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US-12617844-B2 - Antibodies that bind to cleaved form of mutant calreticulin, and diagnostic, preventive, or therapeutic agent for myeloproliferative neoplasm

US12617844B2US 12617844 B2US12617844 B2US 12617844B2US-12617844-B2

Abstract

A diagnostic, preventive, or therapeutic agent may be used for a myeloproliferative neoplasm. An antibody or a functional fragment thereof that binds to a cleaved mutant CALR protein, may include an antigen-recognition site in (a) a polypeptide chain having an amino acid sequence set forth in SEQ ID NO: 1 or (b) a polypeptide chain having an amino acid sequence having deletion, substitution, or addition of one to several amino acids in SEQ ID NO: 1; and a diagnostic, preventive, or therapeutic agent for a myeloproliferative neoplasm containing the antibody.

Inventors

  • Marito ARAKI
  • Yoshihiko KIHARA
  • Norio KOMATSU

Assignees

  • JUNTENDO EDUCATIONAL FOUNDATION
  • MEIJI SEIKA PHARMA CO., LTD.

Dates

Publication Date
20260505
Application Date
20200228
Priority Date
20190228

Claims (12)

  1. 1 . An antibody or a functional fragment thereof that binds to a cleaved mutant CALR polypeptide, said antibody or functional fragment thereof selected from the group consisting of (c), (d), and (e): (c) an antibody in which CDR1, CDR2, and CDR3 of the immunoglobulin VH chain are polypeptides consisting of the amino acid sequences set forth in SEQ ID NOs: 17, 18, and 19, respectively, and CDR1, CDR2, and CDR3 of the immunoglobulin VL chain are polypeptides consisting of the amino acid sequences set forth in SEQ ID NOs: 26, 27, and 28, respectively; (d) an antibody in which CDR1, CDR2, and CDR3 of the immunoglobulin VH chain are polypeptides consisting of the amino acid sequences set forth in SEQ ID NOs: 20, 21, and 22, respectively; and CDR1, CDR2, and CDR3 of the immunoglobulin VL chain are polypeptides consisting of the amino acid sequences set forth in SEQ ID NOs: 29, 30, and 31, respectively; and (e) an antibody in which CDR1, CDR2, and CDR3 of the immunoglobulin VH chain are polypeptides consisting of the amino acid sequences set forth in SEQ ID NOs: 23, 24, and 25, respectively; and CDR1, CDR2, and CDR3 of the immunoglobulin VL chain are polypeptides consisting of the amino acid sequences set forth in SEQ ID NOs: 32, 33, and 34, respectively.
  2. 2 . The antibody or functional fragment thereof of claim 1 , selected from the group consisting of (C), (D), and (E): (C) an antibody comprising an immunoglobulin VH chain consisting of an amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having a deletion, substitution, or addition of up to one amino acid in the amino acid sequence set forth in SEQ ID NO: 5; and an immunoglobulin VL chain consisting of an amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having a deletion, substitution, or addition of up to one amino acid in the amino acid sequence set forth in SEQ ID NO: 8; (D) an antibody comprising an immunoglobulin VH chain consisting of an amino acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence having a deletion, substitution, or addition of up to one amino acid in the amino acid sequence set forth in SEQ ID NO: 6; and an immunoglobulin VL chain consisting of an amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having a deletion, substitution, or addition of up to one amino acid in the amino acid sequence set forth in SEQ ID NO: 9; and (E) an antibody comprising an immunoglobulin VH chain consisting of an amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having a deletion, substitution, or addition of up to one amino acid in the amino acid sequence set forth in SEQ ID NO: 7; and an immunoglobulin VL chain consisting of an amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having a deletion, substitution, or addition of up to one amino acid in the amino acid sequence set forth in SEQ ID NO: 10.
  3. 3 . A composition, comprising: the antibody or functional antibody fragment of claim 1 , formulated for therapy of a myeloproliferative neoplasm (MPN) or formulated for use in a method of diagnosing myeloproliferative neoplasm.
  4. 4 . A method for detecting a mutant CALR polypeptide comprising, contacting the antibody or functional antibody fragment of claim 1 with a sample comprising: a polypeptide consisting of an amino acid sequence set forth in SEQ ID NO: 1 and detecting complex formation, thereby detecting said polypeptide.
  5. 5 . A method for detecting a mutant CALR polypeptide, comprising: contacting the antibody or functional antibody fragment of claim 1 with a polypeptide consisting of an amino acid sequence set forth in SEQ ID NO: 1; and detecting complex formation between the antibody or functional fragment and said polypeptide; wherein complex formation is indicative of presence of the mutant CALR polypeptide.
  6. 6 . The method of detecting of claim 4 , further comprising identifying a therapeutic agent for MPN comprising contacting the therapeutic agent with a polypeptide consisting of an amino acid sequence set forth in SEQ ID NO: 1; and measuring complex formation, wherein complex formation is indicative of a therapeutic effect on MPN.
  7. 7 . A therapeutic method for a myeloproliferative neoplasm (MPN), the method comprising: administering the antibody or functional antibody fragment of claim 1 to a subject in need thereof.
  8. 8 . The antibody or fragment of claim 1 , comprising the antigen-recognition site in the polypeptide chain consisting of the amino acid sequence set forth in SEQ ID NO: 1.
  9. 9 . The antibody or functional fragment of claim 1 , comprising (c) an antibody in which CDR1, CDR2, and CDR3 of the immunoglobulin VH chain are polypeptides consisting of the amino acid sequences set forth in SEQ ID NOs: 17, 18, and 19, respectively; and CDR1, CDR2, and CDR3 of the immunoglobulin VL chain are polypeptides consisting of the amino acid sequences set forth in SEQ ID NOs: 26, 27, and 28, respectively.
  10. 10 . The antibody or functional fragment of claim 1 , comprising (d) an antibody in which CDR1, CDR2, and CDR3 of the immunoglobulin VH chain are polypeptides consisting of the amino acid sequences set forth in SEQ ID NOs: 20, 21, and 22, respectively; and CDR1, CDR2, and CDR3 of the immunoglobulin VL chain are polypeptides consisting of the amino acid sequences set forth in SEQ ID NOs: 29, 30, and 31, respectively.
  11. 11 . The antibody or functional fragment of claim 1 , comprising (e) an antibody in which CDR1, CDR2, and CDR3 of the immunoglobulin VH chain are polypeptides consisting of the amino acid sequences set forth in SEQ ID NOs: 23, 24, and 25, respectively; and CDR1, CDR2, and CDR3 of the immunoglobulin VL chain are polypeptides consisting of the amino acid sequences set forth in SEQ ID NOs: 32, 33, and 34, respectively.
  12. 12 . The method of claim 4 comprising (i) detecting said polypeptide in a biological sample containing a drug.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS The present application is the national stage of international application PCT/JP2020/008434, filed on Feb. 28, 2020, and claims the benefit of the filing date of Japanese Appl. No. 2019-036119, filed on Feb. 28, 2019, the content of each of which is incorporated by reference. REFERENCE TO A SEQUENCE LISTING This application contains a Sequence Listing in computer readable form (CRF) submitted as an ASCII text file named “538416US_051525_ST25.txt”, created on May 15, 2025 and having a size of 24,549 bytes. The contents of the ASCII text file are incorporated herein by reference in their entirety. TECHNICAL FIELD The present invention relates to a diagnostic, preventive, or therapeutic agent for a myeloproliferative neoplasm. BACKGROUND ART In a part of patients with Philadelphia-negative myeloproliferative neoplasms (MPNs), nucleotide deletion or insertion is found in exon 9 of the calreticulin (CALR) gene (Non Patent Literatures 1 and 2). It has been already revealed that the mutant CALR protein produced by a CALR mutant gene has oncogenicity independently causing a myeloproliferative neoplasm (MPN) by constantly activating a thrombopoietin receptor (Non Patent Literatures 3 to 6). The CALR gene mutation found in MPN patients is a frameshift mutation that is necessarily localized in the last exon, and a shift in the amino acid reading frame by the frameshift mutation is always +1. From these things, a sequence that is not present in the wild type is present at the carboxyl-terminal of the mutant CALR protein, and in particular, 44 amino acids of the carboxyl-terminal are common to almost all of the mutant CALR proteins. As an example thereof, FIG. 1 shows comparison of the sequences of carboxyl-terminals of 52-nucleotide deletion type (Del 52), which is the most frequent mutation among CALR gene mutations found in MPN patients, and 5-nucleotide insertion type (Ins 5), which is the next most frequent mutation, with the corresponding region of the wild-type CALR protein. Since the mutant CALR protein causing occurrence of an MPN is expressed in tumor cells, it has been suggested that there is a possibility that the sequence specific to the mutant CALR protein caused by the frameshift mutation becomes a marker for diagnosis or a therapeutic target as a neo-antigen (Patent Literatures 1 and 2). CITATION LIST Patent Literature Patent Literature 1: JP-A-2016-537012Patent Literature 2: WO2016/087514A Non Patent Literature Non Patent Literature 1: Klampfl T, Gisslinger H, Harutyunyan A S, et al., Somatic mutations of calreticulin in myeloproliferative neoplasms, The New England journal of medicine, 2013, 369:2379-90Non Patent Literature 2: Nangalia J, Massie C E, Baxter E J, et al., Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2, The New England journal of medicine, 2013, 369:2391-405Non Patent Literature 3: Araki M, Yang Y, Masubuchi N, et al., Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms, Blood, 2016, 127:1307-16Non Patent Literature 4: Elf S, Abdelfattah N S, Chen E, et al., Mutant Calreticulin Requires Both Its Mutant C-terminus and the Thrombopoietin Receptor for Oncogenic Transformation, Cancer Discov., 2016, 6:368-81Non Patent Literature 5: Marty C, Pecquet C, Nivarthi H, et al., Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis, Blood, 2016, 127:1317-24Non Patent Literature 6: Vainchenker W, Kralovics R., Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms, Blood, 2017, 129:667-79 SUMMARY OF INVENTION Technical Problem However, it was proved that an antibody that recognizes a sequence (neo-antigen) specific to the mutant CALR protein appearing by the frameshift mutation cannot correctly detect the mutant CALR protein in cell extract or on a cell surface. Accordingly, it is an object of the present invention to provide detection methods capable of correctly detecting a mutant CALR gene and a protein of an MPN, a diagnostic, preventive, or therapeutic agent for an MPN, and a method for screening for an MPN therapeutic agent. Solution to Problem The present inventors further performed functional analysis of the mutant CALR proteins and, as a result, found that many of the expressed mutant CALR proteins are cleaved in a sequence (FIG. 1) specific to mutant proteins and that mutant CALR proteins in which most of the sequence inferred as the neo-antigen has been lost are largely expressed. Accordingly, an antibody that specifically recognizes a significantly short amino acid sequence located at the amino-terminal side of the cleavage site in the neo-antigen was produced, and it was verified that cleaved mutant CALR proteins were expressed not only in cultured cells but also in patient peripheral blood cells and patient platelets. Furthermore, it was revealed that by using this antibody, t