can continue DNA synthesis using the salvage pathway of normal cells, and therefore can grow even in HAT selection medium.

[0063] HGPRT-deficient cells and TK-deficient cells can be selected in media containing 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells incorporate these pyrimidine analogs into their DNA and die. On the other hand, cells deficient in these enzymes cannot incorporate these pyrimidine analogs and can survive in the selection medium. In addition, a selection marker called G418 resistance provided by the neomycin resistance gene confers resistance to 2-deoxystreptamine antibiotics (gentamicin analog). Various types of myeloma cells suitable for cell fusion are known.

[0064] For example, myeloma cells including the following cells can be preferably 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.

[0065] Cell fusion between immune cells and myeloma cells is carried out essentially using known methods, for example those described by Kohler and Milstein et al. (Methods Enzymol. (1981) 73: 3-46). More specifically, cell fusion can be carried out, for example, in the presence of a cell fusion promoter in a conventional culture medium. Fusion promoters include, for example, polyethylene glycol (PEG) and Sendai virus (HVJ). If necessary, auxiliary substances such as dimethyl sulfoxide are also added to improve fusion efficiency.

[0066] The ratio of immune cells to myeloma cells may be determined at one's discretion, and is preferably, for example, 1 myeloma cell for every 1-10 immune cells. The culture medium used for cell fusion includes, for example, media suitable for the growth of myeloma cell lines, such as RPMI1640 medium and MEM medium, and other conventional culture media used for this type of cell culture. In addition, it may be preferable to add serum supplements, such as fetal calf serum (FCS), to the culture medium.

[0067] For cell fusion, a predetermined amount of the immune cells and myeloma cells are thoroughly mixed in the above culture medium. Then, a PEG solution (for example, with an average molecular weight of about 1,000 to 6,000) preheated to about 37°C is added thereto at a concentration of generally 30% to 60% (w / v). This is gently mixed to produce the desired fused cells (hybridomas). Then, the above-mentioned appropriate culture medium is gradually added to the cells, which are repeatedly centrifuged to remove the supernatant. In this way, cell fusion agents and the like that are inconvenient for the growth of hybridomas can be removed.

[0068] The hybridomas thus obtained can be selected by culturing them in a conventional selection medium, for example, HAT medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Cells other than the desired hybridoma (non-fused cells) can be killed by continuing the culture in the above-mentioned HAT medium for a sufficient period of time. Typically, the period is several days to several weeks. Hybridomas producing the desired antibody are then screened and single-cloned by the conventional limiting dilution method.

[0069] The hybridomas thus obtained can be selected using a selection medium based on the selection markers possessed by the myeloma used in the cell fusion. For example, HGPRT-deficient cells or TK-deficient cells can be selected by culturing using HAT medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Specifically, when HAT-sensitive myeloma cells are used in the cell fusion, cells that have successfully fused with normal cells can be selectively grown in HAT medium. Cells other than the desired hybridoma (non-fused cells) can be killed by continuing to culture in the above-mentioned HAT medium for a sufficient period of time. Specifically, the desired hybridoma can be selected by culturing for several days to several weeks in general. Next, hybridomas producing the desired antibody are screened and single-cloned by the conventional limiting dilution method.

[0070] Desired antibody can be preferably selected and single cloned by screening method based on known antigen / antibody reaction.For example, HER2 S310F binding monoclonal antibody can bind to HER2 S310F expressed on cell surface.Such monoclonal antibody can be screened by fluorescence activated cell sorting (FACS).FACS is a system that evaluates the binding of antibody to cell surface by analyzing cells contacted with fluorescent antibody using laser beam and measuring the fluorescence emitted from individual cells.

[0071] In order to screen hybridomas that produce the monoclonal antibody of the present invention by FACS, HER2 S310F expressing cells are first prepared. The cells preferably used for screening are mammalian cells that are forced to express HER2 S310F. As a control, non-transformed mammalian cells can be used as host cells to selectively detect the activity of antibodies that bind to cell surface HER2 S310F. Specifically, hybridomas that produce anti-HER2 S310F monoclonal antibodies can be isolated by selecting hybridomas that produce antibodies that bind to cells that are forced to express HER2 S310F but do not bind to host cells.

[0072] Alternatively, the activity of the antibody that binds to the immobilized HER2 S310F-expressing cells can be evaluated based on the principle of ELISA. For example, the HER2 S310F-expressing cells are immobilized in the well of an ELISA plate. The culture supernatant of the hybridoma is contacted with the immobilized cells in the well, and the antibody that binds to the immobilized cells is detected. If the monoclonal antibody is derived from a mouse, the antibody that binds to the cells can be detected using an anti-mouse immunoglobulin antibody. The hybridoma that produces the desired antibody that has antigen binding ability can be selected by the above screening, and they can be cloned by limiting dilution method or the like.

[0073] The monoclonal antibody-producing hybridomas thus prepared can be passaged in conventional culture media and stored long-term in liquid nitrogen.

[0074] The hybridomas described above can be cultured by conventional methods to prepare the desired monoclonal antibodies from the culture supernatant. Alternatively, the hybridomas can be administered to a suitable mammal to grow and prepare the monoclonal antibodies from the ascites. The former method is suitable for preparing antibodies with high purity.

[0075] Antibodies encoded by antibody genes cloned from antibody-producing cells such as the above-mentioned hybridomas can also be preferably used. The cloned antibody gene is inserted into an appropriate vector, which is then introduced into a host to express the antibody encoded by the gene. Methods for isolating antibody genes, inserting the genes 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.

[0076] Preferably, the present invention provides a nucleic acid encoding the antigen-binding molecule or multispecific antigen-binding molecule of the present invention. The present invention also provides a vector into which a nucleic acid encoding an antigen-binding molecule or multispecific antigen-binding molecule is introduced, i.e., a vector containing a nucleic acid. Furthermore, the present invention provides a cell containing a nucleic acid or a vector. The present invention also provides a method for producing an antigen-binding molecule or a multispecific antigen-binding molecule by culturing a cell. Preferably, the present invention provides a method for preparing an antigen-binding molecule or a multispecific antigen-binding molecule, the method comprising a step of expressing a nucleic acid encoding the antigen-binding molecule or the multispecific antigen-binding molecule in a cell. The present invention further provides an antigen-binding molecule or a multispecific antigen-binding molecule produced by the method.

[0077] For example, cDNA encoding the variable region (V region) of anti-HER2 S310F antibody is prepared from hybridoma cells expressing anti-HER2 S310F antibody. For this purpose, total RNA is first extracted from the hybridoma. Methods used to extract mRNA from cells include, for example: - guanidine ultracentrifugation (Biochemistry (1979) 18(24), 5294-5299), and - AGPC method (Anal. Biochem. (1987) 162(1), 156-159).

[0078] The extracted mRNA can be purified using an mRNA Purification Kit (GE Healthcare Bioscience) or the like. Alternatively, kits for directly extracting total mRNA from cells, such as QuickPrep mRNA Purification Kit (GE Healthcare Bioscience), are also commercially available. mRNA can be prepared from hybridomas using such kits. cDNA encoding an antibody V region can be synthesized from the prepared mRNA using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Co.) or the like. In addition, 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) can be used appropriately to synthesize and amplify cDNA. In such a cDNA synthesis process, suitable restriction enzyme sites as described below may be introduced at both ends of the cDNA.

[0079] The cDNA fragment of interest is purified from the resulting PCR product, and then ligated to vector DNA. The recombinant vector thus constructed is introduced into E. coli or the like. After colony selection, the desired recombinant vector can be prepared from the colony-forming E. coli. Then, whether the recombinant vector has the cDNA nucleotide sequence of interest is tested by known methods such as dideoxynucleotide chain termination method.

[0080] The 5'-RACE method, which uses primers to amplify variable region genes, is conveniently used to isolate genes encoding variable regions. First, a 5'-RACE cDNA library is constructed by cDNA synthesis using RNA extracted from hybridoma cells as a template. Commercially available kits, such as the SMART RACE cDNA amplification kit, are suitably used to synthesize the 5'-RACE cDNA library.

[0081] The antibody gene is amplified by PCR using the prepared 5'-RACE cDNA library as a template. The primers for amplifying the mouse antibody gene can be designed based on the known antibody gene sequence. The nucleotide sequence of the primer varies depending on the immunoglobulin subclass. Therefore, it is preferable to determine the subclass in advance using a commercially available kit such as the Iso Strip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics).

[0082] Specifically, the mouse IgG coding gene is isolated using primers that allow the amplification of genes encoding, for example, γ1, γ2a, γ2b, and γ3 heavy chains, and κ and λ light chains. In general, a primer that anneals to a constant region site near the variable region is used as the 3' primer to amplify the IgG variable region gene, while a primer provided in the 5' RACE cDNA library construction kit is used as the 5' primer.

[0083] The PCR product thus amplified is used to reconstruct an immunoglobulin composed of a combination of heavy and light chains. The desired antibody can be selected using the HER2 S310F binding activity of the reconstructed immunoglobulin as an index. For example, when the purpose is to isolate an antibody against HER2 S310F, it is more preferable that the antibody binds specifically to HER2 S310F. The HER2 S310F binding activity can be screened, for example, by the following steps: (1) contacting a HER2 S310F-expressing cell with an antibody comprising a V region encoded by a cDNA isolated from a hybridoma; (2) detecting binding of the antibody to HER2 S310F-expressing cells; (3) selecting an antibody that specifically binds to HER2 S310F-expressing cells; and preferably (4) A step of selecting an antibody that exhibits stronger binding to HER2 S310F expressing cells than to wild-type HER2, or an antibody that binds to HER2 S310F expressing cells but does not bind to wild-type HER2.

[0084] Methods for detecting the binding of an antibody to HER2 S310F expressing cells are known. Specifically, the binding of an antibody to HER2 S310F expressing cells can be detected by the above-mentioned techniques such as FACS. A sample of immobilized HER2 S310F expressing cells is appropriately used to evaluate the binding activity of the antibody.

[0085] A preferred antibody screening method using binding activity as an index also includes a panning method using a phage vector. When an antibody gene is isolated from a heavy and light chain subclass library derived from a polyclonal antibody-expressing cell population, a screening method using a phage vector is advantageous. The genes encoding the variable regions of the heavy and light chains can be linked by a suitable linker sequence to form a single chain Fv (scFv). By inserting a gene encoding an scFv into a phage vector, a phage that displays an scFv on its surface can be produced. The phage is contacted with an antigen of interest. Then, DNA encoding an scFv having a binding activity of interest can be isolated by collecting the phage that binds to the antigen. This process can be repeated as necessary to enrich for scFv having a desired binding activity.

[0086] After isolating the cDNA encoding the V region of the anti-HER2 S310F antibody of interest, the cDNA is digested with a restriction enzyme that recognizes the restriction sites introduced at both ends of the cDNA. A preferred restriction enzyme recognizes and cuts a nucleotide sequence that occurs at a low frequency in the nucleotide sequence of the antibody gene. Furthermore, in order to insert a single copy of the digested fragment in the correct orientation, a restriction site for the enzyme that generates a sticky end is preferably introduced into the vector. The cDNA encoding the V region of the anti-HER2 S310F antibody is digested as described above and inserted into an appropriate expression vector to construct an antibody expression vector. In this case, if the gene encoding the antibody constant region (C region) and the gene encoding the above V region are fused in frame, a chimeric antibody is obtained. In this specification, the term "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 heterochimeric antibodies, human / human allochimeric antibodies are included in the chimeric antibodies of the present invention. A chimeric antibody expression vector can be constructed by inserting the above V region gene into an expression vector that already has a constant region. Specifically, for example, the recognition sequence of the restriction enzyme that excises the above-mentioned V region gene can be appropriately placed at the 5' end of an expression vector carrying DNA encoding the desired antibody constant region (C region). A chimeric antibody expression vector is constructed by fusing in frame two genes digested with the same combination of restriction enzymes.

[0087] To prepare an anti-HER2 S310F monoclonal antibody, the antibody gene is inserted into an expression vector so that the gene is expressed under the control of an expression control region. The expression control region for antibody expression includes, for example, an enhancer and a promoter. In addition, a suitable signal sequence may be added to the amino terminus so that the expressed antibody is secreted outside the cell. In the following example, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 175) is used as the signal sequence. Meanwhile, 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 outside the cell as a mature polypeptide. Then, a suitable host cell is transformed with the expression vector to obtain a recombinant cell expressing the anti-HER2 S310F antibody-encoding DNA.

[0088] DNAs encoding the antibody heavy (H) and light (L) chains are separately inserted into different expression vectors to express the antibody genes. An antibody molecule having H and L chains can be expressed by cotransfecting vectors into which the H and L chain genes are inserted, respectively, into the same host cell. Alternatively, a host cell can be transformed with a single expression vector into which DNAs encoding the H and L chains are inserted (see WO 94 / 11523).

[0089] There are various known host cell / expression vector combinations for antibody preparation by introducing the isolated antibody genes into a suitable host. All of these expression systems are applicable to the isolation of the domains containing the antibody variable regions of the present invention. Suitable eukaryotic cells used as host cells include animal cells, plant cells, and fungal cells. Specifically, animal cells include, for example, the following cells: (1) Mammalian cells: CHO, COS, myeloma, baby hamster kidney (BHK), HeLa, Vero, etc.; (2) Amphibian cells, such as Xenopus oocytes; and (3) Insect cells: sf9, sf21, Tn5, etc.

[0090] In addition, an antibody gene expression system using plant cells derived from the genus Nicotiana, such as Nicotiana tabacum, is known. Callus cultured cells can be appropriately used to transform plant cells.

[0091] In addition, the following cells can be used as fungal cells: Yeasts: Saccharomyces genus, such as Saccharomyces cerevisiae, and Pichia genus, such as Pichia pastoris; and Filamentous fungi: Aspergillus genus, such as Aspergillus niger.

[0092] Furthermore, antibody gene expression systems using prokaryotic cells are also known. For example, when bacterial cells are used, Escherichia coli cells, Bacillus subtilis cells, etc. can be suitably used in the present invention. An expression vector carrying the antibody gene of interest 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.

[0093] In addition to the above host cells, transgenic animals can also be used to produce recombinant antibodies. That is, antibodies can be obtained from animals into which a gene encoding an antibody of interest has been introduced. For example, an antibody gene can be constructed as a fusion gene by inserting it in frame into a gene encoding a protein specifically produced in milk. Goat β-casein, for example, can be used as a protein secreted into milk. A DNA fragment containing a fusion gene into which an antibody gene has been inserted is injected into a goat embryo, and then the embryo is introduced into a female goat. The desired antibody can be obtained as a protein fused with a milk protein from the milk produced by the transgenic goat (or its progeny) born from the embryo recipient goat. In addition, hormones can be administered to the transgenic goat as necessary to increase the volume of milk containing the desired antibody produced by the transgenic goat (Ebert, KM et al., Bio / Technology (1994) 12 (7), 699-702).

[0094] Methods for Producing Humanized Antibodies When the antigen-binding molecule described herein is administered to humans, a domain derived from a genetically engineered antibody or the like that has been artificially modified to reduce heterologous antigenicity to humans can be appropriately used as a domain of the antigen-binding molecule that contains an antibody variable region. Such genetically engineered antibodies include, for example, humanized antibodies. These modified antibodies are appropriately produced by known methods. Furthermore, the binding specificity of a particular antibody can generally be introduced into another antibody by CDR grafting.

[0095] Specifically, humanized antibodies prepared by grafting the CDR of a non-human animal antibody such as a mouse antibody onto a human antibody are known. General genetic engineering techniques for obtaining humanized antibodies are also known. Specifically, for example, overlap extension PCR is known as a method for grafting mouse antibody CDR onto human FR. In overlap extension PCR, a nucleotide sequence encoding the mouse antibody CDR to be grafted is added to a primer for synthesizing a human antibody FR. A primer is prepared for each of the four FRs. When grafting mouse CDR onto human FR, it is usually considered to be advantageous to select a human FR having high identity to the mouse FR in order to maintain CDR function. That is, it is usually preferable to use a human FR that includes an amino acid sequence having high identity to the amino acid sequence of the FR adjacent to the mouse CDR to be grafted.

[0096] The nucleotide sequences to be ligated are designed to be connected in frame with each other. Human FR is synthesized individually using each primer. As a result, a product is obtained in which mouse CDR-encoding DNA is added to each FR-encoding DNA. The nucleotide sequences encoding mouse CDR of each product are designed to overlap with each other. Then, a complementary strand synthesis reaction is carried out to anneal the overlapping CDR regions of the products synthesized using human antibody gene as template. Human FR is ligated through mouse CDR sequence by this reaction.

[0097] The full-length V region gene, in which three CDRs and four FRs are finally ligated, is amplified using a primer that is annealed to its 5' or 3' end and has a suitable restriction enzyme recognition sequence added thereto.The DNA obtained as described above and the DNA encoding the human antibody C region are inserted into an expression vector so that they are ligated in frame, thereby making an expression vector for humanized antibody.After transfecting the recombinant vector into a host to establish recombinant cells, the recombinant cells are cultured, and the DNA encoding humanized antibody is expressed in cell culture to make humanized antibody (see European Patent Publication No. EP 239400 and International Patent Publication No. WO 1996 / 002576).

[0098] By qualitatively or quantitatively measuring and evaluating the antigen-binding activity of the humanized antibody prepared as described above, a human antibody FR that allows the CDR to form a favorable antigen-binding site when ligated through the CDR can be suitably selected. The amino acid residues in the FR may be substituted as necessary so that the CDR of the reshaped human antibody forms a suitable antigen-binding site. For example, amino acid sequence mutations can be introduced into the FR by applying the PCR method used to graft mouse CDRs into human FRs. More specifically, partial nucleotide sequence mutations can be introduced into a primer that anneals to the FR. The nucleotide sequence mutations are introduced into the synthesized FR by using such a primer. By measuring and evaluating the activity of the amino acid substitution mutant antibody that binds to the antigen using the above-mentioned method, a mutant FR sequence having desired characteristics can be selected (Sato, K. et al., Cancer Res. (1993) 53: 851-856).

[0099] Methods for Producing Human Antibodies Alternatively, the desired human antibodies can be obtained by immunizing transgenic animals carrying a full repertoire of human antibody genes (see WO 1993 / 012227; WO 1992 / 003918; WO 1994 / 002602; WO 1994 / 025585; WO 1996 / 034096; WO 1996 / 033735) by DNA immunization.

[0100] Furthermore, techniques for preparing human antibodies by panning using a human antibody library 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. Phages expressing scFvs that bind to an antigen can be selected. The DNA sequence encoding the human antibody V region that binds to the antigen can be determined by analyzing the genes of the selected phage. The DNA sequence of the scFv that binds to the antigen is determined. An expression vector is prepared by fusing the V region sequence in frame with the C region sequence of a desired human antibody and inserting it into a suitable expression vector. The expression vector is introduced into a suitable cell for expression, such as the above-mentioned cells. Human antibodies can be produced by expressing human antibody-encoding genes in cells. These methods are known (see WO 1992 / 001047; WO 1992 / 020791; WO 1993 / 006213; WO 1993 / 011236; WO 1993 / 019172; WO 1995 / 001438; WO 1995 / 015388).

[0101] vector As used herein, the term "vector" refers to a nucleic acid molecule that can propagate another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which it is introduced. Certain vectors can effect expression of a nucleic acid to which it is operatively linked. Such vectors are referred to herein as "expression vectors."

[0102] host cell The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0103] Epitope "Epitope" means an antigenic determinant in an antigen, and refers to an antigenic site to which the antigen-binding domain of an antigen-binding molecule or antibody disclosed herein binds. Thus, for example, an epitope can be defined according to its structure. Alternatively, an epitope may be defined according to the antigen-binding activity of an antigen-binding molecule or antibody that recognizes the epitope. When an antigen is a peptide or polypeptide, an epitope can be specified by the amino acid residues that form the epitope. Alternatively, when an epitope is a glycan, an epitope can be specified by its specific glycan structure.

[0104] A linear epitope is one whose primary amino acid sequence contains the epitope that is recognized. Such linear epitopes typically contain at least 3, and most commonly at least 5, e.g., about 8-10 or 6-20 amino acids in their specific sequence.

[0105] In contrast to linear epitopes, "conformational epitopes" are epitopes in which the primary amino acid sequence that contains the epitope is not the only determinant of the epitope that is recognized (e.g., the primary amino acid sequence of a conformational epitope is not necessarily recognized by the antibody that defines the epitope). Conformational epitopes may contain a greater number of amino acids compared to linear epitopes. The antigen-binding domain that recognizes conformational epitopes recognizes the three-dimensional structure of a 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 conformational epitope are aligned, allowing the epitope to be recognized by the antigen-binding domain. Methods for determining the conformation of an epitope include, but are not limited to, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance, site-directed spin labeling, and electron paramagnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0106] An example of a method for evaluating epitope binding by a test antigen-binding molecule or antibody containing an anti-HER2 S310F antigen-binding domain is described below. According to the following examples, a method for evaluating epitope binding by a test antigen-binding molecule or antibody containing an antigen-binding domain against an antigen other than HER2 S310F can also be appropriately performed.

[0107] For example, whether a test antigen-binding molecule or antibody containing an anti-HER2 S310F antigen-binding domain recognizes a linear epitope in a HER2 S310F molecule can be confirmed, for example, as described below. A linear peptide containing an amino acid sequence forming the extracellular domain of HER2 S310F is synthesized for the above purpose. The peptide can be chemically synthesized, or can be obtained by genetic engineering techniques using a region in HER2 S310F cDNA that codes for an amino acid sequence corresponding to the extracellular domain. Then, a test antigen-binding molecule or antibody containing an anti-HER2 S310F antigen-binding domain is evaluated for its binding activity to a linear peptide containing an amino acid sequence forming the extracellular domain. For example, the binding activity of a polypeptide complex to a peptide can be evaluated by ELISA using an immobilized linear peptide as an antigen. Alternatively, the binding activity to a linear peptide can be evaluated based on the level at which the linear peptide inhibits the binding of an antigen-binding molecule or antibody to HER2 S310F-expressing cells. These tests make it possible to demonstrate the binding activity of antigen-binding molecules or antibodies to linear peptides.

[0108] Whether a test antigen-binding molecule or antibody containing an anti-HER2 S310F antigen-binding domain recognizes a conformational epitope can be evaluated as follows. HER2 S310F-expressing cells are prepared for the above purpose. A test antigen-binding molecule or antibody containing an anti-HER2 S310F antigen-binding domain can be determined to recognize a conformational epitope when it strongly binds to HER2 S310F-expressing cells upon contact, but does not substantially bind to an immobilized linear peptide containing an amino acid sequence forming the extracellular domain of HER2 S310F. In this specification, "does not substantially bind" 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 cells expressing HER2 S310F.

[0109] Methods for assaying the binding activity of a test antigen-binding molecule or antibody containing an anti-HER2 S310F antigen-binding domain to HER2 S310F-expressing cells include, for example, the methods described in Antibodies: A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). Specifically, evaluation can be performed based on the principle of ELISA or fluorescence-activated cell sorting (FACS) using HER2 S310F-expressing cells as antigens.

[0110] In the ELISA format, the binding activity of the test antigen-binding molecule or antibody containing the anti-HER2 S310F antigen-binding domain to HER2 S310F-expressing cells can be quantitatively evaluated by comparing the level of the signal generated by the enzyme reaction. Specifically, the test polypeptide complex is added to an ELISA plate on which HER2 S310F-expressing cells are immobilized. The test antigen-binding molecule or antibody bound to the cells is then detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule or antibody. Alternatively, when using FACS, a dilution series of the test antigen-binding molecule or antibody can be prepared, and the antibody binding titer to HER2 S310F-expressing cells can be determined to compare the binding activity of the test antigen-binding molecule or antibody to HER2 S310F-expressing cells.

[0111] The binding of a test antigen-binding molecule or antibody to an antigen expressed on the surface of cells suspended in a buffer solution or the like can be detected using a flow cytometer. Known flow cytometers include, for example, the following devices: FACSCanto(TM) II FACSAria(trademark) FACSArray™ FACSVantage(TM) SE FACSCalibur™ (all trade names of BD Biosciences) EPICS ALTRA HyperSort Cytomics FC 500 EPICS XL-MCL ADC EPICS XL ADC Cell Lab Quanta / Cell Lab Quanta SC (all trade names of Beckman Coulter)

[0112] A preferred method for assaying the binding activity of a test antigen-binding molecule or antibody containing an anti-HER2 S310F antigen-binding domain to an antigen includes, for example, the following method. First, HER2 S310F-expressing cells are reacted with a 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 appropriately diluted with a suitable buffer to prepare the antigen-binding molecule or antibody at a desired concentration. For example, the antigen-binding molecule or antibody can be used at a concentration within the range of 10 micrograms (μg) / ml to 10 ng / ml. The fluorescence intensity and cell number are then determined using a FACSCalibur (BD). The fluorescence intensity obtained by analysis using CELL QUEST Software (BD), i.e., the geometric mean value, reflects the amount of antibody bound to the cells. That is, the binding activity of the test antigen-binding molecule or antibody, represented by the amount of bound test antigen-binding molecule or antibody, can be determined by measuring the geometric mean (Geo-mean) value.

[0113] Whether the test antigen-binding molecule or antibody that contains anti-HER2 S310F antigen-binding domain shares a common epitope with another antigen-binding molecule or antibody can be evaluated based on the competition between two antigen-binding molecules or antibodies for the same epitope.The competition between antigen-binding molecules or antibodies can be detected by cross-blocking assay, etc.For example, competitive ELISA assay is a preferred cross-blocking assay.

[0114] Specifically, in cross-blocking assay, HER2 S310F protein immobilized on the well of microtiter plate is pre-incubated in the presence or absence of candidate competitive antigen-binding molecule or antibody, and then test antigen-binding molecule or antibody is added thereto.The amount of test antigen-binding molecule or antibody bound to HER2 S310F protein in well is indirectly correlated with the binding ability of candidate competitive antigen-binding molecule or antibody that competes for binding to the same epitope.That is, the higher the affinity of competitive antigen-binding molecule or antibody to the same epitope, the lower the binding activity of test antigen-binding molecule or antibody to the well coated with HER2 S310F protein.

[0115] The amount of the test antigen-binding molecule or antibody bound to the well via HER2 S310F protein can be easily determined by labeling the antigen-binding molecule or antibody in advance. For example, biotin-labeled antigen-binding molecule or antibody is measured using avidin / peroxidase conjugate and a suitable substrate. In particular, cross-blocking assay using enzyme label such as peroxidase is called "competitive ELISA assay". Antigen-binding molecule or antibody can also be labeled with other labeling substances that allow detection or measurement. Specifically, radiolabel, fluorescent label, etc. are known.

[0116] When a candidate competing antigen-binding molecule or antibody is able to block binding by a test antigen-binding molecule or antibody containing an anti-HER2 S310F 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 competing antigen-binding molecule or antibody, the test antigen-binding molecule or antibody is determined to substantially bind to the same epitope bound by the competing antigen-binding molecule or antibody, or to compete for binding to the same epitope.

[0117] When the structure of the epitope bound by a test antigen-binding molecule or antibody containing an anti-HER2 S310F antigen-binding domain 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 the two antigen-binding molecules or antibodies against a peptide prepared by introducing amino acid mutations into the peptide that forms the epitope.

[0118] To measure the above-mentioned binding activity, for example, the binding activity of the test antigen-binding molecule or antibody and the control antigen-binding molecule or antibody to the linear peptide into which the mutation has been introduced is compared in the above-mentioned ELISA format.In addition to the ELISA method, the binding activity to the mutant peptide bound to the column can 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 eluted antigen-binding molecule or antibody in the eluate.For example, a method for adsorbing the mutant peptide to the column in the form of a GST fusion peptide is known.

[0119] Alternatively, if the identified epitope is a conformational 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, HER2 S310F-expressing cells and cells expressing HER2 S310F with a mutation introduced into the epitope are prepared. The test antigen-binding molecule or antibody and the control antigen-binding molecule or antibody are added to a cell suspension prepared by suspending these cells in an appropriate buffer such as PBS. The cell suspension is then appropriately washed with a buffer, and an FITC-labeled antibody that recognizes the test antigen-binding molecule or antibody and the control antigen-binding molecule or antibody is added thereto. The fluorescence intensity and number of cells stained with the labeled antibody are determined using a FACSCalibur (BD). The test antigen-binding molecule or antibody and the control antigen-binding molecule or antibody are appropriately diluted with a suitable buffer and used at the desired concentration. For example, they may be used at a concentration within the range of 10 μg / ml to 10 ng / ml. The fluorescence intensity determined by analysis using CELL QUEST Software (BD), i.e., the geometric mean value, reflects the amount of labeled antibody bound to the cells. That is, the binding activity of the test antigen-binding molecule or antibody and the control antigen-binding molecule or antibody, which is represented by the amount of bound labeled antibody, can be determined by measuring the geometric mean value.

[0120] In the above method, whether an antigen-binding molecule or antibody "does not substantially bind to cells expressing mutant HER2 S310F" 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 HER2 S310F are stained with a labeled antibody. Then, the fluorescence intensity of the cells is determined. When 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 comparison value (ΔGeo-Mean) can be calculated according to the following formula to determine the ratio of the increase in fluorescence intensity as a result of binding by the antigen-binding molecule or antibody. ΔGeo-Mean = Geo-Mean (in the presence of antigen-binding molecules or antibodies) / Geo-Mean (in the absence of antigen-binding molecules or antibodies)

[0121] The geometric mean comparison value determined by the above analysis (ΔGeo-Mean value for mutant HER2 S310F molecules) reflects the amount of the test antigen-binding molecule or antibody bound to cells expressing mutant HER2 S310F, and is compared with the ΔGeo-Mean comparison value reflecting the amount of the test antigen-binding molecule or antibody bound to HER2 S310F-expressing cells.In this case, the concentrations of the test antigen-binding molecule or antibody used to determine the ΔGeo-Mean comparison value for HER2 S310F-expressing cells and cells expressing mutant HER2 S310F are particularly preferably adjusted to be equal or substantially equal.An antigen-binding molecule or antibody that has been confirmed to recognize an epitope in HER2 S310F is used as a control antigen-binding molecule or antibody.

[0122] If the ΔGeo-Mean comparison value of the test antigen-binding molecule or antibody for cells expressing mutant HER2 S310F 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 for cells expressing HER2 S310F, the test antigen-binding molecule or antibody "does not substantially bind to cells expressing mutant HER2 S310F". The formula for determining Geo-Mean (geometric mean) values ​​is described in CELL QUEST Software User's Guide (BD biosciences). If the comparison shows that the comparison values ​​are substantially equivalent, the epitopes for the test antigen-binding molecule or antibody and the control antigen-binding molecule or antibody can be determined to be the same.

[0123] 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 the partners.In addition, "specific" is also used when an antigen-binding domain is specific to a particular epitope among multiple epitopes contained in an antigen.When the epitope that an antigen-binding domain binds to is contained in multiple different antigens, the antigen-binding molecule that contains the antigen-binding domain can bind to various antigens that have that epitope. In the context of the present specification, preferably, the antibodies of the present invention have low or no cross-reactivity to wild-type HER2 and are "specific" for the HER2 S310F mutant. The high specificity of the antibodies of the present invention is characterized by a KD ratio of the KD value for binding to HER2 S310F to the KD value for binding to wild-type HER2 (i.e., KD HER2 S310F / KD 野生型HER2) can be demonstrated. Wild-type HER2 is expressed in normal tissues and is involved in various cellular processes such as proliferation, differentiation, cell migration, and cell survival. Therefore, a potential concern is that non-specific HER2 antibodies that target wild-type HER2 (instead of or in addition to HER2 S310F) may cause severe cytotoxicity and damage to healthy tissues or organs. Specificity to the HER2 S310F mutant is particularly desirable in that the risk of adverse or side effects may be significantly reduced, and higher safety of therapeutic agents containing such "specific" antibodies can be expected.

[0124] Monospecific antigen-binding molecules 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 convenient example of a monospecific antigen-binding molecule is an antigen-binding molecule that comprises a single type of antigen-binding domain. A monospecific antigen-binding molecule can comprise a single antigen-binding domain or multiple antigen-binding domains of the same type. A convenient example of a monospecific antigen-binding molecule is a monospecific antibody. When a monospecific antigen-binding molecule is a monospecific antibody in the form of IgG, the monospecific antibody comprises two antibody variable fragments that have the same antigen-binding specificity. In the present context, preferably, a monospecific antigen-binding molecule or a monospecific antibody of the invention has a single antigen-binding domain or two or more antigen-binding domains that specifically bind to the human HER2 S310F mutant but not (specifically) to wild-type HER2.

[0125] antibody fragment "Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, 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.

[0126] The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain containing an Fc region as defined herein.

[0127] Fragment variable (Fv) As used herein, the term "variable fragment (Fv)" refers to the minimum unit of an antigen-binding domain derived from an antibody, which is composed of a pair of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In 1988, Skerra and Pluckthun found that homogeneous and active antibodies can be prepared from the periplasmic fraction of E. coli by inserting an antibody gene downstream of a bacterial signal sequence and inducing expression of the gene in E. coli (Science (1988) 240(4855), 1038-1041). In the Fv prepared from the periplasmic fraction, VH associates with VL in a manner for binding to an antigen.

[0128] scFv, single chain antibodies, and sc(Fv) 2 As used herein, the terms "scFv", "single-chain antibody" and "sc(Fv)2" all refer to a single polypeptide chain antibody fragment that contains variable regions derived from heavy and light chains but does not contain a constant region. Generally, single-chain antibodies also contain a polypeptide linker between the VH and VL domains, which allows the formation of a desired structure that would allow antigen binding. Single-chain 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 Patent Publication WO 1988 / 001649; U.S. Patent Nos. 4,946,778 and 5,260,203. In certain embodiments, single-chain antibodies can be bispecific and / or humanized.

[0129] An scFv is an antigen-binding domain in which the VH and VL forming the Fv are linked together by a peptide linker (Proc. Natl. Acad. Sci. USA (1988) 85(16), 5879-5883). The VH and VL can be held in close proximity by the peptide linker. sc(Fv)2 is a single-chain antibody in which four variable regions, two VL and two VH, are linked by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231 (1-2), 177-189). The two VH and two VL may be derived from different monoclonal antibodies. Such sc(Fv)2 preferably includes bispecific sc(Fv)2 that recognizes two epitopes present in a single antigen, as disclosed in, for example, 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 scFvs by a linker such as a peptide linker.

[0130] As used herein, the form of the antigen-binding domain forming sc(Fv)2 includes an antibody in which two VH units and two VL units are arranged in the order of VH, VL, VH, and 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 the two VL units is not limited to the above form and may be arranged in any order. Examples of forms 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]

[0131] The molecular form of sc(Fv)2 is also described in detail in WO 2006 / 132352. Following these descriptions, a person skilled in the art can appropriately prepare a desired sc(Fv)2 to produce the peptide complex disclosed herein.

[0132] 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 drug. Alternatively, a glycosylation sequence is preferably inserted into the antigen-binding molecule or antibody so that the glycosylation produces the desired effect.

[0133] Linkers used to link the variable regions of antibodies include any peptide linker that can be introduced by genetic engineering, synthetic linkers, and linkers disclosed in, for example, Protein Engineering, 9 (3), 299-305, 1996. However, in the present invention, peptide linkers are preferred. The length of the peptide linker is not particularly limited, and can be appropriately selected by those skilled in the art according to the purpose. The length is preferably 5 amino acids or more (not particularly limited, the upper limit is generally 30 amino acids or less, preferably 20 amino acids or less), and is particularly preferably 15 amino acids. When sc(Fv)2 contains three peptide linkers, their lengths may all be the same or different.

[0134] For example, such peptide linkers include: Ser Gly Ser Gly Gly Ser Ser Gly Gly Gly Gly Gly Ser (SEQ ID NO: 167) Ser Gly Gly Gly (SEQ ID NO: 168) Gly Gly Gly Gly Ser (SEQ ID NO: 169) Ser Gly Gly Gly Gly (SEQ ID NO: 170) Gly Gly Gly Gly Gly Ser (SEQ ID NO: 171) Ser Gly Gly Gly Gly Gly (SEQ ID NO: 172) Gly Gly Gly Gly Gly Gly Ser (SEQ ID NO: 173) Ser Gly Gly Gly Gly Gly Gly (SEQ ID NO: 174) (Gly Gly Gly Gly Ser (SEQ ID NO: 169)) (Ser Gly Gly Gly Gly (SEQ ID NO: 170)) where n is an integer equal to or greater than 1. The length or sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0135] Synthetic linkers (chemical cross-linkers) are commonly used to cross-link peptides, examples include: N-hydroxysuccinimide (NHS), Disuccinimidyl suberate (DSS), Bis(sulfosuccinimidyl) suberate (BS3), Dithiobis(succinimidyl propionate) (DSP), Dithiobis(sulfosuccinimidyl propionate) (DTSSP), Ethylene glycol bis(succinimidyl succinate) (EGS), Ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), Disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and Bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES) These crosslinkers are commercially available.

[0136] Generally, three linkers are required to link the four antibody variable regions together. The linkers used may be of the same type or of different types.

[0137] Fab, F(ab') 2 , and Fab' A "Fab" consists of a single light chain and the CH1 domain and variable region from a single heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0138] "F(ab')2" or "Fab" refers to an antibody fragment produced by digesting an immunoglobulin (monoclonal antibody) near the disulfide bond present between the hinge regions of each of the two H chains, which is produced by treating the immunoglobulin (monoclonal antibody) with proteases such as pepsin and papain. For example, papain cleaves IgG upstream of the disulfide bond present between the hinge regions of each of the two H chains to produce two homologous antibody fragments, in which the L chain containing VL (L chain variable region) and CL (L chain constant region) is linked to the H chain fragment containing VH (H chain variable region) and CHγ1 (γ1 region in the H chain constant region) via a disulfide bond at their C-terminal regions. Each of these two homologous antibody fragments is called Fab'.

[0139] "F(ab')2" consists of two light chains and two heavy chains containing constant regions of CH1 domain and part of CH2 domain so that disulfide bonds are formed between the two heavy chains. The F(ab')2 disclosed herein can be preferably prepared as follows: A whole monoclonal antibody containing a desired antigen-binding domain is partially digested with a protease such as pepsin; and the Fc fragment is removed by adsorption to a protein A column. The protease is not particularly limited as long as it can cleave the whole antibody in a selective manner to generate F(ab')2 under appropriately set enzyme reaction conditions such as pH. Such proteases include, for example, pepsin and ficin.

[0140] Fc area The term "Fc region" or "Fc domain" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0141] Fc receptors 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 a native human FcR. In some embodiments, the FcR binds to an IgG antibody (gamma receptor), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of those receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, e.g., 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 identified in the future, are encompassed by the term "FcR" herein.

[0142] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for controlling maternal IgG transfer to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and immunoglobulin homeostasis. Methods for measuring binding to FcRn are 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); WO 2004 / 92219 (Hinton et al.)).

[0143] The in vivo binding to human FcRn and the plasma half-life of human FcRn high affinity binding polypeptide can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with polypeptides having variant Fc regions.WO 2000 / 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).

[0144] Fcγ receptors Fcγ receptor refers to a receptor capable of binding to the Fc domain of monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies and includes all members of a family of proteins substantially encoded by Fcγ receptor genes. In humans, this family includes FcγRI (CD64), which includes isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2); and all unspecified human Fcγ receptors, Fcγ receptor isoforms, and allotypes thereof. However, Fcγ receptors are not limited to these examples. Without being limited thereto, Fcγ receptors include those derived from humans, mice, rats, rabbits, and monkeys. Fcγ receptors may be derived 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 unspecified mouse Fcγ receptors, Fcγ receptor isoforms, and allotypes thereof. 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 set forth in SEQ ID NOs: 157 (NM_000566.3) and 152 (NP_000557.1), respectively; the polynucleotide and amino acid sequences of FcγRIIA are set forth in SEQ ID NOs: 158 (BC020823.1) and 153 (AAH20823.1), respectively; the polynucleotide and amino acid sequences of FcγRIIB are set forth in SEQ ID NOs: 159 (BC146678.1), respectively. ) and 154 (AAI46679.1); the polynucleotide and amino acid sequences of FcγRIIIA are shown in SEQ ID NOs: 160 (BC033678.1) and 155 (AAH33678.1); and the polynucleotide and amino acid sequences of FcγRIIIB are shown in SEQ ID NOs: 161 (BC128562.1) and 156 (AAI28563.1), respectively (RefSeq accession numbers are shown in each bracket). Whether an Fcγ receptor has binding activity to the Fc domain of a monoclonal IgG1, IgG2, IgG3, or IgG4 antibody can be assessed by ALPHA screens (Amplified Luminescent Proximity Homogeneous Assay), surface plasmon resonance (SPR)-based BIACORE methods, and others, in addition to the FACS and ELISA formats described above (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0145] On the other hand, "Fc ligand" or "effector ligand" refers to a molecule, and preferably a polypeptide, that binds to an antibody Fc domain to form an Fc / Fc ligand complex. The molecule may be from any organism. Binding of the 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 lectin, mannose receptor, Staphylococcus protein A, Staphylococcus protein B, and viral Fcγ receptors. Fc ligands also include Fc receptor homologs (FcRH), a family of Fc receptors that are homologous to Fcγ receptors (Davis et al., (2002) Immunological Reviews 190, 123-136). Fc ligands also include unidentified molecules that bind to Fc.

[0146] Fcγ receptor binding activity The reduction in binding activity of the Fc domain to any of the Fcγ receptors FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and / or FcγRIIIB can be assessed by the use of the FACS and ELISA formats described above, as well as ALPHA screens (amplified luminescent proximity homogeneous assays) and surface plasmon resonance (SPR)-based BIACORE methods (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0147] The ALPHA screen is carried out by ALPHA technology using two types of beads: donor beads and acceptor beads, which are based on the following principle: 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 when the two beads are located in close proximity. When excited by a laser beam, a photosensitizer in the donor bead converts oxygen around the bead into excited singlet oxygen. When the singlet oxygen diffuses around the donor bead and reaches the nearby acceptor bead, it induces a chemiluminescence reaction in the acceptor bead. This reaction ultimately results in the emission of 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 no chemiluminescence reaction occurs.

[0148] For example, a biotin-labeled antigen-binding molecule or antibody is immobilized on donor beads, and an Fcγ receptor tagged with glutathione S-transferase (GST) is immobilized on acceptor beads. In the absence of a competing antigen-binding molecule or antibody containing a mutant Fc domain, the Fcγ receptor interacts with an antigen-binding molecule or antibody containing a wild-type Fc domain, resulting in a signal at 520 to 620 nm. An antigen-binding molecule or antibody with an untagged mutant Fc domain competes with an antigen-binding molecule or antibody containing a wild-type Fc domain for interaction with the Fcγ receptor. Relative binding affinity can be determined by quantifying the decrease in fluorescence as a result of the competition. Methods for biotinylating an antigen-binding molecule or antibody such as an antibody using sulfo-NHS-biotin or the like are known. Suitable methods for adding a GST tag to an Fcγ receptor include those that include fusing a polypeptide encoding the Fcγ receptor and GST in frame, expressing the fusion gene using a cell into which a vector carrying the gene has been introduced, and then purifying the fusion gene using a glutathione column. The induced signal can be preferably analyzed by fitting to a one-site competition model based on nonlinear regression analysis using software such as, for example, GRAPHPAD PRISM (GraphPad; San Diego).

[0149] One of the substances for observing the interaction is immobilized on the gold film of the sensor chip as a ligand. When light is applied to the back side of the sensor chip so that total reflection occurs at the boundary between the gold film and the glass, the intensity of the reflected light is partially reduced at a certain site (SPR signal). The other substance for observing the interaction is injected onto the surface of the sensor chip as an analyte. 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, dissociation shifts the signal back to its original position). In the Biacore system, the amount of the shift (i.e., the change in mass on the sensor chip surface) is plotted on the ordinate, and the change in mass over time is shown as measured data (sensorgram). The kinetic parameters (association rate constant (ka) and dissociation rate constant (kd)) are determined from the curve of the sensorgram, and the affinity (KD) is determined from the ratio between these two constants. The inhibition assay is preferably used in the BIACORE method. An example of such an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0150] Fc region with reduced Fcγ receptor binding activity As used herein, "reduced Fcγ receptor binding activity" means that, for example, based on the above-mentioned analytical method, the competitive activity of a 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 a control (e.g., wild-type) antigen-binding molecule or antibody. In one embodiment, "reduced Fcγ receptor binding activity" refers to an Fcγ receptor binding activity that is reduced compared to the wild-type Fc domain of IgG1.

[0151] Antigen-binding molecules or antibodies containing the Fc domain of monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies can be used as control antigen-binding molecules or antibodies as appropriate. The Fc domain structures are shown in SEQ ID NO: 148 (RefSeq accession number AAC82527.1 with A added to the N-terminus), SEQ ID NO: 149 (RefSeq accession number AAB59393.1 with A added to the N-terminus), SEQ ID NO: 150 (RefSeq accession number CAA27268.1 with A added to the N-terminus), and SEQ ID NO: 151 (RefSeq accession number AAB59394.1 with A added to the N-terminus). Furthermore, when an antigen-binding molecule or antibody containing an Fc domain mutant of a specific antibody isotype is used as a test substance, the effect of the mutation of the mutant on the Fcγ receptor binding activity is evaluated using an antigen-binding molecule or antibody containing an Fc domain of the same isotype as a control. As described above, antigen-binding molecules or antibodies comprising Fc domain mutants whose Fcγ receptor binding activity has been determined to be reduced are appropriately prepared.

[0152] Such known mutants include, for example, mutants having a deletion of amino acids 231A to 238S (EU numbering) (WO 2009 / 011941), as well as mutants 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).

[0153] Specifically, preferred antigen-binding molecules or antibodies include those that contain an Fc domain having at least one amino acid mutation (e.g., substitution) selected from the following amino acid 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) in the amino acids forming the Fc domain of an antibody of a particular isotype. The antibody isotype from which the Fc domain originates is not particularly limited, and it is possible to use a suitable Fc domain derived from a monoclonal IgG1, IgG2, IgG3, or IgG4 antibody. It is preferable to use an Fc domain derived from an IgG1 antibody.

[0154] Preferred antigen-binding molecules or antibodies include, for example, those that contain an Fc domain having any one of the following substitutions, as identified in the amino acid positions forming the Fc domain of an IgG1 antibody according to EU numbering (each number represents the position of an amino acid residue in EU numbering; the single-letter amino acid code before the number represents the amino acid residue before substitution, while the single-letter amino acid code after the number represents the amino acid residue after substitution): (a) L234F, L235E, P331S; (b) C226S, C229S, P238S; (c) C226S, C229S; or (d)C226S, C229S, E233P, L234V, L235A and those having an Fc domain with a deletion of the amino acid sequence at positions 231-238.

[0155] Further, preferred antigen-binding molecules or antibodies also include an Fc domain having any one of the following substitutions, whose positions are identified according to EU numbering in the amino acids forming the Fc domain of an IgG2 antibody: (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 an amino acid residue in the EU numbering system; the one-letter amino acid code before the number represents the amino acid residue before substitution, while the one-letter amino acid code after the number represents the amino acid residue after substitution.

[0156] Further, preferred antigen-binding molecules or antibodies also include an Fc domain having any one of the following substitutions, whose positions in the amino acids forming the Fc domain of an IgG3 antibody are identified according to EU numbering: (k)F241A; (l) D265A; or (m)V264A Each number represents the position of an amino acid residue in the EU numbering system; the one-letter amino acid code before the number represents the amino acid residue before substitution, while the one-letter amino acid code after the number represents the amino acid residue after substitution.

[0157] Further, preferred antigen-binding molecules or antibodies also include an Fc domain having any one of the following substitutions, whose positions in the amino acids forming the Fc domain of an IgG4 antibody are identified according to EU numbering: (n)L235A, G237A, and E318A; (o) L235E; or (p)F234A and L235A Each number represents the position of an amino acid residue in the EU numbering system; the one-letter amino acid code before the number represents the amino acid residue before substitution, while the one-letter amino acid code after the number represents the amino acid residue after substitution.

[0158] Other preferred antigen-binding molecules or antibodies include, for example, those that comprise an Fc domain in which the amino acid at any of positions 233, 234, 235, 236, 237, 327, 330, or 331 (EU numbering) in the amino acids forming the Fc domain of an IgG1 antibody is substituted with an amino acid at the corresponding position (EU numbering) in a corresponding IgG2 or IgG4.

[0159] Preferred antigen-binding molecules or antibodies also include those that contain an Fc domain in which any one or more of the amino acids at positions 234, 235, and 297 (EU numbering) in the amino acids forming the Fc domain of an IgG1 antibody are substituted with other amino acids. The type of amino acid substituted is not particularly limited; however, antigen-binding molecules or antibodies that contain an Fc domain in which any one or more of the amino acids at positions 234, 235, and 297 are substituted with alanine are particularly preferred.

[0160] Preferred antigen-binding molecules or antibodies also include, for example, those that contain an Fc domain in which the amino acid at position 265 (EU numbering) in the amino acids forming the Fc domain of an IgG1 antibody is substituted with another amino acid. The type of the substituted amino acid is not particularly limited; however, antigen-binding molecules or antibodies that contain an Fc domain in which the amino acid at position 265 is substituted with alanine are particularly preferred.

[0161] Antigen-binding domain that binds to HER2 S310F As used herein, the phrase "antigen-binding domain that binds to HER2 S310F" or "anti-HER2 S310F antigen-binding domain" refers to an antigen-binding domain that specifically binds to the above-mentioned HER2 S310F protein or all or a portion of a partial peptide of the HER2 S310F protein.

[0162] In certain embodiments, the antigen-binding domain that binds to HER2 S310F is a domain that comprises an antibody variable region (antibody light and heavy chain variable regions (VL and VH)). Suitable examples of such domains that comprise antibody light and heavy chain variable regions include "single-chain Fv (scFv)", "single-chain antibody", "Fv", "single-chain Fv2 (scFv2)", "Fab", "F(ab')2", and the like. In a specific embodiment, the antigen-binding domain that binds to HER2 S310F is a domain that comprises an antibody variable fragment. The domain that comprises an antibody variable fragment may be provided from the variable domains of one or more antibodies.

[0163] In certain embodiments, the antigen-binding domain that binds to HER2 S310F comprises the heavy chain variable region and the light chain variable region of an anti-HER2 S310F antibody. In certain embodiments, the antigen-binding domain that binds to HER2 S310F is a domain that comprises a Fab structure.

[0164] Preferably, the anti-HER2 S310F antibody comprises an H chain (H chain variable region) comprising an amino acid sequence and an L chain (L chain variable region) comprising an amino acid sequence as set forth in Table 1, respectively.

[0165] In some embodiments, the anti-HER2 S310F antigen-binding domain specifically binds to the extracellular domain of HER2 S310F (amino acids 23-652 of SEQ ID NO: 166). In some embodiments, the anti-HER2 S310F antigen-binding domain specifically binds to an epitope within the extracellular domain of HER2 S310F that includes the amino acid residue phenylalanine corresponding to the S310F position. In some embodiments, the anti-HER2 S310F antigen-binding domain does not bind to human wild-type HER2, preferably human wild-type HER2 expressed on the surface of human cells. In some embodiments, the anti-HER2 S310F antigen binding domain does not bind or does not substantially bind to wild-type HER2 isoforms from humans, including as disclosed in RefSeq Accession Nos. NP_001005862.1 (receptor tyrosine protein kinase erbB-2 isoform b; SEQ ID NO: 162), NP_001276865.1 (receptor tyrosine protein kinase erbB-2 isoform c; SEQ ID NO: 163), NP_001276866.1 (receptor tyrosine protein kinase erbB-2 isoform d; SEQ ID NO: 164), NP_001276867.1 (receptor tyrosine protein kinase erbB-2 isoform e; SEQ ID NO: 165), NP_004439.2 (receptor tyrosine protein kinase erbB-2 isoform a; SEQ ID NO: 166), preferably expressed on the surface of human cells that do not harbor the HER2 S310F mutation. In some embodiments, the anti-HER2 S310F antigen binding domain binds to the HER2 S310F protein expressed on the surface of a eukaryotic cell. In some embodiments, the anti-HER2 S310F antigen binding domain binds to the HER2 S310F protein expressed on the surface of a cancer cell.

[0166] In a specific embodiment, the antigen binding domain that binds to HER2 S310F comprises any one of the antibody variable fragments shown in Table 1 below.

[0167] (Table 1) HVR, VH, and VL sequences in the antigen-binding domain that binds to HER2 S310F TIFF0007625516000004.tif226166TIFF0007625516000005.tif211166TIFF0007625516000006.tif222166

[0168] In a specific embodiment, the antigen-binding domain that binds to HER2 S310F is a domain comprising an antibody variable fragment that competes for binding to human HER2 S310F with any one of the antibody variable fragments shown in Table 2. In a specific embodiment, the antigen-binding domain that binds to HER2 S310F is a domain comprising an antibody variable fragment that binds to the same epitope in human HER2 S310F as any one of the antibody variable fragments shown in Table 2.

[0169] Alternatively, the antigen-binding domain that binds to HER2 S310F comprises an antibody variable fragment that competes with any one of the above-mentioned antibody variable fragments for binding to human HER2 S310F. Alternatively, the antigen-binding domain that binds to HER2 S310F comprises an antibody variable fragment that binds to the same epitope on human HER2 S310F as any one of the above-mentioned antibody variable fragments binds to.

[0170] Antigen-binding domain that binds to the T cell receptor complex As used herein, the phrase "antigen-binding domain that binds to a T cell receptor complex" or "anti-T cell receptor complex antigen-binding domain" refers to an antigen-binding domain that specifically binds to all or a part of a partial peptide of a T cell receptor complex. The T cell receptor complex may be the T cell receptor itself, or may be an adaptor molecule that constitutes the T cell receptor complex together with the T cell receptor. CD3 is suitable as an adaptor molecule.

[0171] In certain embodiments, the avidity of the antigen-binding domain that binds to the T cell receptor complex is a domain comprising an antibody variable region (antibody light and heavy chain variable regions (VL and VH)). Suitable examples of such domains comprising antibody light and heavy chain variable regions include "single chain Fv (scFv)", "single chain antibody", "Fv", "single chain Fv2 (scFv2)", "Fab", "F(ab')2", and the like. In a specific embodiment, the antigen-binding domain that binds to the T cell receptor complex is a domain comprising an antibody variable fragment. The domain comprising an antibody variable fragment may be provided from the variable domains of one or more antibodies.

[0172] In certain embodiments, the antigen-binding domain that binds to the T cell receptor complex comprises the heavy chain variable region and the light chain variable region of an anti-T cell receptor complex antibody. In certain embodiments, the antigen-binding domain that binds to the T cell receptor complex is a domain that comprises a Fab structure.

[0173] Antigen-binding domain that binds to the T cell receptor As used herein, the phrase "antigen-binding domain that binds to a T cell receptor" or "anti-T cell receptor antigen-binding domain" refers to an antigen-binding domain that specifically binds to all or a portion 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 of the T cell receptor or the constant region of the T cell receptor; however, epitopes present in the constant region are preferred. Examples of constant region sequences include the T cell receptor alpha chain of RefSeq Accession No. CAA26636.1 (SEQ ID NO: 140), the T cell receptor beta chain of RefSeq Accession No. C25777 (SEQ ID NO: 141), the T cell receptor gamma 1 chain of RefSeq Accession No. A26659 (SEQ ID NO: 142), the T cell receptor gamma 2 chain of RefSeq Accession No. AAB63312.1 (SEQ ID NO: 143), and the T cell receptor delta chain of RefSeq Accession No. AAA61033.1 (SEQ ID NO: 144).

[0174] In certain embodiments, the antigen-binding domain that binds to the T cell receptor is a domain comprising an antibody variable region (antibody light and heavy chain variable regions (VL and VH)). Suitable examples of such domains comprising antibody light and heavy chain variable regions include "single chain Fv (scFv)", "single chain antibody", "Fv", "single chain Fv2 (scFv2)", "Fab", "F(ab')2", and the like. In a specific embodiment, the antigen-binding domain that binds to the T cell receptor is a domain comprising an antibody variable fragment. The domain comprising an antibody variable fragment may be provided from the variable domains of one or more antibodies.

[0175] In certain embodiments, the antigen-binding domain that binds to the T cell receptor comprises 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 that binds to the T cell receptor is a domain that comprises a Fab structure.

[0176] Antigen-binding domain that binds to CD3 As used herein, the phrase "antigen-binding domain that binds to CD3" or "anti-CD3 antigen-binding domain" refers to an antigen-binding domain that specifically binds to all or a part of a partial peptide of CD3. The antigen-binding domain that binds to CD3 may be any epitope-binding domain, as long as the epitope is present in the gamma, delta, or epsilon chain sequence that constitutes human CD3. The polynucleotide sequences of the gamma, delta, or epsilon chain structures that constitute CD3 are disclosed in RefSeq accession numbers NM_000073.2, NM_000732.4, and NM_000733.3, and the polypeptide sequences are shown in SEQ ID NOs: 145 (NP_000064.1), 146 (NP_000723.1), and 147 (NP_000724.1) (RefSeq accession numbers are shown in parentheses).

[0177] In certain embodiments, the antigen-binding domain that binds to CD3 is a domain comprising antibody light and heavy chain variable regions (VL and VH). Suitable examples of such domains comprising antibody light and heavy chain variable regions include "single-chain Fv (scFv)", "single-chain antibody", "Fv", "single-chain Fv2 (scFv2)", "Fab", "F(ab')2", and the like. In a specific embodiment, the antigen-binding domain that binds to CD3 is a domain comprising an antibody variable fragment. The domain comprising an antibody variable fragment may be provided from the variable domains of one or more antibodies.

[0178] In certain embodiments, the antigen-binding domain that binds to CD3 comprises a heavy chain variable region and a light chain variable region of an anti-CD3 antibody. In certain embodiments, the antigen-binding domain that binds to CD3 is a domain that comprises a Fab structure.

[0179] The binding activity of the antigen-binding domain of the present invention to bind to CD3 can bind to any epitope as long as the epitope is located within the gamma, delta, or epsilon chain sequence that forms human CD3. In the present invention, preferred antigen-binding domains that bind to CD3 include those that contain 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 epsilon chain of the human CD3 complex. Preferred antigen-binding domains that bind to such CD3 include those that contain the CD3 antibody light chain variable region (VL) and CD3 antibody heavy chain variable region (VH) of the OKT3 antibody (Proc. Natl. Acad. Sci. USA (1980) 77, 4914-4917) or various known CD3 antibodies, such as an antibody having a light chain variable region (VL) of NCBI Accession No. AAB24132 and a heavy chain variable region (VH) of NCBI Accession No. AAB24133 (Int. J. Cancer Suppl. 7, 45-50 (1992)). Several antibodies that bind to various epitopes of human CD3ε, such as the antibody OKT3 (see, e.g., Kung, P. et al, Science 206 (1979) 347-349; Salmeron, A. et al, J Immunol 147 (1991) 3047-3052; US9226962B2), the antibody UCHT1 (see, e.g., Callard, RE et al, Clin Exp Immunol 43 (1981) 497-505; Arnett et al. PNAS 2004), or the antibody SP34 (human-cynomolgus CD3 cross-reactive; see, e.g., Pessano, S. et al, EMBO J 4 (1985) 337-344; Conrad ML, et. al, Cytometry A 71 (2007) WO2015181098 also discloses human-cynomolgus cross-reactive antibodies that specifically bind to human and cynomolgus T cells and activate human T cells, but do not bind to the same epitope as antibody OKT3, antibody UCHT1, and / or antibody SP34.Furthermore, such suitable antigen-binding domains that bind to CD3 include those derived from CD3 antibodies having the desired characteristics obtained by immunizing a desired animal with the gamma, delta or epsilon chains that form human CD3 by the methods described above. Suitable anti-CD3 antibodies from which the antigen-binding domain that binds to CD3 is derived include human antibodies and antibodies that have been appropriately humanized as described above.

[0180] Multispecific antigen binding molecules "Multispecific antigen-binding molecule" refers to an antigen-binding molecule that specifically binds to more than one antigen. In a preferred embodiment, the multispecific antigen-binding molecule of the present invention comprises two or more antigen-binding domains, and different antigen-binding domains specifically bind to different antigens.

[0181] The multispecific antigen-binding molecule of the present invention comprises a first antigen-binding domain that binds to HER2 S310F and a second antigen-binding domain that binds to a T cell receptor complex. A combination of an antigen-binding domain that binds to HER2 S310F selected from those described above in "Antigen-binding domain that binds to HER2 S310F" and an antigen-binding domain that binds to a T cell receptor complex selected from those described above in "Antigen-binding domain that binds to T cell receptor complex" to "Antigen-binding domain that binds to CD3" can be used.

[0182] For example, the first antigen-binding domain is a domain comprising an antibody heavy and light chain variable region, and / or the second antigen-binding domain is a domain comprising an antibody heavy and light chain variable region. Alternatively, 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. Alternatively, 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.

[0183] In a particular embodiment, the present invention provides a multispecific antigen-binding molecule comprising a first antigen-binding domain comprising an antibody variable fragment that binds to HER2 S310F and a second antigen-binding domain comprising an antibody variable fragment that binds to a T cell receptor complex. In a particular embodiment, the present invention provides a bispecific antigen-binding molecule comprising a first antigen-binding domain that binds to HER2 S310F, a second antigen-binding domain that binds to a T cell receptor complex, and a third domain comprising an Fc region having 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 amino acids constituting the Fc regions of SEQ ID NOs: 148 to 151 (IgG1 to IgG4).

[0184] In certain embodiments, the present invention provides a bispecific antibody comprising a first antibody variable fragment that binds to human HER2 S310F and a second antibody variable fragment that binds to CD3. In certain embodiments, the present invention provides a bispecific antibody comprising a first antibody variable fragment that binds to human HER2 S310F, a second antibody variable fragment that binds to CD3, and an Fc region with reduced Fcγ receptor binding activity. In certain embodiments, the present invention provides a bispecific antibody comprising a first antibody variable fragment that binds to human HER2 S310F, a second antibody variable fragment that binds to the CD3ε chain, and an Fc region with reduced Fcγ receptor binding activity compared to naturally occurring IgG Fc region.

[0185] A preferred embodiment of the "multispecific antigen-binding molecule" of the present invention includes a multispecific antibody. When an Fc region with reduced Fcγ receptor binding activity is used as the multispecific antibody Fc region, an Fc region derived from the multispecific antibody may be used as appropriate. Bispecific antibodies are particularly preferred as the multispecific antibody of the present invention. In this case, the 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 types of hybridomas that produce IgG antibodies (Milstein et al., Nature (1983) 305, 537-540).

[0186] Furthermore, the genes of the L and H chains constituting the two types of IgG of interest, i.e., a total of four genes, are introduced into cells and co-expressed to secrete IgG-type bispecific antibodies. However, the number of combinations of H and L chains of IgG that can be produced by these methods is theoretically 10. Therefore, it is difficult to purify IgG containing the desired combination of H and L chains from 10 types of IgG. Furthermore, theoretically, the amount of secreted IgG having the desired combination is significantly reduced, which requires large-scale culture and further increases the production cost.

[0187] Therefore, techniques for promoting association between H chains and between L chains and H chains having desired combinations can be applied to the multispecific antigen-binding molecules of the present invention.

[0188] For example, techniques for suppressing unwanted heavy chain association by introducing electrostatic repulsion at the interface of the second or third constant region (CH2 or CH3) of the antibody heavy chain can be applied to the association of multispecific antibodies (WO2006 / 106905).

[0189] In a method for suppressing unintended H-chain association by introducing electrostatic repulsion at the CH2 or CH3 interface, examples of amino acid residues that contact the interface of other constant regions of the H-chain include regions corresponding to residues at EU numbering positions 356, 439, 357, 370, 399, and 409 in the CH3 region.

[0190] More specifically, examples include antibodies comprising two types of H chain CH3 regions, in which one to three pairs of amino acid residues in a first H chain CH3 region selected from the pairs of amino acid residues shown in (1) to (3) below have the same type of charge: (1) amino acid residues at positions 356 and 439 (EU numbering) in the H chain CH3 region; (2) amino acid residues at positions 357 and 370 (EU numbering) in the H chain CH3 region; and (3) amino acid residues at positions 399 and 409 (EU numbering) in the H chain CH3 region.

[0191] Furthermore, the antibody may be an antibody in which pairs of amino acid residues in a second H chain CH3 region different from the first H chain CH3 region described above are selected from the pairs of amino acid residues described above in (1) to (3), and one to three pairs of amino acid residues corresponding to the pairs of amino acid residues described above in (1) to (3) that have the same type of charge in the first H chain CH3 region have an opposite charge to the corresponding amino acid residues in the first H chain CH3 region described above.

[0192] The amino acid residues shown in (1) to (3) above are located close to each other during association. Those skilled in the art can find positions in the desired H chain CH3 region or H chain constant region that correspond to the above-mentioned amino acid residues in (1) to (3) by homology modeling using commercially available software, and can appropriately modify the amino acid residues at these positions.

[0193] In the above-mentioned antibodies, the "charged amino acid residue" is preferably selected, for example, from amino acid residues included in any one of the following groups: (a) glutamic acid (E) and aspartic acid (D); and (b) Lysine (K), arginine (R), and histidine (H).

[0194] In the above-mentioned antibody, the phrase "having the same charge" means, for example, that all of the two or more amino acid residues are selected from amino acid residues included in any one of the above groups (a) and (b). The phrase "having opposite charges" means, for example, that if at least one amino acid residue among the two or more amino acid residues is selected from amino acid residues included in any one of the above groups (a) and (b), the remaining amino acid residue is selected from amino acid residues included in the other group.

[0195] In a preferred embodiment, the above-mentioned antibody may have a first H chain CH3 region and a second H chain CH3 region cross-linked by a disulfide bond.

[0196] In the present invention, the amino acid residues that are modified are not limited to the above-mentioned amino acid residues in antibody variable region or antibody constant region.Those skilled in the art can identify the amino acid residues that form interfaces in mutant polypeptides or heteromultimers by using commercially available software, such as homology modeling; these amino acid residues can then be modified to control association.

[0197] Other known techniques can also be used for the association of the multispecific antibodies of the present invention. Fc region-containing polypeptides containing different amino acids can be efficiently associated with each other by replacing the amino acid side chains present in one of the antibody H chain Fc regions with a larger side chain (knob) and the amino acid side chains present in the corresponding Fc region of the other H chain with a smaller side chain (hole) to allow the knob to be placed in the hole (WO1996 / 027011; Ridgway JB et al., Protein Engineering (1996) 9, 617-621; Merchant AM et al. Nature Biotechnology (1998) 16, 677-681; and US20130336973).

[0198] In addition, other known techniques can also be used to form the multispecific antibodies of the present invention. The association of polypeptides having different sequences can be efficiently induced by complementary association of CH3 using a chain exchange engineered domain CH3, which is created by converting one part of the H chain CH3 of an antibody to a sequence derived from the corresponding IgA and introducing the corresponding IgA sequence into the complementary part of the H chain CH3 of the other (Protein Engineering Design & Selection, 23; 195-202, 2010). This known technique can also be used to efficiently form the desired multispecific antibody.

[0199] In addition, techniques for antibody production using the association between antibody CH1 and CL and between VH and VL, such as those described in WO 2011 / 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), such as those described in WO2008 / 119353, WO2011 / 131746, WO2015 / 046467, and WO2016159213; techniques for controlling the association between antibody heavy chain CH3, such as those 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, such as those described in WO2012 / 023053; Techniques for producing bispecific antibodies using two bacterial cell lines each expressing one of the chains of an antibody comprising a single H chain and a single L chain, such as those described in (Biotechnology Vol. 31, p 753-758 (2013)), may be used to form multispecific antibodies.

[0200] Alternatively, even if the desired multispecific antibody cannot be efficiently formed, the multispecific antibody of the present invention can be obtained by separating and purifying the desired multispecific antibody from the produced antibodies. For example, a method has been reported for enabling the purification of two types of homomeric forms and the desired heteromeric antibody by ion exchange chromatography by imparting a difference in isoelectric point by introducing amino acid substitutions into the variable regions of two types of H chains (WO2007114325). To date, a method for purifying a heteromeric antibody has been reported in which protein A is used to purify a heterodimeric antibody containing a mouse IgG2a H chain that binds to protein A and a rat IgG2b H chain that does not bind to protein A (WO98050431 and WO95033844). Furthermore, heterodimeric antibodies can themselves be efficiently purified by using H chains that contain substitutions of amino acid residues at positions 435 and 436 (EU numbering), which are the IgG-Protein A binding site, with amino acids such as Tyr and His that result in different Protein A affinities, in order to change the interaction between each of the H chains and Protein A, or by using H chains with different Protein A affinities, and then using a Protein A column.

[0201] Furthermore, an Fc region with improved C-terminal heterogeneity can be appropriately used as the Fc region of the present invention. More specifically, the present invention provides Fc regions prepared by deleting glycine at position 446 and lysine at position 447, as specified by EU numbering, from the amino acid sequences of two polypeptides constituting an Fc region derived from IgG1, IgG2, IgG3, or IgG4.

[0202] A combination of multiple of these techniques, for example, two or more, can be used. Furthermore, these techniques can be appropriately and separately applied to the two H chains to be associated. Furthermore, these techniques can be used in combination with the above-mentioned Fc region having reduced binding activity to Fcγ receptors. Furthermore, the antigen-binding molecule of the present invention may be a molecule that is separately prepared so as to have the same amino acid sequence based on the antigen-binding molecule to be subjected to the above-mentioned modification.

[0203] Preferably, the antigen binding molecule of the present invention may comprise a first antigen binding domain that binds to HER2 S310F and a second antigen binding domain that binds to the T cell receptor complex. In one embodiment, the second antigen binding domain binds to the T cell receptor. In another embodiment, the second antigen binding domain binds to the CD3ε chain. In one embodiment, the first antigen binding domain binds to human HER2 S310F. In a further embodiment, the first antigen binding domain binds to HER2 S310F on the surface of a eukaryotic cell. In one embodiment, the first antigen binding domain binds to human HER2 S310F on the surface of a eukaryotic cell, including a cancer cell.

[0204] The phrase "anti-HER2 S310F arm" as used herein refers to the antibody heavy chain and antibody light chain in a bispecific antibody that bind to HER2 S310F. The phrase "anti-CD3 arm" as used herein refers to the antibody heavy chain and antibody light chain in a bispecific antibody that bind to CD3.

[0205] Preferably, the antigen-binding molecule of the present invention can have cellular cytotoxic activity (also referred to as "cytotoxic activity"). In one embodiment, the cellular cytotoxic activity is T-cell-dependent cellular cytotoxicity (TDCC). In another embodiment, the cytotoxic activity is cellular cytotoxic activity against cells expressing HER2 S310F on their surface. The HER2 S310F-expressing cells may be cancer cells.

[0206] In a preferred aspect, the antibody (or antigen-binding molecule) of the present invention has cytotoxic activity (or cellular cytotoxic activity), or preferably T-cell-dependent cellular cytotoxic activity (TDCC), against HER2 S310F-expressing cells, such as cancer cells. HER2 S310F may be expressed on the surface of such cells. The (cellular) cytotoxic activity 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, TDCC can be measured by lactate dehydrogenase (LDH) release assay as described in Example 5.2. In this assay, target cells (e.g., HER2 S310F-expressing cells) are incubated with T cells (e.g., PBMCs) or expanded T cells in the presence of a test antibody, and the activity of LDH released from target cells killed by T cells is measured using a suitable reagent. Typically, the cytotoxic activity is calculated as a percentage of the LDH activity resulting from incubation with the antibody relative to the LDH activity resulting from 100% killing of the target cells (e.g., lysed by treatment with Triton-X). If the cytotoxic activity calculated as above is higher, the test antibody is determined to have a higher TDCC. Additionally or alternatively, for example, TDCC can also be measured by a real-time cytostatic assay as described in Example 5.2. In this assay, target cells (e.g., HER2 S310F-expressing cells) are incubated with T cells (e.g., PBMCs) or expanded T cells in the presence of the test antibody on a 96-well plate, and the proliferation of the target cells is monitored by methods known in the art, for example, by using a suitable analytical instrument (e.g., xCELLigence Real-Time Cell Analyzer). The percentage of cytostatic inhibition (CGI:%) is calculated as CGI(%)=100-(CI Ab ×100 / CI NoAb It is determined from the cell index value according to the formula given as "CI Ab ” represents the cell index value of the well containing the antibody at a particular experimental time, and “CI NoAb" represents the average cell index value of wells not containing antibody. If the CGI rate of an antibody is high, i.e., has a significantly positive value, it can be said that the antibody has TDCC activity and is more preferable in the present invention.

[0207] In a preferred aspect, the antibody (or antigen-binding molecule) of the present invention has T cell activation activity. T cell activation can be assayed by methods known in the art, such as using an engineered T cell line (e.g., Jurkat / NFAT-RE Reporter Cell Line (T Cell Activation Bioassay, Promega)) that expresses a reporter gene (e.g., luciferase) in response to its activation. In this method, target cells (e.g., HER2 S310F-expressing cells) are cultured with T cells in the presence of a test antibody, and then the level or activity of the expression product of the reporter gene is measured by a suitable method as an indicator of T cell activation. When the reporter gene is a luciferase gene, the luminescence resulting from the reaction between luciferase and its substrate may be measured as an indicator of T cell activation. If the T cell activation measured as above is higher, the test antibody is determined to have a higher T cell activation activity. In a preferred embodiment, the multispecific antigen binding molecule of the invention comprises one or more polypeptide chains as listed in Table 3.

[0208] Chimeric antigen receptors (CARs) Another aspect of the present invention relates to immune cells expressing HER2 S310F targeting chimeric antigen receptors (CARs) and their uses. In some embodiments, the CAR of the present invention comprises a first antigen binding domain of the present invention that specifically binds to HER2 S310F, preferably one of the antigen binding domains as described in Table 2. In certain aspects, the CAR comprises a scFv specific for HER2 S310F, or any natural or artificial moiety that can specifically bind to HER2 S310F. In certain embodiments, the CAR utilizes a single chain variable fragment (scFv) specific for HER2 S310F. In certain embodiments, the CAR utilizes a scFv specific for HER2 S310F that is derived from a monoclonal antibody. The CAR may be a second or third generation CAR, but in a specific embodiment, the CAR is not a third generation CAR and only comprises one costimulatory endodomain.

[0209] In a specific embodiment, an individual is given a certain type of immunotherapy, such as immune cells that recognize HER2 S310F, to treat and prevent HER2 S310F-positive cancer. In a specific embodiment, the immune cells are T cells, NK cells, or NKT cells. Other effector cells include those that can naturally exhibit anti-tumor activity or are modified to exhibit this effect. In a specific embodiment, the immune cells include HER2 S310F-specific CARs, and may be modified to be other than HER2 S310F-specific CARs. In a specific embodiment, the HER2 S310F-specific CARs include scFvs derived from any of the antigen-binding molecules listed in Table 2. Another genetic modification of immune cells is to express one or more chemokine receptors, in order to utilize them, for example, to enhance T cell homing to tumor sites. In a specific embodiment of CAR T cells, one or more stimulatory cytokines can be transiently expressed. In certain embodiments, HER2 S310F-CAR T cells can be conferred resistance to the suppressive tumor microenvironment. Additionally, genetic modifications that increase safety, such as inducible suicide genes (e.g., caspase-9) and / or inhibitory receptors to limit T cell effector function to the tumor site, may avoid "on-target / off cancer" toxicity.

[0210] In certain embodiments, an individual in need thereof, e.g., an individual known to have or suspected of having HER2 S310F positive cancer, is provided with a therapeutically effective amount of immune cells encompassed by the present disclosure. In certain embodiments, an individual is provided with at least 1×10 4 / m 2 ~1×10 10 / m 2HER2 S310F-CAR T cells may be given in a single dose, although other doses may be utilized. Multiple administrations of cells may be provided to the individual. In certain embodiments, with or without lymphodepleting chemotherapy or irradiation prior to T cell transfer. In certain embodiments, there is no post-treatment infusion of cytokines such as IL2, but in alternative embodiments, there is a post-treatment infusion of cytokines. In specific embodiments, the HER2 S310F-CAR immune cells can be combined with one or more additional immunological cancer therapies, such as checkpoint antibodies, immunomodulators, or vaccines to increase T cell activation and extend in vivo survival. Other cancer therapies, such as surgery, radiation, drug therapy, and / or hormone therapy, may also be used.

[0211] In one embodiment, there is a polynucleotide encoding a HER2 S310F chimeric antigen receptor, and the chimeric antigen receptor may comprise a transmembrane domain selected from the group consisting of CD3-zeta, CD28, CD8, 4-1BB, CTLA4, CD27, and combinations thereof. In some embodiments, the chimeric antigen receptor comprises only one costimulatory endodomain, while in certain embodiments, the chimeric antigen receptor comprises more than one costimulatory endodomain. In certain embodiments, the chimeric antigen receptor comprises a costimulatory molecule endodomain selected from the group consisting of CD28, CD27, 4-1BB, OX40 ICOS, Myd88, CD40, and combinations thereof. The chimeric antigen receptor may comprise a scFv specific for HER2 S310F, selected from the group consisting of trastuzumab, FRP5, scFv800E6, F5cys, pertuzumab, and combinations thereof.

[0212] In certain aspects, there is an expression vector comprising a polynucleotide encompassed by the present disclosure, which may be a viral vector, such as a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector, or may be a non-viral vector.

[0213] In certain embodiments, there are cells that contain polynucleotides or expression vectors as encompassed by the present disclosure.In specific embodiments, the cells are immune cells, such as T cells, NK cells, or NKT cells.The cells may be specific to another antigen, including tumor antigens in some cases.In specific embodiments, the cells are pp65CMV-specific T cells, CMV-specific T cells, EBV-specific T cells, varicella virus-specific T cells, influenza virus-specific T cells, and / or adenovirus-specific T cells.

[0214] In one embodiment, there is a method of treating an individual for cancer, comprising providing the individual with a therapeutically effective amount of any of a plurality of cells as encompassed by the present disclosure. In a specific embodiment, the cancer is HER2 S310F positive. The cancer may be refractory or recurrent. In a specific embodiment, the cancer is sarcoma or glioblastoma. The sarcoma may be, for example, osteosarcoma. The dose may be formulated in another manner, such as per weight or per age. In certain embodiments, the therapeutically effective amount of the plurality of cells is at least 1×10 4 / m 2 , 1×10 5 / m 2 , 1×10 6 / m 2 , 1×10 7 / m 2 , 1×10 8 / m 2 , 1×10 9 / m 2 , or 1 × 10 10 / m 2 In a specific embodiment, the therapeutically effective amount of the plurality of cells is at a dose of 1×10 10 / m 2 , 1×10 9 / m 2 , 1×10 8 / m 2 , 1×10 7 / m 2 , 1×10 6 / m 2 , 1×10 5 / m 2 , or 1 × 10 4 / m 2 In certain embodiments, the method is performed without or with administration of one or more cytokines and without or with lymphodepletion therapy, and is at a dose of 1×10 4 / m 2 ~1×10 10 / m 2 The cytokines may be IL2, IL7, IL12, and / or IL15.

[0215] In certain embodiments, the use of immune cells expressing HER2 S310F-specific chimeric antigen receptor is carried out ex vivo.For example, immune cells may be exposed to one or more cells, one or more tissues, and / or one or more organs ex vivo, so that the cells target cells with HER2 S310F, including HER2 S310F-expressing cancer cells.In a specific embodiment, HER2 S310F-specific chimeric antigen receptor-expressing immune cells are used to process one or more cells, one or more tissues, and / or one or more organs ex vivo.In a specific example, tissues or organs can be removed from some or all HER2 S310F-expressing cancer cells by ex vivo exposing an effective amount of HER2 S310F-specific chimeric antigen receptor-expressing immune cells to each tissue or organ. As an example, bone marrow can be exposed ex vivo to HER2 S310F-specific chimeric antigen receptor-expressing immune cells prior to transplantation, or HER2 S310F-specific chimeric antigen receptor-expressing immune cells can be used to process organs bearing malignancies prior to introduction into a host in need thereof.

[0216] A method for generating immune cells expressing HER2 S310F-specific chimeric antigen receptor is contemplated herein. In a specific embodiment, immune cells that are engineered to express HER2 S310F-specific chimeric antigen receptor are obtained from another party, including commercial or skilled artisans, or are isolated from an individual treated with HER2 S310F-specific chimeric antigen receptor immune cells or are isolated from another individual. Immune cells may be modified to express HER2 S310F-specific chimeric antigen receptor using standard means in the art, such as during transduction of a polynucleotide encoding HER2 S310F-specific chimeric antigen receptor. When the obtained or isolated immune cells are genetically modified to express HER2 S310F-specific chimeric antigen receptor and also contain a second genetic modification (e.g., express another chimeric antigen receptor or another type of non-natural receptor), the order in which the genetic modifications can be performed may be any order. Any polynucleotide encoding a HER2 S310F-specific chimeric antigen receptor or another type of receptor may be transduced into cells using a vector, such as a viral vector or a non-viral vector. The viral vector may be a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or the like.

[0217] In a specific embodiment, the cell is an immune cell that transiently expresses one or more chemokine receptors, e.g., the chemokine receptor is a receptor for a chemokine expressed by the cancer. In a specific embodiment, the chemokine is CXCL1, CXCL8, CCL2, and / or CCL17. The individual may be provided with a therapeutically effective amount of additional cancer therapy, e.g., administered to the individual before, during, and / or after the individual is administered the plurality of cells. In a specific embodiment, the additional therapy includes surgery, drug therapy, chemotherapy, radiation, immunotherapy, or a combination thereof. In a specific embodiment, the individual is administered lymphodepleting therapy before the plurality of cells is administered, although in some embodiments, the individual is not administered lymphodepleting therapy before the plurality of cells is administered.

[0218] In certain embodiments, the immunotherapy comprises one or more checkpoint antibodies, such as checkpoint antibodies that recognize CTLA4, PD-1, PD-L1, TIM3, BLTA, VISTA, and / or LAG3. In certain embodiments, the cells comprise an inhibitory receptor.

[0219] In one embodiment, a kit comprising a polynucleotide as encompassed by this disclosure, an expression vector as encompassed by this disclosure, and / or a cell as encompassed by this disclosure, wherein the polynucleotide, expression vector, and / or cell are contained in a suitable container.

[0220] In certain embodiments, there are HER2 S310F chimeric antigen receptor modified CMV-specific T cells for use in cancer.

[0221] The present invention further relates to a method for treating a patient for a disease, comprising administering to the patient an effective amount of the engineered cells of the present disclosure.Various diseases, including cancer, can be treated according to the present invention.In some embodiments, the cancer or cancer cell is bladder cancer, cervical cancer, colorectal cancer (CRC), non-small cell lung cancer (NSCLC), esophageal cancer, head and neck cancer, skin cancer (melanoma), bile duct cancer, kidney cancer, stomach cancer, small intestine cancer, liver cancer, uterine cancer, duodenal cancer, breast cancer, gallbladder cancer, preferably characterized by the expression of HER2 S310F.In a preferred embodiment, the cancer or cancer cell is bladder cancer with HER2 S310F.

[0222] In some embodiments, the method comprises administering to a human patient a pharmaceutical composition comprising an effective anti-tumor amount of a population of human T cells, wherein the human T cells of the population comprise human T cells comprising a nucleic acid sequence as described in this disclosure.

[0223] cancer The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancer include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal carcinoma, 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, liver cancer, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0224] "HER2 S310F" positive cancer includes cancer cells characterized by expression of HER2 S310F gene, mRNA, or protein, which can be identified, for example, by next generation sequencing (NGS) or real-time polymerase chain reaction (RT-PCR), or any known protein detection method or assay known in the art (e.g., ELISA). In some embodiments, the cancer or cancer cells are bladder cancer, cervical cancer, colorectal cancer (CRC), non-small cell lung cancer (NSCLC), esophageal cancer, head and neck cancer, skin cancer (melanoma), bile duct cancer, kidney cancer, stomach cancer, small intestine cancer, liver cancer, uterine cancer, duodenal cancer, breast cancer, gallbladder cancer, preferably characterized by expression of HER2 S310F (e.g., Nat Med. 2017 Jun;23(6):703-713). In a preferred embodiment, the cancer or cancer cells are bladder cancer with HER2 S310F.

[0225] tumor The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as referred to herein.

[0226] Ovarian cancer "Ovarian cancer" refers to a heterogeneous group of malignant tumors originating from the ovary. Approximately 90% of malignant ovarian tumors are epithelial in origin; the remainder are germ cell and stromal tumors. Epithelial ovarian tumors are classified into the following histological subtypes: serous adenocarcinoma (accounting for about 50% of epithelial ovarian tumors); endometrioid adenocarcinoma (approximately 20%); mucinous adenocarcinoma (approximately 10%); clear cell carcinoma (approximately 5-10%); and Brenner (transitional cell) tumors (relatively rare). The prognosis for ovarian cancer, the sixth most common cancer in women, is usually poor, with 5-year survival rates ranging from 5-30%. For reviews of ovarian cancer, see Fox et al. (2002) "Pathology of epithelial ovarian cancer," in Ovarian Cancer ch. 9 (Jacobs et al., eds., Oxford University Press, New York); Morin et al. (2001) "Ovarian Cancer," in Encyclopedic Reference of Cancer, pp.654-656 (Schwab, ed., Springer-Verlag, New York). The present invention contemplates methods of diagnosing or treating any of the above epithelial ovarian tumor subtypes, particularly the serous adenocarcinoma subtype.

[0227] Stomach cancer The terms "gastric cancer" or "gastric tumor" or "stomach tumor" or "stomach cancer" refer 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.

[0228] Breast tumors The term "breast tumor" or "breast cancer" refers to any tumor or cancer of the breast, including, for example, invasive or ductal carcinoma in situ, invasive or lobular carcinoma in situ, adenocarcinomas such as medullary carcinoma, colloid carcinoma, and papillary carcinoma; as well as less common forms such as cystosarcoma phyllodes, sarcoma, squamous cell carcinoma, and carcinosarcoma.

[0229] Colorectal tumors The term "colorectal tumor" or "colorectal cancer" refers to any tumor or cancer of the large intestine, including the colon (the large intestine from the cecum to the rectum) and rectum, including, for example, adenocarcinoma, as well as less common forms such as lymphoma and squamous cell carcinoma.

[0230] Gastric tumors The term "gastric tumor" or "gastric 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.

[0231] Complement-dependent cytotoxicity "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that is bound to a cognate antigen. To assess complement activation, a CDC assay may be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Polypeptide variants with altered Fc region amino acid sequences (polypeptides with variant Fc regions) and increased or decreased C1q binding capacity are described, for example, in U.S. Patent No. 6,194,551 B1 and WO 1999 / 51642. See also, for example, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0232] Immunoconjugates The present invention also provides immunoconjugates comprising an antigen-binding molecule of the present invention herein (e.g., an anti-HER2 S310F antibody) conjugated to one or more cytotoxic agents, e.g., chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins of bacterial, fungal, plant, or animal origin, enzymatically active toxins, or fragments thereof), or radioisotopes.

[0233] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including, but not limited to, maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent EP 0 425 235). B1); auristatins such as the monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998) see, for example, daunomycin or anthracyclines such as doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2003)); (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0234] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0235] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate.Various radioisotopes are available for making radioconjugates.Examples include 211At, 131I, 125I, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P, 212Pb, and Lu radioisotopes.When using radioconjugates for detection, they can comprise radioactive atoms for scintigraphy examination, such as Tc-99m or 123I, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0236] Conjugates of antibodies and cytotoxic agents may be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionuclides to antibodies. See WO94 / 11026. The linker may be a "cleavable linker" that facilitates the release of the cytotoxic drug in cells. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) may be used.

[0237] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, such conjugates prepared with cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0238] Pharmaceutical preparations The term "pharmaceutical formulation" refers to a preparation that is in a form such that it allows the biological activity of the active ingredients contained therein to be effective, and that does not contain any additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0239] Pharmaceutically acceptable carriers "Pharmaceutically acceptable carrier" refers to an ingredient, other than an active ingredient, in a pharmaceutical formulation that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0240] treatment As used herein, "treatment" (and its grammatical variations, such as "treat" or "treating") refers to a clinical intervention that seeks to modify the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical disease. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological effects of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and achieving remission or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of disease or to slow the progression of disease.

[0241] In one aspect, the present invention is based in part on a multispecific antigen-binding molecule comprising a first domain comprising a first antibody variable region that binds to HER2 S310F and a second domain comprising a second antibody variable region that binds to T cell receptor complex, and its use.The antigen-binding molecule and antibody of the present invention are useful for diagnosing or treating cancer, for example, ovarian tumor, non-small cell lung cancer, gastric cancer, and liver cancer in particular.

[0242] Pharmaceutical Compositions The pharmaceutical composition of the present invention, the therapeutic agent for inducing cellular cytotoxic activity, the cell growth inhibitor, or the anticancer agent of the present invention may be formulated with various types of multispecific antigen-binding molecules, if necessary. For example, the cytotoxic effect against cells expressing an antigen can be enhanced by a cocktail of multiple multispecific antigen-binding molecules of the present invention.

[0243] If necessary, the multispecific antigen-binding molecules of the present invention may be encapsulated in microcapsules (microcapsules made of hydroxymethylcellulose, gelatin, poly[methyl methacrylate], etc.) and made into components of colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) (see, for example, "Remington's Pharmaceutical Sciences 16th edition", Oslo Ed. (1980)). In addition, methods for preparing agents as sustained release agents are 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 Application (EP) Nos. EP 58481 and EP 133988; Biopolymers (1983) 22, 547-556).

[0244] The pharmaceutical composition, cytostatic agent, or anticancer agent of the present invention may be administered to a patient either orally or parenterally. Parenteral administration is preferred. Specifically, such administration methods include injection, nasal administration, intrapulmonary administration, and transdermal administration. Injection includes, for example, intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. For example, the pharmaceutical composition, therapeutic agent for inducing cellular cytotoxic activity, cytostatic agent, or anticancer agent of the present invention may be administered locally or systemically by injection. Furthermore, an appropriate administration method may be selected according to the age and symptoms of the patient. The administered dose may be selected, for example, from the range of 0.0001 mg to 1,000 mg per kg of body weight for each administration. Alternatively, the dose may be selected, for example, from the range of 0.001 mg / body to 100,000 mg / body per patient. However, the dose of the pharmaceutical composition of the present invention is not limited to these doses.

[0245] The pharmaceutical composition 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 pharma- ceutically acceptable carriers and additives. Examples include, but are not limited to, surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, and correctives, and other commonly used carriers can be suitably used. 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, saccharose, carboxymethyl cellulose, corn starch, inorganic salts, etc.

[0246] Preferably, the pharmaceutical composition of the present invention comprises the multispecific antigen-binding molecule of the present invention. In one aspect, the composition is a pharmaceutical composition for use in inducing cellular cytotoxic activity. In another aspect, the composition is a pharmaceutical composition for use in treating or preventing cancer. The pharmaceutical composition of the present invention can be used to treat or prevent cancer. Thus, 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. In a preferred embodiment, the cancer is bladder cancer, cervical cancer, colorectal cancer (CRC), non-small cell lung cancer (NSCLC), esophageal cancer, head and neck cancer, skin cancer (melanoma), bile duct cancer, kidney cancer, gastric cancer, small intestine cancer, liver cancer, uterine cancer, duodenal cancer, breast cancer, gallbladder cancer, preferably characterized by the expression of HER2 S310F. In a preferred embodiment, the cancer is bladder cancer with HER2 S310F.

[0247] Therapeutic Methods and Compositions Any of the antigen binding molecules provided herein (eg, anti-HER2 S310F antibodies, anti-HER2 S310F x CD3 bispecific antibodies) can be used in therapeutic methods.

[0248] In one aspect, an antigen-binding molecule of the present invention (e.g., anti-HER2 S310F antibody, anti-HER2 S310F x CD3 bispecific antibody) is provided for use as a medicament. In a further aspect, an antigen-binding molecule is provided for use in the treatment of cancer. In a particular embodiment, an antigen-binding molecule is provided for use in a method of treatment. In a particular embodiment, the present invention provides an antigen-binding molecule for use in a method of treating an individual with cancer, the method comprising administering to the individual an effective amount of the antigen-binding molecule. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In a further embodiment, the present invention provides an antigen-binding molecule for use in, e.g., blocking ligand-independent dimerization of HER2 S310F, suppressing proliferation and / or growth of HER2 S310F-expressing cancer cells. An "individual" according to any of the above embodiments is preferably a human. In one embodiment, the cancer or cancer cells are characterized by expression of HER2 S310F. In another embodiment, the cancer or cancer cell is bladder cancer, cervical cancer, colorectal cancer (CRC), non-small cell lung cancer (NSCLC), esophageal cancer, head and neck cancer, skin cancer (melanoma), bile duct cancer, kidney cancer, stomach cancer, small intestine cancer, liver cancer, uterine cancer, duodenal cancer, breast cancer, gallbladder cancer, preferably characterized by the expression of HER2 S310F. In a preferred embodiment, the cancer or cancer cell is bladder cancer with HER2 S310F.

[0249] In a further aspect, the present invention provides the use of an antigen-binding molecule (e.g., an anti-HER2 S310F antibody, an anti-HER2 S310F x CD3 bispecific antibody) in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of cancer. In a further embodiment, the medicament is for use in a method of treating cancer comprising administering an effective amount of the medicament to an individual having cancer. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In a further embodiment, the medicament is for, e.g., blocking ligand-independent dimerization of HER2 S310F, inhibiting proliferation and / or growth of HER2 S310F-expressing cancer cells. In a further embodiment, the medicament is for use in a method of, for example, inhibiting ligand-independent dimerization of HER2 S310F and inhibiting proliferation and / or growth of HER2 S310F-expressing cancer cells in an individual, comprising administering to the individual an effective amount of the medicament to inhibit ligand-independent dimerization of HER2 S310F and inhibit proliferation and / or growth of HER2 S310F-expressing cancer cells. An "individual" according to any of the above embodiments may be a human. In one embodiment, the cancer or cancer cells are characterized by expression of HER2 S310F. In another embodiment, the cancer or cancer cells are bladder cancer, cervical cancer, colorectal cancer (CRC), non-small cell lung cancer (NSCLC), esophageal cancer, head and neck cancer, skin cancer (melanoma), bile duct cancer, kidney cancer, gastric cancer, small intestine cancer, liver cancer, uterine cancer, duodenal cancer, breast cancer, gallbladder cancer, preferably characterized by expression of HER2 S310F. In a preferred embodiment, the cancer or cancer cells are bladder cancer with HER2 S310F.

[0250] In a further aspect, the present invention provides a method for treating cancer. In one embodiment, the method comprises administering to an individual having such cancer an effective amount of an antigen binding molecule of the present invention (e.g., anti-HER2 S310F antibody, anti-HER2 S310F x CD3 bispecific antibody). In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. An "individual" according to any of the above embodiments may be a human. In one embodiment, the cancer or cancer cells are characterized by expression of HER2 S310F. In another embodiment, the cancer or cancer cells are bladder cancer, cervical cancer, colorectal cancer (CRC), non-small cell lung cancer (NSCLC), esophageal cancer, head and neck cancer, skin cancer (melanoma), bile duct cancer, kidney cancer, gastric cancer, small intestine cancer, liver cancer, uterine cancer, duodenal cancer, breast cancer, gallbladder cancer, preferably characterized by expression of HER2 S310F. In a preferred embodiment, the cancer or cancer cells are bladder cancer with HER2 S310F.

[0251] In a further aspect, the present invention provides a pharmaceutical formulation comprising any of the antigen binding molecules of the present invention provided herein (e.g., anti-HER2 S310F antibody, anti-HER2 S310F x CD3 bispecific antibody), for use in any of the above-mentioned therapeutic methods. In one embodiment, the pharmaceutical formulation comprises any of the antigen binding molecules of the present invention provided herein (e.g., anti-HER2 S310F antibody, anti-HER2 S310F x CD3 bispecific antibody) and a pharma- ceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the antigen binding molecules of the present invention provided herein (e.g., anti-HER2 S310F antibody, anti-HER2 S310F x CD3 bispecific antibody) and at least one additional therapeutic agent, for example, as described below.

[0252] Combination therapy The antigen binding molecules of the present invention (e.g., anti-HER2 S310F antibodies, anti-HER2 S310F x CD3 bispecific antibodies) can be used either alone or in combination with other agents in therapy. For example, the antibodies of the present invention may be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an additional other cytotoxic agent, chemotherapeutic agent, or anti-cancer agent, or a compound that enhances the effect of anti-HER2 S310F therapy in the patient. Such agents include, for example: Any of the anti-HER2 antibodies or inhibitors known in the art, such as trastuzumab or pertuzumab, trastuzumab emtansine, lapatinib or neratinib; Alkylating agents or agents having alkylating properties, such as cyclophosphamide (CTX; e.g., cytoxan®), chlorambucil (CHL; e.g., leukeran®), cisplatin (CisP; e.g., platinol®), busulfan (e.g., myleran®), melphalan, carmustine (BCNU), streptozotocin, triethylenemelamine (TEM), mitomycin C, and the like; tumor necrosis factors; interferons alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or immunomodulators, including MIP-1, MIP-1beta, MCP-1, RANTES, and other chemokines; Antimetabolites, such as methotrexate (MTX), etoposide (VP16; e.g., vepesid®), 6-mercaptopurine (6 MP), 6-thioguanine (6TG), cytarabine (Ara-C), 5-fluorouracil (5-FU), capecitabine (e.g., Xeloda®), dacarbazine (DTIC), and the like; Antibiotics, such as actinomycin D, doxorubicin (DXR; e.g., adriamycin®), daunorubicin (daunomycin), bleomycin, mithramycin, and the like; and Alkaloids, e.g., Vinca alkaloids such as vincristine (VCR), vinblastine, etc.; Other anti-tumor agents, such as paclitaxel (e.g., taxol®) and paclitaxel derivatives, cytostatics, glucocorticoids such as dexamethasone (DEX; e.g., decadron®) and corticosteroids such as prednisone, nucleoside enzyme inhibitors such as hydroxyurea, amino acid depleting enzymes such as asparaginase, leucovorin and other folic acid derivatives, and similar miscellaneous anti-tumor agents.

[0253] The following agents may also be used as additional agents: arnifostine (e.g., ethyol®), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, lomustine (CCNU), doxorubicin lipoprotein (e.g., doxil®), gemcitabine (e.g., gemzar®), daunorubicin lipoprotein (e.g., daunoxome®), procarbazine, mitomycin, docetaxel (e.g., taxotere®), aldesleukin, carboplatin, oxaliplatin, cladribine, camptothecin, CPT 11 (irinotecan), 10-hydroxy-7-ethyl-camptothecin (SN38), floxuridine, fludarabine, ifosfamide, idarubicin, mesna, interferon beta, interferon alpha, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, and chlorambucil.

[0254] In one embodiment, anti-hormonal agents may also be used in combination with the antigen binding molecules of the present invention for the treatment of HER2 S310F positive cancer or metastasis of HER2 S310F positive cancer in patients. As used herein, the term "anti-hormonal agent" includes natural or synthetic organic compounds or peptide compounds that act to control or inhibit hormone action on tumors. Anti-hormonal agents include, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazole, other aromatase inhibitors, 42-hydroxytamoxifen, trioxyphene, ketoxifene, LY. steroid receptor antagonists, antiestrogens, such as 117018, onapristone, and toremifene (e.g., Fareston®); antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharma- ceutically acceptable salts, acids, or derivatives of any of the above; agonists and / or antagonists of glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH) and LHRH (luteinizing hormone-releasing hormone); LHRH agonists, commercially available as Zoladex® (AstraZeneca), goserelin acetate, an LHRH antagonist; D-alaninamide N-acetyl-3-(2-naphthalenyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridinyl)-D-alanyl-L-seryl-N-6-(3-pyridinylcarbonyl)-L-lysyl-N-6-(3-pyridinylcarbonyl)-D-lysyl-L-leucyl-N-6-(1-methylethyl)-L-lysyl-L-proline (e.g., Antide®, Ares-Serono); ganirelix acetate, an LHRH antagonist; cyproterone acetate (CPA), a steroidal antiandrogen, and megestrol acetate, available commercially as Megace® (Bristol-Myers Oncology); Eulexin® (Schering Corp.), a nonsteroidal antiandrogen flutamide (2-methyl-N-[4,20-nitro-3-(trifluoromethyl)phenylpropanamide); the nonsteroidal antiandrogen nilutamide (5,5-dimethyl-3-[4-nitro-3-(trifluoromethyl-4'-nitrophenyl)-4,4-dimethyl-imidazolidine-dione); as well as antagonists against other nonpermissive receptors such as antagonists against RAR (retinoic acid receptor), RXR (retinoid X receptor), TR (thyroid receptor), VDR (vitamin-D receptor), etc. .

[0255] The combination therapy as described above includes combined administration (two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which the administration of the antibody of the present invention can be performed prior to, simultaneously with, and / or subsequent to the administration of one or more additional therapeutic agents. In one embodiment, the administration of the antigen-binding molecule and the administration of the additional therapeutic agent are performed within about one month, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other. The antigen-binding molecule of the present invention (e.g., anti-HER2 S310F antibody, anti-HER2 S310F x CD3 bispecific antibody) can also be used in combination with radiation therapy.

[0256] The antigen-binding molecules of the present invention (e.g., anti-HER2 S310F antibodies, anti-HER2 S310F x CD3 bispecific antibodies) (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, pulmonary, and intranasal administration, and, if desired for localized treatment, intralesional administration. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, injections such as intravenous or subcutaneous injections, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or repeated administration over various time points, bolus administration, and pulse infusion.

[0257] The antigen-binding molecules of the present invention (e.g., anti-HER2 S310F antibodies, anti-HER2 S310F x CD3 bispecific antibodies) are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the delivery site of the agent, the method of administration, the schedule of administration, and other factors known to medical practitioners. The antigen-binding molecules are optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antigen-binding molecules present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and routes of administration as described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route determined empirically / clinically appropriate. It will be appreciated that any of the above formulations or treatment methods may be carried out using the immunoconjugates of the invention instead of, or in addition to, the antigen-binding molecules of the invention.

[0258] The present invention also provides a method for damaging or inhibiting cell proliferation of HER2 S310F-expressing cells by contacting the cells with a monospecific or multispecific antigen-binding molecule of the present invention that binds to HER2 S310F (e.g., anti-HER2 S310F antibody, anti-HER2 S310F x CD3 bispecific antibody). The monoclonal antibody that binds to HER2 S310F is described above as the monospecific or multispecific antigen-binding molecule of the present invention contained in the therapeutic agent, cell proliferation inhibitor, and anticancer agent for inducing cellular cytotoxicity of the present invention. The cells to which the monospecific or multispecific antigen-binding molecule of the present invention binds are not particularly limited as long as they express HER2 S310F. Specifically, in the present invention, preferred cancer antigen-expressing cells include HER2 S310F positive cancers, including cancer cells characterized by the expression of HER2 S310F gene, mRNA, or protein, which can be identified, for example, by next-generation sequencing (NGS) or real-time polymerase chain reaction (RT-PCR), or any known protein detection method or assay known in the art (e.g., ELISA). In some embodiments, the cancer or cancer cells are bladder cancer, cervical cancer, colorectal cancer (CRC), non-small cell lung cancer (NSCLC), esophageal cancer, head and neck cancer, skin cancer (melanoma), bile duct cancer, kidney cancer, stomach cancer, small intestine cancer, liver cancer, uterine cancer, duodenal cancer, breast cancer, gallbladder cancer, preferably characterized by the expression of HER2 S310F (e.g., Nat Med. 2017 Jun;23(6):703-713). In a preferred embodiment, the cancer or cancer cells are bladder cancer with HER2 S310F.

[0259] In the present invention, "contact" can be performed, for example, by adding the multispecific antigen-binding molecule of the present invention to the culture medium of cells expressing HER2 S310F cultured in vitro. In this case, the multispecific antigen-binding molecule to be added can be used in an appropriate form, such as a solution or a solid prepared by lyophilization. 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 only the multispecific antigen-binding molecule, or may be a solution containing, for example, the above-mentioned surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow-promoting substances, and corrective agents. The concentration to be 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.

[0260] In another embodiment of the present invention, "contact" can also be performed by administration to a non-human animal transplanted with HER2 S310F-expressing cells in vivo, or to an animal having cancer cells that endogenously express HER2 S310F. The administration method can be oral or parenteral. Parenteral administration is particularly preferred. Specifically, parenteral administration methods include injection, nasal administration, pulmonary administration, and transdermal administration. Injection includes, for example, intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. For example, the pharmaceutical composition of the present invention, the therapeutic agent for inducing cellular cytotoxic activity, the cytostatic agent, or the anticancer agent can be administered locally or systemically by injection. Furthermore, an appropriate administration method can be selected according to the age and symptoms 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 only the multispecific antigen-binding molecule, or may be a solution containing, for example, the above-mentioned surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, and corrective agents. The administered dose can be selected, for example, from the range of 0.0001 to 1,000 mg per kg of body weight for each administration. Alternatively, the dose can be selected from the range of 0.001 to 100,000 mg / body for each patient. However, the dose of the multispecific antigen-binding molecule of the present invention is not limited to these examples.

[0261] The following method is preferably used as a method for evaluating or determining cellular cytotoxic activity by contacting the multispecific antigen-binding molecule of the present invention with HER2 S310F-expressing cells to which the antigen-binding domain forming the multispecific antigen-binding molecule of the present invention binds. Methods for evaluating or determining cellular 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 of inducing T cell-mediated cellular cytotoxic activity 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 cytotoxic activity assay, a multispecific antigen-binding molecule whose antigen-binding domain binds to an antigen different from HER2 S310F and not expressed in cells is used as a control multispecific antigen-binding molecule. The control multispecific antigen-binding molecule is assayed in the same manner. The activity is then evaluated by testing whether the multispecific antigen-binding molecule of the present invention exhibits stronger cytotoxic activity than a control multispecific antigen-binding molecule.

[0262] Meanwhile, in vivo antitumor efficacy is 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 implanted intradermally or subcutaneously into a non-human animal subject. Then, from the day of implantation or thereafter, a test multispecific antigen-binding molecule is administered intravenously or intraperitoneally every day or at intervals of several days. Tumor size is measured over time. The difference in the change in tumor size can be defined as cytotoxic activity. As in the in vitro assay, a control multispecific antigen-binding molecule is administered. 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.

[0263] To evaluate or determine the effect of contacting the multispecific antigen-binding molecule of the present invention with the antigen-binding domain forming the multispecific antigen-binding molecule to inhibit the proliferation of cells expressing the antigen that binds, the MTT method and the measurement of the incorporation of isotope-labeled thymidine into cells are preferably used.On the other hand, to evaluate or determine the activity of inhibiting cell proliferation in vivo, the same method as above for evaluating or determining in vivo cytotoxicity can be preferably used.

[0264] The present invention also provides kits containing the multispecific antigen-binding molecules of the present invention or multispecific antigen-binding molecules produced by the methods of the present invention for use in the methods of the present invention. The kits may be packaged with additional pharma- ceutically acceptable carriers or vehicles, or instruction manuals explaining how to use the kit, or the like.

[0265] In addition, the present invention relates to a multispecific antigen-binding molecule of the present invention, or a multispecific antigen-binding molecule produced by a method of the present invention, for use in a method of the present invention.

[0266] Methods and Compositions for Diagnosis and Detection In certain embodiments, any of the antigen binding molecules of the present invention provided herein (e.g., anti-HER2 S310F antibody) are useful for detecting the presence of HER2 S310F in biological samples.The term "detecting" as used herein includes quantitative or qualitative detection.In certain embodiments, the biological sample includes cells or tissues, such as bladder tissues.

[0267] In one embodiment, an anti-HER2 S310F antibody is provided for use in a method of diagnosis or detection. In a further aspect, a method is provided for detecting the presence of HER2 S310F in a biological sample. In certain embodiments, the method comprises contacting a biological sample with an anti-HER2 S310F antibody as described herein under conditions that allow the binding of the anti-HER2 S310F antibody to HER2 S310F, and detecting whether a complex is formed between the anti-HER2 S310F antibody and HER2 S310F. Such a method may be an in vitro method or an in vivo method. In one embodiment, when HER2 S310F is a biomarker for patient selection, the anti-HER2 S310F antibody is used to select subjects who are eligible for treatment with the anti-HER2 S310F antibody. In one embodiment, the anti-HER2 S310F antibody is used in a method for diagnosing cancer in an individual or predicting cancer treatment response in an individual, comprising determining the presence of HER2 S310F in a sample isolated from the patient. In one embodiment, the cancer or cancer cell is characterized by the expression of HER2 S310F.In another embodiment, the cancer or cancer cell is bladder cancer, cervical cancer, colorectal cancer (CRC), non-small cell lung cancer (NSCLC), esophageal cancer, head and neck cancer, skin cancer (melanoma), bile duct cancer, kidney cancer, stomach cancer, small intestine cancer, liver cancer, uterine cancer, duodenal cancer, breast cancer, gallbladder cancer, preferably characterized by the expression of HER2 S310F.In a preferred embodiment, the cancer or cancer cell is bladder cancer with HER2 S310F.

[0268] In certain embodiments, a labeled anti-HER2 S310F antibody is provided.Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as indirectly detectable moieties, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions.Exemplary labels include, but are not limited to, radioisotopes 32P, 14C, 125I, 3H, and 131I, fluorophores, such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase, such as firefly luciferase and bacterial luciferase (US Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β- Galactosidase, glucoamylase, lysozyme, monosaccharide oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with enzymes that use hydrogen peroxide to oxidize dye precursors (e.g., HRP, lactoperoxidase, or microperoxidase), biotin / avidin, spin labels, bacteriophage labels, stable free radicals, etc.

[0269] All documents cited herein are hereby incorporated by reference. EXAMPLES

[0270] The following are examples of methods and compositions of the present invention. In light of the general description provided above, it will be understood that various other embodiments may be practiced.

[0271] Example 1. Expression and purification of human HER2 S310F extracellular domain (ECD) and wild-type HER2 ECD A synthetic polypeptide (SEQ ID NO:1) containing amino acids 23-646 of human HER2 S310F ECD with a rabbit Fc tag at its C-terminus was transiently expressed using the Expi293 cell line (Thermo Fisher). Conditioned medium expressing the synthetic polypeptide was applied to a column packed with MabSelect SuRe resin (GE Healthcare) and eluted with Na acetate. Fractions containing the synthetic polypeptide were collected and subsequently subjected to a Superdex 200 gel filtration column (GE healthcare) equilibrated with 1xD-PBS. Fractions containing the synthetic polypeptide were then pooled and stored at -80°C.

[0272] SEQ ID NO:1 (shown below) illustrates the amino acid sequence from 23 to 646 of human HER2 S310F ECD (the F amino acid residue corresponding to position 310 of HER2 is bold and underlined) with a linker (bold) at its C-terminus followed by a rabbit Fc tag (underlined), including the signal sequence (italics, first 19 amino acid residues). The signal sequence is not part of the mature polypeptide chain. TIFF0007625516000007.tif119166

[0273] A synthetic polypeptide (SEQ ID NO:2) containing amino acids 23-646 of human HER2 S310F ECD with a FLAG tag at its C-terminus was transiently expressed using the Expi293 cell line (Thermo Fisher). 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). Fractions containing the synthetic polypeptide were collected and subsequently subjected to a Superdex 200 gel filtration column (GE healthcare) equilibrated with 1xD-PBS. Fractions containing the synthetic polypeptide were then pooled and stored at -80°C.

[0274] SEQ ID NO:2 (shown below) illustrates the amino acid sequence from 23 to 646 of human HER2 S310F ECD (the F amino acid residue corresponding to position 310 of HER2 is bold and underlined) with a linker (bold) at its C-terminus followed by a FLAG tag (underlined), including a signal sequence (italics, first 19 amino acid residues). The signal sequence is not part of the mature polypeptide chain. TIFF0007625516000008.tif91166

[0275] A synthetic polypeptide (SEQ ID NO:3) containing amino acids 23-646 of human wild-type HER2 ECD with a twin Strep tag at its C-terminus was transiently expressed using the Expi293 cell line (Thermo Fisher). Conditioned medium expressing the synthetic polypeptide was applied to a column packed with Strep Tactin resin (IBA Lifesciences) and eluted with Desthiobiotin (Sigma). Fractions containing the synthetic polypeptide were collected and subsequently subjected to a Superdex 200 gel filtration column (GE healthcare) equilibrated with 1xD-PBS. Fractions containing the synthetic polypeptide were then pooled and stored at -80°C.

[0276] SEQ ID NO:3 (shown below) illustrates the amino acid sequence from 23 to 646 of human wild-type HER2 ECD (the S amino acid residue corresponding to position 310 of HER2 is bold and underlined) with a linker (bold) at its C-terminus followed by a twin Strep tag (underlined), including the signal sequence (italics, first 19 aa). The signal sequence is not part of the mature polypeptide chain. TIFF0007625516000009.tif92166

[0277] Example 2. Establishment of FreeStyle 293 cell lines expressing human HER2 S310F or wild-type HER2 FreeStyle 293 cells were transfected with pcDNA3.1 (+) Hygro-HER2 S310F or pcDNA3.1 (+) Hygro-wild-type HER2 expression vectors using 293fectin and selected with hygromycin B (10 micrograms (μg) / ml). FreeStyle 293 cells stably expressing human HER2 S310F and human wild-type HER2 were sorted (FACS aria III, BD) by staining with anti-HER2 antibody (Biolegend, 324402), expanded in culture, and cryopreserved for future use.

[0278] Example 3. Generation and screening of anti-HER2 S310F monospecific antibodies Anti-HER2 S310F antibodies were generated through both protein and DNA immunization.

[0279] For protein immunization, three NZW rabbits were first immunized intradermally (ID) with 100 μg of recombinant human HER2 S310F ECD protein with rabbit Fc tag. Two weeks after the first immunization, three more weekly doses of the same immunogen were given (50 μg / dose / rabbit). One week after the last immunization, spleens and blood from the immunized rabbits were collected for B cell isolation.

[0280] For DNA immunization, three NZW rabbits were immunized with gold particles coated with pCXND3-HER2 S310F plasmid (1 μg DNA per shot for a total of 40 shots) by the Helios Gene Gun system (Bio-Rad) according to the manufacturer's instructions (J Virol. 2013 Sep;87(18):10232-43). Concurrently, a combination of intradermal (100 μg DNA per site for a total of four sites on the leg and shoulder) and intramuscular (200 μg DNA per site for a total of four sites on two legs) electroporations of pCXND3-HER2 S310F plasmid were performed as described in Vaccine. 2008 Apr 16;26(17):2100-10; J Pharmacol Toxicol Methods. 2008 Jul-Aug;58(1):27-31; and Int J Pharm. 2005 Apr 27;294(1-2):53-63. Immunizations were repeated weekly for seven doses, followed by blood and spleen collection.

[0281] B cells were stained with recombinant human HER2 S310F ECD protein with a FLAG tag and recombinant human wild-type HER2 ECD protein with a twin-strep tag. HER2 S310F ECD-specific B cells were sorted with a flow cytometry (FCM) cell sorter (FACSAria™ III, BD Biosciences) and plated in 96-well plates at a density of 1 cell / well together with 25,000 cells / well of EL4 cells (European Collection of Cell Cultures) pretreated with mitomycin C (Sigma) in culture medium supplemented with rabbit T cell conditioned medium as described in WO2016098356A1. After 7-12 days of culture, B cell culture supernatants were collected for further analysis and cell pellets were cryopreserved.

[0282] In total, 6015 B cell supernatants were screened, and B cell supernatants containing antibodies that specifically bind to HER2 S310F were identified by flow cytometry analysis and ELISA. FreeStyle 293 cells transiently expressing human HER2 S310F and human wild-type HER2 were first stained with Violet and Far Red CellTrace dyes (Invitrogen). Equal numbers of FreeStyle 293 cells overexpressing HER2 S310F (Violet), FreeStyle 293 cells overexpressing wild-type HER2 (Far Red), and parental FreeStyle 293 cells (unstained) were mixed together, stained with B cell supernatants and anti-rabbit IgG PE antibody, and analyzed by iQUE screener PLUS (IntelliCyt). Antibodies from 20 B cell supernatants showed stronger binding to HER2 S310F-overexpressing FreeStyle 293 cells than to parental FreeStyle 293 cells and wild-type HER2-overexpressing FreeStyle 293 cells. Concurrently, the binding of antibodies in B cell supernatants to recombinant HER2 S310F ECD protein and wild-type HER2 ECD protein was evaluated by ELISA. 61 B cell lines showed stronger binding to recombinant HER2 S310F ECD protein than to wild-type HER2 ECD protein. 84 B cell lines, along with 3 additional controls, were selected for antibody gene cloning and further analysis (MHR0001-0084).

[0283] RNA was extracted from the corresponding cell pellets by using ZR-96 Quick-RNA Kit (ZYMO RESEARCH). The DNA of the heavy and light chain variable regions was amplified by reverse transcription PCR and cloned into an expression vector with a human heavy chain constant region sequence (SEQ ID NO: 133) and an expression vector containing a human light chain constant region sequence (SEQ ID NO: 136), respectively. The recombinant antibodies were transiently expressed in FreeStyle 293-F cells according to the manufacturer's instructions (Life technologies) and purified using the AssayMAP Bravo platform with Protein A cartridge (Agilent).

[0284] The binding of these 84 purified antibodies to HER2 S310F was confirmed again by flow cytometry analysis using FreeStyle 293 cells carrying HER2 S310F and FreeStyle 293 cells carrying wild-type HER2. Twelve antibodies that showed specific binding to HER2 S310F were selected for downstream analysis (Table 2).

[0285] Table 2. Anti-HER2 S310F monospecific antibodies TIFF0007625516000010.tif132165

[0286] Example 4. Binding analysis of anti-HER2 S310F monospecific antibody Figure 1 shows the binding of anti-HER2 S310F antibody to FreeStyle 293 transfectants expressing HER2 S310F and wild-type HER2, as determined by FCM analysis. Figure 2 shows the binding of anti-HER2 S310F antibody to 5637 cancer cell line with HER2 S310F (Proc Natl Acad Sci US A. 2012 Sep 4;109(36):14476-81), OV90 cancer cell line with wild-type HER2, and OVCAR3 cancer cell line with wild-type HER2, as determined by FCM analysis. Anti-HER2 S310F antibody was incubated with each cell line for 30 minutes at 4°C and washed with washing buffer (Nacalai Tesque, Cat. L3P8406). Then, goat F(ab')2 anti-human IgG, mouse ads-PE (Southern Biotech, Cat. 2043-09) was added and incubated for 30 minutes in the dark at 4°C, followed by washing. Data acquisition was performed using LSR Fortessa X-20 (BD Biosciences) and analyzed with FlowJo software (Tree Star). Figure 1 shows that all anti-HER2 S310F antibodies made in Example 3, namely MHR0007, MHR0009, MHR0010, MHR0012, MHR014, MHR0015, MHR0016, MHR0017, MHR0018, MHR0019, MHR0060 / MHR0060', and MHR0082, bind to FreeStyle 293 transfectants expressing HER2 S310F. Figure 2 shows that anti-HER2 S310F antibodies such as MHR0009, MHR0010, MHR0016, MHR0019, and MHR0060 / MHR0060' bind to 5637 bladder cancer cell lines carrying HER2 S310F. The binding activity of each antibody is represented by the mean fluorescence intensity value (MFI).

[0287] Example 5. Functional evaluation of anti-HER2 S310F / CD3 bispecific antibody Example 5.1. Measurement of T cell activation activity of anti-HER2 S310F / CD3 bispecific antibody Anti-HER2 S310F / CD3 bispecific antibodies were generated using HER2 S310F monospecific antibodies such as MHR0009, MHR0010, MHR0016, MHR0019, and MHR0060 listed in Table 3 and anti-CD3 antibodies using conventional methods published in WO2015046467A1 or Sci Rep. 2017 Apr 3;7:45839. The sequences of the HER2 S310F binding arms in the anti-HER2 S310F / CD3 bispecific antibodies are shown in Table 3. The generated bispecific antibodies contain a silent Fc with reduced affinity for Fcγ receptors.

[0288] Table 3. HER2 S310F binding arms in anti-HER2 S310F / CD3 bispecific antibodies TIFF0007625516000011.tif236163

[0289] The described anti-CD3 antibodies are also used to generate control bispecific antibodies, such as HER2-1 / CD3 and HER2-2 / CD3 and KLH / CD3 bispecific antibodies. The sequences of the wild-type HER2 binding arm or KLH binding arm in the control bispecific antibodies are shown in Table 4. The generated bispecific antibodies contain a silent Fc with reduced affinity for Fcγ receptors.

[0290] Table 4. Wild-type HER2-binding arms or KLH-binding arms in control bispecific antibodies TIFF0007625516000012.tif210123

[0291] Figure 3 shows the T cell activation activity of anti-HER2 S310F / CD3 bispecific antibodies in the presence of FreeStyle 293 transfectants expressing HER2 S310F or wild-type HER2, as determined by the luciferase assay system using GloResponse™ NFAT-luc2 Jurkat cells as effector cells. 20,000 target cells and 100,000 effector cells (E / T=5) seeded in a 96-well white culture plate were incubated with various concentrations of antibodies for 24 hours at 37°C and 5% CO2. Luminescence (RLU) was measured using the Bio-Glo™ Luciferase Assay System (Promega, Cat. G7941), and T cell activation activity was calculated based on the fold change in RLU using the following equation: Fold change in RLU = RLU induced by antibody / RLU induced by no antibody control

[0292] The bispecific antibodies MHR0009 / CD3, MHR0010 / CD3, MHR0016 / CD3, MHR0019 / CD3, and MHR0060 / CD3 induced T cell activation in the presence of FreeStyle 293 transfectants expressing HER2 S310F, but not in transfectants expressing wild-type HER2, demonstrating that T cell activation induced by MHR0009 / CD3, MHR0010 / CD3, MHR0016 / CD3, MHR0019 / CD3, and MHR0060 / CD3 is significantly specific for the HER2 S310F mutant.

[0293] Example 5.2. Measurement of T cell-dependent cytotoxicity of anti-HER2 S310F / CD3 bispecific antibody Figure 4 shows the T cell-dependent cytotoxicity of anti-HER2 S310F / CD3 bispecific antibodies against FreeStyle 293 transfectants expressing HER2 S310F or wild-type HER2. Cytotoxicity was assessed by LDH assay using human PBMCs (StemCell, Cat. 70025). 20,000 target cells and 200,000 human PBMCs (E / T=10) were seeded into each well of a 96-well U-bottom plate and incubated with various concentrations of antibodies for 24 hours at 37°C and 5% CO2. Target cell killing was measured by LDH cytotoxicity detection kit (Takara Bio). The cytotoxicity (%) of each antibody was calculated using the following formula: Cytotoxic activity (%)=(ABC)×100 / (DC) "A" represents the absorbance of wells treated with antibody and PBMC, "B" represents the average absorbance value of effector PBMC only, "C" represents the average absorbance value of untreated target cells only, and "D" represents the average value of wells lysed with Triton-X. Background absorbance has been accounted for and subtracted.

[0294] The bispecific antibodies MHR0009 / CD3, MHR0010 / CD3, MHR0016 / CD3, MHR0019 / CD3, and MHR0060 / CD3 showed cytotoxic activity against FreeStyle 293 transfectants expressing HER2 S310F, but did not induce cytotoxic activity against FreeStyle 293 transfectants expressing wild-type HER2. That is, MHR0009 / CD3, MHR0010 / CD3, MHR0016 / CD3, MHR0019 / CD3, and MHR0060 / CD3 showed T cell-dependent cytotoxic activity against FreeStyle 293 cells that was highly specific for the HER2 S310F mutant.

[0295] FIG. 5 shows the T cell-dependent cytotoxicity activity of anti-HER2 S310F / CD3 bispecific antibodies against 5637 bladder cancer cell line with HER2 S310F (Proc Natl Acad Sci US A. 2012 Sep 4;109(36):14476-81) or NCI-H441 lung cancer cell line with wild-type HER2 as target cells. Cytotoxicity was evaluated by the rate of cell proliferation inhibition using xCELLigence Real-Time Cell Analyzer (ACEA Biosciences). Target cells were seeded at the desired density in E-Plate 96 (ACEA Biosciences) to start measuring cell proliferation over 24 hours, after which 50 microliters (μL) of each test antibody and expanded T cells were added at an E / T ratio of 10. The effector T cells used were isolated from human PBMCs (Stemcell, Cat. 70025) using a T cell isolation kit (Stemcell, Cat. 17951) and activated with CD3 / CD28 beads (Invitrogen, Cat. 1132D). Expanded T cells were harvested at the end of 7 days of culture. Cell proliferation was monitored after 92 hours of treatment at 37°C and 5% CO2. Cytostatic inhibition (CGI) rate (%) was determined using the following equation: The cell index value obtained from the xCELLigence Real-Time Cell Analyzer used in the calculation was a normalized value, where the cell index value at the time point immediately before the addition of the antibody was defined as 1. CGI rate (%)=(AB)×100 / (A-1) A represents the mean Cell Index value in wells without antibody treatment (containing only target cells and human T cells) and B represents the mean Cell Index value of target wells. Studies were performed in duplicate.

[0296] The bispecific antibodies MHR0009 / CD3, MHR0010 / CD3, MHR0016 / CD3, MHR0019 / CD3, and MHR0060 / CD3 showed cytotoxic activity against the 5637 cell line (Figure 5). In particular, MHR0009 / CD3 and MHR0010 / CD3 showed the strongest cytotoxic activity against the 5637 cell line. On the other hand, none of them showed cytotoxic activity against the H441 cell line (Figure 5). That is, it was demonstrated that the T cell-dependent cytotoxic activity against bladder cancer cells exhibited by MHR0009 / CD3, MHR0010 / CD3, MHR0016 / CD3, MHR0019 / CD3, and MHR0060 / CD3 was significantly specific to the HER2 S310F mutant.

[0297] Example 5.3. Affinity measurements of anti-HER2 S310F / CD3 bispecific antibodies The binding affinity of anti-HER2 S310F / CD3 bispecific antibodies binding to human HER2 S310F was determined at 37 °C using a Biacore 8K instrument (GE Healthcare). Anti-human Fc (GE Healthcare) was immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). All antibodies and analytes were prepared in ACES pH 7.4 containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3. Each antibody was captured on the sensor surface by anti-human Fc. The antibody capture level aimed for 200 resonance units (RU). Recombinant human HER2 S310F was injected at 400–25 nM prepared by 2-fold serial dilutions and then dissociated. The sensor surface was regenerated with 3 M MgCl2 for each cycle. Binding affinities were determined by processing the data and fitting to a 1:1 binding model using Biacore 8K Evaluation software (GE Healthcare). Binding specificity binding to wild-type HER2 was assessed as above by injection of recombinant wild-type HER2 at 400-25 nM prepared by two-fold serial dilutions. The binding affinities of the anti-HER2 S310F / CD3 bispecific antibody, as well as wild-type HER2 / CD3 bispecific antibodies such as HER2-1 / CD3 and HER2-2 / CD3 as controls, to recombinant HER2 S310F and wild-type HER2 are shown in Table 5.

[0298] Table 5. Affinity of bispecific antibodies to human HER2 S310F or wild-type HER2 TIFF0007625516000013.tif75166

[0299] In Table 5, "nd" means that the binding response is too low and therefore the KD cannot be determined based on it. For MHR0009 / CD3, MHR0010 / CD3, MHR0016 / CD3, MHR0019 / CD3, and MHR0060 / CD3, the KD value for wild-type HER2 cannot be calculated for this reason. This indicates that the specificity of these bispecific antibodies for the HER2 S310F mutant is remarkably high.

[0300] The foregoing invention has been described in some detail by way of illustration and illustration, for purposes of clarity of understanding, but the illustrations and illustrations should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entireties. [Industrial Applicability]

[0301] The present invention provides an antigen-binding molecule that exhibits high specificity and binding activity to HER2 S310F and has low or no cross-reactivity to wild-type HER2. Such mutant-specific anti-HER2 antigen-binding molecules can be developed as promising agents that demonstrate excellent specificity (i.e., minimal off-target toxicity) to tumor cells expressing tumor-specific mutations, such as for cancer treatment and / or diagnosis.

[0302] The present invention further provides a novel multispecific antigen-binding molecule that has strong antitumor activity, an excellent safety profile that does not induce cytokine storms or the like independent of cancer antigens, and has a long half-life in blood. A cytotoxic activity inducer containing the antigen-binding molecule of the present invention as an active ingredient can target HER2 S310F-expressing cells and tumor tissues containing these cells and induce cell damage. Administering the multispecific antigen-binding molecule of the present invention to patients not only has a high level of safety, but also enables desirable treatment that reduces physical burden and is very convenient.

Claims

1. An antigen-binding molecule comprising a first antigen-binding domain that specifically binds to human HER2 having an S310F mutation (HER2 S310F) but does not bind to wild-type human HER2, wherein the antigen-binding molecule is a monoclonal antibody.

2. The antigen-binding molecule of claim 1, which inhibits ligand-independent dimerization of human HER2 S310F.

3. The antigen-binding molecule of claim 1 or 2, further comprising a second antigen-binding domain that specifically binds to a T cell receptor complex molecule.

4. The antigen-binding molecule of claim 3, wherein the T cell receptor complex molecule is CD3, preferably the human CD3ε chain.

5. The antigen-binding molecule of claim 3 or 4, which is a multispecific antigen-binding molecule, preferably a bispecific antibody.

6. The antigen-binding molecule according to any one of claims 3 to 5, further comprising an Fc domain having reduced Fcγ receptor binding activity compared to a wild-type Fc domain of IgG1.

7. The antigen-binding molecule according to any one of claims 3 to 6, having T cell-dependent cytotoxicity (TDCC).

8. The antigen-binding molecule of any one of claims 1 to 7, wherein the first antigen-binding domain and / or the second antigen-binding domain comprises a Fab, Fv, scFv, or VHH structure.

9. The antigen-binding molecule according to any one of claims 1 to 8, wherein the first antigen-binding domain and / or the second antigen-binding domain comprises a Fab structure.

10. The antigen-binding molecule of any one of claims 1 to 9, wherein the first antigen-binding domain comprises any one of the following antibody variable regions: (a1) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 29, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 41, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 53, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 65, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 77, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 89; (a2) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 30, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 42, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 54, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 66, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 78, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 90; (a3) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 31, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 43, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 67, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 79, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 91; (a4) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 44, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 56, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 68, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 80, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 92; (a5) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 33, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 45, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 57, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 69, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 81, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 93; (a6) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 46, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 58, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 70, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 82, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 94; (a7) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 35, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 47, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 59, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 71, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 83, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 95; (a8) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 36, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 60, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 72, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 84, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 96; (a9) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 37, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 49, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 61, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 73, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 85, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 97; (a10) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 38, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 50, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 62, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 74, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 86, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 98; (a11) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 39, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 51, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 63, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 75, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 87, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 99; (a12) an antibody variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 40, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 52, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 64, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 76, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 100; (b1) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 5, and a light chain variable domain (VL) of SEQ ID NO: 17; (b2) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 6, and a light chain variable domain (VL) of SEQ ID NO: 18; (b3) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 7, and a light chain variable domain (VL) of SEQ ID NO: 19; (b4) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 8, and a light chain variable domain (VL) of SEQ ID NO: 20; (b5) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 9, and a light chain variable domain (VL) of SEQ ID NO: 21; (b6) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 10, and a light chain variable domain (VL) of SEQ ID NO: 22; (b7) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 11, and a light chain variable domain (VL) of SEQ ID NO: 23; (b8) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 12, and a light chain variable domain (VL) of SEQ ID NO: 24; (b9) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 13, and a light chain variable domain (VL) of SEQ ID NO: 25; (b10) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 14, and a light chain variable domain (VL) of SEQ ID NO: 26; (b11) an antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 15, and a light chain variable domain (VL) of SEQ ID NO: 27; (b12) An antibody variable region comprising a heavy chain variable domain (VH) of SEQ ID NO: 16, and a light chain variable domain (VL) of SEQ ID NO:

28.

11. The antigen-binding molecule of any one of claims 1 to 10 for use in the treatment, prevention, or diagnosis of cancer.

12. The antigen-binding molecule for use according to claim 11 , wherein the cancer is characterized by expression of HER2 S310F.

13. The antigen-binding molecule for use according to claim 11 or 12, wherein the cancer is bladder cancer, cervical cancer, colorectal cancer (CRC), non-small cell lung cancer (NSCLC), esophageal cancer, head and neck cancer, skin cancer (melanoma), bile duct cancer, kidney cancer, gastric cancer, small intestine cancer, liver cancer, uterine cancer, duodenal cancer, breast cancer, or gallbladder cancer.

14. A method for preparing the antigen-binding molecule according to any one of claims 1 to 13, comprising expressing a nucleic acid encoding the antigen-binding molecule in an ex vivo cell.

Images