RU2815960C2 - Antibodies which bind to split form of mutant calreticulin, and agent for diagnosing, preventing or treating myeloproliferative growth - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to an antibody or its functional fragment, which binds to a split mutant CALR protein, as well as to a composition containing same. Also disclosed is the use of said antibody or fragment thereof in detecting mutant CALR protein, in screening for detecting a therapeutic agent for treating myeloproliferative neoplasm associated with mutant CALR protein, as well as in diagnosing myeloproliferative neoplasm associated with mutant CALR protein.

EFFECT: invention is effective for preventing or treating myeloproliferative neoplasm associated with the mutant CALR protein.

10 cl, 18 dwg, 6 ex

Description

Field of technology to which the invention relates

[0001]

The present invention relates to an agent for the diagnosis, prevention or treatment of a myeloproliferative neoplasm.

Prerequisites for creating the invention

[0002]

In a subset of patients with Philadelphia-negative myeloproliferative neoplasms (MPN), a nucleotide deletion or insertion was found in exon 9 of the calreticulin gene (CALR) (Non-Patent Literature 1 and 2). It has already been found that the mutant CALR protein produced by the mutant CALR gene has oncogenicity, independently causing myeloproliferative neoplasm (MPN) by persistently activating the thrombopoietin receptor (Non-Patent Literature 3-6).

[0003]

The CALR gene mutation found in MPN patients is a frameshift mutation that is necessarily located in the last exon, and the amino acid reading frame shift by frameshift mutation is always +1. It follows that a sequence not present in the wild type is present at the carboxy terminus of the mutant CALR protein, and in particular the carboxy terminus 44 amino acids are common to almost all mutant CALR proteins. As an example, FIG. 1 shows a comparison of the carboxy terminus sequences of the 52 nucleotide deletion type (Del 52), which is the most common mutation of the CALR gene mutations found in MPN patients, and the 5 nucleotide insertion type (Ins 5), which is the next most common mutation. with the corresponding region of the wild-type CALR protein.

Since the mutant CALR protein causing MPN is expressed in tumor cells, the possibility has been proposed that a sequence specific for the mutant CALR protein caused by a frameshift mutation would become a marker for diagnosis or a therapeutic target as a neoantigen (Patent Literature 1 and 2).

List of cited documents

Patent literature

[0004]

Patent Literature 1: JP-A-2016-537012

Patent Literature 2: WO2016/087514A

Non-patent literature

[0005]

Non-patent literature 1: 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

Non-patent literature 2: 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

Non-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: 1307-16

Non-Patent Literature 4: 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

Non-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: 1317-24

Non-patent literature 6: Vainchenker W, Kralovics R., Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms, Blood, 2017, 129: 667-79

The essence of the invention

Technical challenge

[0006]

However, it has been proven that an antibody that recognizes a sequence (neoantigen) specific for a mutant CALR protein resulting from a frameshift mutation cannot correctly detect the mutant CALR protein in a cell extract or on the cell surface.

Accordingly, it is an object of the present invention to provide detection methods capable of accurately detecting mutant CALR gene and MPN protein, means for diagnosing, preventing or treating MPN, and a screening method for identifying a therapeutic agent for treating MPN.

The solution of the problem

[0007]

The present inventors also performed a functional analysis of mutant CALR proteins and, as a result, discovered that many of the expressed mutant CALR proteins are cleaved in sequence (Fig. 1) specific for the mutant proteins, and that the mutant CALR proteins are largely expressed, in which most the sequence predicted to be a neoantigen is lost. Accordingly, an antibody was generated that specifically recognizes a substantially short amino acid sequence located at the amino-terminal side of the cleavage site in the neoantigen, and it was confirmed that the cleaved mutant CALR proteins were expressed not only in cultured cells, but also in patient peripheral blood cells and platelets patient. In addition, it was found that by using this antibody, the detection sensitivity for mutant cell surface CALR protein was significantly improved and, in addition, excellent therapeutic effect against MPN was achieved. Based on these results, it was found that for the diagnosis or treatment of patients with MPN who have a CALR gene mutation, pharmaceutical products could be developed that demonstrate higher efficacy by detecting or targeting not only the full-length type (non-cleavage type) but also the mutant CALR proteins, including cleavage types. Moreover, there was good reason to believe that the effect of a pharmaceutical product targeting a sequence that is lost by cleavage could be increased by preventing the cleavage of the mutant CALR protein, and the present invention has been made.

[0008]

Thus, the present invention provides the following [1]-[14].

[0009]

[1] An antibody or functional fragment thereof that binds to a cleaved mutant CALR protein comprising an antigen recognition site on (a) a polypeptide chain consisting of the amino acid sequence of SEQ ID NO:1, or on (b) a polypeptide chain consisting of the amino acid sequence with the deletion, substitution or addition of one or more amino acids in SEQ ID NO:1.

[2] An antibody or a functional fragment thereof that binds to a cleaved mutant CALR protein in accordance with aspect [1], wherein the antibody or a functional fragment thereof is selected from the following (c), (d) and (e):

(c) an antibody wherein the CDR1, CDR2 and CDR3 immunoglobulin VH chains are polypeptides consisting of the amino acid sequences of SEQ ID NOs: 17, 18 and 19, respectively, or amino acid sequences with the deletion, substitution or addition of one or more amino acids in amino acid sequences SEQ ID NO:17, 18 and 19; and CDR1, CDR2 and CDR3 of the immunoglobulin VL chain are polypeptides consisting of the amino acid sequences SEQ ID NO: 26, 27 and 28, respectively, or amino acid sequences with the deletion, substitution or addition of one or more amino acids in the amino acid sequences SEQ ID NO: 26, 27 and 28;

(d) an antibody wherein the CDR1, CDR2 and CDR3 immunoglobulin VH chains are polypeptides consisting of the amino acid sequences of SEQ ID NOs: 20, 21 and 22, respectively, or amino acid sequences with the deletion, substitution or addition of one or more amino acids in amino acid sequences SEQ ID NO:20, 21 and 22; and CDR1, CDR2 and CDR3 of the immunoglobulin VL chain are polypeptides consisting of the amino acid sequences SEQ ID NO: 29, 30 and 31, respectively, or amino acid sequences with the deletion, substitution or addition of one or more amino acids in the amino acid sequences SEQ ID NO: 29, 30 and 31; And

(e) an antibody wherein the CDR1, CDR2 and CDR3 immunoglobulin VH chains are polypeptides consisting of the amino acid sequences of SEQ ID NO: 23, 24 and 25, respectively, or amino acid sequences with the deletion, substitution or addition of one or more amino acids in amino acid sequences SEQ ID NO:23, 24 and 25; and CDR1, CDR2 and CDR3 of the immunoglobulin VL chain are polypeptides consisting of the amino acid sequences SEQ ID NO: 32, 33 and 34, respectively, or amino acid sequences with the deletion, substitution or addition of one or more amino acids in the amino acid sequences SEQ ID NO: 32, 33 and 34.

[3] An antibody or functional fragment thereof that binds to a cleaved mutant CALR protein according to aspect [1] or [2], selected from the following (C), (D) and (E):

(C) an antibody comprising an immunoglobulin VH chain consisting of the amino acid sequence of SEQ ID NO:5, or an amino acid sequence with the deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO:5, and an immunoglobulin VL chain consisting of from the amino acid sequence of SEQ ID NO:8, or an amino acid sequence with a deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO:8;

(D) an antibody comprising an immunoglobulin VH chain consisting of the amino acid sequence of SEQ ID NO:6, or an amino acid sequence with the deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO:6, and an immunoglobulin VL chain consisting of from the amino acid sequence of SEQ ID NO:9, or an amino acid sequence with a deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO:9; And

(E) an antibody comprising an immunoglobulin VH chain consisting of the amino acid sequence of SEQ ID NO:7, or an amino acid sequence with the deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO:7, and an immunoglobulin VL chain consisting of from the amino acid sequence of SEQ ID NO:10, or an amino acid sequence with a deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO:10.

[4] An antibody or functional fragment thereof that competes with an antibody of aspect [2] or [3] for binding to a portion of the amino acid sequence of SEQ ID NO:1 in a cleaved mutant CALR protein.

[5] A pharmaceutical composition comprising an antibody or a functional fragment thereof according to any one of aspects [1]-[4].

[6] The pharmaceutical composition according to aspect [5], wherein the pharmaceutical composition is an agent for the diagnosis, prevention or treatment of a myeloproliferative neoplasm.

[7] A method for detecting a myeloproliferative neoplasm-associated mutant CALR protein, comprising detecting the following polypeptide (a) or (b) in a biological sample:

(a) a polypeptide consisting of the amino acid sequence of SEQ ID NO:1; or

(b) a polypeptide consisting of an amino acid sequence with deletion, substitution or addition of one or more amino acids in SEQ ID NO:1.

[8] A method for detecting a mutant CALR protein in accordance with aspect [7], wherein detection of polypeptide (a) or (b) is immunological detection using an antibody or a functional fragment thereof in accordance with any of aspects [1]-[4].

[9] A method of screening for a therapeutic agent for a myeloproliferative neoplasm, comprising screening for a drug that binds to the following polypeptide (a) or (b):

(a) a polypeptide consisting of the amino acid sequence of SEQ ID NO:1; or

(b) a polypeptide consisting of an amino acid sequence with the deletion, substitution or addition of one or more amino acids in SEQ ID NO:1.

[10] A method for diagnosing a myeloproliferative neoplasm, comprising detecting the following polypeptide (a) or (b) in a biological sample:

(a) a polypeptide consisting of the amino acid sequence of SEQ ID NO:1; or

(b) a polypeptide consisting of an amino acid sequence with deletion, substitution or addition of one or more amino acids in SEQ ID NO:1.

[11] The diagnostic method according to aspect [10], wherein the detection of polypeptide (a) or (b) is immunological detection using an antibody or a functional fragment thereof according to any one of aspects [1]-[4].

[12] A method for preventing or treating a myeloproliferative neoplasm, comprising administering an antibody or a functional fragment thereof according to any one of aspects [1]-[4].

[13] An antibody or functional fragment thereof according to any one of aspects [1]-[4] for use in the diagnosis of a myeloproliferative neoplasm.

[14] Use of an antibody or a functional fragment thereof in accordance with any of aspects [1]-[4] for the preparation of an agent for diagnosing a myeloproliferative neoplasm.

Effects of the invention

[0010]

By using an antibody having an antigen recognition site (epitope) in the above polypeptide chain (a) or (b), the mutant CALR protein caused by cleavage of the mutant CALR protein can be detected, and the detection sensitivity of the mutant CALR protein on the cell surface can be significantly improved. Accordingly, the detection sensitivity of MPN is greatly improved by using the MPN diagnostic tool of the present invention. In addition, specific prophylaxis or treatment of MPN can be performed using this antibody. In addition, a therapeutic agent for MPN can be screened to identify a drug that binds to polypeptide (a) or (b).

Brief description of drawings

[0011]

[Fig. 1] Fig. 1 is a diagram showing the characteristics of mutant CALR proteins. The CALR gene mutation responsible for the development of myeloproliferative neoplasms in patients is a +1 frameshift mutation, and the amino acid sequence characteristic of mutant proteins is found in the carboxy-terminal region of the mutant CALR proteins produced by the mutation. SP: signal sequence, N:N domain, and P:P domain. The arrow indicates the site at which amino acid sequences that differ from the wild type (WT) begin.

[Fig. 2] Fig. 2 is a diagram showing antibodies that recognize sequences on the amino-terminal side of a CALR protein and the carboxy-terminal side of mutant CALR proteins. An antibody that recognizes a sequence on the amino-terminal side of a CALR protein is a commercially available antibody (anti-CALR antibody) that recognizes an amino-terminal sequence on the amino-terminal side of the mutation site of mutant CALR proteins. In addition, an antibody that recognizes the carboxy-terminal sequence of each mutant CALR protein is a commercially available antibody (anti-mutant CALR antibody) that recognizes an amino acid sequence that is newly formed on the carboxy-terminal side of the CALR protein as a result of mutation of the nucleotide sequence.

[Fig. 3] Fig. 3 is a graph showing the results of immunoblot analysis of proteins secreted from UT-7/TPO cells (Komatsu N. et al., Blood, 1996, 87, 4552-4556) using the antibody FIG. 2. Since the vec is a cell transfected with expression vector only, endogenous CALR protein is found as in UT-7/TPO cell/CALR (WT). In Del 52 type and Ins type 5, a mutant CALR protein having a molecular weight less than that of the full-length mutant CALR protein detected by the anti-mutant CALR antibody is detected only by the anti-CALR antibody (asterisk in the figure). In particular, because the full-length Ins 5 mutant CALR protein is electrophoresed to essentially the same position as the endogenous wild-type CALR protein, the Ins 5 type cannot be specifically detected by the anti-CALR antibody.

[Fig. 4] Fig. 4 is a schematic diagram showing proteins in which FLAG tags are fused to the amino (N) termini and carboxy (C) termini of typical mutant CALR proteins, Del 52 type and Ins type 5. Mutant type-specific sequences are shown in black and FLAG tags are shown as slanted lines.

[Fig. 5] Fig. 5 is a graph showing the results of measuring the amounts of mutant CALR proteins on cell surfaces by flow cytometry using an anti-FLAG antibody. In cells expressing a mutant CALR protein that has a FLAG tag at the N-terminus, at which the tag remains regardless of the presence or absence of cleavage, a strong signal was detected compared with cells in which the C-terminal FLAG is cut off as a result of cleavage occurring in a sequence specific for the mutant CALR protein. In this case, a difference is observed due to the fact that the expression level of type 5 Del is lower than the expression level of type 5 Ins.

[Fig. 6] Fig. 6 shows an antibody that recognizes a mutant type-specific sequence at the amino-terminal side of a protein cleavage site produced into a mutant CALR sequence-specific protein (black).

[Fig. 7] Fig. 7 shows the results of the flow cytometry cell analysis used in FIG. 3, using the antibody of FIG. 6. The difference in recognition that is caused by the positions at which the FLAG tag is fused has disappeared. In this case, a difference is observed that the expression level of type 5 Del 52 is lower than the expression level of type 5 Ins.

[Fig. 8] Fig. 8 is a graph showing the detection of split-type CALR protein in a patient's myeloproliferative neoplasm cells. Cell extracts obtained from the patient's cells were analyzed by immunoblotting using an antibody that recognizes the amino acid sequence at the amino-terminal side of the cleavage site of the protein produced in the sequence-specific mutant CALR protein. Expression of a cleaved mutant CALR protein, which can be recognized by an antibody, was detected in peripheral blood mononuclear cells and platelets from patients with myeloproliferative neoplasms with a mutation in the CALR gene.

[Fig. 9] Fig. 9 is a graph showing that clone B3, C6 and G1 antibodies all recognize full-length and cleaved mutant CALR proteins. The diagram shows the results of immunoblot analysis of proteins secreted from UT-7/TPO cells using clone B3 antibody and clone C6 and G1 antibodies. Both the full-length types, Del 52 type and Ins 5 type, each fused to a FLAG tag downstream of the signal sequence at the amino terminus, and the split types, having lower molecular weights, were also detected by any of the B3, C6 and G1 antibodies, both in anti-FLAG antibody.

[Fig. 10] Fig. 10 is a graph showing that clone B3, C6 and G1 antibodies all recognize mutant CALR proteins on cell surfaces. The diagram shows the results of measuring the amounts of mutant CALR proteins on the cell surfaces of UT-7/TPO cells expressing mutant CALR protein type Del 52 or Ins type 5 fused to a FLAG tag, and UT-7/TPO/vec cells transfected with the expression vector only , by flow cytometry using antibody clone B3 and antibodies clone C6 and G1. Moreover, since the expression level of type 5 Del is lower than the expression level of type 5 Ins, there is a difference in signal intensity due to the type of mutation.

[Fig. 11] Fig. 11 is a graph showing the results of evaluating the binding activity of clone B3, C6 and G1 antibodies to the mutant CALR protein. The binding specificity of clone B3 antibodies, clone C6 and G1 antibodies, and rat IgG as a control to the Del 52 mutant CALR protein was assessed by ELISA. Concentration-dependent specific binding to the mutant CALR protein was detected in all clone B3, C6, and G1 antibodies.

[Fig. 12] Fig. 12 is a graph showing the results of evaluating the binding strength of an antibody of clone B3 to an antigen used for antibody production by surface plasmon resonance. The binding strength of the clone B3 antibody and the antigen used to produce the antibody was assessed by surface plasmon resonance.

[Fig. 13A] FIG. 13A is a graph showing the results of identification of antigen recognition sequences of clone B3, C6 and G1 antibodies by immunoblotting. Proteins each containing an alanine (A) substituting one amino acid in the Del 52 type CALR mutant protein shown in the diagram were expressed and the reactivity of the corresponding antibodies to the proteins was assessed by immunoblotting. Intact means a Del 52 mutant CALR protein that does not have an amino acid substitution. The amino acids outlined with a thick black line are the antigenic sequences used to produce antibodies.

[Fig. 13B] FIG. 13B is a graph showing the results of identification of antigen recognition sequences of clone B3, C6 and G1 antibodies by ELISA. The reactivity of the corresponding antibodies against the same proteins shown in Fig. 13A was assessed by ELISA. Intact means a type 52 mutant CALR protein that has an amino acid substitution. The amino acids outlined with a thick black line are the antigenic sequences used to produce antibodies.

[Fig. 14] Fig. 14 is a diagram showing the amino acid sequences of the heavy chain variable regions of antibodies (B3, C6 and G1) of the present invention.

[Fig. 15] Fig. 15 is a diagram showing the amino acid sequences of the light chain variable regions of antibodies (B3, C6 and G1) of the present invention.

[Fig. 16] Fig. 16 is a diagram showing the nucleotide sequences of the heavy chain variable regions of antibodies (B3, C6 and G1) of the present invention.

[Fig. 17] Fig. 17 is a diagram showing the nucleotide sequences of light chain variable regions of antibodies (B3, C6 and G1) of the present invention.

[Fig. 18] Fig. 18 is a graph showing the therapeutic effect on the MPN mouse model using the B3 mouse chimeric antibody. B3 chimeric antibody or vehicle (PBS) was administered to a model mouse model of myeloproliferative neoplasm (CALR Del 52 mice) obtained by transplantation of hematopoietic stem cells expressing the mutant CALR protein Del 52 type, or to control mice after transplantation of hematopoietic stem cells transfected with a control vector, each week, starting from the 9th week after transplantation, and assessed the effect of suppressing thrombocytosis, characteristic of a mouse model of myeloproliferative neoplasm.

Description of Embodiments

[0012]

The polypeptide (a) or (b) of the present invention, which is used in a method for detecting MPN-associated mutant CALR protein, and is used in a diagnostic method and as a target for the prevention or treatment of MPN, is a polypeptide remaining on the carboxy-terminal side after cleavage of the sequence (44 amino acids common to the mutant CALR proteins in Fig. 1) specific to the mutant CALR proteins.

The wild type CALR protein includes the amino acid sequence (SEQ ID NO:2) shown in FIG. 1. In addition, the 52-nucleotide deletion type (Del 52), which is the most common mutation among CALR gene mutations, includes the amino acid sequence (SEQ ID NO:3) shown in FIG. 1. In addition, the 5-nucleotide insertion type (Ins 5), which is the next most common mutation among CALR gene mutations, includes the amino acid sequence (SEQ ID NO:4) shown in FIG. 1. Moreover, the area enclosed by a frame in FIG. 1 is the amino acid sequence common to most mutant CALR proteins. The cleaved mutant CALR protein includes only 13 amino acids on the amino-terminal side of this general region. The present inventors have discovered that this common region is cleaved to produce cleaved mutant CALR proteins, and that most of the common region is lost from the mutant CALR proteins.

[0013]

Polypeptide (a) consists of the amino acid sequence of SEQ ID NO:1. The amino acid sequence having the deletion, substitution or addition of one to more amino acids in the form of polypeptide (b) is preferably an amino acid sequence having the deletion, substitution or addition of one to four amino acids, more preferably an amino acid sequence having the deletion, substitution or addition of one - three amino acids in SEQ ID NO:1. More specifically, an amino acid sequence having a deletion of three amino acids from the carboxy-terminal side of SEQ ID NO:1 is more preferred. Moreover, the identity between the amino acid sequence of the polypeptide (b) 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.

[0014]

The method for detecting MPN-associated mutant CALR protein and the method for diagnosing, preventing or treating MPN of the present invention uses an antibody or a functional fragment thereof that binds to polypeptide (a) or (b) in a biological sample.

[0015]

The antibody may be any antibody having an antigen recognition site (epitope) on the polypeptide chain (a) or (b), and may bind only to the cleaved mutant CALR protein or may bind to both the cleaved mutant CALR protein and the full length mutant protein CALR, and the antibody is preferably a "mutant CALR protein" specific antibody that binds to both the cleaved mutant CALR protein and the full length mutant CALR protein. Specific examples of the antibody include antibodies selected from the following (c), (d) and (e):

(c) an antibody wherein the CDR1, CDR2 and CDR3 immunoglobulin VH chains are polypeptides consisting of the amino acid sequences of SEQ ID NOs: 17, 18 and 19, respectively, or amino acid sequences with the deletion, substitution or addition of one or more amino acids in amino acid sequences SEQ ID NO:17, 18 and 19; and CDR1, CDR2 and CDR3 of the immunoglobulin VL chain are polypeptides consisting of the amino acid sequences SEQ ID NO: 26, 27 and 28, respectively, or amino acid sequences with the deletion, substitution or addition of one or more amino acids in the amino acid sequences SEQ ID NO: 26, 27 and 28;

(d) an antibody wherein the CDR1, CDR2 and CDR3 immunoglobulin VH chains are polypeptides consisting of the amino acid sequences of SEQ ID NOs: 20, 21 and 22, respectively, or amino acid sequences with the deletion, substitution or addition of one or more amino acids in amino acid sequences SEQ ID NO:20, 21 and 22; and CDR1, CDR2 and CDR3 of the immunoglobulin VL chain are polypeptides consisting of the amino acid sequences SEQ ID NO: 29, 30 and 31, respectively, or amino acid sequences with the deletion, substitution or addition of one or more amino acids in the amino acid sequences SEQ ID NO: 29, 30 and 31; and

(e) an antibody wherein the CDR1, CDR2 and CDR3 immunoglobulin VH chains are polypeptides consisting of the amino acid sequences of SEQ ID NO: 23, 24 and 25, respectively, or amino acid sequences with the deletion, substitution or addition of one or more amino acids in amino acid sequences SEQ ID NO:23, 24 and 25; and CDR1, CDR2 and CDR3 of the immunoglobulin VL chain are polypeptides consisting of the amino acid sequences SEQ ID NO: 32, 33 and 34, respectively, or amino acid sequences with the deletion, substitution or addition of one or more amino acids in the amino acid sequences SEQ ID NO: 32, 33 and 34.

[0016]

An amino acid sequence having a deletion, substitution or addition of one to more amino acids in each of the amino acid sequences respectively presented in SEQ ID NO: 17-34 is preferably an amino acid sequence having a deletion, substitution or addition of one to four amino acids, more preferably an amino acid a sequence having a deletion, substitution or addition of one to three amino acids, and even more preferably an amino acid sequence having a deletion, substitution or addition of one or two amino acids in SEQ ID NO: 17-34. In addition, the identity between each of the amino acid sequences respectively presented in SEQ ID NO: 17-34 and the amino acid sequence having a deletion, substitution or addition of one or more amino acids of the corresponding amino acid sequence is preferably 80% or greater, more preferably 85% or more and even more preferably 90% or more.

[0017]

The antibody having an antigen recognition site (epitope) on the polypeptide chain (a) or (b) is even more preferably an antibody selected from the following (C), (D) and (E):

(C) an antibody comprising an immunoglobulin VH chain consisting of the amino acid sequence of SEQ ID NO:5, or an amino acid sequence with the deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO:5, and an immunoglobulin VL chain consisting of from the amino acid sequence of SEQ ID NO:8, or an amino acid sequence with a deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO:8;

(D) an antibody comprising an immunoglobulin VH chain consisting of the amino acid sequence of SEQ ID NO:6, or an amino acid sequence with the deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO:6, and an immunoglobulin VL chain consisting of from the amino acid sequence of SEQ ID NO:9, or an amino acid sequence with a deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO:9; And

(E) an antibody comprising an immunoglobulin VH chain consisting of the amino acid sequence of SEQ ID NO:7, or an amino acid sequence with the deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO:7, and an immunoglobulin VL chain consisting of from the amino acid sequence of SEQ ID NO:10, or an amino acid sequence with a deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO:10.

[0018]

An amino acid sequence having a deletion, substitution or addition of one to more amino acids in each of the amino acid sequences respectively presented in SEQ ID NO: 5-10 is preferably an amino acid sequence having a deletion, substitution or addition of one to ten amino acids, more preferably an amino acid a sequence having a deletion, substitution or addition of one to eight amino acids, and even more preferably an amino acid sequence having a deletion, substitution or addition of one to five amino acids, in SEQ ID NO:5-10. In addition, the identity between each of the amino acid sequences respectively presented in SEQ ID NO:5-10 and the amino acid sequence having a deletion, substitution or addition of one or more amino acids of the corresponding amino acid sequence of SEQ ID NO:5-10 is preferably 80% or more, more preferably 85% or more and even more preferably 90% or more.

[0019]

The antibody of the present invention includes the sequences of each CDR of antibodies (c)-(e) or the sequences of the VH region and VL region of the immunoglobulin antibodies (C)-(E), and the amino sequences of regions other than these regions are not particularly limited. Accordingly, the antibody of the present invention may be a non-rat mammalian antibody, such as a human antibody, or may be a humanized antibody. More specifically, the antibody may be a chimeric antibody comprising the heavy chain and light chain variable regions of a non-human mammalian antibody, such as a rat, and the heavy chain and light chain constant regions of a human antibody, and such antibody can be produced by ligating DNA encoding the variable region of a rat antibody, with DNA encoding the constant region of a human antibody, incorporating it into an expression vector, and introducing the vector into a host for production. A humanized antibody is also called a reshaped human antibody and is produced by transplanting the CDR of a non-human mammalian antibody, such as a rat antibody, into the CDR of a human antibody, and the general gene recombination method used for this is also known. In a specific example, a DNA sequence designed to ligate the CDR of a rat antibody to the framework region (FR) of a human antibody is synthesized from several oligonucleotides prepared to include a portion overlapping the terminal portion by PCR. The resulting DNA is ligated to DNA encoding the constant region of a human antibody, which is then incorporated into an expression vector, and the expression vector is administered to a host to produce a humanized antibody (see European Patent Application Publication No. EP239400 and International Patent Application Publication WO96/02576). The human antibody FR that is ligated through the CDR is selected such that the CDR forms a favorable antigen binding site. If necessary, an amino acid from the FR variable region of an antibody can be replaced so that the CDR of the reshaped human antibody forms the corresponding antigen binding site (Sato, K. et al., Cancer Res., 1993, 53, 851-856).

[0020]

In addition, methods for producing human antibodies are also known. For example, human lymphocytes are sensitized in vitro with the desired antigen or a cell expressing the desired antigen, and the sensitized lymphocytes are fused with human myeloma cells, such as U266, to produce the desired human antibody having antigen binding activity (see JP-B-1-59878 ). In addition, the desired human antibody can be obtained by immunizing a transgenic animal having the entire repertoire of human antibody genes with the desired antigen (see WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096 and WO 96/33735). In addition, a panning method for producing a human antibody using a library of human antibodies is also known. For example, the variable region of a human antibody is expressed as a single chain antibody (scFv) on the surface of a phage by phage display, and the phage that binds to the antigen can be selected. By analyzing the gene of the selected phage, 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 has been determined, a human antibody can be produced by preparing a suitable expression vector containing this sequence. Such methods are already well known and reference may be made to WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438 and WO 95/15388.

[0021]

The class of the antibody is not particularly limited, and antibodies having any of the isotypes such as IgG, IgM, IgA, IgD and IgE are included. Considering, for example, ease of purification, IgG is preferred.

[0022]

Examples of functional fragments include small molecule antibodies such as antibody fragments and modified antibodies. Specific examples of antibody fragments include Fab, Fab', F(ab') ₂ , Fv, scFv and diabodies.

[0023]

Moreover, with respect to the antibodies of the present invention, the antibody that is used to detect polypeptide (a) or (b) can be any antibody that binds to these polypeptides. Alternatively, the antibody that is used for the prevention or treatment of MPN is preferably an antibody that binds to polypeptide (a) or (b) and has cytotoxic activity.

[0024]

The antibody that binds to the cleaved mutant CALR protein may be any antibody that binds to a polypeptide chain comprising the sequence set forth in SEQ ID NO:1, and is preferably an antibody that competes with at least one of the above antibodies and their functional fragments for binding to part of the amino acid sequence of SEQ ID NO:1, in the cleaved mutant CALR protein.

[0025]

Here, the biological sample used in the detection method is, for example, a biological sample taken from a subject and includes polypeptide (a) or (b). In particular, examples of samples include whole blood, plasma, serum, lymphocytes, lymph fluid, platelets, mononuclear cells, granulocytes, body fluids such as saliva and urine, and tissue samples (bone marrow fluid and bone marrow tissue) obtained by bone marrow biopsy brain

[0026]

Examples of the polypeptide detection method (a) or (b) include liquid chromatography, mass spectrometry, an immunological method and a method using a low or medium molecular weight molecule having a specific binding ability, or a nucleic acid or a nucleic acid derivative, and an immunological method preferred given its simplicity and high sensitivity.

[0027]

The immunological method is preferably an immunological method using an antibody or a functional fragment thereof comprising an antigen recognition site (epitope) on a polypeptide chain (a) or (b). The epitope in polypeptide chain (a) or (b) may be any sequence present in polypeptide chain (a) or (b), and is preferably a peptide comprising three or more consecutive amino acids in polypeptide chain (a) or (b ), and more preferably a peptide comprising five or more consecutive amino acids in the polypeptide chain (a) or (b).

[0028]

The antibody used in the immunological method may be any antibody produced using a peptide comprising, for example, three or more consecutive amino acids in the polypeptide chain (a) or (b) as an antigen, and may be a polyclonal antibody or a monoclonal antibody, and a monoclonal antibody is preferred. A monoclonal antibody can be produced, for example, by producing antibody-producing cells by sensitizing with a peptide comprising three or more consecutive amino acids in the polypeptide chain (a) or (b), fusing the antibody-producing cells with myeloma cells to produce hybridomas, selecting a clone, producing the target antibody, and propagation of this cell.

[0029]

As the immunological method, for example, any one of immunochromatography, enzyme-linked immunostaining assay (EIA), radioimmunoassay (RIA), coagulation method, nephelometry, flow cytometry, tissue immunostaining and immunoblotting such as Western blotting can be used. In this case, immunoblotting is a technique in which a polypeptide is transferred to a membrane and the polypeptide on the membrane is detected by an antibody. When detecting an antibody, for example, an enzyme tag or a fluorescent tag is used.

If polypeptide (a) or (b) is detected in a biological sample, it can be concluded that the biological sample is a biological sample obtained from a patient with a myeloproliferative neoplasm.

[0031]

The MPN diagnostic agent of the present invention contains an antibody having an antigen recognition site (epitope) in a polypeptide chain (a) or (b). This antibody is preferably the antibody described above.

Moreover, the MPN diagnostic tool of the present invention may include, for example, a buffer and a measurement protocol in addition to an antibody.

[0032]

A pharmaceutical composition, such as an agent for the prevention or treatment of MPN, of the present invention at least contains the antibody described above and can be prepared by formulation together with a pharmaceutically acceptable carrier, for example, by mixing, dissolving, emulsifying, encapsulating or lyophilizing.

[0033]

The MPN as a target for prevention or therapy using the pharmaceutical composition of the present invention is preferably an MPN in which a mutant CALR is detected.

[0034]

Suitable compositions for oral administration are, for example, a liquid prepared by dissolving an effective amount of an antibody of the present invention in a diluent such as water or saline, a capsule, granule, powder or tablet containing an effective amount of the antibody in the form of a solid or granule, suspension , obtained by suspending an effective amount of the antibody in a suitable dispersion medium, and an emulsion obtained by dispersing and emulsifying a solution of an effective amount of the antibody in a suitable dispersion medium.

[0035]

For parenteral administration, the antibody of the present invention can be formulated in a dosage form such as an injection, suspension, emulsion, cream, ointment, inhalation form and suppository, together with a pharmaceutically acceptable solvent, excipient, binder, stabilizer, dispersant, etc. . To prepare an injectable preparation, the antibody of the present invention can be dissolved in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution or physiological saline. In addition, the pharmaceutical product of the present invention may form, for example, a suspension, solution or emulsion in an oily or aqueous vehicle. Alternatively, the antibody of the present invention is obtained in the form of a powder, and an aqueous solution or suspension can be prepared using sterile water or the like. before use. For inhalation administration, the antibody of the present invention is ground into a powder and mixed with an appropriate base, such as lactose or starch, to form a powder mixture. A suppository can be prepared by mixing the antibody of the present invention with a conventional suppository base such as cocoa butter. Moreover, the therapeutic agent of the present invention can be prepared as a sustained release composition by encapsulation using a polymer matrix or the like.

[0036]

The method for screening for a therapeutic agent for MPN of the present invention is characterized in that it screens for an antibody, protein, low or medium molecular weight molecule, nucleic acid or nucleic acid derivative that recognizes polypeptide (a) or (b). In particular, the development of an antibody drug having antibody-dependent cytotoxic activity or complement-dependent cytotoxic activity using an antibody that recognizes polypeptide (a) or (b) is illustrated. The above-described antibody of the present invention is an example of an antibody drug obtained by the screening method of the present invention. In addition, an example is provided of the development of an antitumor agent that delivers, for example, a low or medium molecular weight compound, a nucleic acid or nucleic acid derivative, or a protein having a cytotoxic effect specifically to tumor cells by producing a low or medium molecular weight compound. molecular weight, nucleic acid or nucleic acid derivative, or protein that specifically binds to polypeptide (a) or (b).

Examples

[0037]

The present invention will now be described in more detail with reference to examples, but it is not limited to these examples.

[0038]

[Test example 1]

(1) Materials and test method

[1] Anti-CALR antibody

Rabbit monoclonal antibody clone D3E6 (Cat# 12238), manufactured by Cell Signaling Technology, Inc.

[0039]

[2] Antibody against mutant CALR

Mouse monoclonal antibody clone CAL2 (Cat# DIA-CAL), manufacturer Dianova GmbH

[0040]

[3]An antibody that recognizes a mutant CALR protein at the amino-terminal side of the cleavage site

A peptide having a cysteine at the carboxy-terminal or amino-terminal end of the amino acid sequence (RRMMRTKMR) included in SEQ ID NO:1 was synthesized and conjugated to a carrier protein, keyhole limpet hemocyanin (KLH). The conjugate was used to immunize 8-week-old female WKY/Izm rats and lymphocytes were collected after boost immunization. Lymphocytes were fused with SP2 murine myeloma cells using the PEG method and hybridomas were obtained by culturing in a selective medium. The specificity of the antibody in the culture supernatant was determined by ELISA using the immunized peptide or purified protein to produce hybridomas (clones B3, C6 and G1) producing antibodies that bind to mutant CALR proteins, including the cleaved type. Antibodies (B3 antibody, C6 antibody and G1 antibody) produced by these hybridomas were purified and used in the tests.

[0041]

[4] Test method

The human megakaryoblastic leukemia cell line UT-7/TPO was transfected with a vector expressing mutant CALR protein type Del 52 or Ins 5, a vector expressing wild-type CALR as a control, or an empty vector. The resulting UT-7/TPO/CALR Del 52 and UT-7/TPO/CALR Ins 5 neoplastically proliferating cells, UT-7/TPO/CALR (WT) cells and UT-7/TPO/vec cells, respectively, were seeded at density of 6.0 x 10 ⁵ cells/ml in Opti-MEM medium (Thermo Fisher Scientific KK, Cat# 31985070) and cultured in 5% CO ₂ at 37°C for 32 hours. Here, UT-7/TPO/CALR WT and UT-7/TPO/vec cells were cultured in medium containing 10 ng/ml thrombopoietin (TPO). Culture supernatants were obtained by removing cells from culture solutions by centrifugation (15000g × 10 min, 4°C), each of the resulting fractions was subjected to heat treatment in the presence of a reducing agent, then subjected to electrophoresis in polyacrylamide gel with sodium dodecyl sulfate, transferred to a polyvinylidene fluoride (PVDF) membrane by electrotransfer and reacted with TBS-T solution (0.1% Tween-20, 150 mM sodium chloride, 50 mM Tris-HCl, pH 7.5) containing 5% skim milk at room temperature for 1 hour followed by reaction at 4°C overnight with a 5% BSA/TBS-T solution containing an antibody that recognizes both wild type and mutant (rabbit monoclonal antibody D3E6, Cell Signaling Technology, Inc., Cat# 12238), or commercial an available antibody that recognizes the carboxy-terminal sequence of the mutant type (mouse monoclonal antibody CAL2, Dianova GmbH, Cat# DIA-CAL-250). After washing with TBS-T solution, reaction was carried out in a solution of 5% skim milk/TBS-T containing peroxidase-labeled anti-rabbit IgG antibody (Santa Cruz Biotechnology, Inc. Cat# sc-2030) or peroxidase-labeled anti-mouse IgG antibody (Santa Cruz Biotechnology, Inc. Cat# sc-2005), at room temperature for 1 hour. The PVDF membrane was washed with TBS-T solution and then reacted with peroxidase fluorescent reagent (Thermo Fisher Scientific KK, Cat# 34094). Signal detection was carried out using an image converting device FUSION (manufactured by Vilber-Lourmat SA).

[0042]

Del 52 type mutant CALR protein having a histidine tag inserted downstream of the signal sequence at the amino terminus was expressed in HEK293T cells and the culture supernatant containing the secreted mutant CALR protein was obtained by centrifugation (15,000 g x 10 min, 4°C). The resulting supernatant was applied to a HisTrap column (GE Healthcare, Cat# 17531901), the column was washed with purification buffer (0.3 M sodium chloride, 25 mM Tris-HCl, pH 7.4) containing 20 mM imidazole, and the mutant CALR protein was purified purification buffer containing 50 mM imidazole. The purified protein was reacted with 2-nitro-5-thiocyanobenzoic acid (NTCB) to cyanate the cysteine residue and then fragmented under alkaline conditions. The mass of protein fragments was determined using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer, and the cleavage site was determined from the peptide sequence information of the Del 52 mutant CALR protein.

[0043]

Del 52 type or Ins type 5 mutant CALR protein tagged with a FLAG tag downstream of the signal sequence at the amino terminus or tagged with a FLAG tag at the carboxy terminus was expressed in UT-7/TPO cells and the cells were cultured using Dulbecco's modified Iscove's medium (IMDM) containing 10% inactivated fetal bovine serum in 5% CO ₂ at 37°C. Cells (1x10 ⁵ ) in the proliferative stage were washed with PBS (phosphate buffer containing 137 mm sodium chloride and 27 mm potassium chloride) and then reacted with a mouse antibody to the DYKDDDDK tag sequence (FUJIFILM Wako Pure Chemical Corporation, Cat# 014-22383 ) or a rat anti-mutant CALR antibody that recognizes the amino-terminal side of the cleavage site, in a 2% FBS/PBS solution on ice for 30 minutes. After interaction, cells were washed with 2% FBS/PBS solution and then reacted with anti-mouse IgG-Alexa Fluor 647 (Thermo Fisher Scientific KK, Cat# A21235) and anti-rat IgG-Alexa Fluor 647 (Thermo Fisher Scientific KK, Cat# A21247), respectively, as secondary antibodies in a 2% FBS/PBS solution on ice for 30 minutes. After completion of the reaction, cells were collected by centrifugation and exposed to PBS containing 2% paraformaldehyde on ice for 15 minutes. Finally, the cells were washed by adding 2% FBS/PBS solution, then centrifugation was performed (400 g × 5 min, 4°C) and the supernatant was discarded. 300 ml of 2% FBS/PBS solution was then added to the cells and signals were quantified by flow cytometry using FACS Calibur (manufactured by BD biosciences).

[0044]

Human peripheral blood mononuclear cells were obtained by centrifuging (500 g × 10 min, 4°C) peripheral blood collected in a Spitz tube containing anticoagulant, suspending the resulting cell fraction in PBS, and then applying the suspension to Lymphosep lymphocyte separation solution (MP biomedicals, catalog number 11444815), performing centrifugation (1500 rpm 30 min, room temperature), to obtain buffy coat, adding PBS solution to the resulting buffy coat to resuspend, then performing centrifugation (1500 rpm 10 min, room temperature) and removing supernatant. The obtained peripheral blood mononuclear cells were suspended in RIPA solution (150 mM sodium chloride, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 2 mM sodium orthovanadate (V), 10 mM β-glycerophosphoric acid, 1 μg/ml aprotinin , 2 µg/ml E-64, 1 µg/ml leupeptin, 0.67 µg/ml bestatin, 0.67 µg/ml pepstatin, 43.5 µg/ml PMSF, 20 mM Tris-HCl, pH 7.4) , and then treated with ultrasound followed by centrifugation (15000 g × 15 min, 4°C). The supernatant was collected as a cell extract. The resulting cell extract was heat-treated in the presence of a reducing agent, then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride (PVDF) membrane by electrotransfer, and reacted with a TBS-T solution containing 5% skim milk at room temperature for 1 hour. followed by reaction with a 5% BSA/TBS-T solution containing a rat anti-mutant CALR antibody that recognizes the amino-terminal side of the cleavage site at 4°C overnight. After rinsing with TBS-T solution, react with 5% skim milk/TBS-T solution containing peroxidase-labeled anti-rat IgG antibody (Santa Cruz Biotechnology, Inc., Cat# sc-2006) at room temperature for 1 hour . The PVDF membrane was washed with TBS-T solution and then reacted with a peroxidase luminescent reagent. Signal detection was carried out using an image converting device FUSION.

[0045]

Platelets were obtained by centrifuging peripheral blood collected in a Spitz tube containing EDTA (500 g × 10 min, 4°C), subjecting the resulting supernatant to centrifugation (500 g × 10 min, 4°C)) and washing the resulting pellet with 500 μl of purification solution for platelets (10 mM sodium citrate, 150 mM sodium chloride, 1 mM EDTA, 1% dextrose, pH 7.4) twice. The resulting platelets were suspended in a RIPA solution and then treated with ultrasound followed by centrifugation (15,000 g × 15 min, 4°C). The supernatant was collected as a cell extract. The resulting cell extract was heat-treated in the presence of a reducing agent, then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride (PVDF) membrane by electrotransfer, and reacted with a TBS-T solution containing 5% skim milk at room temperature for 1 hour. followed by reaction with a 5% BSA/TBS-T solution containing a rat anti-mutant CALR antibody (antibody B3), which recognizes the amino-terminal side of the cleavage site, at 4°C overnight. After washing with TBS-T solution, react with 5% skim milk/TBS-T solution containing peroxidase-labeled anti-rat IgG antibody (Santa Cruz Biotechnology, Inc. Cat# sc-2006) at room temperature for 1 hour. The PVDF membrane was washed with TBS-T solution and then reacted with a peroxidase luminescent reagent. Signal detection was carried out using an image converting device FUSION.

[0046]

(2) Result

CALR proteins contained in culture supernatants of UT-7/TPO/CALR Del 52 and UT-7/TPO/CALR Ins 5 neoplastically proliferating cells, UT-7/TPO/CALR (WT) cells as control and UT-7/ TPO/vec cells that were generated by transfecting the human megakaryoblastic leukemia cell line UT-7/TPO with a vector expressing Del 52 or Ins type 5 mutant CALR protein, a vector expressing wild-type CALR, and an empty vector, respectively, were assessed using an antibody that recognizes both wild type and mutant type (monoclonal antibody D3E6, Cell Signaling Technology, Inc., Cat# 12238), and a commercially available antibody that recognizes the carboxy-terminal sequence of the mutant type (monoclonal antibody CAL2, Dianova GmbH, Cat# DIA-CAL-250) (Fig. 2). As a result, immunoblotting using an antibody (antibody B3) that recognizes wild-type and mutant CALR proteins revealed bands that could not be recognized by the mutant antibody, in addition to endogenous CALR, in cells expressing the mutant CALR protein (Fig. 3).

[0047]

Of the peptide fragments obtained by fragmentation using NTCB at the amino-terminal side of the cysteine residue, the peptide fragments found only in the cleaved type of CALR mutant protein (Del 52 type was used) had molecular weights of 25827.7 and 25418.4. As the cleavage sites of the Del 52 mutant CALR protein, the site between methionine residue 380 and arginine residue 381 and the site between methionine residue 377 and arginine residue 378 were identified by comparing the above numerical values and the calculated molecular masses. , determined from the position of the cleaved cysteine residue.

[0048]

Del 52 type or Ins type 5 mutant CALR protein tagged with a FLAG tag downstream of the signal sequence at the amino terminus or tagged with a FLAG tag at the carboxy terminus was expressed in UT-7/TPO cells and cell surface expression of the mutant CALR protein was analyzed by flow cytometry using anti-FLAG antibody. In cells expressing a mutant CALR protein having a FLAG tag at the amino terminus, a stronger signal was observed compared to the signal in cells expressing a mutant CALR protein having a carboxy terminus FLAG tag (Fig. 4 and 5). The cell lines were then analyzed by flow cytometry as shown in FIG. 4 and 5, using an antibody generated by immunization with a peptide including a sequence specific to the amino-terminal side of the cleavage site of the mutant CALR protein, which resulted in the disappearance of the difference in the quantitative level of detection due to the positions of the FLAG tag insertion (Fig. 6 and 7). Based on this result, it was shown that the establishment of an antigen recognition site on the carboxy-terminal side of the sequence specific for the mutant CALR protein remaining after cleavage is important for the development of a more sensitive test reagent or a more potent therapeutic agent.

[0049]

Indeed, when cell extracts of peripheral blood mononuclear cells and platelets obtained from CALR mutation-positive patients were analyzed by immunoblotting using an antibody specific for the amino-terminal side of the mutant CALR cleavage site, expression of the cleaved mutant CALR protein was observed as in the cell lines (Fig. . 8). These results showed that the same cleavage actually occurs in patient cells, and that installing an antigen recognition site on the carboxy-terminal side remaining after cleavage is important for creating a more sensitive test reagent or a more potent therapeutic agent.

[0050]

[Test example 2]

(1) Confirmation that three antibodies recognize full-length and cleaved mutant CALR proteins

UT-7/TPO cells expressing type 52 mutant CALR protein or type 5 Ins mutant CALR protein having a FLAG tag inserted downstream of the signal sequence at the amino terminus were cultured and culture supernatants containing secreted mutant CALR proteins were obtained by centrifugation (1600g × 5 min, 4°C). The resulting culture supernatants were each heat-treated in the presence of SDS and a reducing agent, then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride (PVDF) membrane by electrotransfer, and reacted with a TBS-T solution containing 5% skim milk at room temperature in for 1 hour followed by interaction with a 5% BSA/TBS-T solution containing a rat antibody, clone B3, C6 or G1, or a mouse antibody to the DYKDDDDK tag sequence (FUJIFILM Wako Pure Chemical Corporation, Cat# 014-22383), at 4 °C overnight. After washing with TBS-T solution, the reaction products were reacted with a 5% skim milk/TBS-T solution containing peroxidase-labeled goat anti-rat IgG antibody (Jackson Immuno Research Inc., Cat# 112-035-003) or peroxidase-labeled goat anti-mouse IgG antibody (Jackson Immuno Research Inc., Cat# 115-035-003), at room temperature for 1 hour. PVDF membranes were washed with TBS-T solution and then reacted with a peroxidase luminescent reagent. Signal detection was carried out using an image converting device FUSION.

As a result, as shown in FIG. 9, it was confirmed that antibodies from all clones B3, C6 and G1 recognized full-length and cleaved mutant CALR proteins.

[0051]

(2) Confirmation that three antibodies recognize mutant CALR proteins on cell surfaces

Mutant CALR protein Del 52 type or Ins type 5, tagged with a FLAG tag downstream of the signal sequence at the amino terminus, was expressed in UT-7/TPO cells and the cells were cultured using IMDM medium containing 10% fetal bovine serum in 5 % CO ₂ at 37°C. Cells (1x10 ⁵ ) in the proliferative stage were washed with PBS and then reacted with a mouse antibody to the DYKDDDDK tag sequence (FUJIFILM Wako Pure Chemical Corporation, Cat# 014-22383) or a rat antibody clone B3, C6 or G1 in a solution of 2% FBS /PBS on ice for 30 minutes. After interaction, cells were washed with 2% FBS/PBS and then reacted with anti-mouse IgG-Alexa Fluor 647 (Thermo Fisher Scientific KK, Cat# A21235) and anti-rat IgG-Alexa Fluor 647 (Thermo Fisher Scientific KK, Cat# A21247), respectively, as secondary antibodies in a solution of 2% FBS/PBS on ice for 30 minutes. After completion of the reaction, cells were collected by centrifugation and exposed to PBS containing 4% paraformaldehyde on ice for 15 minutes. Finally, the cells were washed by adding 2% FBS/PBS solution, then centrifugation was performed (400 g × 5 min, 4°C) and the supernatant was discarded. 300 ml of 2% FBS/PBS solution was then added to the cells and signals were quantified by flow cytometry using FACS Calibur (manufactured by BD biosciences).

As a result, as shown in FIG. 10, antibodies from all clones B3, C6, and G1 were confirmed to recognize the mutant CALR protein on cell surfaces.

[0052]

[Test Example 3]

(Assessment of antibody avidity to antigen)

(1) Antigen avidity assessment by ELISA

In an ELISA plate (Sumitomo Bakelite Co., Ltd., Cat# MS-8896F), 100 ng per well of purified Del 52 mutant CALR protein or BSA was added as a control and reacted with PBS containing 5 mg/ml BSA at room temperature. temperature for 1 hour. Wells were washed with PBS and then reacted with PBS containing 0, 6.25, 12.5, 25, 50, 100, 200, 400, or 800 ng/ml clone B3, C6, or G1 antibody or rat IgG (Santa Cruz Biotechnology , Inc. Cat# 2026) and 5 mg/ml BSA, at room temperature for 1 hour. Wells were washed with PBS and then reacted with PBS containing 16 ng/ml peroxidase-labeled goat anti-rat IgG antibody (Jackson Immuno Research Inc., Cat# 112-035-003) and 5 mg/ml BSA, at room temperature in within 1 hour. After washing the wells with TBS-T solution, TMB solution (Nacalai Tesque, Cat# 05298-80) was added to the wells and left to react at room temperature for 15 minutes and then 1 M aqueous sulfuric acid solution was added to the wells. The absorbance was measured at 450 nm and 620 nm and the difference between the absorbances was determined. The difference in BSA as a control was subtracted from each difference.

As a result, as shown in FIG. 11, it is demonstrated that the antibodies (B3, C6 and G1) of the present invention all bind to the mutant CALR protein even at low concentrations.

[0053]

(2) Assessment of antigen avidity by surface plasmon resonance

Sensor chips (GE Healthcare, Cat# BR100530) for surface plasmon resonance analysis were loaded into a Biacore T200 surface plasmon resonance analyzer (GE Healthcare) and equilibrated with HBS-EP buffer (GE Healthcare, Cat# BR100669). Clone B3 antibody, adjusted to a concentration of 10 μg/ml with Acetate 5.5 (GE Healthcare, Cat# BR100352), was immobilized using an amine coupling kit (GE Healthcare, Cat# BR100050) in an immobilized amount of 200 RU. Surface plasmon resonance was then measured using a peptide containing a cysteine at the amino terminus (RRMMRTKMR) included in SEQ ID NO:1, adjusted to concentrations of 20, 10, 5, 2.5 and 1.25 nM with HBS- Buffer EP and dissociation constant were obtained using Biacore T200 Evaluation Software.

As a result, as shown in FIG. 12, it was demonstrated that the antibody of the present invention binds strongly to the mutant CALR protein.

[0054]

[Test example 4]

(Identification of the antigen recognition site of an antibody)

Antibodies of clone B3, C6 and G1 were confirmed to recognize the amino acid sequence used as antigen on the mutant CALR protein. The reactivity of the corresponding antibodies against mutant CALR proteins, each containing an alanine (A) replacing one amino acid at each position of the amino acids used as the antigen and the amino acids around it, was assessed by immunoblotting (Fig. 13A) and ELISA (Fig. 13B) .

[0055]

(1) Immunoblotting

Del 52 mutant CALR proteins, each containing an alanine (A) substituting one amino acid at each amino acid position in the region from threonine 367 (T) to arginine 378 (R), or a protein having no substitutions, were expressed in HEK 293T cells and culture supernatants containing secreted mutant CALR proteins were obtained by centrifugation (1600 g × 5 min, 4°C). The resulting protein solutions were each heat-treated in the presence of SDS and a reducing agent, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride (PVDF) membrane by electrotransfer, and reacted with a TBS-T solution containing 5% skim milk at room temperature for 1 hour followed by reaction with a 5% BSA/TBS-T solution containing peroxidase-labeled clone B3, C6 or G1 antibody or mouse anti-CALR antibody (Abcam plc., Cat# ab22683) at 4°C overnight. The PVDF membrane that was reacted with the mouse anti-CALR antibody was washed with TBS-T solution and then reacted with a 5% skim milk/TBS-T solution containing peroxidase-labeled anti-mouse IgG antibody (Jackson Immuno Research Inc., Cat# 115-035-003), at room temperature for 1 hour. All PVDF membranes were then washed with TBS-T solution and then reacted with peroxidase fluorescent reagent (Thermo Fisher Scientific K.K., Cat# 34094). Signal detection was carried out using an image converting device FUSION (manufactured by Vilber-Lourmat S.A.).

As a result, as shown in FIG. 13A, it has been demonstrated that the antibodies of the present invention recognize the site between the 1st arginine and the 9th arginine of SEQ ID NO:1.

[0056]

(2) ELISA

The ELISA plate to which the mutant CALR proteins obtained as described above were bound was reacted with PBS containing 5 mg/ml BSA at room temperature for 1 hour. Wells were washed with PBS and then reacted with PBS containing 200 ng/ml clone B3, C6, or G1 antibody or mouse anti-CALR antibody (Santa Cruz Biotechnology, Inc., Cat# sc-373863) and 5 mg/ml BSA, at room temperature for 1 hour. Wells were washed with PBS and then reacted with PBS containing 16 ng/ml peroxidase-labeled goat anti-rat IgG antibody (Jackson Immuno Research Inc., Cat# 112-035-003) or goat anti-mouse IgG antibody (Santa Cruz Biotechnology , Inc., Cat# sc-2005) and 5 mg/ml BSA, at room temperature for 1 hour. After washing the wells with TBS-T solution, TMB solution was added to the wells and left to react at room temperature for 15 minutes and then 1 M aqueous sulfuric acid solution was added to the wells. The absorbance was measured at 450 nm and 620 nm and the difference between the absorbance was determined.

As a result, as shown in FIG. 13B, the antibodies of the present invention have been demonstrated to recognize the site between the 1st arginine and the 9th arginine of SEQ ID NO:1.

[0057]

[Test Example 5]

(Determination of sequence information of the antibody of the present invention)

As information about the sequence of the heavy chain and light chain of the antibodies (B3, C6 and G1) of the present invention, the full-length cDNA sequences of the heavy chain and light chain were determined by obtaining mRNA from antibody-producing cells using the PureLinc RNA Mini kit (Thermo Fisher Scientific K.K., Cat# 12183025), cDNA synthesis using the resulting mRNAs as templates and reverse transcription primers specific for the heavy chain and light chain, by rapidly amplifying the ends of the cDNA, then cloning these cDNAs into plasmids and analyzing the plasmids using sequencing Sanger.

The results are shown in Fig. 14-17 and SEQ ID NO:5-34.

[0058]

Fig. 14 shows the amino acid sequences of the heavy chain variable regions of antibodies (B3, C6 and G1) of the present invention. Fig. 15 shows the amino acid sequences of the light chain variable regions of antibodies (B3, C6 and G1) of the present invention. Fig. 16 shows the nucleotide sequences of the heavy chain variable regions of antibodies (B3, C6 and G1) of the present invention. Fig. 17 shows the nucleotide sequences of the light chain variable regions of antibodies (B3, C6 and G1) of the present invention.

[0059]

SEQ ID NO:5 is the amino acid sequence of the heavy chain variable region of the B3 antibody. SEQ ID NO:6 is the amino acid sequence of the C6 heavy chain variable region of the antibody. SEQ ID NO:7 is the amino acid sequence of the G1 antibody heavy chain variable region. SEQ ID NO:8 is the amino acid sequence of the antibody B3 light chain variable region. SEQ ID NO:9 is the amino acid sequence of the C6 light chain variable region of the antibody. SEQ ID NO:10 is the amino acid sequence of the G1 light chain variable region of the antibody.

SEQ ID NO:11 is the nucleotide sequence of the heavy chain variable region of the B3 antibody. SEQ ID NO:12 is the nucleotide sequence of the C6 heavy chain variable region of the antibody. SEQ ID NO:13 is the nucleotide sequence of the antibody G1 heavy chain variable region. SEQ ID NO:14 is the nucleotide sequence of the antibody B3 light chain variable region. SEQ ID NO:15 is the nucleotide sequence of the C6 light chain variable region of the antibody. SEQ ID NO:16 is the nucleotide sequence of the antibody G1 light chain variable region.

[0060]

SEQ ID NO:17 is the amino acid sequence of CDR1 VH antibody B3. SEQ ID NO:18 is the CDR2 amino acid sequence of VH antibody B3. SEQ ID NO:19 is the CDR3 amino acid sequence of VH antibody B3. SEQ ID NO:20 is the amino acid sequence of CDR1 VH antibody C6. SEQ ID NO:21 is the CDR2 amino acid sequence of the C6 VH antibody. SEQ ID NO:22 is the CDR3 amino acid sequence of the C6 VH antibody. SEQ ID NO:23 is the amino acid sequence of CDR1 VH antibody G1. SEQ ID NO:24 is the CDR2 amino acid sequence of the G1 antibody VH. SEQ ID NO:25 is the CDR3 amino acid sequence of the G1 antibody VH. SEQ ID NO:26 is the amino acid sequence of CDR1 VL antibody B3. SEQ ID NO:27 is the amino acid sequence CDR2 of VL antibody B3. SEQ ID NO:28 is the CDR3 amino acid sequence of VL antibody B3. SEQ ID NO:29 is the amino acid sequence CDR1 of VL antibody C6. SEQ ID NO:30 is the CDR2 amino acid sequence of the C6 antibody VL. SEQ ID NO:31 is the CDR3 amino acid sequence of the C6 VL antibody. SEQ ID NO:32 is the amino acid sequence of CDR1 VL antibody G1. SEQ ID NO:33 is the CDR2 amino acid sequence of the VL antibody G1. SEQ ID NO:34 is the CDR3 amino acid sequence of the VL antibody G1.

[0061]

[Test Example 6]

(Evaluation of therapeutic effect)

The chimeric B3 antibody was prepared by fusing the variable regions of the heavy chain and light chain of the antibody clone B3 and the constant regions of the heavy chain and light chain of the corresponding murine IgG2a and was administered to a model mouse model of myeloproliferative neoplasm obtained by grafting hematopoietic stem cells expressing the mutant CALR gene type Del 52, and the therapeutic effect of the chimeric antibody was evaluated.

An expression vector containing a B3 murine heavy chain-light chain chimeric gene ligated to a gene sequence encoding a clone B3 antibody heavy chain variable region and a murine IgG2a constant region, or a gene sequence encoding a clone B3 antibody light chain variable region and a murine IgG2a constant region. immunoglobulin κ were transfected into ExpiCHO-S cells (Thermo Fisher Scientific KK, Cat# A29127) and cells were cultured using ExpiCHO expression medium (Thermo Fisher Scientific KK, Cat# A2910001) in 5% CO ₂ at 37°C for 10 days. Culture supernatants were collected by centrifugation (4000 g × 30 min, 4°C) and each applied to a HiTrap Protein G HP column (GE Healthcare, Cat# 29048581) to attach the chimeric antibody to the column, then eluted using 0.1 M Glycine-HCl ( pH 2.7). The eluted fractions, neutralized with 1 M Tris-HCl (pH 9.0), were dialyzed against PBS to obtain the B3 mouse chimeric antibody.

Bone marrow transplanted mouse models were used to evaluate the therapeutic effect of the B3 mouse chimeric antibody. Bone marrow cells were purified from the femurs of 8- to 10-week-old congenic B6.CD45.1 mice (Sankyo Labo Service Corporation, Inc.), and c-kit-positive cells labeled with APC-labeled anti-c-kit antibody were isolated. and also isolated LSK cells that were Ter119, CD4, CD8, Gr-1, CD45R, CD3e and CD11b negative and CD117 and Sca1 positive. Cells were infected with a retrovirus containing a pMSCV-IRES-GFP vector expressing the mutant Del 52 CALR gene or a control vector. At 72 hours postinfection, 4000 LSK cells per mouse were transplanted into 8- to 10-week-old C57BL/6J mice (Oriental Yeast Co., Ltd.) irradiated twice at 6 Gy. After some time, the number of platelets in the peripheral blood was counted using an automatic multichannel blood cell counter (Sysmex Corporation, Cat# pocH-100iV Diff). Two months after transplantation, after confirmation of thrombocytosis, which is a phenotype of myeloproliferative neoplasm caused by the expression of a mutant CALR gene, B3 mouse chimeric antibody or vehicle (PBS) was administered at 250 μg per mouse every week for 9 weeks after transplantation, and the effect of suppressing thrombocytosis was assessed , a B3-specific mouse chimeric antibody.

As a result, as shown in FIG. 18, the antibody of the present invention showed excellent therapeutic effect on MPN.

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LIST OF SEQUENCES

<110> JUNTENDO EDUCATIONAL FOUNDATION

<120> ANTIBODIES THAT BIND TO THE CLEAVED FORM OF THE MUTANT

CALRETICULIN, AND A MEANS FOR DIAGNOSIS, PREVENTION OR

TREATMENT OF MYELOPROLIFERATIVE NEOPLASIS

<130> JUSM0023

<150> JP 2019-036119

<151> 2019-02-28

<160> 34

<170> PatentIn version 3.5

<210> 1

<211> 13

<212> Protein

<213> Homo sapiens

<400> 1

Arg Arg Met Met Arg Thr Lys Met Arg Met Arg Arg Met

1 5 10

<210> 2

<211> 417

<212> Protein

<213> Homo sapiens

<400> 2

Met Leu Leu Ser Val Pro Leu Leu Leu Gly Leu Leu Gly Leu Ala Val

1 5 10 15

Ala Glu Pro Ala Val Tyr Phe Lys Glu Gln Phe Leu Asp Gly Asp Gly

20 25 30

Trp Thr Ser Arg Trp Ile Glu Ser Lys His Lys Ser Asp Phe Gly Lys

35 40 45

Phe Val Leu Ser Ser Gly Lys Phe Tyr Gly Asp Glu Glu Lys Asp Lys

50 55 60

Gly Leu Gln Thr Ser Gln Asp Ala Arg Phe Tyr Ala Leu Ser Ala Ser

65 70 75 80

Phe Glu Pro Phe Ser Asn Lys Gly Gln Thr Leu Val Val Gln Phe Thr

85 90 95

Val Lys His Glu Gln Asn Ile Asp Cys Gly Gly Gly Tyr Val Lys Leu

100 105 110

Phe Pro Asn Ser Leu Asp Gln Thr Asp Met His Gly Asp Ser Glu Tyr

115 120 125

Asn Ile Met Phe Gly Pro Asp Ile Cys Gly Pro Gly Thr Lys Lys Val

130 135 140

His Val Ile Phe Asn Tyr Lys Gly Lys Asn Val Leu Ile Asn Lys Asp

145 150 155 160

Ile Arg Cys Lys Asp Asp Glu Phe Thr His Leu Tyr Thr Leu Ile Val

165 170 175

Arg Pro Asp Asn Thr Tyr Glu Val Lys Ile Asp Asn Ser Gln Val Glu

180 185 190

Ser Gly Ser Leu Glu Asp Asp Trp Asp Phe Leu Pro Pro Lys Lys Ile

195 200 205

Lys Asp Pro Asp Ala Ser Lys Pro Glu Asp Trp Asp Glu Arg Ala Lys

210 215 220

Ile Asp Asp Pro Thr Asp Ser Lys Pro Glu Asp Trp Asp Lys Pro Glu

225 230 235 240

His Ile Pro Asp Pro Asp Ala Lys Lys Pro Glu Asp Trp Asp Glu Glu

245 250 255

Met Asp Gly Glu Trp Glu Pro Pro Val Ile Gln Asn Pro Glu Tyr Lys

260 265 270

Gly Glu Trp Lys Pro Arg Gln Ile Asp Asn Pro Asp Tyr Lys Gly Thr

275 280 285

Trp Ile His Pro Glu Ile Asp Asn Pro Glu Tyr Ser Pro Asp Pro Ser

290 295 300

Ile Tyr Ala Tyr Asp Asn Phe Gly Val Leu Gly Leu Asp Leu Trp Gln

305 310 315 320

Val Lys Ser Gly Thr Ile Phe Asp Asn Phe Leu Ile Thr Asn Asp Glu

325 330 335

Ala Tyr Ala Glu Glu Phe Gly Asn Glu Thr Trp Gly Val Thr Lys Ala

340 345 350

Ala Glu Lys Gln Met Lys Asp Lys Gln Asp Glu Glu Gln Arg Leu Lys

355 360 365

Glu Glu Glu Glu Asp Lys Lys Arg Lys Glu Glu Glu Glu Ala Glu Asp

370 375 380

Lys Glu Asp Asp Glu Asp Lys Asp Glu Asp Glu Glu Asp Glu Glu Asp

385 390 395 400

Lys Glu Glu Asp Glu Glu Glu Asp Val Pro Gly Gln Ala Lys Asp Glu

405 410 415

Leu

<210> 3

<211> 411

<212> Protein

<213> Homo sapiens

<400> 3

Met Leu Leu Ser Val Pro Leu Leu Leu Gly Leu Leu Gly Leu Ala Val

1 5 10 15

Ala Glu Pro Ala Val Tyr Phe Lys Glu Gln Phe Leu Asp Gly Asp Gly

20 25 30

Trp Thr Ser Arg Trp Ile Glu Ser Lys His Lys Ser Asp Phe Gly Lys

35 40 45

Phe Val Leu Ser Ser Gly Lys Phe Tyr Gly Asp Glu Glu Lys Asp Lys

50 55 60

Gly Leu Gln Thr Ser Gln Asp Ala Arg Phe Tyr Ala Leu Ser Ala Ser

65 70 75 80

Phe Glu Pro Phe Ser Asn Lys Gly Gln Thr Leu Val Val Gln Phe Thr

85 90 95

Val Lys His Glu Gln Asn Ile Asp Cys Gly Gly Gly Tyr Val Lys Leu

100 105 110

Phe Pro Asn Ser Leu Asp Gln Thr Asp Met His Gly Asp Ser Glu Tyr

115 120 125

Asn Ile Met Phe Gly Pro Asp Ile Cys Gly Pro Gly Thr Lys Lys Val

130 135 140

His Val Ile Phe Asn Tyr Lys Gly Lys Asn Val Leu Ile Asn Lys Asp

145 150 155 160

Ile Arg Cys Lys Asp Asp Glu Phe Thr His Leu Tyr Thr Leu Ile Val

165 170 175

Arg Pro Asp Asn Thr Tyr Glu Val Lys Ile Asp Asn Ser Gln Val Glu

180 185 190

Ser Gly Ser Leu Glu Asp Asp Trp Asp Phe Leu Pro Pro Lys Lys Ile

195 200 205

Lys Asp Pro Asp Ala Ser Lys Pro Glu Asp Trp Asp Glu Arg Ala Lys

210 215 220

Ile Asp Asp Pro Thr Asp Ser Lys Pro Glu Asp Trp Asp Lys Pro Glu

225 230 235 240

His Ile Pro Asp Pro Asp Ala Lys Lys Pro Glu Asp Trp Asp Glu Glu

245 250 255

Met Asp Gly Glu Trp Glu Pro Pro Val Ile Gln Asn Pro Glu Tyr Lys

260 265 270

Gly Glu Trp Lys Pro Arg Gln Ile Asp Asn Pro Asp Tyr Lys Gly Thr

275 280 285

Trp Ile His Pro Glu Ile Asp Asn Pro Glu Tyr Ser Pro Asp Pro Ser

290 295 300

Ile Tyr Ala Tyr Asp Asn Phe Gly Val Leu Gly Leu Asp Leu Trp Gln

305 310 315 320

Val Lys Ser Gly Thr Ile Phe Asp Asn Phe Leu Ile Thr Asn Asp Glu

325 330 335

Ala Tyr Ala Glu Glu Phe Gly Asn Glu Thr Trp Gly Val Thr Lys Ala

340 345 350

Ala Glu Lys Gln Met Lys Asp Lys Gln Asp Glu Glu Gln Arg Thr Arg

355 360 365

Arg Met Met Arg Thr Lys Met Arg Met Arg Arg Met Arg Arg Thr Arg

370 375 380

Arg Lys Met Arg Arg Lys Met Ser Pro Ala Arg Pro Arg Thr Ser Cys

385 390 395 400

Arg Glu Ala Cys Leu Gln Gly Trp Thr Glu Ala

405 410

<210> 4

<211> 430

<212> Protein

<213> Homo sapiens

<400> 4

Met Leu Leu Ser Val Pro Leu Leu Leu Gly Leu Leu Gly Leu Ala Val

1 5 10 15

Ala Glu Pro Ala Val Tyr Phe Lys Glu Gln Phe Leu Asp Gly Asp Gly

20 25 30

Trp Thr Ser Arg Trp Ile Glu Ser Lys His Lys Ser Asp Phe Gly Lys

35 40 45

Phe Val Leu Ser Ser Gly Lys Phe Tyr Gly Asp Glu Glu Lys Asp Lys

50 55 60

Gly Leu Gln Thr Ser Gln Asp Ala Arg Phe Tyr Ala Leu Ser Ala Ser

65 70 75 80

Phe Glu Pro Phe Ser Asn Lys Gly Gln Thr Leu Val Val Gln Phe Thr

85 90 95

Val Lys His Glu Gln Asn Ile Asp Cys Gly Gly Gly Tyr Val Lys Leu

100 105 110

Phe Pro Asn Ser Leu Asp Gln Thr Asp Met His Gly Asp Ser Glu Tyr

115 120 125

Asn Ile Met Phe Gly Pro Asp Ile Cys Gly Pro Gly Thr Lys Lys Val

130 135 140

His Val Ile Phe Asn Tyr Lys Gly Lys Asn Val Leu Ile Asn Lys Asp

145 150 155 160

Ile Arg Cys Lys Asp Asp Glu Phe Thr His Leu Tyr Thr Leu Ile Val

165 170 175

Arg Pro Asp Asn Thr Tyr Glu Val Lys Ile Asp Asn Ser Gln Val Glu

180 185 190

Ser Gly Ser Leu Glu Asp Asp Trp Asp Phe Leu Pro Pro Lys Lys Ile

195 200 205

Lys Asp Pro Asp Ala Ser Lys Pro Glu Asp Trp Asp Glu Arg Ala Lys

210 215 220

Ile Asp Asp Pro Thr Asp Ser Lys Pro Glu Asp Trp Asp Lys Pro Glu

225 230 235 240

His Ile Pro Asp Pro Asp Ala Lys Lys Pro Glu Asp Trp Asp Glu Glu

245 250 255

Met Asp Gly Glu Trp Glu Pro Pro Val Ile Gln Asn Pro Glu Tyr Lys

260 265 270

Gly Glu Trp Lys Pro Arg Gln Ile Asp Asn Pro Asp Tyr Lys Gly Thr

275 280 285

Trp Ile His Pro Glu Ile Asp Asn Pro Glu Tyr Ser Pro Asp Pro Ser

290 295 300

Ile Tyr Ala Tyr Asp Asn Phe Gly Val Leu Gly Leu Asp Leu Trp Gln

305 310 315 320

Val Lys Ser Gly Thr Ile Phe Asp Asn Phe Leu Ile Thr Asn Asp Glu

325 330 335

Ala Tyr Ala Glu Glu Phe Gly Asn Glu Thr Trp Gly Val Thr Lys Ala

340 345 350

Ala Glu Lys Gln Met Lys Asp Lys Gln Asp Glu Glu Gln Arg Leu Lys

355 360 365

Glu Glu Glu Glu Asp Lys Lys Arg Lys Glu Glu Glu Glu Ala Glu Asp

370 375 380

Asn Cys Arg Arg Met Met Arg Thr Lys Met Arg Met Arg Arg Met Arg

385 390 395 400

Arg Thr Arg Arg Lys Met Arg Arg Lys Met Ser Pro Ala Arg Pro Arg

405 410 415

Thr Ser Cys Arg Glu Ala Cys Leu Gln Gly Trp Thr Glu Ala

420 425 430

<210> 5

<211> 119

<212> Protein

<213> Rattus norvegicus

<400> 5

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Asn

20 25 30

Phe Ile His Trp Ile Lys Gln Gln Pro Gly Asn Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Phe Pro Gly Asp Gly Asp Thr Glu Tyr Asn Gln Lys Phe

50 55 60

Asn Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Asn Tyr Asn Tyr Glu Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Val Met Val Thr Val Ser Ser

115

<210> 6

<211> 121

<212> Protein

<213> Rattus norvegicus

<400> 6

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Phe Val His Trp Ile Lys Gln Gln Pro Gly Asp Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Asp Thr Glu Tyr Asn His Lys Phe

50 55 60

Asn Gly Lys Ser Thr Leu Thr Ala Asp Arg Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Asn Tyr Tyr Asp Gly Arg Glu Val Met Asp Ala Trp Gly

100 105 110

Gln Gly Ala Ser Val Thr Val Ser Ser

115 120

<210> 7

<211> 121

<212> Protein

<213> Rattus norvegicus

<400> 7

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Phe Val His Trp Ile Lys Gln Gln Pro Gly Asp Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Asp Thr Glu Tyr Asn Gln Lys Phe

50 55 60

Asn Gly Lys Ala Thr Leu Thr Ala Asp Arg Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Asn Tyr Tyr Asp Gly Arg Glu Val Met Asp Ala Trp Gly

100 105 110

Gln Gly Ala Ser Val Thr Val Ser Ser

115 120

<210> 8

<211> 107

<212> Protein

<213> Rattus norvegicus

<400> 8

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Thr Val Ser Ile Glu Cys Leu Ala Ser Glu Asp Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile

35 40 45

Phe Ala Ala Asn Arg Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Ser Gly Met Gln Pro

65 70 75 80

Glu Asp Glu Gly Asp Tyr Phe Cys Leu Gln Gly Ser Lys Phe Pro Tyr

85 90 95

Thr Phe Gly Pro Gly Thr Lys Leu Glu Leu Asn

100 105

<210> 9

<211> 112

<212> Protein

<213> Rattus norvegicus

<400> 9

Asp Val Val Leu Thr Gln Thr Pro Pro Thr Leu Ser Ala Thr Ile Gly

1 5 10 15

Gln Ser Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Glu Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Ser Val Ser Asn Leu Glu Ser Gly Val Pro

50 55 60

Asn Arg Phe Ser Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Gly Met Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Thr His Gly Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 10

<211> 112

<212> Protein

<213> Rattus norvegicus

<400> 10

Asp Val Val Leu Thr Gln Thr Pro Pro Thr Leu Ser Ala Thr Ile Gly

1 5 10 15

Gln Ser Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Glu Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Cys

35 40 45

Pro Gln Leu Leu Ile Tyr Ser Val Ser Asn Leu Glu Ser Gly Val Pro

50 55 60

Asn Arg Phe Ser Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Gly Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Met Gln Gly

85 90 95

Thr His Gly Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 11

<211> 357

<212> DNA

<213> Rattus norvegicus

<400> 11

caggtacagc tgcagcaatc tggggctgaa ctggtgaagc ctgggtcctc agtgaaaatt

60

tcctgcaagg cttctggcta caccttcacc cgtaacttta tacactggat aaaacagcag

120

cctggaaatg gccttgagtg gattgggtgg atttttcctg gagatggtga tacagagtac

180

aatcaaaagt tcaatgggaa ggcaacactc actgcagaca aatcgtccag cacagcctat

240

atgcagctca gcagcctgac atctgaggac tctgcagtct atttctgtgc aagaggaaat

300

tacaactacg agtactttga ttactggggc caaggagtca tggtcacagt ctcctca

357

<210> 12

<211> 363

<212> DNA

<213> Rattus norvegicus

<400> 12

caggtacagc tgcagcaatc tggggctgaa ctggtgaagc ctgggtcctc agtgaaaatt

60

tcctgcaagg cttctggcta caccttcacc agttactttg tgcactggat aaaacagcag

120

cctggagatg gccttgagtg gattgggtgg atttatcctg gagatggtga tacagagtac

180

aatcacaagt tcaatgggaa gtcaacactc actgcagaca gatcctccag tacagcctat

240

atgcagctca gcagcctgac atctgaggac tctgcagtct atttctgtgc aagagggaat

300

tactatgatg gtcgggaagt tatggatgcc tggggtcaag gagcttcagt cactgtctcc

360

tca

363

<210> 13

<211> 363

<212> DNA

<213> Rattus norvegicus

<400> 13

caggtacagc tgcagcaatc tggggctgaa ctggtgaagc ctgggtcctc agtgaaaatt

60

tcctgcaagg cttctggcta caccttcacc agttactttg tgcactggat aaaacagcag

120

cctggagatg gccttgagtg gattgggtgg atttatcctg gagatggtga tacagagtac

180

aatcaaaagt tcaatgggaa ggcaacactc actgcagaca gatcctccag tacagcctat

240

atgcagctca gcagcctgac atctgaggac tctgcagtct atttctgtgc aagagggaat

300

tactatgatg gtcgggaagt tatggatgcc tggggtcaag gagcttcagt cactgtctcc

360

tca

363

<210> 14

<211> 321

<212> DNA

<213> Rattus norvegicus

<400> 14

gacatccaga tgacacagtc tccggcttcc ctgtctgcat ctctgggaga aactgtctcc

60

atcgagtgtc tagcaagtga ggacatttac agttatttag catggtatca acagaagcca

120

gggaaatctc ctcagctcct gatctttgct gcaaataggt tgcaagatgg ggtcccatca

180

cggttcagtg gcagtggatc tggcacacag ttttctctca agatcagcgg catgcaacct

240

gaagatgaag gggattattt ctgtctacag ggttccaagt ttccgtacac ctttggacct

300

gggaccaagc tggaactgaa c

321

<210> 15

<211> 336

<212> DNA

<213> Rattus norvegicus

<400> 15

gatgttgtgc tgacccagac tccacccact ttatcggcta ccattggaca atcggtctcc

60

atctcttgca ggtcaagtca gagtctctta gatagtgatg gagaaaccta tttaaattgg

120

ttgctacaga ggccaggcca atctccacag cttctaattt attcggtctc caacctggaa

180

tctggggtcc ccaacaggtt cagtggcagt gggtcagaaa cagatttcac actcaaaatc

240

agtggaatgg aggctgaaga tttgggagtt tactactgca tgcaagctac ccatggtccg

300

tacacgtttg gagctgggac caagctggaa ctgaaa

336

<210> 16

<211> 336

<212> DNA

<213> Rattus norvegicus

<400> 16

gatgttgtgc tgacccagac tccacccact ttatcggcta ccattggaca atcggtctcc

60

atctcttgca ggtcaagtca gagtctctta gatagtgatg gagaaaccta tttaaattgg

120

ttgctacaga ggccaggcca atgtccacag cttctaattt attcggtatc caacctggaa

180

tctggggtcc ccaacaggtt cagtggcagt gggtcagaaa cagatttcac actcaaaatc

240

agtggagtgg aggctgaaga tttgggagtt tactactgca tgcaaggtac ccatggtccg

300

tacacgtttg gagctgggac caagctggaa ctgaaa

336

<210> 17

<211> 10

<212> Protein

<213> Rattus norvegicus

<400> 17

Gly Tyr Thr Phe Thr Arg Asn Phe Ile His

1 5 10

<210> 18

<211> 17

<212> Protein

<213> Rattus norvegicus

<400> 18

Trp Ile Phe Pro Gly Asp Gly Asp Thr Glu Tyr Asn Gln Lys Phe Asn

1 5 10 15

Gly

<210> 19

<211> 10

<212> Protein

<213> Rattus norvegicus

<400> 19

Gly Asn Tyr Asn Tyr Glu Tyr Phe Asp Tyr

1 5 10

<210> 20

<211> 10

<212> Protein

<213> Rattus norvegicus

<400> 20

Gly Tyr Thr Phe Thr Ser Tyr Phe Val His

1 5 10

<210> 21

<211> 17

<212> Protein

<213> Rattus norvegicus

<400> 21

Trp Ile Tyr Pro Gly Asp Gly Asp Thr Glu Tyr Asn His Lys Phe Asn

1 5 10 15

Gly

<210> 22

<211> 12

<212> Protein

<213> Rattus norvegicus

<400> 22

Gly Asn Tyr Tyr Asp Gly Arg Glu Val Met Asp Ala

1 5 10

<210> 23

<211> 10

<212> Protein

<213> Rattus norvegicus

<400> 23

Gly Tyr Thr Phe Thr Ser Tyr Phe Val His

1 5 10

<210> 24

<211> 17

<212> Protein

<213> Rattus norvegicus

<400> 24

Trp Ile Tyr Pro Gly Asp Gly Asp Thr Glu Tyr Asn Gln Lys Phe Asn

1 5 10 15

Gly

<210> 25

<211> 12

<212> Protein

<213> Rattus norvegicus

<400> 25

Gly Asn Tyr Tyr Asp Gly Arg Glu Val Met Asp Ala

1 5 10

<210> 26

<211> 11

<212> Protein

<213> Rattus norvegicus

<400> 26

Leu Ala Ser Glu Asp Ile Tyr Ser Tyr Leu Ala

1 5 10

<210> 27

<211> 7

<212> Protein

<213> Rattus norvegicus

<400> 27

Ala Ala Asn Arg Leu Gln Asp

15

<210> 28

<211> 9

<212> Protein

<213> Rattus norvegicus

<400> 28

Leu Gln Gly Ser Lys Phe Pro Tyr Thr

15

<210> 29

<211> 16

<212> Protein

<213> Rattus norvegicus

<400> 29

Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Glu Thr Tyr Leu Asn

1 5 10 15

<210> 30

<211> 7

<212> Protein

<213> Rattus norvegicus

<400> 30

Ser Val Ser Asn Leu Glu Ser

15

<210> 31

<211> 9

<212> Protein

<213> Rattus norvegicus

<400> 31

Met Gln Ala Thr His Gly Pro Tyr Thr

15

<210> 32

<211> 16

<212> Protein

<213> Rattus norvegicus

<400> 32

Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Glu Thr Tyr Leu Asn

1 5 10 15

<210> 33

<211> 7

<212> Protein

<213> Rattus norvegicus

<400> 33

Ser Val Ser Asn Leu Glu Ser

15

<210> 34

<211> 9

<212> Protein

<213> Rattus norvegicus

<400> 34

Met Gln Gly Thr His Gly Pro Tyr Thr

15

<---

Claims (22)

1. An antibody or functional fragment thereof that binds to a cleaved mutant CALR protein selected from the following (a), (b) and (c): (a) an antibody wherein the immunoglobulin VH chain CDR1, CDR2 and CDR3 are polypeptides consisting of the amino acid sequences of SEQ ID NO: 17, 18 and 19, respectively; and CDR1, CDR2 and CDR3 of the immunoglobulin VL chain are polypeptides consisting of the amino acid sequences of SEQ ID NO: 26, 27 and 28, respectively; (b) an antibody wherein the immunoglobulin VH chain CDR1, CDR2 and CDR3 are polypeptides consisting of the amino acid sequences of SEQ ID NO: 20, 21 and 22, respectively; and CDR1, CDR2 and CDR3, immunoglobulin VL chain are polypeptides consisting of the amino acid sequences of SEQ ID NO: 29, 30 and 31, respectively; And (c) an antibody wherein the immunoglobulin VH chain CDR1, CDR2 and CDR3 are polypeptides consisting of the amino acid sequences of SEQ ID NO: 23, 24 and 25, respectively; and CDR1, CDR2 and CDR3 of the immunoglobulin VL chain are polypeptides consisting of the amino acid sequences of SEQ ID NO: 32, 33 and 34, respectively. 2. An antibody or a functional fragment thereof that binds to the cleaved mutant CALR protein of claim 1, wherein the antibody or a functional fragment thereof is selected from the following (A), (B) and (C): (A) antibody containing an immunoglobulin VH chain consisting of the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence with the deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 5, and an immunoglobulin VL chain consisting of the amino acid sequence SEQ ID NO: 8 or an amino acid sequence with a deletion, substitution or addition of one or more amino acids in the amino acid sequence SEQ ID NO: 8; (B) antibody containing an immunoglobulin VH chain consisting of the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence with the deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 6, and an immunoglobulin VL chain consisting of the amino acid sequence SEQ ID NO: 9 or an amino acid sequence with a deletion, substitution or addition of one or more amino acids in the amino acid sequence SEQ ID NO: 9; And (C) an antibody containing an immunoglobulin VH chain consisting of the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence with the deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 7, and an immunoglobulin VL chain consisting of the amino acid sequence SEQ ID NO: 10 or an amino acid sequence with a deletion, substitution or addition of one or more amino acids in the amino acid sequence SEQ ID NO: 10. 3. Pharmaceutical composition for the prevention or treatment of myeloproliferative neoplasm associated with a mutant CALR protein, containing an antibody or a functional fragment thereof according to claim 1 or 2. 4. Pharmaceutical composition for the diagnosis of myeloproliferative neoplasm associated with a mutant CALR protein, containing an antibody or a functional fragment thereof according to claim 1 or 2. 5. A method for detecting mutant CALR protein, comprising detecting a polypeptide in a biological sample using an antibody or a functional fragment thereof according to claim 1 or 2. 6. A method of screening for the identification of a therapeutic agent for the treatment of a myeloproliferative neoplasm associated with a mutant CALR protein, including screening for the identification of a drug using an antibody or a functional fragment thereof according to claim 1 or 2. 7. A method for diagnosing a myeloproliferative neoplasm associated with a mutant CALR protein, including detection of a polypeptide in a biological sample using an antibody or a functional fragment thereof according to claim 1 or 2. 8. A method for preventing or treating a myeloproliferative neoplasm associated with a mutant CALR protein, comprising administering an antibody or a functional fragment thereof according to any one of claims 1 or 2. 9. Use of an antibody or a functional fragment thereof according to claim 1 or 2 for the diagnosis of a myeloproliferative neoplasm associated with a mutant CALR protein. 10. Use of an antibody or a functional fragment thereof according to claim 1 or 2 to obtain an agent for diagnosing a myeloproliferative neoplasm associated with a mutant CALR protein.