Medicine comprising combination of Anti-mutant-CALR antibody and another drug

Abstract

The purpose of the present invention is to provide a medicine exhibiting a satisfactory anti-cancer effect by combining an antibody capable of binding to a mutant CALR protein with another anti-cancer agent.　Provided is a medicine for preventing and / or treating cancer, the medicine comprising a combination of (A) an antibody capable of binding specifically to a mutant calreticulin protein or a functional fragment thereof and (B) at least one drug selected from an alkylating agent, a platinum preparation, an antimetabolite, a ribonucleotide reductase inhibitor, a nucleotide analogue, a topoisomerase inhibitor, a microtubule assembly inhibiting drug, an anti-tumor antibiotic, an interferon, a cytokine preparation, a molecule targeting drug, a nucleic acid synthesis inhibitor, a JAK inhibitor and an immuno-oncology drug.

Description

Medicine comprising a combination of an anti-mutated CALR antibody and another drug

[0001] The present invention relates to a pharmaceutical comprising an antibody having specificity for a mutant CALR protein or a functional fragment thereof in combination with another drug.

[0002] In some patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), base deletions or insertions are found in exon 9 of the calreticulin (CALR) gene (Non-Patent Documents 1 and 2). It has already been shown that the mutant CALR protein produced by this CALR mutant gene constitutively activates the thrombopoietin (TPO) receptor, thereby independently causing myeloproliferative neoplasms (MPNs) (Non-Patent Documents 3 to 6).

[0003] CALR gene mutations found in MPN patients are always frameshift mutations at a localized location in the final exon, and mutant CALR proteins have sequences at their C-terminus that are not present in the wild-type. In particular, the C-terminal 44 amino acids are common to almost all mutant CALR proteins. As an example, Figure 1 shows a comparison of the C-terminal sequences of the most common CALR gene mutation found in MPN patients, the 52-base deletion (Del 52), and the second most common, the 5-base insertion (Ins 5), with those of the corresponding region in the wild-type CALR protein. Because mutant CALR proteins that cause MPN are expressed in tumor cells, it has been suggested that sequences specific to mutant CALR proteins resulting from frameshift mutations could serve as neoantigens, diagnostic markers, or therapeutic targets (Patent Documents 1 and 2).

[0004] Furthermore, functional analysis of mutant CALR proteins revealed that, in addition to those represented by Del 52 and Ins 5, there exist truncated mutant CALR proteins in which the mutant CALR protein is truncated within a sequence specific to the mutant CALR protein, resulting in the loss of most of the sequence thought to be the neoantigen. We have previously demonstrated that the production of antibodies that specifically recognize a very short amino acid sequence located N-terminal to the cleavage site in the neoantigen, and antibodies that bind to an amino acid sequence further C-terminal to that, results in a therapeutic effect against MPN. Furthermore, by taking advantage of the properties of antibodies that can specifically bind to two types of antigens, we have obtained an anti-CALR / CD3 bispecific antibody as a more effective therapeutic agent, with the aim of enhancing antitumor activity.

[0005] Meanwhile, although alkylating agents, platinum compounds, metabolic antagonists, ribonucleotide reductase inhibitors, nucleotide analogs, topoisomerase inhibitors, microtubule polymerization inhibitors, Bcl2 inhibitors, antitumor antibiotics, interferons, cytokine preparations, molecularly targeted drugs, nucleic acid synthesis inhibitors, JAK inhibitors, and cancer immunotherapy drugs have been reported as small molecule cancer therapeutic agents, none of which have satisfactory efficacy or safety issues such as bone marrow suppression and blood toxicity.

[0006] International Publication No. WO 2015 / 036599 International Publication No. WO 2016 / 087514 International Publication No. WO 1999 / 054440 International Publication No. WO 2019 / 178362 International Publication No. WO 2020 / 175689

[0007] Klampfl T,Gisslinger H,Harutyunyan AS,et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. The New England journal of medicine.2013;369:2379-90.Nangalia J,Massie CE,Baxter EJ,et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. The New England journal of medicine.2013;369:2391-405.Araki M,Yang Y,Masubuchi N,et al. Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms. Blood.2016;127:1307-16.Elf S,Abdelfattah NS,Chen E,et al. Mutant Calreticulin Requires Both Its Mutant C-terminus and the Thrombopoietin Receptor for Oncogenic Transformation. Cancer Discov.2016;6:368-81.Marty C,Pecquet C,Nivarthi H,et al. Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis. Blood.2016;127:1317-24.Vainchenker W,Kralovics R. Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms. Blood.2017;129:667-79.

[0008] An object of the present invention is to provide a pharmaceutical agent that exhibits a sufficient antitumor effect by using an antibody that binds to a mutant CALR protein in combination with another anticancer drug.

[0009] Therefore, the present inventors have conducted studies to solve the above-mentioned problems, and have found that the antitumor effect is dramatically improved and excellent therapeutic effects are obtained by using an antibody that specifically recognizes a mutant amino acid sequence of CALR in combination with a specific anticancer drug. They have also found that a pharmaceutical comprising a combination of an antibody that specifically binds to a mutant CALR protein and a specific anticancer drug is useful as a more effective pharmaceutical for preventing and / or treating cancer, and have completed the present invention.

[0010] That is, the present invention provides the following (1) to (10): (1) A pharmaceutical for preventing and / or treating cancer, comprising a combination of (A) an antibody or a functional fragment thereof that specifically binds to a mutant calreticulin protein, and (B) one or more drugs selected from alkylating agents, platinum compounds, antimetabolites, ribonucleotide reductase inhibitors, nucleotide analogs, topoisomerase inhibitors, microtubule polymerization inhibitors, Bcl2 inhibitors, antitumor antibiotics, interferons, cytokine preparations, molecularly targeted drugs, nucleic acid synthesis inhibitors, JAK inhibitors, and cancer immunotherapeutic drugs. (2) The pharmaceutical according to (1), wherein component (A) is an antibody or a functional fragment thereof that does not bind to a wild-type calreticulin protein and has high affinity for a mutant calreticulin protein. (3) The pharmaceutical according to (1) or (2), wherein component (A) is an antibody or a functional fragment thereof that binds to a mutant amino acid sequence represented by SEQ ID NO: 2 or 3, or a sequence showing 80% or more homology to said sequence. (4) The pharmaceutical according to any one of (1) to (3), wherein component (A) is one or more antibodies or functional fragments thereof selected from a human antibody, a humanized antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody of an animal-derived antibody and a human antibody, Fab, Fab', F(ab')2, a single-chain antibody (scFv), and a diabody. (5) The pharmaceutical according to any one of (1) to (4), wherein component (B) is one or more drugs selected from a JAK inhibitor, a nucleic acid synthesis inhibitor, an interferon, a cytokine preparation, a molecular-targeted drug, and a cancer immunotherapy drug. (6) The pharmaceutical according to any one of (1) to (5), wherein component (B) is one or more drugs selected from ruxonitilib, hydroxyurea, interferon α, and a cancer immune checkpoint inhibitor. (7) The medicine according to any one of (1) to (6), wherein the cancer is selected from lymphoma, leukemia, multiple myeloma, myeloproliferative neoplasm (MPN), digestive cancer (esophageal cancer, gastric cancer, duodenal cancer, etc.), liver cancer, biliary tract cancer (gallbladder / bile duct cancer, etc.), pancreatic cancer, small intestine cancer, large intestine cancer (colorectal cancer, colon cancer, rectal cancer, etc.), gastrointestinal stromal tumor, lung cancer (non-small cell lung cancer, small cell lung cancer), and renal cancer.(8) Use of a combination of (A) an antibody or a functional fragment thereof that specifically binds to a mutant calreticulin protein and (B) one or more drugs selected from alkylating agents, platinum preparations, antimetabolites, ribonucleotide reductase inhibitors, nucleotide analogs, topoisomerase inhibitors, microtubule polymerization inhibitors, Bcl2 inhibitors, antitumor antibiotics, interferons, cytokine preparations, molecularly targeted drugs, nucleic acid synthesis inhibitors, JAK inhibitors, and cancer immunotherapeutic drugs, for the manufacture of a pharmaceutical for preventing and / or treating cancer. (9) For the prevention and / or treatment of cancer, a combination of (A) an antibody or a functional fragment thereof that specifically binds to a mutant calreticulin protein and (B) one or more drugs selected from alkylating agents, platinum preparations, antimetabolites, ribonucleotide reductase inhibitors, nucleotide analogs, topoisomerase inhibitors, microtubule polymerization inhibitors, Bcl2 inhibitors, antitumor antibiotics, interferons, cytokine preparations, molecularly targeted drugs, nucleic acid synthesis inhibitors, JAK inhibitors, and cancer immunotherapeutic drugs. (10) A method for preventing and / or treating cancer, characterized by administering in combination (A) an antibody or a functional fragment thereof that specifically binds to a mutant calreticulin protein and (B) one or more drugs selected from alkylating agents, platinum preparations, antimetabolites, ribonucleotide reductase inhibitors, nucleotide analogs, topoisomerase inhibitors, microtubule polymerization inhibitors, Bcl2 inhibitors, antitumor antibiotics, interferons, cytokine preparations, molecularly targeted drugs, nucleic acid synthesis inhibitors, JAK inhibitors, and cancer immunotherapeutic drugs.

[0011] The antibody used in the present invention has an antigen recognition site (epitope) in the mutant CALR polypeptide chain, and when used in combination with the anticancer drug, it can more effectively damage cancer cells and dramatically improve the antitumor effect than when either drug is administered alone. Therefore, the pharmaceutical of the present invention can be used to specifically prevent and / or treat various cancers, particularly diseases expressing mutant CALR.

[0012] This figure shows the characteristics of mutant CALR proteins. The CALR gene mutation that causes the onset of MPN patients is a +1 frameshift mutation, and the resulting mutant CALR protein has a common amino acid sequence found at its C-terminus. SP: signal sequence, N: N domain, P: P domain. The arrow indicates the beginning of the amino acid sequence that differs from the wild-type (WT). This figure shows the combined effect of an anti-mutant CALR / CD3 bispecific antibody (antibody No. 1) and hydroxyurea. UT-7 / TPO / Ins5-Luc cells (target cells) expressing mutant CALR and luciferase were cultured with human peripheral blood T cells in the presence of a CALR / CD3 bispecific antibody and hydroxyurea, and the cell viability of the target cells was measured using luciferase activity as an indicator to evaluate T cell-dependent cellular cytotoxicity (TDCC). The cell viability of each well was calculated, with the chemiluminescence value of the group without a CALR / CD3 bispecific antibody and concomitant drug set at 100%. The combined effect of an anti-mutant CALR / CD3 bispecific antibody (antibody no. 2) and hydroxyurea is shown. The combined effect of an anti-mutant CALR / CD3 bispecific antibody (antibody no. 3) and hydroxyurea is shown.

[0033] Figure 1 shows the combined effect of anti-mutated CALR / CD3 bispecific antibody (antibody No. 4) and hydroxyurea.

[0034] Figure 1 shows the combined effect of anti-mutated CALR / CD3 bispecific antibody (antibody No. 5) and hydroxyurea.

[0035] Figure 1 shows the combined effect of anti-mutated CALR / CD3 bispecific antibody (antibody No. 6) and hydroxyurea.

[0036] Figure 1 shows the combined effect of anti-mutated CALR / CD3 bispecific antibody (antibody No. 7) and hydroxyurea.

[0037] Figure 1 shows the combined effect of anti-mutated CALR / CD3 bispecific antibody (antibody No. 8) and hydroxyurea.

[0038] Figure 1 shows the combined effect of anti-mutated CALR / CD3 bispecific antibody (antibody No. 9) and hydroxyurea.

[0039] Figure 2 shows the combined effect of anti-mutated CALR / CD3 bispecific antibody (antibody No. 1) and ruxolitinib in in vitro efficacy.UT-7 / TPO / Ins5 / GFP-Luc cells (target cells) expressing mutant CALR and luciferase were cultured with human peripheral blood T cells in the presence of a CALR / CD3 bispecific antibody and ruxolitinib. The cell viability of the target cells was measured using luciferase activity as an indicator, and T cell-dependent cellular cytotoxicity (TDCC) was evaluated. The cell viability of each well was calculated, with the chemiluminescence value of the group containing the CALR / CD3 bispecific antibody (antibody no. 1) and no concomitant drug set at 100%. The combined effect of an anti-mutant CALR / CD3 bispecific antibody (antibody no. 7) and ruxolitinib is shown. The combined effect of an anti-mutant CALR / CD3 bispecific antibody (antibody no. 8) and ruxolitinib is shown. Figure 1 shows the combined effect of an anti-mutant CALR / CD3 bispecific antibody (antibody No. 9) and ruxolitinib. Figure 2 shows the combined effect of an anti-mutant CALR / CD3 bispecific antibody (antibody No. 1) and interferon α. ​​UT-7 / TPO / Ins5-Luc cells (target cells) expressing mutant CALR and luciferase were cultured with human peripheral blood T cells in the presence of a CALR / CD3 bispecific antibody and interferon α (IFN-α). The cell viability of the target cells was measured using luciferase activity as an indicator, and T cell-dependent cellular cytotoxicity (hereinafter referred to as TDCC) was evaluated. The cell viability of each well was calculated assuming that the chemiluminescence value of the group not containing the CALR / CD3 bispecific antibody and the concomitant drug was 100%.

[0033] Figure 1 shows the combined effect of anti-mutated CALR / CD3 bispecific antibody (antibody No. 4) and IFN-α.

[0034] Figure 1 shows the combined effect of anti-mutated CALR / CD3 bispecific antibody (antibody No. 5) and IFN-α.

[0035] Figure 1 shows the combined effect of anti-mutated CALR / CD3 bispecific antibody (antibody No. 6) and IFN-α.

[0036] Figure 1 shows the combined effect of anti-mutated CALR / CD3 bispecific antibody (antibody No. 8) and IFN-α.

[0037] Figure 1 shows the combined effect of anti-mutated CALR / CD3 bispecific antibody (antibody No. 9) and IFN-α.

[0013] The present invention provides a method for detecting and diagnosing a mutant CALR protein associated with MPN, and has discovered that cancer can be effectively treated and / or prevented by using an antibody that recognizes 13 amino acids (SEQ ID NO: 4) in the C-terminal polypeptide ( FIG. 1 ) remaining after cleavage in a sequence specific to the mutant CALR protein, which is used as a preventive or therapeutic agent for MPN, in combination with an antibody that recognizes a sequence further C-terminal thereto (SEQ ID NO: 5), in combination with the anticancer drug. That is, the present invention provides a pharmaceutical for preventing and / or treating cancer, particularly a pharmaceutical for preventing and / or treating MPN, comprising a combination of (A) an antibody that specifically recognizes a mutant CALR protein and (B) the anticancer drug.

[0014] Component (A) used in the pharmaceutical of the present invention is an antibody that specifically recognizes a mutant CALR protein. As used herein, the term "antibody or a functional fragment thereof" refers to a protein that specifically binds to a mutant CALR, including a monoclonal antibody, a chimeric antibody produced by genetic recombination technology, a humanized antibody, a human antibody, a bispecific antibody, a multispecific antibody, an engager, Fab, Fab', F(ab')2, a single-chain antibody (scFv), a diabody, a minibody, and the like, a protein fragment thereof, and a derivative containing the protein fragment.

[0015] Monoclonal antibody forms include IgG, IgM, IgA, IgE, and IgD of humans and non-human animals. Furthermore, the form of bispecific and multispecific antibodies is not particularly limited and may be any form known in the art, or any other form as long as they retain specificity for two or more antigens. Bispecific antibodies are broadly classified into IgG-like and small molecule types. IgG-like types are forms that retain the Fc region. Examples of IgG-like antibody forms include, but are not limited to, CrossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-into-holes, knobs-into-holes common LC, SEEDbody, Triomab, κλ-body, DVD-Ig, IgG-scFv, and DuoBody. Since IgG-like antibodies retain the Fc region, they are expected to exhibit effector functions such as ADCC and CDC, facilitate purification, improve stability, and extend blood half-life. On the other hand, small molecule forms are typically formed using an Fv region consisting of a heavy chain variable region and a light chain variable region as their basic constituent. Examples of small molecule antibody forms include, but are not limited to, diabodies (Db), BiTEs, DARTs, TandAb, scDb, triple bodies, miniantibodies, minibodies, scFvs, tandem scFvs, and F(ab')2. Due to their size, small molecule antibodies are expected to have improved tissue permeability and high productivity. Other examples include modified antibodies in which the amino acid sequence has been deleted, substituted, or added while retaining antigen-binding ability, those in which a portion of the sugar chain has been deleted or added, those in which a linker or the like has been added, those fused with other proteins, and antibody-drug conjugates (ADCs) in which an antibody and a small molecule drug are linked via a linker.

[0016] The antibody of the present invention has a domain that specifically binds to a mutant calreticulin protein. The domain competes with an antibody that binds to an epitope of the mutant calreticulin protein of SEQ ID NO: 4 or 5. Here, the mutant calreticulin protein preferably has an antigen recognition site in a polypeptide chain consisting of the amino acid sequence represented by SEQ ID NO: 4 or 5, or in a polypeptide chain consisting of the amino acid sequence of SEQ ID NO: 4 or 5 in which one or several amino acids have been deleted, substituted, or added.

[0017] The domain does not bind to the wild-type calreticulin protein, but has high affinity for the mutant calreticulin protein.

[0018] Truncated CALR proteins include polypeptide (i) consisting of the amino acid sequence represented by SEQ ID NO: 4 or 5, and polypeptide (ii) consisting of the amino acid sequence represented by SEQ ID NO: 4 or 5 in which one or several amino acids have been deleted, substituted, or added. In (ii), an amino acid sequence in which one to four amino acids have been deleted, substituted, or added is preferred, and an amino acid sequence in which one to three amino acids have been deleted, substituted, or added is more preferred. Furthermore, the identity between the amino acid sequence of polypeptide (ii) and the amino acid sequence of SEQ ID NO: 1 is preferably 80% or more, more preferably 85% or more, and even more preferably 90% or more.

[0019] The antibody binding domain may be any antibody having an antigen recognition site (epitope) in the polypeptide chain of (i) or (ii), but may be one that binds only to a truncated mutant CALR protein, or one that binds to both a truncated mutant CALR protein and a full-length mutant CALR protein, and is preferably an antibody specific to a "mutant CALR protein" that binds to both a truncated mutant CALR protein and a full-length mutant CALR protein.

[0020] Such binding domains are preferably the heavy chain variable region and light chain variable region, as well as the heavy chain complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3) and light chain complementarity determining regions (CDR-L1, CDR-L2, and CDR-L3) of the anti-mutated CALR antibodies (clones B3, C6, and G1 antibodies) discovered by the present inventors.

[0021] In bispecific antibodies, the first domain is preferably the heavy chain variable region and light chain variable region of the anti-mutated CALR antibody (clone B3, C6, and G1 antibody), and further the heavy chain complementarity determining region (CDR-H1, CDR-H2, and CDR-H3) and light chain complementarity determining region (CDR-L1, CDR-L2, and CDR-L3). The second domain in bispecific antibodies is preferably a domain that specifically binds to the CD3 antigen, and is preferably the heavy chain variable region and light chain variable region derived from an existing anti-CD3 antibody, and further the heavy chain complementarity determining region (CDR-H1, CDR-H2, and CDR-H3) and light chain complementarity determining region (CDR-L1, CDR-L2, and CDR-L3) derived from a known anti-CD3 antibody. Examples of existing anti-CD3 antibodies include OKT3 and UCTH1.

[0022] Preferred examples of the antibodies of the present invention are the heavy chain variable regions and light chain variable regions, as well as the heavy chain complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3) and light chain complementarity determining regions (CDR-L1, CDR-L2, and CDR-L3) of anti-mutated CALR antibodies (clones B3, C6, and G1 antibodies), and examples of their amino acid sequences are shown in Table 1. Table 1 also shows the heavy chain variable regions and light chain variable regions, as well as the heavy chain complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3) and light chain complementarity determining regions (CDR-L1, CDR-L2, and CDR-L3) of anti-mutated CALR antibodies (clones B3, C6, and G1 antibodies) and anti-CD3 antibodies (OKT3, UCHT1), as preferred examples of the first domain and second domain of the bispecific antibody.

[0023]

[0024] These regions can be produced as recombinant antibodies by genetic engineering techniques based on the sequence information of antibodies obtained by immunizing animals with a partial or full-length truncated mutant CALR protein or by in vitro sensitization of lymphocytes and fusing them with myelomas, or antibodies obtained from antibody libraries such as phage display. Sequence information for anti-CD3 antibodies can be obtained from existing information. Specifically, antibody heavy and light chain genes can be synthesized from the obtained genetic information, inserted into a vector (e.g., a plasmid), and then introduced into host cells (e.g., CHO cells, HEK cells, etc.). The host cells can then be cultured, allowing recombinant antibodies to be collected from the culture. Codon optimization is preferred when synthesizing the antibody heavy and light chain genes.

[0025] Preferred bispecific antibodies of the present invention include bispecific antibodies in which the CDRs of the first domain have (a) an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 14-16 (VHCDR) and an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 17-19 (VLCDR), (b) an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 20-22 (VHCDR) and an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 23-25 ​​(VLCDR), or (c) an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 26-28 (VHCDR) and an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 29-31 (VLCDR).

[0026] Further examples include bispecific antibodies in which the first domain has (d) a VH region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 6, and a VL region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 7, (e) a VH region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 8, and a VL region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 9, or (f) a VH region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 10, and a VL region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 11.

[0027] Also preferred are bispecific antibodies in which the CDRs of the second domain have an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 32-34 (VHCDR) and an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 35-37 (VLCDR), or an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 219-221 (VHCDR) and an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 222-224 (VLCDR). Furthermore, a bispecific antibody in which the second domain comprises a VH region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 12, and a VL region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 13, or a bispecific antibody in which the second domain comprises a VH region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 217, and a VL region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 218, is preferred.

[0028] More preferred bispecific antibodies of the present invention include those in which the CDR of the first domain has (a) an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NOs: 14-16 (VHCDR) and an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NOs: 17-19 (VLCDR), (b) an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NOs: 20-22 (VHCDR) and an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NOs: 23-25 ​​(VLCDR), or (c) an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NOs: 26-28 (VHCDR), and an amino acid sequence having 80% or more homology to the amino acid sequence shown in SEQ ID NO: 29-31 (VLCDR); and a bispecific antibody in which the CDR of the second domain has an amino acid sequence having 80% or more homology to the amino acid sequence shown in SEQ ID NO: 32-34 (VHCDR) and an amino acid sequence having 80% or more homology to the amino acid sequence shown in SEQ ID NO: 35-37 (VLCDR), or an amino acid sequence having 80% or more homology to the amino acid sequence shown in SEQ ID NO: 219-221 (VHCDR) and an amino acid sequence having 80% or more homology to the amino acid sequence shown in SEQ ID NO: 222-224 (VLCDR).Further preferred bispecific antibodies include those in which the first domain comprises (d) a VH region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 6, and a VL region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 7, (e) a VH region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 8, and a VL region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 9, or (f) a VH region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 10, and a VL region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 11. and a VL region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 11, and the second domain has (g) a VH region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 12, and a VL region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 13, or (h) a VH region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 217, and a VL region having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 218.

[0029] As described above, the antibody of the present invention may be in any form, including an IgG-like form or a low-molecular-weight form, as long as it retains specificity for the two antigens. Specifically, it may have an Fc region or a heterocyclic heavy chain.

[0030] Specific examples of the bispecific antibody include antibodies selected from the following (I) to (IX): (I) a bispecific antibody consisting of an H chain comprising the amino acid sequence set forth in SEQ ID NO: 174 and an L chain comprising the amino acid sequence set forth in SEQ ID NO: 202 (Antibody 1); (II) a bispecific antibody consisting of an H chain comprising the amino acid sequence set forth in SEQ ID NO: 186 and an L chain comprising the amino acid sequence set forth in SEQ ID NO: 201 (Antibody 2); (III) a bispecific antibody consisting of an H chain comprising the amino acid sequence set forth in SEQ ID NO: 174 and an L chain comprising the amino acid sequence set forth in SEQ ID NO: 201 (Antibody 3); (IV) a bispecific antibody consisting of an H chain comprising the amino acid sequence set forth in SEQ ID NO: 180 and an L chain comprising the amino acid sequence set forth in SEQ ID NO: 202 (Antibody 4); (V) a bispecific antibody consisting of an H chain comprising the amino acid sequence set forth in SEQ ID NO: 181 and an L chain comprising the amino acid sequence set forth in SEQ ID NO: 202 (Antibody 5); and (VI) a bispecific antibody consisting of an H chain comprising the amino acid sequence set forth in SEQ ID NO: 161 and an L chain comprising the amino acid sequence set forth in SEQ ID NO: 171 (Antibody 6). (VII) A bispecific antibody (Antibody 7) consisting of an H chain comprising the amino acid sequence set forth in SEQ ID NO: 225 and an L chain comprising the amino acid sequence set forth in SEQ ID NO: 202. The antibodies (I) to (VII) above all have the same structure, and are bispecific antibodies in which a polypeptide is formed in which a single-chain Fv that specifically binds to CD3 is fused via a peptide linker to the C-terminus of the heavy chain of a Fab fragment that specifically binds to a mutant CALR, and an Fc is further fused to the polypeptide. (VIII) A bispecific antibody (Antibody 8) consisting of the amino acid sequence set forth in SEQ ID NO: 226. (IX) A bispecific antibody (Antibody 9) consisting of an H chain comprising the amino acid sequence set forth in SEQ ID NO: 227 and an L chain comprising the amino acid sequence set forth in SEQ ID NO: 228, in which the N-terminus of the light chain of an IgG that specifically binds to a mutant CALR is fused via a peptide linker to the C-terminus of the single-chain Fv that specifically binds to the CD3 antigen.

[0031] Preferred specific examples of bispecific antibodies of the present invention have the sequences described above, and the amino acid sequences of regions other than these regions are not particularly limited. Furthermore, the antibodies of the present invention may be humanized antibodies and antibodies from mammals other than humans. More specifically, they may be chimeric antibodies consisting of the heavy and light chain variable regions of an antibody from a mammal other than humans, such as a rat, and the heavy and light chain constant regions of a human antibody. Such antibodies can be obtained by linking DNA encoding the variable regions of a rat antibody to DNA encoding the constant regions of a human antibody, incorporating this into an expression vector, and introducing it into a host for production. Examples of humanized antibodies include VH1-5 and VL1-5 shown below (Table 2).

[0032]

[0033] Other methods for obtaining human antibodies are also known. For example, human lymphocytes can be sensitized in vitro with a desired antigen or cells expressing the desired antigen, and the sensitized lymphocytes can be fused with human myeloma cells, such as U266, to obtain a desired human antibody with antigen-binding activity (see Japanese Patent Publication No. 1-59878). Alternatively, a desired human antibody can be obtained by immunizing a transgenic animal carrying a full repertoire of human antibody genes with the desired antigen (see WO93 / 12227, WO92 / 03918, WO94 / 02602, WO94 / 25585, WO96 / 34096, and WO96 / 33735). Furthermore, a technique for obtaining human antibodies by panning using a human antibody library is also known. For example, the variable regions of human antibodies can be expressed on the surface of phages as single-chain fragments (scFv) by phage display, and phages that bind to the antigen can be selected. By analyzing the genes of the selected phages, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. Once the DNA sequence of the scFv that binds to the antigen is known, the sequence can be used to construct an appropriate expression vector to obtain a human antibody. These methods are already well known, and reference can be made to WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, and WO95 / 15388.

[0034] Bispecific antibodies can be produced, for example, by constructing a bispecific antibody expression vector using DNA encoding an anti-mutated CALR antibody and an anti-CD3 antibody and an expression vector, and then introducing the expression vector into CHO cells or HEK293 cells to obtain transformed cells. The culture medium obtained by culturing these transformed cells can be purified by chromatography. Methods for producing bispecific antibodies are already known, and reference can be made to JP2019022497, JP2017137329, and JP2015110628.

[0035] The class of the antibody is not particularly limited, and includes antibodies having any isotype, such as IgG, IgM, IgA, IgD, or IgE. Considering ease of purification, IgG is preferred.

[0036] Examples of domain structures include minibodies such as antibody fragments and modified antibodies. Specific examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, and diabodies. In the Examples described below, the heavy chain constant region of human IgG with amino acid mutations at 228, 233-238, 265, 268, 309, 318, and 328-331, which are believed to suppress binding affinity to Fc receptors, and amino acid mutations at 349, 354, 366, 368, and 407 for heterocyclic heavy chains are shown, but are not limited to these.

[0037] Among the antibodies of the present invention, antibodies that bind to the polypeptides (i) or (ii) above may bind to these polypeptides. On the other hand, antibodies used in the prevention and / or treatment of MPNs are preferably antibodies that bind to the polypeptides (i) or (ii) above and have cytotoxic activity.

[0038] The antibody that binds to a truncated mutant CALR protein may be any antibody that binds to a polypeptide chain having the sequence shown in SEQ ID NO: 2 or 3, but is preferably an antibody that competes with at least one of the above antibodies or functional fragments thereof in binding to the amino acid sequence portion of the mutant CALR protein shown in SEQ ID NO: 2 or 3.

[0039] Examples of component (B) include one or more drugs selected from alkylating agents, platinum preparations, antimetabolites, ribonucleotide reductase inhibitors, nucleotide analogs, topoisomerase inhibitors, microtubule polymerization inhibitors, Bcl2 inhibitors, antitumor antibiotics, interferons, cytokine preparations, molecular targeted drugs, nucleic acid synthesis inhibitors, JAK inhibitors, and cancer immunotherapy drugs. Of these, one or more drugs selected from JAK inhibitors, antimetabolites (including ribonucleotide reductase inhibitors), nucleic acid synthesis inhibitors, interferons, cytokine preparations, molecular targeted drugs, and cancer immunotherapy drugs are preferred.

[0040] Alkylating agents include cyclophosphamide, ifosfamide, dacardin, temozolomide, nimustine, busulfan, ranimustine, etc. Platinum drugs include cisplatin, carboplatin, oxaliplatin, etc. Antimetabolites, ribonucleotide reductase inhibitors, and nucleotide analogs include hydroxyurea, methotrexate, 5-FU, tegafur, 6-mercaptopurine, capecitabine, pendostatin, etc. Topoisomerase inhibitors include doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone, pirarubicin, amrubicin, etoposide, and irinotecan, etc. Bcl2 inhibitors include Beneclexta, etc. Antitumor antibiotics include mitomycin C, actinomycin D, bleomycin, beplomycin, and zinostatin steamer, etc. Examples of microtubule polymerization inhibitors include paclitaxel and docetaxel. Examples of JAK inhibitors include tofacitinib, baricitinib, peficitinib, upadacitinib, filgotinib, agrocitinib, and ruxolitinib. Examples of interferons include interferon-α, interferon-β, and interferon-γ. Examples of cytokine preparations include interleukin 2. Examples of nucleic acid synthesis inhibitors include azathioprion, mizoribine, and cyclophosphamide. Examples of molecularly targeted drugs and cancer immunotherapeutic drugs include antibody drugs such as anti-EGFR antibodies, anti-HER2 antibodies, anti-VEGF antibodies, anti-VEGFR antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies; EGFR agonists such as gefitinib, erlotinib, afatinib, osimertinib, and dakocitinib; HER2 agonists such as lapatinib; mTOR agonists such as eperolimus, temsirolimus, and sirolimus; and cancer immune checkpoint inhibitors such as anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, and CTLA-4-Ig (cancer immune checkpoint inhibitors are also classified as molecularly targeted drugs). Examples of anti-PD-1 antibodies include nivolumab and pembrolizumab. Examples of anti-PD-L1 antibodies include atezolizumab, durvalumab, and avelumab.Examples of anti-CTLA-4 antibodies include ipilimumab and tremelimumab. Examples of CTLA-4-Ig include abatacept. Among these components (B), one or more drugs selected from ruxolitinib, hydroxyurea, interferon α, and cancer immune checkpoint inhibitors are particularly preferred.

[0041] As shown in the Examples below, when component (A) and component (B) are used in combination, the antitumor effect against various cancers is enhanced, making them useful as a pharmaceutical for preventing and / or treating cancer. Furthermore, component (A) is useful as an agent for enhancing the antitumor effect of component (B). Furthermore, component (B) is useful as an agent for enhancing the antitumor effect of component (A).

[0042] The form of the combination pharmaceutical of the present invention is not particularly limited, and specific examples include the following forms (i) and (ii): (i) a form of a single preparation (combined preparation) containing both component (A) and component (B). (ii) a form for separately administering a preparation containing component (A) and a preparation containing component (B). In the case of form (ii), the preparations may be administered simultaneously or separately at an appropriate time interval, and an appropriate administration regimen can be adopted so as to achieve the desired cancer prevention and / or treatment effect. In a form for separately administering a preparation containing component (A) and a preparation containing component (B), both preparations can also be provided as a kit preparation containing both preparations in a single package.

[0043] Specific examples of cancers that are the subject of the present invention include lymphoma, leukemia, multiple myeloma, myeloproliferative neoplasm (MPN), head and neck cancer, digestive cancer (esophageal cancer, gastric cancer, duodenal cancer, etc.), liver cancer, biliary tract cancer (gallbladder / bile duct cancer, etc.), pancreatic cancer, small intestine cancer, large intestine cancer (colorectal cancer, colon cancer, rectal cancer, etc.), gastrointestinal stromal tumor, lung cancer (non-small cell lung cancer, small cell lung cancer), breast cancer, ovarian cancer, uterine cancer (cervical cancer, endometrial cancer, etc.), kidney cancer, bladder cancer, prostate cancer, skin cancer, etc. Here, cancer includes not only primary tumors but also cancer that has metastasized to other organs (such as the liver). The pharmaceutical agent of the present invention is more useful for lymphoma, leukemia, multiple myeloma, myeloproliferative neoplasms (MPN), digestive cancers (esophageal cancer, gastric cancer, duodenal cancer, etc.), liver cancer, biliary tract cancer (gallbladder / bile duct cancer, etc.), pancreatic cancer, small intestine cancer, large intestine cancer (colorectal cancer, colon cancer, rectal cancer, etc.), gastrointestinal stromal tumor, lung cancer (non-small cell lung cancer, small cell lung cancer, etc.), renal cancer, etc., and is further useful for lymphoma, leukemia, multiple myeloma, myeloproliferative neoplasms (MPN), etc., and is even more useful for cancers in which a mutant CALR is detected, in particular MPN.

[0044] As described above, the pharmaceutical of the present invention may be in the form of a composition containing component (A), a composition containing component (B), or a composition containing component (A) and component (B). These compositions can be formulated and produced by mixing, dissolving, emulsifying, encapsulating, lyophilizing, or the like, component (A) and / or (B) together with a pharmaceutically acceptable carrier.

[0045] Suitable formulations for oral administration include liquid preparations in which an effective amount of component (A) and / or component (B) is dissolved in a diluent such as water or physiological saline; capsules, granules, powders, or tablets containing an effective amount as a solid or granule; suspensions in which an effective amount is suspended in a suitable dispersion medium; and emulsions in which a solution in which an effective amount is dissolved is dispersed and emulsified in a suitable dispersion medium.

[0046] For parenteral administration, component (A) and / or component (B) can be formulated into dosage forms such as injection solutions, suspensions, emulsions, creams, ointments, inhalants, and suppositories together with pharmaceutically acceptable solvents, excipients, binders, stabilizers, dispersants, etc. For injection formulations, component (A) and / or component (B) can be dissolved in an aqueous solution, preferably a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. Furthermore, the medicament of the present invention can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle. Alternatively, component (A) and / or component (B) can be prepared in powder form, and an aqueous solution or suspension can be prepared using sterile water or the like before use. For inhalation administration, component (A) and / or component (B) can be powdered and mixed with a suitable base such as lactose or starch to form a powder mixture. Suppository formulations can be prepared by mixing component (A) and / or component (B) with a conventional suppository base such as cocoa butter. Furthermore, the medicament of the present invention can be encapsulated in a polymer matrix or the like to be formulated as a sustained-release preparation.

[0047] When a commercially available product is used, component (B) can be administered according to its dosage and administration. Component (A) is preferably administered at a dose of 0.3 mg or more, more preferably 0.3 mg to 300 mg, and even more preferably 3 mg to 300 mg.

[0048] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples in any way.

[0049] Test Example 1: Preparation of antibodies that recognize mutant CALR proteins, including truncated forms. Peptides containing cysteine ​​added to the C- or N-terminus of Dianova mouse monoclonal antibody clone CAL2 (Cat#DIA-CAL) or the amino acid sequence contained in SEQ ID NOs: 2-5 were synthesized and conjugated to the carrier protein keyhole limpet hemocyanin (KLH). These were then used to immunize 8-week-old female WKY / Izm rats. Lymphocytes were then collected after a booster immunization. The lymphocytes were fused with mouse myeloma SP2 cells using the PEG method and cultured in selective medium to obtain hybridomas. The specificity of antibodies in the culture supernatant was screened by ELISA using the immunizing peptide or purified protein to obtain hybridomas (clones B3, C6, and G1) that produced antibodies that bind to mutant CALR proteins, including truncated forms. The antibodies produced from these hybridomas were purified and used in the tests (clone B3 antibody, C6 antibody, and G1 antibody).

[0050] Test Example 2: Preparation of mutant CALR-expressing cells UT-7 / TPO CALR Del 52 cells and UT-7 / TPO CALR Ins 5 cells, which grow tumorously by expressing the Del 52 or Ins 5 mutant CALR protein in the human megakaryoblastic leukemia cell line UT-7 / TPO cells, and UT-7 / TPO vec cells, which were transfected with only the vector used for gene transfer as a control, were cultured at 6.0 × 10 5 The cells were seeded in OptiMEM medium (Thermo Fisher) at a density of 10 cells / mL and cultured for 32 hours at 37°C in the presence of 5% CO. UT-7 / TPO vec cells were cultured in a medium containing 10 ng / mL TPO.

[0051] Test Example 3 Confirmation of Recognition of Mutant CALR Protein UT-7 / TPO cells expressing Del 52 or Ins 5 mutant CALR with a FLAG tag inserted downstream of the N-terminal signal sequence were cultured, and the culture supernatant containing the secreted mutant CALR protein was prepared by centrifugation (1,600 g × 5 minutes, 4°C). The resulting culture supernatant was heat-treated in the presence of SDS and a reducing agent, and then developed on a gel by SDS-polyacrylamide electrophoresis. The resulting protein was then electrically transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was then reacted with a TBS-T solution containing 5% skim milk at room temperature for 1 hour, followed by overnight reaction with a 5% BSA / TBS-T solution containing rat clone B3, C6, or G1 antibodies or a mouse anti-DYKDDDDK tag antibody (Fujifilm Wako Pure Chemical Industries, Ltd.) at 4°C. After washing with TBS-T solution, the membranes were incubated with peroxidase-labeled goat anti-rat IgG antibody (Jackson ImmunoResearch Inc.) or peroxidase-labeled goat anti-mouse IgG antibody (Jackson ImmunoResearch Inc.) in 5% skim milk / TBS-T solution at room temperature for 1 hour. After washing the PVDF membrane with TBS-T solution, the membranes were reacted with a peroxidase luminescent reagent, and the resulting signals were detected using a FUSION imaging system. The results confirmed that clones B3, C6, and G1 antibodies all recognized both the full-length and truncated mutant CALR proteins, while CAL2 recognized a sequence closer to the C-terminus than the full-length and truncated mutant CALR proteins.

[0052] [Test Example 4] Determination of Sequence Information of the Antibody of the Present Invention The sequence information of the heavy and light chains of the antibody of the present invention was determined by preparing mRNA from antibody-producing cells using the PureLinc RNA Mini Kit (Thermo Fisher). Using the obtained mRNA as a template and heavy-chain or light-chain-specific reverse transcription primers, cDNA was synthesized by the rapid amplification of cDNA ends method and then cloned into a plasmid. The obtained plasmid was subjected to Sanger sequencing to determine the full-length sequences of the heavy and light chain cDNAs.

[0053] [Example 1] Preparation of bispecific antibodies Materials and test methods Bispecific antibody expression vectors were constructed using DNA encoding bispecific antibodies based on the sequence information of clones B3, C6, and G1 antibodies and anti-CD3 antibodies (Table 1) and an expression vector (pcDNA3.4, Thermo Fisher). The expression vectors were introduced into CHO cells or HEK293 cells using an ExpiFectamine CHO Transfection Kit (Thermo Fisher) or ExpiFectamine 293 Transfection Kit (Thermo Fisher). The transformed cells were cultured, and the cells were removed by centrifugation and filtration, and the culture medium was collected. Antibody purification was carried out by a combination of affinity chromatography using nickel-conjugated agarose and gel filtration chromatography, a combination of Protein A affinity chromatography and gel filtration chromatography, or a combination of affinity chromatography using CaptureSelect kappa XL resin (Thermo Fisher) and gel filtration chromatography. Furthermore, bispecific antibodies (antibody 32: SEQ ID NOs: 114 and 115, antibody 33: SEQ ID NOs: 116 and 117, antibody 34: SEQ ID NOs: 118 and 119) and B3 chimeric antibody (SEQ ID NOs: 52 and 64) based on the sequence information described in Patent Document 4 and Patent Document 2 were prepared using the same method as above.

[0054] Example 2 Confirmation of Recognition of Mutant CALR Protein on Cell Surface by Flow Cytometry Analysis Materials and Test Methods UT-7 / TPO vec cells transfected with only the vector used for gene transfer as a control, UT-7 / TPO CALR Ins 5 cells expressing mutant CALR, and UT-7 / TPO CALR Del 52 cells were cultured in IMDM medium containing 10% inactivated fetal bovine serum (FBS) at 37°C in the presence of 5% CO. 5 x 10 cells in the proliferation phase were cultured. 4After centrifugation (400g x 5 minutes, 4°C), various antibodies were prepared in an FBS / PBS solution at a concentration of 5 μg / mL and allowed to react on ice for 30 minutes. After the reaction, the cells were washed twice with MACS buffer, and then an anti-human Fc antibody was prepared as a secondary antibody in an FBS / PBS solution at a concentration of 2.5 μg / mL and allowed to react on ice for 30 minutes. After the reaction was completed, the cells were washed twice by adding MACS buffer, centrifuging (400g x 5 minutes, 4°C), and discarding the supernatant. The signal was quantified by flow cytometry analysis using a BD LSRFortessa X-20 (BD Biosciences).

[0055] Representative anti-truncated mutant CALR-CD3 bispecific antibodies prepared using the VH chain of SEQ ID NO: 6, the VL chain of SEQ ID NO: 3, the VH chain of SEQ ID NO: 8, and the VL chain of SEQ ID NO: 9 were used to perform flow cytometry analysis on [a] UT-7 / TPO vec cells, [b] UT-7 / TPO CALR Ins 5 cells, and [c] UT-7 / TPO CALR Del 52 cells.

[0056] [Example 3] Confirmation of recognition of CD3 antigen on cell surface by flow cytometry analysis Materials and test method Peripheral blood mononuclear cells (PBMCs) isolated from healthy human donors were cultured for 2 days on plates coated with anti-CD3 antibody (Takara Bio) for 4 to 24 hours, and then cultured for an additional 10 to 21 days in RPMI-1640 medium (Sigma-Aldrich) containing IL-2 and 10% FBS to induce T-LAK cells. 5 x 10 T-LAK cells 5After centrifugation (400 g × 5 minutes, 4°C), an anti-truncated mutant CALR-CD3 bispecific antibody prepared using the VH chain of SEQ ID NO: 2, the VL chain of SEQ ID NO: 3, the VH chain of SEQ ID NO: 8, and the VL chain of SEQ ID NO: 9 was prepared in an FBS / PBS solution at a concentration of 5 μg / mL and allowed to react on ice for 30 minutes. After the reaction, the cells were washed twice with MACS buffer, and then an anti-human Fc antibody was prepared as a secondary antibody in an FBS / PBS solution at a concentration of 5 μg / mL and allowed to react on ice for 30 minutes. After the reaction was completed, the cells were washed twice by adding MACS buffer, centrifuging (400 g × 5 minutes, 4°C), and discarding the supernatant. The signal was quantified by flow cytometry analysis using a BD LSRFortessa X-20 (BD Biosciences). As a result, it was confirmed that the bispecific antibody specifically recognizes the CD3 antigen expressed on the surface of T-LAK cells.

[0057] Example 4 Evaluation of in vitro cytotoxic activity (Materials and test method) UT-7 / TPO / Ins5-Luc cells, which are target cells, were cultured with T cells in the presence of a bispecific antibody and hydroxyurea, ruxolitinib, or interferon α, and the cell viability of the target cells was measured using luciferase activity as an indicator to evaluate T cell-dependent cellular cytotoxicity (hereinafter referred to as TDCC). T cells were isolated and enriched from healthy donor blood using RosetteSep Human T Cell Enrichment Cocktail (STEMCELL technologies) or from healthy donor peripheral blood mononuclear cells (PBMCs) using a Pan T cell isolation kit (Miltenyi biotech). To each well of a 384-well plate, 20 μL of T cell suspension, 20 μL of target cells, 5 μL of anti-bispecific antibody dilution, and 5 μL of combination drug dilution were added (final volume 50 μL / well, 5 × 10 T cells). 4 cells / well, target cells 5x10 3After 48 hours of incubation at 37°C and 5% CO2 (25 cells / well, E / T = 10), 25 μL of Steady-Glo Luciferase Assay reagent was added to each well, and the chemiluminescence of each well was measured. The chemiluminescence value of the control well containing no bispecific antibody or concomitant drug was set at 100%, and the cell viability of each well was calculated. The combination effect was evaluated using the combination index (CI). CI calculations were based on the Chou-Talalay method (Chou T.C. & Talalay, P.: Adv. Enz. Regul. 22:27-55, 1984) and were performed using CompuSyn software (Combosyn, Inc.). A CI of less than 0.7 indicates a synergistic effect, and a CI of less than 0.3 indicates a strong synergistic effect. A CI of around 1 indicates an additive effect. Table 3 shows representative CI values, which are the CI values ​​for the lowest concentration combinations at which cytotoxic activity exceeds 85% and a combined effect is observed.

[0058] As shown in Figures 2-A to 4 and Table 3, the combination of the antibody of the present invention and the above-mentioned drug exhibited cytotoxic activity against UT-7 / TPO CALR Ins 5 cells expressing mutant CALR.

[0059]

Claims

1. A pharmaceutical for preventing and / or treating cancer, comprising a combination of (A) an antibody or a functional fragment thereof that specifically binds to a mutant calreticulin protein and (B) one or more drugs selected from alkylating agents, platinum preparations, antimetabolites, ribonucleotide reductase inhibitors, nucleotide analogs, topoisomerase inhibitors, microtubule polymerization inhibitors, Bcl2 inhibitors, antitumor antibiotics, interferons, cytokine preparations, molecularly targeted drugs, nucleic acid synthesis inhibitors, JAK inhibitors, and cancer immunotherapeutic drugs.

2. The pharmaceutical according to claim 1, wherein component (A) is an antibody or a functional fragment thereof that does not bind to a wild-type calreticulin protein but has high affinity for a mutant calreticulin protein.

3. 3. The pharmaceutical according to claim 1, wherein component (A) is an antibody or a functional fragment thereof that binds to a mutant amino acid sequence represented by SEQ ID NO: 2 or 3 or a sequence showing 80% or more homology to said sequence.

4. The component (A) is a human antibody, a humanized antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody of an animal-derived antibody and a human antibody, Fab, Fab', F(ab') 2 3. The pharmaceutical according to claim 1 or 2, which is one or more antibodies or functional fragments thereof selected from the group consisting of a single-chain antibody (scFv) and a diabody.

5. 3. The pharmaceutical composition according to claim 1, wherein component (B) is one or more drugs selected from the group consisting of JAK inhibitors, nucleic acid synthesis inhibitors, interferons, cytokine preparations, molecular targeted drugs, and cancer immunotherapeutic drugs.

6. Ingredient (B) is ruxonitirib, hydroxyurea, interferon α and immunosuppressant. The pharmaceutical composition according to claim 1 or 2, which is one or more drugs selected from immune checkpoint inhibitors.

7. 3. The pharmaceutical composition according to claim 1 or 2, wherein the cancer is selected from lymphoma, leukemia, multiple myeloma, myeloproliferative neoplasm (MPN), digestive cancer (esophageal cancer, gastric cancer, duodenal cancer), liver cancer, biliary tract cancer (gallbladder / bile duct cancer), pancreatic cancer, small intestine cancer, large intestine cancer (colorectal cancer, colon cancer, rectal cancer), gastrointestinal stromal tumor, lung cancer (non-small cell lung cancer, small cell lung cancer), and renal cancer.