Composition comprising Anti-mutant CALR antibody and method for producing antibody

Abstract

The problem addressed by the present invention is to produce a novel antibody recognizing a mutant CALR protein at a site distinct from the sites recognized by conventional antibodies, and to provide pharmaceuticals using the same. The present invention provides a composition for treating a tumor, the composition comprising an antibody or an antigen-binding fragment thereof that binds to a region N-terminal to a mutation site in a mutant calreticulin and binds to the mutant calreticulin present on the cell surface. The present invention also provides a method for producing an antibody that binds to a region N-terminal to a mutation site in a mutant calreticulin, said method comprising a step for screening: an antibody that binds to the mutant calreticulin present on a cell surface; or an antibody that binds to a dimer of the mutant calreticulin.

Description

Compositions containing anti-mutant CALR antibodies and methods for producing the antibodies

[0001] This application claims priority from Japanese Patent Application No. 2024-108582, filed July 5, 2024, the entire disclosure of which is expressly incorporated herein by reference.

[0002] The present invention relates to a composition for treating tumors, comprising an antibody or an antigen-binding fragment thereof that binds to the N-terminal side of a mutation site in mutant calreticulin and binds to the mutant calreticulin on a cell surface. The present invention also relates to a method for producing an antibody that binds to the N-terminal side of a mutation site in mutant calreticulin, the method comprising the step of screening for an antibody that binds to mutant calreticulin on a cell surface or an antibody that binds to a mutant calreticulin dimer.

[0003] Some patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) harbor base deletions (Del52 type) or insertions (Ins5 type) in exon 9 of the calreticulin (CALR) gene (M Araki et al. Homomultimerization of mutant calreticulin is a prerequisite for MPL binding and activation. Leukemia (2019) 33:122-131.; N Papadopoulos et al. Oncogenic CALR mutant C-terminus mediates dual binding to the thrombopoietin receptor triggering complex dimerization and activation. Nature Communications, 2023.14:1881.). CALR gene mutations found in MPN patients are always frameshift mutations at a localized location in the final exon, and the amino acid reading frame shift due to the frameshift mutation is always +1. Based on these findings, the C-terminus of mutant CALR proteins contains a sequence not present in the wild-type, and in particular, the C-terminal 44 amino acids are common to almost all mutant CALR proteins (WO 2015 / 036599). Furthermore, mutant CALR proteins produced from CALR mutant genes bind to thrombopoietin (TPO) receptors intracellularly and are presented on the cell surface together with the TPO receptor. Furthermore, binding of mutant CALR proteins to the TPO receptor constitutively activates the TPO receptor, which is believed to have tumorigenic properties that cause myeloproliferative neoplasms (MPNs). This suggests that sequences specific to mutant CALR proteins may serve as diagnostic markers or therapeutic targets (WO 2016 / 087514, WO 2019 / 178362).

[0004] It is known that many of the mutant CALR proteins that arise due to the frameshift mutation are truncated within the sequence of the C domain of the mutant CALR protein and exist as truncated forms (M. Araki et al., supra). WO 2020 / 175689 discloses an antibody that recognizes an amino acid sequence common to truncated mutant CALR proteins present in the C domain. WO 2022 / 045247 discloses a bispecific antibody having a first domain that specifically binds to a mutant CALR protein and a second domain that specifically binds to the CD3 antigen, and that this bispecific antibody has cytotoxic activity against cells expressing the mutant CALR protein.

[0005] Mutation patterns of the CALR gene include Ins5 type and Del52 type, and the three-dimensional structures of Ins5 type or Del52 type mutant CALR proteins and the TPO receptor have recently been predicted. It has been suggested that the N domain of these mutant CALR proteins binds to the TPO receptor via immature N-glycans, and that the sequence specific to the mutant CALR proteins present on the C-terminus is embedded inside the TPO receptor (N Papadopoulos et al., supra).

[0006] However, antibodies that bind to the mutation site of a mutant CALR protein include antibodies with insufficient binding affinity or cytotoxic activity, and there has been a need to produce an antibody that recognizes a mutant CALR protein using an approach different from conventional methods. Therefore, an object of the present invention is to produce a novel antibody that recognizes a site different from that of conventional antibodies that recognize a mutant CALR protein, and to provide a pharmaceutical using the same.

[0007] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that by screening antibodies that bind to mutant CALR proteins on the cell surface among antibodies that bind to the N-terminal side of the mutation site in mutant CALR, it is possible to produce anti-mutant CALR antibodies that bind to mutant CALR proteins and exhibit cytotoxic activity regardless of the type of mutation, and have completed the present invention. Specifically, the present invention relates to a composition containing the specific antibodies shown below and a method for producing the antibodies.

[0008] The present invention provides the following: [1] A composition for treating a tumor, comprising an antibody or an antigen-binding fragment thereof that binds to the N-terminal side of the mutation site in mutant calreticulin and binds to the mutant calreticulin on a cell surface. [2] The composition according to [1], wherein the antibody or antigen-binding fragment thereof binds to the N-terminal side of the mutation site in the N domain, P domain, or C domain of mutant calreticulin. [3] The antibody or antigen-binding fragment thereof has a binding affinity (K Ｄ[4] The composition according to [1] or [2], which binds to mutant calreticulin at the N-terminal side of the mutation site of mutant calreticulin. [4] A method for producing an antibody that binds to mutant calreticulin on the cell surface or to a mutant calreticulin dimer, comprising a step of screening for an antibody that binds to the N-terminal side of the mutation site of the mutant calreticulin. [5] The method for producing an antibody according to [4], which comprises an additional screening step of selecting an antibody that binds to a polypeptide comprising the N-terminal side of the mutation site of mutant calreticulin in the screening step, or selecting an antibody that binds to a polypeptide comprising the N-terminal side of the mutation site of mutant calreticulin. [6] The method for producing an antibody according to [4] or [5], which further comprises a step of immunizing an animal with a polypeptide comprising the N-terminal side of the mutation site of mutant calreticulin. [7] A method for treating a tumor in a subject, comprising administering to the subject an antibody or an antigen-binding fragment thereof that binds to the N-terminal side of the mutation site of mutant calreticulin and binds to the mutant calreticulin on the cell surface. [8] An antibody or an antigen-binding fragment thereof that binds to the N-terminal side of a mutation site in a mutant calreticulin and binds to the mutant calreticulin on a cell surface, for use in treating a tumor. [9] Use of an antibody or an antigen-binding fragment thereof that binds to the N-terminal side of a mutation site in a mutant calreticulin and binds to the mutant calreticulin on a cell surface, in the manufacture of a medicament for treating a tumor.

[0009] FIG. 1 is a schematic diagram of the CALR protein. The amino acid sequences of the C-terminal side of the C domain containing the frameshift mutation site are shown for wild-type (WT), mutant Ins5, and Del52. WT: amino acid sequence from positions 347 to 400 of SEQ ID NO: 1; mutant Ins5: amino acid sequence from positions 347 to 413 of SEQ ID NO: 2; Del52: amino acid sequence from positions 347 to 394 of SEQ ID NO: 3. The starting points of the arrows on mutant Ins5 and Del52 indicate the frameshift mutation site. FIG. 2 is a histogram of flow cytometry analysis evaluating the binding of the anti-calreticulin antibody (hereinafter referred to as anti-CALR antibody) obtained in Example 1 to wild-type CALR protein localized in cells. FIG. 3 is a histogram of flow cytometry analysis evaluating the binding of the anti-CALR antibody obtained in Example 1 to cells transfected with mutant CALR proteins or control cells transfected with a vector. Figure 4 shows the binding affinity (K ) of seven anti-mutated CALR antibodies confirmed to bind to mutant CALR proteins in Example 3 to UT-7 / TPO / Ins5 cells (A and B) or UT-7 / TPO / Del52 cells (C and D). Ｄ Figure 5 shows a binding saturation curve for determining the binding activity (GeoMFI) of UT-7 / TPO / Ins5 cells and UT-7 / TPO / Del52 cells (value). The vertical axis represents the geometric mean fluorescence intensity (GeoMFI), and the horizontal axis represents the antibody concentration. The GeoMFI of UT-7 / TPO / Ins5 cells and UT-7 / TPO / Del52 cells was plotted against the antibody concentration after subtracting the nonspecific binding to UT-7 / TPO / vec cells. Figure 5 shows a graph showing the cytotoxic activity of T cells induced by a T cell-inducing bispecific antibody having a mutant CALR recognition portion against (A) UT-7 / TPO / Ins5 cells, (B) UT-7 / TPO / Del52 cells, or (C) UT-7 / TPO / vec cells. Figure 6 shows a graph showing the results of epitope mapping. The binding ability of each antibody, CAL3004 (C), CAL3010 (D), and CAL3012 (E), to each of the synthetic peptides 1 to 35 was evaluated by ELISA. Diluent (A) and IgG (B) were used as negative controls.

[0010] The following description of the present invention may be based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits. In this specification, the amino acid positions of an amino acid sequence are counted from the N-terminus to the C-terminus, with the N-terminal residue of the amino acid sequence shown in the specified SEQ ID NO: being position 1. For example, position 100 refers to the 100th amino acid residue from the N-terminus.

[0011] Calreticulin is a calcium-binding protein present in the endoplasmic reticulum (ER) and is known to be involved in a variety of functions, such as maintaining calcium homeostasis within the ER, assisting protein folding, and regulating immune responses. In humans, it is encoded by the CALR gene. Calreticulin consists of an N domain, a P domain, and a C domain, and the wild-type calreticulin has an endoplasmic reticulum retention signal sequence, KDEL, at its C-terminus (Figure 1). In the amino acid sequence of wild-type calreticulin shown in SEQ ID NO: 1, the N domain is located at positions 1-180 (SEQ ID NO: 4), the P domain is located at positions 181-291 (SEQ ID NO: 5), and the C domain is located at positions 292-400.

[0012] The Del52 type (52-base deletion type; Type 1: c.1092_1143del) of the CALR gene mutation is the most common in MPN patients. The next most common mutation is the Ins5 type (5-base insertion type; Type 2: c.1154_1155insTTGTC). These two CALR mutations have been reported to account for 80% of all CALR mutations (Marito Araki and Norio Komatsu, Cancer Sci 108 (2017) 1907-1912, doi: 10.1111 / cas.13327). The C-terminal end of the mutant calreticulin Del52 and Ins5 types contains a novel sequence that differs from wild-type calreticulin (Figure 1).

[0013] As used herein, "calreticulin" and "CALR protein" are used interchangeably. A wild-type calreticulin or wild-type CALR protein refers to a wild-type calreticulin, and a mutant calreticulin or mutant CALR protein refers to a mutant calreticulin or mutant CALR protein. As used herein, mutant calreticulin or mutant CALR protein refers to any mutant type, without being limited to the Del52 or Ins5 type mutant types.

[0014] An antibody typically has a structure in which two heavy chains (H chains) and two light chains (L chains) are bound together. These light and heavy chains are linked by disulfide bonds (SS bonds) to form a heterodimer, and two such heterodimers are further linked to form a Y-shaped heterotetramer. Typically, the heavy chain consists of a heavy chain variable region VH, heavy chain constant regions CH1, CH2, and CH3, and a hinge region located between CH1 and CH2, while the light chain consists of a light chain variable region VL and a light chain constant region CL. The variable regions include complementarity-determining regions (CDRs) and framework regions (FRs). The light chain and heavy chain variable regions each contain three CDRs (heavy chain CDR1-3 and light chain CDR1-3) and four FRs (heavy chain FR1-4 and light chain FR1-4). Methods for identifying CDRs are known, and can be found, for example, in the IMGT / V-QUEST Search page (https: / / www.imgt.org / IMGT_vquest / input), Brochet, X. et al., Nucl. Acids Res. 36, W503-508 (2008), and Giudicelli, V., Brochet, X., Lefranc, M.-P., Cold Spring Harb Protoc. 2011 Jun 1;2011(6). pii: nb.prot5633. doi: 10.1101 / pdb.prot5633. PMID: 21632778. Any other means known in the art can also be used as long as similar results are obtained (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institutes of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342: 877). The antibody may be of any type (e.g., immunoglobulin (Ig) G, IgE, IgM, IgD, IgA, and IgY).

[0015] As used herein, examples of antibody fragments include Fab and F(ab').２ Examples of F(ab') include, but are not limited to, Fv, scFv, and the like. Fab refers to an antibody fragment in which the VL-CL and VH-CH1 domains are linked by a disulfide bond. F(ab') ２ refers to an antibody fragment in which two Fab fragments are linked by a disulfide bond at the hinge region. An Fv fragment consists of a VL and a VH. An scFv is a fusion protein in which a VH and a VL are connected by a linker peptide (approximately 10 to 25 amino acids in length). An antibody fragment may be an antigen-binding fragment.

[0016] A multispecific antibody contains at least two antigen-recognition moieties, which bind to different epitopes (antigenic determinants). "Bispecific" means that the antibody specifically binds to two different epitopes.

[0017] As used herein, "recognize," when used in the context of an antigen and an antibody, refers to an antibody or antigen-binding fragment thereof interacting with (e.g., binding to) an antigen or epitope. An epitope may be linear or conformational. An "epitope" refers to the site on an antigen to which an antibody or antigen-binding fragment of the present invention binds. An epitope can be formed from contiguous amino acids or noncontiguous amino acids arranged by tertiary folding of a protein. An epitope typically contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. To identify the binding epitope of an antibody, epitope mapping methods based on the structure of the antigen protein can be used. Epitope mapping is a technique for identifying the amino acid sequence region of an antigen recognized by an antibody, and includes synthetic peptide scanning, alanine scanning mutagenesis, truncation mutant analysis, and the like. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography, cryo-electron microscopy (cryo-EM), and two-dimensional nuclear magnetic resonance analysis. See, for example, "Epitope Mapping Protocols in Methods in Molecular Biology," Vol. 66, G. E. Morris, Ed. (1996).

[0018] As used herein, the term "specifically bind" means selectively binding to a specific antigen and can be distinguished from non-specific interactions. The binding ability of an antigen-binding molecule to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR) techniques, etc.

[0019] (Composition) The present invention is a composition for treating tumors, comprising an antibody or an antigen-binding fragment thereof that binds to the N-terminal side of the mutation site in mutant calreticulin and binds to the mutant calreticulin on the cell surface.

[0020] The compositions of the present invention can be used to treat tumors. Examples of tumors that can be treated with the compositions of the present invention include, but are not limited to, tumors caused by mutant calreticulin, more specifically, tumors caused by frameshift mutations in exon 9 of the CALR gene. In certain embodiments, tumors caused by mutant calreticulin include myeloproliferative neoplasms (MPNs), particularly Philadelphia chromosome-negative MPNs. MPNs are diseases characterized by excessive growth or proliferation of hematopoietic cells in the bone marrow or by the excessive proliferation of fibrous tissue, which pushes hematopoietic cells out of the bone marrow. MPN patients to be treated may have essential thrombocythemia (ET) and primary myelofibrosis (PMF).

[0021] The antibodies and antigen-binding fragments thereof contained in the compositions of the present invention (hereinafter referred to as the antibodies and antigen-binding fragments thereof of the present invention) bind to the N-terminal side of the mutation site of mutant calreticulin and can bind to the mutant calreticulin on the cell surface, thereby therapeutically targeting cells expressing mutant calreticulin.

[0022] The mutation site of mutant calreticulin refers to a site where a mutation has occurred in the amino acid sequence compared to wild-type calreticulin. The mutation site can be identified by aligning the amino acid sequences of mutant calreticulin and wild-type calreticulin. For the alignment, for example, Clustal W or Clustal X version 2.0 (Larkin, MA, et al., (2007) Bioinformatics, 23:2947-2948.) can be used. For example, mutant calreticulin Ins5 type differs from wild-type calreticulin on the C-terminal side from amino acid position 368 onwards in the amino acid sequence shown in SEQ ID NO: 2. Mutant calreticulin Del52 type differs from wild-type calreticulin on the C-terminal side from amino acid position 350 onwards in the amino acid sequence shown in SEQ ID NO: 3. For mutant calreticulin other than the Ins5 type and Del52 type, the mutation site can be identified by alignment with wild-type calreticulin, and the N-terminal side of the mutation site can be determined.

[0023] It has been suggested that mutant calreticulin forms a homodimer and is presented on the cell surface together with the TPO receptor (M. Araki et al., supra), that the N domain binds to the TPO receptor via an immature N-glycan, and that a sequence specific to mutant calreticulin present on the C-terminus is embedded inside the TPO receptor (N. Papadopoulos et al., supra). That is, in one embodiment, the mutant calreticulin may have a frameshift mutation in, for example, the C domain that forms a homodimer, and as a result, bind to the TPO receptor and be presented on the cell surface.

[0024] For example, the antibody of WO 2020 / 175689 recognizes the mutation site C-terminal to the mutation site, but it is believed that this portion is not exposed to the outside, making it difficult or impossible for the antibody to bind. Without being bound by any particular theory, the antibody or antigen-binding fragment thereof of the present invention binds to the N-terminal side of the mutation site in mutant calreticulin, and this portion does not extend inside the TPO receptor. Therefore, it is believed that the antibody or antigen-binding fragment thereof is less susceptible to steric hindrance by the TPO receptor and can easily bind to mutant calreticulin. Furthermore, the antibody or antigen-binding fragment thereof of the present invention binds to mutant calreticulin on the cell surface, and is believed to bind to a site that is less susceptible to steric hindrance even when mutant calreticulin forms a homodimer.

[0025] In the Examples described below, it is shown that the antibodies or antigen-binding fragments of the present invention bind to mutant calreticulin and exhibit cytotoxic activity regardless of the type of mutation. The compositions of the present invention can treat tumors caused by mutant calreticulin regardless of the type of mutation. In one embodiment, the compositions of the present invention can treat any tumor that has mutant calreticulin that is displayed on the cell surface by binding to a TPO receptor, for example, can treat both Ins5-associated tumors and Del52-associated tumors of mutant calreticulin.

[0026] In one embodiment, the mutant calreticulin has a mutation in the C domain, and the antibody or antigen-binding fragment thereof of the present invention can bind to the N-terminal side of the mutation site in the N-domain, P-domain, or C-domain of the mutant calreticulin. It has been suggested that the portion N-terminal to the mutation site in the C-domain does not extend into the TPO receptor (N Papadopoulos et al., supra).

[0027] In one embodiment, the mutant calreticulin has a mutation at position 350 or later of the amino acid sequence shown in SEQ ID NO: 1 (e.g., position 368 or 350 of the amino acid sequence shown in SEQ ID NO: 1), and the antibodies and antigen-binding fragments thereof of the present invention can bind to any of the portions of positions 1 to 367 or positions 1 to 349 (the N-terminal side of the mutation site) of the amino acid sequence shown in SEQ ID NO: 1. More specifically, the antibodies and antigen-binding fragments thereof may bind to any of the portions of positions 1 to 180, or any of the portions of positions 171 to 292 or positions 181 to 291 of the amino acid sequence shown in SEQ ID NO: 1.

[0028] The epitope of mutant calreticulin recognized by the antibody and its antigen-binding fragment of the present invention can be identified, for example, by epitope mapping. In epitope mapping, for example, a set of peptides approximately 20 amino acids in length is designed and synthesized to encompass the sequence N-terminal to the mutation site of the CALR protein (the portion corresponding to positions 1 to 349 of SEQ ID NO: 1) and to ensure that the amino acid sequences of the peptides partially overlap. This peptide set can then be used to screen the binding ability of the antibody and its antigen-binding fragment of the present invention. In the Examples described below, 35 types of peptides were synthesized and used for screening. The synthetic peptides used in the Examples consist of the amino acid sequences set forth in any one of SEQ ID NOs: 132 to 166. Epitope mapping analysis using synthetic peptides can particularly identify linear epitopes. In addition, when an antibody that binds to the N-terminal side of the mutation site in the CALR protein cannot identify a linear epitope by epitope mapping analysis, it is considered that the antibody recognizes a three-dimensional structure formed on the N-terminal side of the mutation site in the CALR protein, i.e., the epitope of the antibody is conformational.

[0029] Epitope mapping can be used to demonstrate that the antibodies and antigen-binding fragments of the present invention specifically bind to a peptide consisting of an amino acid sequence having at least 80%, 90%, or 95% sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 132 to 166, preferably to a peptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 132 to 166. It can also be shown that the antibodies and antigen-binding fragments of the present invention recognize an epitope comprising or consisting of 3 to 6, 3 to 7, 3 to 8, or 3 to 9 residues in the amino acid sequence set forth in any one of SEQ ID NOs: 132 to 166. It can also be shown that the antibodies and antigen-binding fragments of the present invention bind to a continuous or discontinuous epitope in the amino acid sequence set forth in any one of SEQ ID NOs: 132 to 166. The antibodies and antigen-binding fragments thereof of the present invention can be shown to recognize an epitope comprising or consisting of at least 3, 4, 5, 6, 7, 8, or 9 consecutive bases in the amino acid sequence set forth in any one of SEQ ID NOs: 132 to 166.

[0030] In one embodiment, the antibody or antigen-binding fragment thereof of the invention has a binding affinity (K Ｄ ) can bind to mutant calreticulin. As described above, the antibody or antigen-binding fragment thereof of the present invention binds to a portion that is not susceptible to steric hindrance, and is therefore thought to be able to bind to mutant calreticulin with such high binding affinity. The binding affinity of the antibody or antigen-binding fragment thereof to a specific antigen can be determined by measuring the dissociation constant K Ｄ It can be determined by calculating

[0031] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is a multispecific antibody, such as a bispecific antibody, or an antibody-drug conjugate. A multispecific antibody, such as a bispecific antibody, can be, for example, a T cell engager, and can attract T cells to target cells to kill them. The antibody-drug conjugate comprises an antibody or antigen-binding fragment thereof linked via a linker to a toxin, such as maytansines, hemiasterlins, amanitins, auristatins, calicheamicins, or duocarmycins, and can deliver the toxin payload to target cells to kill them.

[0032] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention may kill target cells through antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. Furthermore, the antibody or antigen-binding fragment thereof of the present invention may comprise a defucosylated Fc domain. Antibodies comprising a defucosylated Fc domain can induce enhanced antibody-dependent cellular cytotoxicity (ADCC) effect against target cells.

[0033] In certain embodiments, a composition for treating tumors of the present invention comprises an anti-mutated CALR antibody or an antigen-binding fragment thereof comprising any of the sets of VH and VL domains comprising the CDR amino acid sequences listed in Table 2 below. In certain embodiments, a composition for treating tumors of the present invention comprises a multispecific antibody, such as a bispecific antibody, having a portion that recognizes mutant calreticulin, wherein the portion that recognizes mutant calreticulin comprises any of the sets of VH and VL domains comprising the CDR amino acid sequences listed in Table 2 below.

[0034] (Method for producing antibodies) The present invention is a method for producing antibodies that bind to the N-terminal side of the mutation site of mutant calreticulin, comprising a step of screening for antibodies that bind to mutant calreticulin on the cell surface or antibodies that bind to a mutant calreticulin dimer.

[0035] Because the N-terminal region of the mutation site in mutant calreticulin typically contains a wild-type sequence, it has not previously been considered a target region for treating tumors caused by mutant calreticulin. However, the present inventors have discovered that the N-terminal region of the mutation site in mutant calreticulin can be a target region for tumor therapy. Mutant calreticulin forms a dimer and is presented on the surface of tumor cells. The N-terminal region of the mutation site in mutant calreticulin is presumed to be less susceptible to steric hindrance. Therefore, by screening for antibodies that bind to mutant calreticulin on the cell surface or to a dimer of mutant calreticulin, it is possible to produce antibodies that bind to the N-terminal region of the mutation site in mutant calreticulin. These antibodies can be used to treat tumors caused by mutant calreticulin, regardless of the type of mutation.

[0036] The screening step defined by the present invention (a step of screening for antibodies that bind to mutant calreticulin on the cell surface or to a mutant calreticulin dimer) can be carried out by methods commonly used in the art without particular limitation, and may include, for example, evaluating candidate antibodies for binding to cells expressing mutant calreticulin on their cell surface and selecting antibodies that bind to the cells, or selecting antibodies that bind to a plate on which a mutant calreticulin dimer has been immobilized. Cells that express mutant calreticulin on their cell surface can be prepared, for example, using the human megakaryoblastic leukemia cell line UT-7 / TPO (Komatsu N. et al., Blood 87:4552-4560(1996)) described in WO 2020 / 175689. UT-7 / TPO / CALR Del52 cells (herein referred to as UT-7 / TPO / Del52 cells) and UT-7 / TPO / CALR Ins5 cells (herein referred to as UT-7 / TPO / Ins5 cells) described in WO 2020 / 175689 are cells in which the Del52 or Ins5 form of UT-7 / TPO is expressed, respectively, and grow in a neoplastic manner. In UT-7 / TPO / Del52 cells and UT-7 / TPO / Ins5 cells, mutant calreticulin forms a homodimer, and it is presumed that the polypeptide N-terminal to the mutation site of the mutant calreticulin is exposed on the outside of the TPO receptor on the cell surface. By screening candidate antibodies for their binding activity to both UT-7 / TPO / Del52 cells and UT-7 / TPO / Ins5 cells, it is possible to obtain anti-mutated CALR antibodies that bind to mutant calreticulin regardless of the type of mutation. In another embodiment, in the screening step, the binding ability of candidate antibodies can also be evaluated using cells collected from a patient that express mutant calreticulin on their cell surface.

[0037] In one embodiment, the method for producing an antibody of the present invention can include (a) selecting an antibody that binds to a polypeptide comprising a region N-terminal to the mutation site in mutant calreticulin in the screening step, or (b) an additional screening step of selecting an antibody that binds to a polypeptide comprising a region N-terminal to the mutation site in mutant calreticulin. That is, a one-step screening step (embodiment a) can be employed in which antibodies that bind to mutant calreticulin on the cell surface or antibodies that bind to a mutant calreticulin dimer are screened to select an antibody that binds to a polypeptide comprising a region N-terminal to the mutation site in mutant calreticulin. Alternatively, a multi-step screening step (embodiment b) can be employed before or after the screening step (particularly before the screening step) to select an antibody that binds to a polypeptide comprising a region N-terminal to the mutation site in mutant calreticulin. The additional screening step may include, for example, evaluating B cells obtained from the immunized animal, hybridomas prepared therefrom, or their culture supernatants for binding to a polypeptide comprising the N-terminal side of the mutation site in mutant calreticulin (hereinafter sometimes referred to as the "target polypeptide"), and selecting B cells, hybridomas, or culture supernatants that test positive for binding to the target polypeptide. The additional screening step may also include biopanning (affinity selection), which involves incubating phages displaying an antigen recognition site prepared by a phage display method with the target polypeptide immobilized on a plastic surface such as an immunotube or magnetic beads, and then washing away phages that do not bind to the target polypeptide. The additional screening step may also include evaluating candidate antibodies prepared from B cells, hybridomas, culture supernatants, or phages selected as a result of another screening for binding to the target polypeptide, and selecting antibodies that test positive for binding to the target polypeptide. Each screening may be performed using ELISA (enzyme-linked immunosorbent assay), flow cytometry, or the like.

[0038] In one embodiment, the mutant calreticulin has a mutation at position 350 or later in the amino acid sequence set forth in SEQ ID NO: 1. Thus, a polypeptide containing the N-terminal side of the mutation site of mutant calreticulin, which is used as a target polypeptide for screening, comprises the amino acid sequence of positions 1 to 349 of SEQ ID NO: 1, and may be a full-length or fragment thereof. In a specific embodiment, the polypeptide comprises at least 5 to 349 contiguous amino acid residues of the amino acid sequence of positions 1 to 349 of SEQ ID NO: 1, for example, at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 300 contiguous amino acid residues. The target polypeptide used in each screening may be the same, or a fragment of the target polypeptide used in a previous screening may be used in a subsequent screening. The target polypeptide for screening may be the same as the immunogenic polypeptide used for animal immunization, which will be described later, or may be a fragment of the immunogenic polypeptide.

[0039] In one embodiment, the method for producing an antibody of the present invention further comprises the step of immunizing an animal with a polypeptide comprising the N-terminal side of the mutation site of a mutant calreticulin.

[0040] Immunogenic polypeptides can be prepared by purification and isolation (fractionation, chromatography, etc.) from animal tissues (including body fluids), tissue-derived cells, cell cultures, genetic recombination, in vitro protein expression, chemical synthesis (e.g., solid-phase peptide synthesis methods such as Fmoc synthesis and Boc synthesis), etc. Immunogenic polypeptides can also be expressed as fusion proteins with secretion signals, intracellular localization signals, affinity purification tags, or partner peptides.

[0041] The immunogenic polypeptide can comprise the full-length amino acid sequence of the mutant calreticulin N-terminal to the mutation site (corresponding to the amino acid sequence of positions 1 to 349 of SEQ ID NO: 1), or a fragment thereof. In a specific embodiment, the immunogenic polypeptide comprises at least 10 to 349 contiguous amino acid residues of the amino acid sequence of positions 1 to 349 of SEQ ID NO: 1, for example, at least 10 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 300 contiguous amino acid residues.

[0042] The immunogenic polypeptide can be chemically coupled to a carrier protein such as keyhole limpet hemocyanin (KLH), and can be administered to an animal in the presence of an adjuvant.

[0043] The animal to be immunized may be selected from the group including mice, rats, rabbits, sheep, non-human primates, goats, horses, and poultry.

[0044] For example, a method based on single B cell technology can be used to produce antibodies. In antibody production methods based on single B cell technology, B cells are obtained from an animal, such as a rabbit, immunized with an antigen and screened for binding to a target polypeptide. B cells expressing an antibody that recognizes the target polypeptide are isolated into single cells using a cell sorter, the antibody gene is sequenced, and monoclonal antibodies can be produced based on the sequence information. Alternatively, B cells can be isolated from an immunized animal, isolated into single cells in a microchamber, secreted, and screened for binding to the target polypeptide, and the antibody gene can be sequenced from B cells secreting an antibody that recognizes the target polypeptide.

[0045] Antibodies can also be produced using the hybridoma method, phage display method, etc. In the hybridoma method, B cells collected from the spleen or lymph nodes of an animal, particularly a rat or mouse, immunized with an immunogen are fused with immortalized cells, such as myeloma cells, to produce hybridomas, and hybridomas that produce antibodies reactive with the target polypeptide are selected. Monoclonal antibodies can then be produced using the selected hybridomas. Human antibodies can be obtained by using mice transfected with human antibody genes. To obtain monoclonal antibodies from hybridomas, the hybridomas can be cultured according to conventional methods and the culture supernatant obtained, or the hybridomas can be administered to a compatible mammal to grow and the ascites obtained. For details of the hybridoma method, see, for example, Meyaard et al. (1997) Immunity 7:283-290; Wright et al. (2000) Immunity 13:233-242; Kaithamana et al. (1999) J. Immunol. 163:5157-5164.

[0046] Phage display is a technique for selecting antibodies with affinity for a target molecule using a phage antibody library in which antibody variable regions are functionally displayed on phages (see, for example, Smith, GP et al., Science 228:1315-1317, 1985). The antibody genes contained in the phages are sequenced, and monoclonal antibodies can be produced based on the sequence information. Human antibodies with desired binding properties can be generated by using a human antibody library as the phage antibody library.

[0047] (Anti-mutated CALR antibodies) The present invention relates to antibodies or antigen-binding fragments thereof obtained by carrying out the antibody production method of the present invention. As shown in the Examples below, at least 13 types of anti-mutated CALR antibodies were obtained by carrying out the antibody production method of the present invention, and the amino acid sequences of seven types of anti-mutated CALR antibodies are disclosed herein. In one embodiment, the present invention relates to anti-mutated CALR antibodies having the VH and VL amino acid sequences shown in the table below. The nucleotide sequences encoding the VH and VL of each antibody are not particularly limited, and may each be the nucleotide sequences of the SEQ ID NOs listed in the "SEQ ID NO of nucleotide sequence" column in the table below.

[0048] The heavy and light chain CDRs of the above seven anti-mutated CALR antibodies are shown in the table below.

[0049] In one embodiment, the anti-mutated CALR antibody or antigen-binding fragment thereof of the present invention is: (a) an anti-mutated CALR antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable (VH) domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 67, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68, and a light chain variable (VL) domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 69, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 70, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 71; (b) an anti-mutated CALR antibody or an antigen-binding fragment thereof, comprising: a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 72, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 73, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 74, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 75, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 76, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 77; (c) (d) an anti-mutated CALR antibody or antigen-binding fragment thereof, comprising: a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 81, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 82, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 83; (d) an anti-mutated CALR antibody or antigen-binding fragment thereof, comprising: a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 84, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 85, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 86, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 87, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 88, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 89;(e) an anti-mutated CALR antibody or antigen-binding fragment thereof, comprising: a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 90, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 91, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 92, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 93, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 94, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 95; (f) an anti-mutated CALR antibody or antigen-binding fragment thereof, comprising: a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 96, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 97, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 98, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 99, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 100, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 101; An anti-mutated CALR antibody or antigen-binding fragment thereof, comprising: a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 102, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 103, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 104, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 105, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 106, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 107;

[0050] In certain embodiments, the anti-mutated CALR antibody or antigen-binding fragment thereof comprises the following VH domain and VL domain, respectively: (a) a VH domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 10, and a VL domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 11; (b) (c) a VH domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 12, and a VL domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 13; (d) a VH domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 14, and a VL domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 15; (e) comprising a VH domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 16, and a VL domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 17; a VH domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 18, and a VL domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 19;(f) a VH domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 20, and a VL domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 21; (g) a VH domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 22, and a VL domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 23;

[0051] As used herein, "percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with those in the reference polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished by a variety of methods within the skill of one in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0052] In certain embodiments, amino acid sequence variants of the antibody are generated to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants can be generated by introducing appropriate mutations (deletions, insertions, and substitutions) into the nucleotide sequence encoding the anti-mutated CALR antibody or by peptide synthesis. Depending on the properties to be improved, the mutations can be introduced into the complementarity-determining regions (CDRs) or framework regions (FRs) of the antibody. In one embodiment, the mutations are in the CDRs. In one embodiment, the mutations are in the FRs. Amino acid sequence variants are screened for retention or improvement of binding affinity, retention or improvement of antigen specificity, reduction of immunogenicity, etc.

[0053] The antibody or antigen-binding fragment thereof of the present invention can be humanized. Humanization methods are known, and can be performed, for example, by grafting the CDRs in the VH and VL of an antibody derived from a non-human animal between the FRs of the VH and VL of a human antibody. For example, overlap extension PCR can be used as a method for grafting the CDRs of a non-human antibody into the FRs of a human antibody by genetic recombination. In general, when grafting non-human CDRs, such as mouse CDRs, into human FRs, it is considered advantageous to select human FRs that are highly identical to the mouse FRs in order to maintain CDR function. In other words, it is generally preferable to use human FRs whose amino acid sequences are highly identical to the amino acid sequences of the FRs adjacent to the mouse CDR to be grafted.

[0054] The antibody or antigen-binding fragment thereof of the present invention can be chimerized. A chimerized antibody is one in which the variable region of an antibody derived from a non-human animal is linked to the constant region of a human antibody.

[0055] The Examples described below specifically demonstrate that 13 types of anti-mutant CALR antibodies bind to mutant calreticulin regardless of the type of mutation. The anti-mutant CALR antibodies CAL3001, 3002, 3005, 3006, 3008, 3009, 3013, 3014, and 3015 bound to P-Fc, which was designed to induce immunity against the P domain, and are therefore considered to be antibodies that bind to the P domain. The anti-mutant CALR antibodies CAL3004, 3010, 3011, and 3012 did not bind to P-Fc but bound to NPC-His, which contains a sequence N-terminal to the mutation site in the CALR protein (the portion corresponding to positions 1 to 349 in SEQ ID NO: 1). These antibodies are therefore considered to be antibodies that bind to the N domain or the C domain portion contained in NPC-His, or antibodies that recognize a three-dimensional structure formed N-terminal to the mutation site in the CALR protein. Therefore, the anti-mutated CALR antibody defined by the present invention can be used to target cells expressing mutant calreticulin.

[0056] In the Examples described below, epitope mapping analysis was performed on CAL3004, one of the anti-mutant CALR antibodies. In epitope mapping, for example, a set of peptides approximately 20 amino acids in length (synthetic peptide Nos. 1-35, SEQ ID NOs: 132-166) was designed and synthesized to encompass the sequence N-terminal to the mutation site of the CALR protein (the portion corresponding to positions 1-349 of SEQ ID NO: 1), with the amino acid sequences of each peptide partially overlapping. The binding ability of the anti-mutant CALR antibody was evaluated using ELISA. As a result, CAL3004 showed a strong and specific reaction with synthetic peptide No. 30 (corresponding CALR sequence: EYSPDPSIYAYDNFGVLGLD; SEQ ID NO: 161), suggesting that this peptide contains the epitope of CAL3004. CAL3004 reacted strongly and specifically ... The CAL3004 antibody did not react with synthetic peptides No. 29 or No. 31, which contain amino acid sequences overlapping by 10 residues with the amino acid sequence of SEQ ID NO: 30, suggesting that the epitope of CAL3004 is a sequence near the center of synthetic peptide No. 30. For example, it was predicted that the epitope region is contained in the DPSIYAYDNFG (SEQ ID NO: 167) portion, which is the central portion of the amino acid sequence set forth in SEQ ID NO: 161, from positions 5 to 15. This region spans the P and C domains of CALR.

[0057] Furthermore, epitope mapping analysis was performed on the CAL3005 and CAL3011 antibodies in the same manner as described above. The results showed that CAL3005 exhibited binding affinity to synthetic peptides No. 29-32, with a particularly strong and specific reactivity to No. 31 (corresponding CALR sequence: GYDNFGVLGLDLWQVKSGTI; SEQ ID NO: 162). CAL3011 also exhibited a strong and specific reactivity to synthetic peptide No. 30 (corresponding CALR sequence: EYSPDPSIYAYDNFGVLGLD; SEQ ID NO: 161). These results suggest that CAL3005 recognizes a broad range corresponding to synthetic peptides No. 29-32, that its major binding region is particularly contained in synthetic peptide No. 31, and that synthetic peptide No. 30 contains the epitope of CAL3011.

[0058] In one embodiment, the antibody and antigen-binding fragment thereof of the present invention can specifically bind to a peptide consisting of an amino acid sequence having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 161, preferably can specifically bind to a peptide consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 161, more preferably can specifically bind to a peptide consisting of an amino acid sequence having at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 161, and even more preferably can specifically bind to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 161.

[0059] In one embodiment, the antibodies and antigen-binding fragments thereof of the present invention can recognize an epitope in the amino acid sequence set forth in SEQ ID NO: 161. In a specific embodiment, the antibodies and antigen-binding fragments thereof can recognize an epitope comprising or consisting of 3 to 6, 3 to 7, 3 to 8, or 3 to 9 residues in the amino acid sequence set forth in SEQ ID NO: 161. In a specific embodiment, the antibodies and antigen-binding fragments thereof can bind to a linear epitope in the amino acid sequence set forth in SEQ ID NO: 161. The antibodies and antigen-binding fragments thereof can recognize an epitope comprising or consisting of at least 3, 4, 5, 6, 7, 8, or 9 consecutive bases in the amino acid sequence set forth in SEQ ID NO: 161.

[0060] In one embodiment, the antibody and antigen-binding fragment thereof of the present invention can specifically bind to a peptide consisting of an amino acid sequence having at least 80% sequence identity with the amino acid sequence set forth in positions 2 to 20 of SEQ ID NO: 162, preferably can specifically bind to a peptide consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in positions 2 to 20 of SEQ ID NO: 162, more preferably can specifically bind to a peptide consisting of an amino acid sequence having at least 95% sequence identity with the amino acid sequence set forth in positions 2 to 20 of SEQ ID NO: 162, and even more preferably can specifically bind to a peptide consisting of the amino acid sequence set forth in positions 2 to 20 of SEQ ID NO: 162.

[0061] In one embodiment, the antibody and antigen-binding fragment thereof of the present invention can recognize an epitope in the amino acid sequence set forth in positions 2 to 20 of SEQ ID NO: 162. In a specific embodiment, the anti-mutated CALR antibody of the present invention can recognize an epitope comprising or consisting of 3 to 6, 3 to 7, 3 to 8, or 3 to 9 residues in the amino acid sequence set forth in positions 2 to 20 of SEQ ID NO: 162. In a specific embodiment, the antibody and antigen-binding fragment thereof of the present invention can bind to a linear epitope in the amino acid sequence set forth in positions 2 to 20 of SEQ ID NO: 162. The antibody and antigen-binding fragment thereof of the present invention can recognize an epitope comprising or consisting of at least 3, 4, 5, 6, 7, 8, or 9 consecutive residues in the amino acid sequence set forth in positions 2 to 20 of SEQ ID NO: 162.

[0062] In one embodiment, the antibody and antigen-binding fragment thereof of the present invention can specifically bind to a peptide having an amino acid sequence having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 167 (DPSIYAYDNFG), preferably can bind to a peptide consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 167, and more preferably can bind to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 167.

[0063] In one embodiment, the antibodies and antigen-binding fragments thereof of the present invention can recognize an epitope in the amino acid sequence set forth in SEQ ID NO: 167. In a specific embodiment, the antibodies and antigen-binding fragments thereof can recognize an epitope comprising or consisting of 3 to 6, 3 to 7, 3 to 8, or 3 to 9 residues in the amino acid sequence set forth in SEQ ID NO: 167. In a specific embodiment, the antibodies and antigen-binding fragments thereof can bind to a linear epitope in the amino acid sequence set forth in SEQ ID NO: 167. The antibodies and antigen-binding fragments thereof can recognize an epitope comprising or consisting of at least 3, 4, 5, 6, 7, 8, or 9 consecutive bases in the amino acid sequence set forth in SEQ ID NO: 167.

[0064] In one embodiment, the antibody and antigen-binding fragment thereof of the present invention can specifically bind to a peptide consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 168 (TWIHPEIDNPEYSPDPSIYAYDNFGVLGLDLWQVKSGTIFDNFLITNDEA), preferably can specifically bind to a peptide consisting of an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 168, more preferably can specifically bind to a peptide consisting of an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 168, and even more preferably can specifically bind to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 168. The amino acid sequence set forth in SEQ ID NO: 168 is a region spanning the P domain and the C domain.

[0065] In one embodiment, the antibody and antigen-binding fragment thereof of the present invention can recognize an epitope in the amino acid sequence set forth in SEQ ID NO: 168. In a specific embodiment, the antibody and antigen-binding fragment thereof can recognize an epitope comprising or consisting of 3 to 6, 3 to 7, 3 to 8, or 3 to 9 residues in the amino acid sequence set forth in SEQ ID NO: 168. The antibody and antigen-binding fragment thereof can recognize an epitope comprising or consisting of at least 3, 4, 5, 6, 7, 8, or 9 consecutive residues in the amino acid sequence set forth in SEQ ID NO: 168. In a specific embodiment, the antibody and antigen-binding fragment thereof can recognize a discontinuous epitope in the amino acid sequence set forth in SEQ ID NO: 168.

[0066] Furthermore, in the Examples described below, epitope mapping analysis was performed on CAL3010 and CAL3012 in the same manner as described above. Compared to negative controls (a diluent-only group or a normal human IgG group), no synthetic peptides were observed to exhibit significant binding activity. The Examples also demonstrate that CAL3010 and CAL3012 bind to an antigen comprising the N domain, P domain, and a portion of the C domain (an antigen comprising a portion corresponding to positions 1 to 349 of SEQ ID NO: 1), but not to the P domain alone. Therefore, rather than a specific subsequence, both antibodies are thought to recognize a conformational structure formed by the portion from the N domain to the C domain N-terminal to the mutation site of CALR. In other words, in one embodiment, the antibody and antigen-binding fragment thereof of the present invention can recognize a conformational epitope formed by the portion from the N domain to the C domain N-terminal to the mutation site of CALR (e.g., the portion corresponding to positions 1 to 349 of SEQ ID NO: 1).

[0067] In the Examples described below, it was shown that CAL3004, 3006, 3008, 3010, 3012, 3013, and 3014 all bound to mutant CALR proteins displayed on the cell surface in a concentration-dependent manner. In particular, CAL3004, 3010, and 3012 showed high binding affinities (K ) of 0.1 to 0.2 nM to mutant CALR proteins expressed on the cell surface. Ｄ In one embodiment, the antibodies or antigen-binding fragments thereof of the invention have been shown to bind with a binding affinity (K value) of less than 20 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, or less than 0.1 nM. Ｄ ) can bind to mutant calreticulin.

[0068] The present invention also relates to a multispecific antibody, such as a T cell-inducing bispecific antibody that targets mutant calreticulin. The multispecific antibody of the present invention comprises: (1) a portion that recognizes an antigen expressed on a T cell; and (2) a portion that recognizes mutant calreticulin.

[0069] The multispecific antibodies of the present invention can recruit T cells (effector cells) to tumor cells expressing mutant calreticulin and induce cellular responses such as T cell cytokine production or cytotoxic activity. In the present invention, antigens expressed on T cells include, but are not limited to, CD3, CD4, CD8, CD25, CD28, CD127, CD152, CXCR3, CCR4, TRBC1, and TRBC2. The moiety that recognizes antigens expressed on T cells can be prepared from an antibody or an antigen-binding fragment thereof that has agonistic activity and can activate T cells.

[0070] The portion that recognizes mutant calreticulin (hereinafter referred to as the mutant CALR recognition portion) can be prepared from an antibody or its antigen-binding fragment that binds to the N-terminal side of the mutation site in mutant calreticulin, produced by carrying out the antibody production method of the present invention.

[0071] In one embodiment, the antigen expressed on T cells is CD3. The CD3-recognizing portion (hereinafter referred to as the CD3-recognizing portion) can be prepared from, for example, the anti-CD3 antibody described in U.S. Pat. No. 7,635,475 (SEQ ID NOs: 43 and 44 in U.S. Pat. No. 7,635,475). In one embodiment, the mutant CALR-recognizing portion can be prepared from, for example, the seven types of antibodies obtained in the Examples described below.

[0072] The multispecific antibody of the present invention comprises at least (1) a CD3-recognition portion and (2) a mutant CALR-recognition portion, wherein the (1) CD3-recognition portion comprises a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 108, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 109, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 110, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 111, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 112, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 113, and the (2) mutant CALR-recognition portion comprises a VH domain and a VL domain selected from any of (a) to (g) below: (a) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 67, and a VH domain comprising a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68, and a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 69, (b) a VL domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 72, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 73, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 74, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 75, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 76, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 77; (c) a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 81, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 82, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 83;(d) a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 84, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 85, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 86, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 87, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 88, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 89; (e) a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 90, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 91, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 92, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 93, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 94, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 95; (f) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 96, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 97, and (g) a VH domain comprising a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 98, and a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 99, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 100, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 101; (g) a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 102, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 103, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 104, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 105, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 106, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 107.;

[0073] In a specific embodiment, (1) the CD3 recognition portion comprises a VH domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 114, and a VL domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 115. Nucleotide sequences encoding the amino acid sequences set forth in SEQ ID NOs: 114 and 115 are not particularly limited, and may, for example, be the nucleotide sequences set forth in SEQ ID NOs: 130 and 131, respectively.

[0074] In a specific embodiment, (2) the mutant CALR recognition portion comprises a VH domain and a VL domain of any of (a) to (g) below: (a) a VH domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 10, and a VL domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 11. (b) a VH domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 12, and a VL domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 13. (c) a VH domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 14, and a VL domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 15. (d) a VH domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 16, and a VL domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 17. (e) a VH domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 18, and a VL domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 19.(f) a VH domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 20, and a VL domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 21. (g) a VH domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 22, and a VL domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 23.

[0075] The amino acid mutations (deletions, insertions, and substitutions) to the amino acid sequence can occur in the complementarity determining regions (CDRs) or framework regions (FRs). In one embodiment, the mutations occur in the CDRs. In one embodiment, the mutations occur in the FRs of the antibody.

[0076] In certain embodiments, amino acid sequence variants of multispecific antibodies are generated to improve the binding affinity and / or other biological properties of the CD3 recognition portion and / or mutant CALR recognition portion. Amino acid sequence variants can be generated, for example, by introducing appropriate mutations (deletions, insertions, and substitutions) into the nucleotide sequence encoding the anti-CD3 antibody or mutant CALR recognition portion, or by peptide synthesis. Mutations can be introduced into the CDRs or FRs of the antibody, depending on the property to be improved. Amino acid sequence variants are screened for retained or improved binding affinity, retained or improved antigen specificity, reduced immunogenicity, etc.

[0077] In certain embodiments, the format of the multispecific antibodies of the present invention may be any one of the structures / formats described in Brinkmann and Kontermann, "The making of bispecific antibodies," MABs, vol. 9, pp. 182-212, 2017, or Ulrich H. Weidle et al., "The Intriguing Options of Multispecific Antibody Formats for Treatment of Cancer," Cancer Genomics & Proteomics, January 2013, 10 (1) 1-18. Examples include, but are not limited to, cross-Mab, IgG-dssFv2, DVD, IgG-dsFv, IgG-scFab, scFab-dssFv, Fv2-Fc, Fab-scFv2, Fab-scFv, Fab-scFv-Fc, BiTE (scFv-scFv), diabody, DART, etc.

[0078] In a specific embodiment, in the multispecific antibody of the present invention, (1) the T cell antigen recognition portion is an scFv, and (2) the mutant CALR recognition portion is a Fab fragment, and the scFv is linked to the C-terminus of the heavy chain of the Fab fragment via a peptide linker. In a further specific embodiment, the multispecific antibody has (1) the T cell antigen recognition portion is an scFv, and (2) the mutant CALR recognition portion is an scFv, and the two scFvs are linked via a peptide linker.

[0079] A "peptide linker" is a peptide containing about 2 to 20 amino acids, e.g., (G ４ S) ｎ , (SG ４ ) ｎ or G ４ (SG ４ ) ｎwhere "n" is generally an integer from 1 to 10. Typically, the peptide linker is selected from the group consisting of GGGGS (SEQ ID NO: 116), GGGGSGGGGS (SEQ ID NO: 117), GGGGSGGGGSGGGGGS (SEQ ID NO: 118), SGGGGSGGGGG (SEQ ID NO: 119) and GGGGSGGGGSGGGG (SEQ ID NO: 120), although the sequences GSPGSSSSGSGS (SEQ ID NO: 121), (G ４ S) ４ , GSGSGSG (SEQ ID NO: 122), GSGSGNGS (SEQ ID NO: 123), GGSGSGSG (SEQ ID NO: 124), GGSGSG (SEQ ID NO: 125), GGSG (SEQ ID NO: 126), GGSGNGSG (SEQ ID NO: 127), GGNGSGSG (SEQ ID NO: 128) and GGNGSG (SEQ ID NO: 129) may also be used.

[0080] In certain embodiments, the multispecific antibody further comprises (3) an Fc domain. In even more specific embodiments, the Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain, and preferably an IgG1 Fc domain. In even more specific embodiments, the IgG1 Fc domain polypeptide can have one or more amino acid mutations, particularly amino acid mutations that alter the binding ability to Fc receptors, and more preferably amino acid mutations that decrease the binding ability to Fc receptors.

[0081] In certain embodiments, the heavy chain of the antibody is of human IgG1 isotype and can have a mutation of leucine at positions 234 and 235 to alanine, L234AL235A (LALA mutation), or a corresponding mutation. These mutations can occur at equivalent positions in other isotypes and subtypes. The LALA mutation abolishes binding to complement component (C1q) and Fc gamma receptor (FcγR), thereby preventing in vitro FcγR-mediated coactivation of innate immune effector cells, including natural killer (NK) cells, monocytes / macrophages, and neutrophils, without altering functional binding to FcRn (fetal Fc receptor) (Clin Cancer Res (2016) 22 (13): 3286-3297.). Thus, multispecific antibodies with an Fc domain containing the LALA mutation lack complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) activity, and the only immune effector cells involved may be T cells.

[0082] In certain embodiments, the multispecific antibodies of the invention are monovalent with respect to both the antigen expressed on T cells and the CALR antigen. In certain embodiments, the multispecific antibodies of the invention are bivalent with respect to both the antigen expressed on T cells and the CALR antigen. In certain embodiments, the multispecific antibodies of the invention are trivalent with respect to both the antigen expressed on T cells and the CALR antigen. In certain embodiments, the multispecific antibodies of the invention are monovalent with respect to the antigen expressed on T cells and bivalent with respect to the CALR antigen. In certain embodiments, the multispecific antibodies of the invention are bivalent with respect to the antigen with agonistic activity expressed on T cells and monovalent with respect to the CALR antigen.

[0083] In a specific embodiment, the multispecific antibody of the present invention has two structures (i.e., bivalent + bivalent) in which (1) an scFv that recognizes an antigen expressed on T cells and (2) a Fab fragment that recognizes CALR are linked by a peptide linker, and further has an Fc domain (Fab-scFv-Fc).

[0084] The present invention also relates to a composition for treating tumors comprising the above-mentioned multispecific antibody, in particular any of the above-mentioned seven types of multispecific antibody.

[0085] The present invention further provides nucleic acid molecules encoding the heavy and light chains of the anti-mutated CALR antibodies and multispecific antibodies of the present invention. The nucleic acid molecules of the present invention may be RNA, DNA, or cDNA.

[0086] The nucleic acid molecules of the present invention may be in the form of a vector, may be present in a vector, and / or may be part of a vector such as a plasmid, cosmid, or YAC. The vector may in particular be an expression vector, which may provide for expression of the multispecific antibodies of the present invention in a host cell, host organism, and / or expression system. Expression vectors typically contain at least one nucleic acid of the present invention operably linked to one or more appropriate expression control elements (e.g., promoters, enhancers, terminators, etc.). The selection of elements and their sequences for expression in a particular host is within the knowledge of those skilled in the art. Specific examples of regulatory elements and other elements useful or essential for the expression of the heavy and light chains of the multispecific antibodies of the present invention include promoters, enhancers, terminators, integration elements, selection markers, leader sequences, reporter genes, etc.

[0087] The nucleic acid molecules of the present invention can be prepared or obtained by known methods (e.g., by automated DNA synthesis and / or recombinant DNA techniques) based on the information on the amino acid sequences of the heavy and light chains of the anti-mutated CALR antibodies of the present invention and the multispecific antibodies of the present invention disclosed herein, and / or can be isolated from suitable natural sources.

[0088] The present invention includes hosts that express or are capable of expressing one or more of the heavy and light chains of the anti-mutated CALR antibodies of the present invention and the multispecific antibodies of the present invention, and / or provides nucleic acids or vectors of the present invention. Preferred hosts of the present invention are bacterial, fungal, or mammalian cells.

[0089] Suitable bacteria include gram-negative bacterial strains (eg, E. coli, Proteus, and Pseudomonas) and / or gram-positive bacterial strains (eg, Bacillus, Streptomyces, Staphylococcus, and Lactococcus).

[0090] Suitable fungi include species of the genera Trichoderma, Neurospora and / or Aspergillus; or species of the genus Saccharomyces, such as Saccharomyces cerevisiae, Schizosaccharomyces, such as Schizosaccharomyces pombe, Pichia, such as Pichia pastoris and Pichia methanolica, and / or species of Hansenula. Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, and / or COS cells.

[0091] However, amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins can also be used in the present invention.

[0092] The heavy and light chains of the anti-mutated CALR antibodies of the invention and the multispecific antibodies of the invention can be produced intracellularly as described above and then isolated from the host cells, and optionally further purified, or can be produced extracellularly (e.g., in the medium in which the host cells are cultured) and then isolated from the medium, and optionally further purified.

[0093] Methods and reagents for recombinant production of polypeptides are known in the art, such as particular appropriate expression vectors, transformation or transfection methods, selectable markers, methods for inducing protein expression, culture conditions, etc. Similarly, protein isolation and purification techniques suitable for the methods of making the multispecific antibodies of the invention are well known to those of skill in the art.

[0094] However, the anti-mutated CALR antibodies of the present invention and the multispecific antibodies of the present invention can also be obtained by other protein production methods known in the art, such as chemical synthesis, including solid-phase or liquid-phase synthesis.

[0095] The multispecific antibodies of the present invention can recruit CD3-positive T cells and induce cytotoxicity and / or cell death of tumor cells. They can bind to mutant calreticulin and exhibit cytotoxic activity regardless of the type of mutation. The cytotoxic activity of a multispecific antibody can be determined in vitro by culturing target tumor cells with the multispecific antibody in the presence of effector cells and measuring the viability of the target tumor cells. For example, activated T cells (e.g., T-LAK) can be used as effector cells.

[0096] The present invention relates to a method for treating tumors, comprising administering to a subject an antibody or an antigen-binding fragment thereof that binds to the N-terminal side of a mutation site in mutant calreticulin and binds to the mutant calreticulin on the cell surface. As used herein, the term "subject" refers to a mammal, particularly a primate, and more particularly a human. In a specific embodiment, the method for treating tumors of the present invention comprises administering to a subject an anti-mutated CALR antibody or an antigen-binding fragment thereof, which antibody comprises any of the sets of VH and VL domains comprising the CDR amino acid sequences listed in Table 2. In a specific embodiment, the method for treating tumors of the present invention comprises administering to a subject a multispecific antibody having a portion that recognizes mutant calreticulin, wherein the portion that recognizes mutant calreticulin comprises any of the sets of VH and VL domains comprising the CDR amino acid sequences listed in Table 2.

[0097] The present invention relates to an antibody or an antigen-binding fragment thereof that binds to the N-terminal side of a mutation site in a mutant calreticulin and binds to the mutant calreticulin on a cell surface, for use in tumor treatment.

[0098] The present invention relates to the use of an antibody or an antigen-binding fragment thereof that binds to the N-terminal side of a mutation site in a mutant calreticulin and binds to the mutant calreticulin on a cell surface in the manufacture of a medicament for treating a tumor.

[0099] The composition for treating tumors of the present invention can be provided as a pharmaceutical composition. The pharmaceutical composition can further include a pharmaceutically acceptable carrier. The formulation of a pharmaceutical composition with a pharmaceutically acceptable carrier is known in the art (e.g., Remington: The Science and Practice of Pharmacy (23rd edition (2020)). Pharmaceutically acceptable carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0100] The composition of the present invention is preferably produced under conditions that comply with regulations for manufacturing and quality control of pharmaceuticals and quasi-drugs (good manufacturing practice, GMP).

[0101] In one embodiment, the composition of the present invention is a liquid such as a solution, suspension, emulsion, microemulsion, or gel. The pharmaceutical composition may be an aqueous formulation and may contain at least 50% (w / w) water. In one embodiment, the composition of the present invention is in a dosage form suitable for injection. The injection may be, for example, subcutaneous, intramuscular, intraperitoneal, intravitreal, or intravenous.

[0102] In one embodiment, the compositions of the present invention may be in solid dosage form, for example in freeze-dried or spray-dried form, which may be administered, for example, by adding solvents and / or diluents prior to administration.

[0103] The compositions of the present invention may be provided in packaging such as syringes, vials, or infusion bags. The syringes may contain the compositions in lyophilized form (which must be solubilized, for example, with water for injection, before administration) or in aqueous form. Other solid dosage forms may include powders, granules, tablets, and capsules.

[0104] The dosage and interval of administration of the compositions of the present invention may be adjusted individually to provide plasma levels of the antibody sufficient to maintain therapeutic effects. The dosage varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is approximately 0.1 to 50 mg / kg / day. Therapeutically effective plasma levels can be achieved by administering multiple doses daily. Plasma levels can be measured, for example, by HPLC.

[0105] The compositions of the present invention can be administered in combination with other agents used in chemotherapy, radiation, and / or cancer immunotherapy. In combination therapy, the compositions of the present invention and one or more agents or therapies can be administered simultaneously or sequentially. When administered simultaneously or sequentially, all agents or therapies can be administered by the same route or by different routes.

[0106] As used herein, "co-administration" refers to the administration of two or more drugs or therapies separated by a time interval of a few minutes to a few seconds or less. For example, two drugs or therapies are administered about 15 minutes to 1 minute or less apart. As used herein, "sequential administration" refers to the administration of two drugs or therapies separated by a time interval of a few minutes, a few hours, a few days, or a few weeks. For example, two drugs or therapies are administered at intervals of 15 minutes or more, 30 minutes or more, 60 minutes or more, or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, or 2 weeks, 3 weeks, or 4 weeks.

[0107] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

[0108] Example 1: Preparation of anti-CALR antibodies In this example, an antibody was prepared that binds to the N-terminal side of the mutation site of the mutant CALR protein. (1) Preparation and purification of recombinant CALR antigen used for animal immunization Two types of peptides containing the amino acid sequences of the following regions of the wild-type CALR protein were designed and used as recombinant antigens. (i) NPC-His (SEQ ID NO: 6): N, P, and C domains (portions corresponding to positions 1 to 349 of SEQ ID NO: 1) (containing a His tag at the N-terminus) (ii) P-Fc (SEQ ID NO: 8): mainly the P domain (portions corresponding to positions 171 to 292 of SEQ ID NO: 1) (containing an Fc tag and a His tag at the C-terminus)

[0109] Recombinant antigens were constructed by expressing the nucleotide sequences set forth in SEQ ID NOs: 7 (NPC-His) and 9 (P-Fc) with signal sequences added using expression vectors. Specifically, recombinant CALR antigen expression vectors were constructed using DNA encoding the recombinant CALR antigen and an expression vector (pcDNA3.4, Thermo Fisher Scientific). The expression vectors were transfected into HEK293 cells at a DNA content of 1 μg per mL of culture medium using ExpiFectamine 293 Reagent (Thermo Fisher Scientific). The day after transfection, Expi293 Feed and ExpiFectamine 293 Transfection Enhancer 1 and 2 were added, and the cells were cultured at 37°C for 7 days. The cells were removed from the culture medium by centrifugation and filtration, and the culture supernatant was collected. The antigen was purified by affinity chromatography using nickel-conjugated agarose and gel filtration chromatography, or by dialysis using a cellulose membrane.

[0110] (2) Antibody Production Rabbits (New Zealand White (Kbl: NZW)) were immunized four times at two-week intervals with the recombinant antigen NPC-His or P-Fc. For the first immunization, 1 mL of each antigen prepared at 1 mg / mL was mixed with 1 mL of Freund's complete adjuvant (FCA) to form an emulsion, and 2 mL of this emulsion was administered subcutaneously. From the second immunization onwards, 1 mL of each antigen prepared at 1 mg / mL was mixed with 1 mL of Freund's incomplete adjuvant (FIA), and 1 mL of this emulsion was administered subcutaneously. One week after the fourth immunization, whole blood was collected and the blood was recovered.

[0111] B cells were isolated from the collected blood by standard methods and screened for their binding to the antigen used for immunization (NPC-His or P-Fc). Specifically, the isolated B cells were mixed with fluorescently labeled antigen, and the stained B cells, i.e., B cells expressing antibodies that recognize the antigen, were sorted using a cell sorter. The B cells obtained in this manner were lysed into single cells, and the gene sequences of the antibodies were analyzed. Fabs were prepared based on the obtained sequences by outsourcing to iBody.

[0112] (3) Confirmation of antibody binding activity Using the Fab prepared in (2) above, differences in binding activity to each antigen were evaluated using ELISA. Specifically, each antigen was immobilized on an ELISA plate, and the obtained candidate antibody was reacted. The candidate antibody bound to the antigen was detected using an HRP (horseradish peroxidase)-labeled antibody, and the color was developed using a TMB (3,3',5,5'-tetramethylbenzidine) substrate, and the absorbance (450 nm) was measured. Binding was evaluated when the absorbance was 0.3 or higher. The results are shown in Tables 3-1 and 3-2.

[0113]

[0114] P-Fc was designed to induce immunity against the P domain. CAL3001, 3002, 3005, 3006, 3008, 3009, 3013, 3014, and 3015 bound to P-Fc and are therefore considered to be antibodies that bind to the P domain. CAL3004, 3010, 3011, and 3012 did not bind to P-Fc but bound to NPC-His, and are therefore considered to be antibodies that bind to the N domain or C domain. These antibodies were confirmed to have high binding affinity to mutant calreticulin on the cell surface in Example 4, which will be described later, and are therefore considered to be highly likely to be antibodies that bind to the N domain and are less susceptible to steric hindrance during binding.

[0115] Example 2: Preparation of rabbit chimeric anti-CALR monoclonal antibody In this example, a rabbit chimeric anti-CALR monoclonal antibody was prepared from the anti-CALR antibody obtained in Example 1. Specifically, a heavy chain (HC) was designed by linking the heavy chain variable region of the anti-CALR antibody, human IgG heavy chain constant region 1, human IgG heavy chain constant region 2, and human IgG heavy chain constant region 3, and a light chain (LC) was designed by combining the light chain variable region of the anti-CALR antibody and a human IgG light chain constant region. An HC expression vector and an LC expression vector were prepared using DNA encoding the HC or LC of the anti-CALR antibody and an expression vector (pcDNA3.4, Thermo Fisher Scientific), respectively. These expression vectors were mixed at a molar ratio of 1:1 and transfected into CHO cells at 0.8 μg of DNA per mL of culture medium using ExpiFectamine CHO Reagent (Thermo Fisher Scientific). ExpiCHO Feed and ExpiFectamine CHO Enhancer were added the day after and 5 days after transfection. The culture temperature was 37°C for 1 day, and from the following day, culture was continued at 32°C for 6 to 13 days. Cells were removed from the culture medium by filtration using diatomaceous earth (DE) filter aid (Zaltoclear DY Lab was used), and the culture supernatant was collected. Then, anti-CALR antibodies were purified from the culture supernatant using a combination of Protein A affinity chromatography and gel filtration chromatography to produce candidate antibodies CAL3001, CAL3002, CAL3004 to CAL3006, and CAL3008 to CAL3015 in the form of rabbit chimeric antibodies.

[0116] Example 3: Screening for binding to wild-type CALR protein In this example, the candidate antibodies prepared in Example 2 were screened for their ability to bind to wild-type CALR protein. By co-incubating the antibodies with cells, their ability to bind to wild-type CALR protein localized within cells was analyzed. Reagents used: PBS (phosphate-buffered saline) (Nacalai Tesque, 14249-24), FBS (fetal bovine serum) (Gibco, A5256701), Fixable Viability Dye eFluor780 (Invitrogen, 65-0865-14), Methanol (Nacalai Tesque, 2191535), Fluorescein Labeling Kit-NH2 (Dojindo Laboratories, LK01).

[0117] Method Fluorescein Labeling Kit-NH ２Candidate antibodies to be screened were fluorescently labeled using the protocol. JM cells, a human T cell-derived cell line, were centrifuged (300g x 5 minutes), the supernatant removed, and washed with 2% FBS / PBS. Fixable Viability Dye eFluor 780 solution diluted with 2% FBS / PBS was added and incubated at 4°C for 15 minutes to stain dead cells. After incubation, the cells were washed twice by adding 2% FBS / PBS, centrifuging (500g x 2 minutes), and discarding the supernatant. The supernatant was removed, and ice-cold 90% methanol was added and incubated on ice for 15 minutes to permeabilize the cells. After incubation, the cells were centrifuged (500g x 2 minutes), the supernatant discarded, and washed twice with 2% FBS / PBS. Fluorescently labeled candidate antibodies were diluted to 10 nM in 2% FBS / PBS, added to the cells, and incubated at 4°C for at least 30 minutes. As an isotype control, a fluorescently labeled human IgG antibody (abcam, ab206198) was adjusted to 10 nM and added ("Isotype control" group). A group without antibody (2% FBS / PBS only) was also set up as the "unstained" group. The cells were washed twice by adding 2% FBS / PBS, centrifuging (500 g x 2 minutes), and discarding the supernatant. Signals were quantified by flow cytometry analysis using a BD FACSymphony A1 (BD Biosciences). Flow Jo_v10.6.1 was used as the analysis software.

[0118] Results Of the antibodies prepared in Example 2, CAL3001, 3002, 3004, 3005, 3006, 3008, 3009, 3010, 3011, 3012, 3013, 3014, and 3015 were confirmed to bind to the wild-type CALR protein localized inside the cells (Figure 2).

[0119] Example 4: Screening for binding to mutant CALR proteins In this example, the candidate antibodies prepared in Example 2 were analyzed for their ability to bind to mutant CALR proteins displayed on the cell surface of UT-7 / TPO cells. Reagents used: Hank's Balanced Salt Solution (HBSS) (Gibco, 14025-076), FBS (Gibco, A5256701), HEPES (Nacalai Tesque, 17557-94), APC-labeled anti-human IgG antibody: Allophycocyanin (APC), AffiniPure TM F(ab')2 Fragment Goat Anti-Human IgG, Fcγ fragment specific (Jackson ImmunoResearch Inc., 109-136-170) Fixable Viability Dye eFluor780 (Invitrogen, 65-0865-14)

[0120] UT-7 / TPO / Ins5 cells and UT-7 / TPO / Del52 cells were prepared by transfecting the mutant CALR protein Ins5 type (SEQ ID NO: 2) or Del52 type (SEQ ID NO: 3) into the human megakaryoblastic leukemia cell line UT-7 / TPO (Komatsu N. et al., supra). Furthermore, UT-7 / TPO / vec cells (vector control) were prepared by transfecting only the vector used for gene transfection. UT-7 / TPO cell lines (UT-7 / TPO / Ins5 cells, UT-7 / TPO / Del52 cells, UT-7 / TPO / vec cells) were centrifuged (300 g × 5 minutes), and the supernatant was removed. Candidate antibodies and a negative control, an anti-human CD3 antibody (OKT3 antibody) were added at 10 nM in 5% FBS / 10 mM Hepes / HBSS, and the cells were incubated at room temperature for 1 hour. A control well containing medium alone was designated the "unstained" group. After the incubation, the cells were washed twice by adding 2% FBS / PBS, centrifuging (400 g × 3 minutes), and discarding the supernatant. An APC-labeled anti-human IgG antibody and FVD780 were added in 2% FBS / PBS, and the cells were incubated on ice for 1 hour. The cells were washed twice by adding 2% FBS / PBS, centrifuging (400 g x 3 min), discarding the supernatant, and then analyzing with a BD FACSymphony.TM Signals were quantified by flow cytometry analysis using a BD Biosciences ELISA Kit (Flow Jo_v10.6.1) and analytical software.

[0121] Results: The candidate antibodies prepared in Example 2 were evaluated for binding to mutant CALR proteins displayed on the cell surface. Seven antibodies, CAL3004, 3006, 3008, 3010, 3012, 3013, and 3014, were observed to bind. These seven antibodies bound to both UT-7 / TPO / Ins5 cells and UT-7 / TPO / Del52 cells, which express the Ins5 and Del52 types, respectively, but did not bind to UT-7 / TPO / Vec cells, which do not express mutant CALR proteins (Figure 3). These results indicate that antibodies that bind to mutant CALR proteins on the cell surface were selected from among antibodies that recognize wild-type CALR proteins. These antibodies are expected to exert antitumor effects through ADCC activity, etc.

[0122] Example 5: Binding affinity In this example, the binding affinity of CAL3004, 3006, 3008, 3010, 3012, 3013, and 3014, which were observed to bind to mutant CALR proteins in Example 4, and the B3 antibody (B3hIgG) described in WO 2020 / 175689, to mutant CALR proteins displayed on the cell surface was evaluated.

[0123] Methods: UT-7 / TPO cell lines (UT-7 / TPO / Ins5 cells, UT-7 / TPO / Del52 cells, UT-7 / TPO / vec cells) were centrifuged (300g x 5 minutes), the supernatant removed, and each antibody to be evaluated was added at 0.1 nM to 30 nM in 5% FBS / 10 mM Hepes / HBSS and incubated at room temperature for 1 hour. After incubation, the cells were washed twice by adding 2% FBS / PBS, centrifuging (400g x 3 minutes), and discarding the supernatant. APC-labeled anti-human IgG antibody and FVD780 were added in 2% FBS / PBS, and incubated on ice for 1 hour. The cells were washed twice by adding 2% FBS / PBS, centrifuging (400g x 3 minutes), and discarding the supernatant. Then, the cells were analyzed by BD FACSymphony. TMSignals were quantified by flow cytometry analysis using a flow cytometer (BD Biosciences) with Flow Jo v10.6.1 software. Binding saturation curves were calculated using Prism Software (GraphPad Software Inc.) and the K Ｄ The values ​​(binding affinity) were calculated and the results are shown in the table below.

[0124] CAL3004, 3006, 3008, 3010, 3012, 3013, and 3014 all bound to the mutant CALR protein in a concentration-dependent manner. In particular, CAL3004, 3010, and 3012 showed high binding affinity (K ) of 0.1 to 0.2 nM for the mutant CALR protein expressed on the cell surface. Ｄ It was shown that they bind with a small value.

[0125] Example 6: Preparation of bispecific antibodies In this example, T cell-inducing bispecific antibodies were prepared from the anti-mutated CALR antibodies (CAL3004, 3006, 3008, 3010, 3012, 3013, and 3014) that were found to bind to mutant CALR proteins on the cell surface in Example 4. Specifically, a heavy chain (HC) was designed by linking the heavy chain variable region of the anti-mutated CALR antibody, human IgG heavy chain constant region 1, a portion that binds to CD3 (heavy chain variable region and light chain variable region of a known anti-CD3 antibody), human IgG heavy chain constant region 2, and human IgG heavy chain constant region 3, with appropriate linker sequences interposed therebetween, and a light chain (LC) was designed by linking the light chain variable region of the anti-mutated CALR antibody and human IgG light chain constant region (for the structure of the bispecific antibody, see Structure 1 in Figure 6 of WO 2022 / 045247 and Figure 1a in mAbs, 2017, 9(2):240-256. doi:10.1080 / 19420862.2016.1270492). The CD3-binding moiety was designed based on the sequence information of the anti-CD3 antibody described in US Pat. No. 7,635,475 (US Pat. No. 7,635,475 Diabody-type bispecific antibody SEQ ID No. 43, SEQ ID No. 44).

[0126] The table below shows the amino acid sequences of the HC and LC of the bispecific antibodies, their SEQ ID NOs, and the SEQ ID NOs of the nucleotide sequences encoding the HC or LC.

[0127] DNA encoding the HC or LC of the bispecific antibody and an expression vector (pcDNA3.4, Thermo Fisher Scientific) were used to prepare HC expression vectors and LC expression vectors, respectively. These expression vectors were mixed at a molar ratio of 1:1 and transfected into CHO cells at 0.8 μg of DNA per mL of culture medium using ExpiFectamine CHO Reagent (Thermo Fisher Scientific). The day after and 5 days after transfection, ExpiCHO Feed and ExpiFectamine CHO Enhancer were added, and the cells were cultured at 32 ° C for 10 to 12 days. The cells were removed from the culture medium by centrifugation and filtration, and the culture supernatant was collected. Then, bispecific antibodies were purified from the culture supernatant using a combination of affinity chromatography using nickel-conjugated agarose and gel filtration chromatography, or a combination of Protein A affinity chromatography and gel filtration chromatography.

[0128] Example 7: Cytotoxic activity In this example, the cytotoxic activity of T cells induced by the bispecific antibody prepared in Example 5 was analyzed. The following reagents were used: HBSS (Hank's Balanced Salt Solution) (Gibco, 14025092), RPMI1640 Medium, no phenol red (Gibco, 11835-030), Probenecid Water Soluble (Invitrogen, P36400), Calcein-AM (Invitrogen, C1430), Lysis Solution 10X (Promega, G1821).

[0129] Bispecific antibody-mediated cytotoxicity of T cells against UT-7 cells. Method: Bispecific antibodies were adjusted to a final concentration of 0.1 pM to 30 nM in RPMI non-phenolic medium containing 5 mM probenecid and 0.5% FBS (assay medium) and added to a 96-well round-bottom culture plate. Control wells contained assay medium only. UT-7 / TPO / Ins5, UT-7 / TPO / Del52, and UT-7 / TPO / vec cells were harvested from the culture medium and incubated with 10 μM calcein-AM supplemented with 5 mM probenecid at 37°C for 30 minutes to label the target cells with calcein. After incubation, the cells were washed once with HBSS and suspended in assay medium. After recovering the required amount of effector cells (T-LAK cells) from the culture medium, the cells were counted and suspended in assay medium. Effector cells and labeled target cells were added to a 96-well round-bottom culture plate containing the bispecific antibody at a ratio of 10:1. The plate containing the bispecific antibody, labeled target cells, and effector cells was incubated at 25°C for 1 hour at 5% CO ２ The cells were incubated at 37°C for 2 hours. The 96-well round-bottom culture plate was centrifuged (300 g, 5 minutes), and the supernatant was collected. The fluorescence of calcein eluted from the labeled target cells contained in the supernatant was measured using a plate reader (BioTek, SYNERGY H1). The calcein fluorescence value was used as an index to calculate the cytotoxicity rate of the target cells, and bispecific antibody-dependent cytotoxicity was evaluated.

[0130] The cytotoxicity rate was calculated as follows: The minimum eluted fluorescence value of labeled target cells (lysis rate = 0%) was obtained from wells in which labeled target cells and effector cells were incubated without any bispecific antibody. The maximum eluted fluorescence value (lysis rate = 100%) was obtained by adding 10x Lysis Solution to wells under the same conditions as those for the minimum eluted fluorescence value. The cytotoxicity rate was calculated using the following formula: Cytotoxicity rate = [(eluted fluorescence value of target cells in the presence of bispecific antibody) - (minimum eluted fluorescence value)] / [(maximum eluted fluorescence value) - (minimum eluted fluorescence value)]. Sigmoidal dose-response curves were calculated using Prism Software (GraphPad Software Inc.), and the EC50 value (50% effective concentration) and Emax value (maximum activity value) were calculated.

[0131] Results As shown in Table 6 and Figures 5A to 5C, all of the bispecific antibodies used in the evaluation were found to induce concentration-dependent cytotoxic activity and exhibit antitumor activity.

[0132] Example 8: Epitope mapping (1) To identify the binding sequences of each of the antibodies CAL3004, CAL3010, and CAL3012, 35 types of peptides shown in Table 7 were synthesized and epitope mapping was performed by evaluating the differences in binding affinity to these peptides using ELISA. Each synthetic peptide was designed to cover the entire sequence N-terminal to the mutation site of the CALR protein (the portion corresponding to positions 1 to 349 of SEQ ID NO: 1), and to have partial overlapping amino acid sequences, and in some cases, to adjust the terminal amino acid residues for ease of synthesis.

[0133]

[0134] Specifically, each synthetic peptide was immobilized as an antigen on an ELISA plate, and each of the above antibodies, appropriately diluted with diluent (PBS containing 0.1% (w / v) BSA and 0.05% (v / v) Tween-20), was reacted with the plate using standard methods. The antibody bound to the antigen was detected with an HRP-labeled antibody (Anti-Human IgG (H+L), HRP Conjugate, Promega). Color was developed using TMB (3,3',5,5'-tetramethylbenzidine) substrate, and the absorbance (450 nm) was measured using a microplate reader, Spectramax 190 (Molecular Devices Co.). As negative controls, groups were prepared in which only the diluent or normal human IgG (Fujifilm Wako Pure Chemical Industries) was used instead of each of the above antibodies. These results are shown in Figures 6(A) to 6(E).

[0135] CAL3004 showed a strong and specific reaction with synthetic peptide No. 30 (corresponding CALR sequence: EYSPDPSIYAYDNFGVLGLD; SEQ ID NO: 161), suggesting that this peptide contains the epitope of CAL3004. CAL3004 did not react with synthetic peptides No. 29 or No. 31, which contain amino acid sequences overlapping 10 residues with the amino acid sequence of synthetic peptide No. 30, suggesting that the epitope of CAL3004 is a sequence near the center of synthetic peptide No. 30. This region spans the P and C domains of CALR, and since the P-Fc used in Example 1 contained only a portion of this region, it is believed that CAL3004 did not bind to P-Fc.

[0136] For CAL3010 and CAL3012, no synthetic peptides were observed to exhibit significant binding activity compared to the negative controls (diluent only group or normal human IgG group). Therefore, it is believed that both antibodies recognize the three-dimensional structure formed by the portion from the N domain to the C domain on the N-terminal side of the CALR mutation site. These results are not inconsistent with the possibility discussed in Example 1.

[0137] (2) Epitope mapping was also performed on the CAL3005 and CAL3011 antibodies in the same manner as described in (1) above. As a result, CAL3005 exhibited binding affinity to synthetic peptides No. 29 to 32, with a particularly strong and specific reaction to No. 31 (corresponding CALR sequence: GYDNFGVLGLDLWQVKSGTI; SEQ ID NO: 162). CAL3011 also exhibited a strong and specific reaction to synthetic peptide No. 30 (corresponding CALR sequence: EYSPDPSIYAYDNFGVLGLD; SEQ ID NO: 161). These results suggest that CAL3005 recognizes a broad range corresponding to synthetic peptides No. 29 to 32, that its major binding region is particularly contained in synthetic peptide No. 31, and that synthetic peptide No. 30 contains the epitope of CAL3011.

Claims

1. A composition for treating tumors, comprising an antibody or an antigen-binding fragment thereof that binds to the N-terminal side of the mutation site in mutant calreticulin and binds to the mutant calreticulin on the cell surface.

2. The composition according to claim 1, wherein the antibody or antigen-binding fragment thereof binds to the N-terminal side of the mutation site in the N domain, P domain, or C domain of the mutant calreticulin.

3. The antibody or antigen-binding fragment thereof has a binding affinity (K Ｄ 3. The composition of claim 1 or 2, which binds to mutant calreticulin at the .

4. A method for producing an antibody that binds to the N-terminal side of the mutation site of the mutant calreticulin, comprising a step of screening for an antibody that binds to mutant calreticulin on the cell surface or an antibody that binds to a mutant calreticulin dimer.

5. The method for producing an antibody described in claim 4, wherein the screening step selects an antibody that binds to a polypeptide that includes the N-terminal side of the mutation site in mutant calreticulin, or includes an additional screening step of selecting an antibody that binds to a polypeptide that includes the N-terminal side of the mutation site in mutant calreticulin.

6. A method for producing an antibody according to claim 4 or 5, further comprising the step of immunizing an animal with a polypeptide comprising the N-terminal side of the mutation site of the mutant calreticulin.

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