JP2017095417A - Anticancer agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To find a new cancer antigen that can be a target even in HER2-negative breast cancer and provide a new choice for cancer treatment.SOLUTION: An anticancer agent is a complex of a polypeptide that can specifically recognize human CD239, and an agent having cytotoxicity.SELECTED DRAWING: Figure 2

Description

  The present invention provides an anticancer agent comprising a polypeptide that specifically recognizes CD239, and a pharmaceutical composition containing the same. The present invention also provides a new marker for detecting CD239-expressing cancer cells and a carrier for delivering a drug having cytotoxicity against CD239-expressing cancer cells.

  CD239 is a cell membrane single-pass glycoprotein known as one of the immunoglobulin superfamily existing on the cell surface. It has an extracellular domain consisting of five immunoglobulin-like domains, and is a Lutheran blood group glycoprotein (Lu) (NCBI Accession No: NP_005572.2) or a cell adhesion molecule (B-CAM) ) (NCBI Accession No: NP_001013275.1) (Non-Patent Documents 1 and 2), the intracellular domain of Lu consists of 59 residues, whereas B-CAM contains 40 residues of Lu. It is missing.

  CD239 was also discovered as an antigen whose expression is increased in ovarian cancer (Non-patent Document 2). Since then, it has been clarified that it is a specific receptor for laminin α5 chain (Non-patent Document 3) and has been shown to be involved in embolization of sickle cell disease (Non-patent Document 4).

  In addition, CD239 has been reported to be involved in cancer cell motility (Non-Patent Document 5), and it has also been reported that expression is increased in skin cancer and hepatocellular carcinoma (Non-Patent Documents 6 and 7). There is no report about the expression in. Moreover, there was no report related to the proliferation of cancer cells, and it was not expected as a target molecule for drug discovery.

  On the other hand, molecules involved in cancer cell proliferation and cancer immunity have attracted attention as molecular targets for antibody drugs. In Japan, trastuzumab, an anti-HER2 (humanized) monoclonal antibody, was approved for HER2-positive cases as a new option in breast cancer treatment in 2001 (Patent Document 1). Also, trastuzumab emtansine (DM1), which is a tubulin polymerization inhibitor emtansine bound to trastuzumab with a highly stable linker, is an antibody-drug that is indicated for HER2-positive inoperable or recurrent breast cancer It is approved as a conjugate (ADC: antibody-drug conjugate) and is the second-line standard therapy for HER2-positive breast cancer (Patent Document 2). The mechanism of action of this therapeutic agent is that trastuzumab exerts inhibition of HER2 signaling and antibody-dependent cytotoxicity, and also delivers DM1 directly into HER2-positive cancer cells.

  However, HER2-positive cases account for only 20-30% of all malignant breast cancers, and there is a problem that there are cases of treatment resistance. At present, development of chemotherapeutic agents and linkers that bind to existing antibody drugs is underway, while new target antigens and antibodies for cancer suitable for ADC are being sought.

Special table 2002-544238 Special table 2014-502596

Proc. Natl. Acad. Sci. USA, 1995, Vol. 92, pp. 5496-5500 Cancer Res. , 1994, Vol. 54, pp. 5761-5765 J. Biol. Chem. , 2002, Vol. 277, pp. 44864-44869 Transfusion, 2006, Vol. 46, pp. 668-677 J. Biol. Chem. , 2013, Vol. 288, pp. 30990-31001 J. Cutan. Pathol. , 2000, Vol. 27, pp. 108-111 Exp. Cell Res. , 2008, Vol. 314, pp. 2579-2590

  Antibody-drug conjugates (ADCs) are very effective in the treatment of cancer because they can deliver drugs directly to cancer cells, but the antibodies used are limited to those on cell surface antigens, whereas many anticancer drugs are In order to show a damaging action in a cell, an antigen that is internalized in the cell is required. That is, a target antigen suitable for ADC drug discovery is required to have a balance of three properties of “expression level”, “internalization”, and “dynamic behavior”, and the antibody is ideally human.

  One object of the present invention is to search for a new antigen that has the above-mentioned characteristics and can be a target even in HER2-negative breast cancer. Another object of the present invention is to develop a human recombinant antibody against a new target antigen. If such an antibody is obtained, it can be expected as a new treatment option not only for Her2-positive breast cancer having resistance to trastuzumab but also for all cancers including breast cancer.

  The present inventors have clarified that CD239 is strongly expressed in breast cancer tissues that have not been reported so far, in various studies of various protein molecules present on the surface of cancer cells. Antibodies that specifically recognize CD239 bound with high affinity to breast cancer cells expressing CD239, independent of the presence or absence of HER2 expression. Furthermore, as a result of preparing and examining an anti-CD239 antibody to which diphtheria toxin is bound, it was found that this antibody is internalized in cells that highly express CD239 and is effective in suppressing the growth of cancer cells.

That is, the present invention provides the following.
1. An anticancer agent, which is a complex of a polypeptide capable of specifically recognizing human CD239 and a cytotoxic drug.
2. 2. The anticancer agent according to 1 above, wherein the polypeptide is an antibody that specifically binds to human CD239, or an antigen-binding fragment or antigen-binding derivative thereof.
3. The polypeptide is the following (a) to (i):
(A) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1,
VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3,
And VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4,
VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6
An antibody or antigen-binding fragment or antigen-binding derivative thereof,
(B) an antibody heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having 5 amino acid additions, substitutions and deletions to the amino acid sequence;
And an antibody comprising an antibody light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having 5 amino acid additions, substitutions and deletions to the amino acid sequence, or an antigen-binding fragment or antigen thereof Binding derivatives,
(C) an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 18, or a polypeptide consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence,
(D) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 19,
VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 20 and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 21
An antibody or antigen-binding fragment or antigen-binding derivative thereof,
(E) An antibody or antigen-binding fragment thereof comprising an antibody heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22, or an amino acid sequence having 5 amino acid additions, substitutions and deletions with respect to the amino acid sequence Or an antigen-binding derivative,
(F) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 23 or an amino acid sequence having 90% or more sequence identity to the amino acid sequence,
(G) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 24,
VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 25, and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 26,
VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 27,
VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 28, and VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 29,
An antibody or antigen-binding fragment or antigen-binding derivative thereof,
(H) an antibody heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence having 5 amino acid additions, substitutions and deletions with respect to the amino acid sequence;
And an antibody comprising an antibody light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having addition, substitution and deletion of 5 or less amino acids to the amino acid sequence, or an antigen-binding fragment or antigen thereof 3. The anticancer agent according to 2 above, which is selected from a binding derivative and (i) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 32 or an amino acid sequence having 90% or more sequence identity to the amino acid sequence.
4). 4. The anticancer agent according to any one of 1 to 3 above, wherein the drug is diphtheria toxin.
5. 5. The anticancer agent according to any one of 1 to 4 above, which has a growth inhibitory effect on breast cancer.
6). 6. A pharmaceutical composition comprising the anticancer agent according to any one of 1 to 5 above.
7). The following (a) to (i):
(A) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1,
VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3,
And VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4,
VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6
An amino acid sequence comprising
(B) an antibody heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having 5 amino acid additions, substitutions and deletions to the amino acid sequence;
And an amino acid sequence comprising an antibody light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having addition, substitution and deletion of 5 or less amino acids to the amino acid sequence,
(C) an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 18, or an amino acid sequence having 90% or more sequence identity to the amino acid sequence,
(D) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 19,
VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 20 and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 21
An amino acid sequence comprising
(E) an amino acid sequence comprising the antibody heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 22, or an amino acid sequence having additions, substitutions and deletions of 5 or less amino acids to the amino acid sequence;
(F) the amino acid sequence represented by SEQ ID NO: 23, or an amino acid sequence having 90% or more sequence identity to the amino acid sequence,
(G) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 24,
VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 25, and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 26,
VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 27,
VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 28, and VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 29,
An amino acid sequence comprising
(H) an antibody heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence having 5 amino acid additions, substitutions and deletions with respect to the amino acid sequence;
And an amino acid sequence comprising an antibody light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having additions, substitutions and deletions of 5 or less amino acids to the amino acid sequence, and (i) SEQ ID NO: A recombinant protein having an amino acid sequence selected from the amino acid sequence shown by 32 or an amino acid sequence having 90% or more sequence identity to the amino acid sequence.
8). 8. The recombinant protein according to 7 above, further comprising a signal peptide consisting of an amino acid sequence selected from SEQ ID NOs: 33 to 36.
9. A polynucleotide encoding the recombinant protein according to 7 or 8 above.
10. 10. An expression vector comprising the polynucleotide according to 9 above.
11. A host cell comprising the expression vector according to 10 above.
12 9. A method for producing a recombinant protein according to 7 or 8, comprising culturing the host cell according to 11 above and recovering the expressed protein.
13. A marker for detecting a CD239-expressing cancer cell, comprising a polypeptide capable of specifically recognizing human CD239.
14 14. The marker according to 13, wherein the polypeptide is the recombinant protein according to 7 above.
15. A carrier for delivering an agent having cytotoxicity against CD239-expressing cancer cells, comprising a polypeptide capable of specifically recognizing human CD239.
16. 16. The carrier according to 15 above, wherein the polypeptide is the recombinant protein according to 7 above.

  The anticancer agent of the present invention can suppress the growth of cancer cells that highly express CD239. The polypeptide provided in the present invention can be used as a marker for CD239-expressing cancer cells and as a carrier for delivering a drug to cancer cells.

2 shows CD239 and Her2 expression in human breast cancer tissue. (A) Example of fluorescent immunostaining in breast cancer tissue using mouse anti-CD239 monoclonal antibody (87207) and rabbit anti-Her2 monoclonal antibody. In patient number 22, strong expression of Her2 and CD239 was observed. In patient number 8, strong expression of only CD239 was observed. 2 shows CD239 and Her2 expression in human breast cancer tissue. (B) Expression of CD239 and Her2 in human breast cancer tissue that was pathologically diagnosed. The strong expression was classified as ++, the intermediate expression was classified as ++, the weak expression was classified as +, and the case where the expression was not expressed was classified as-. (C) Results of plotting each human breast cancer tissue with the level of CD239 and Her2 expression. The expression of CD239 and Her2 in a human breast cancer cell line (SKBR3 cell) is shown. (A) Flow cytometry was performed using mouse anti-CD239 monoclonal antibody (87207; left panel) and anti-Her2 monoclonal antibody (Herceptin; right panel). A solid line histogram shows each antibody, and a gray histogram shows a control (no antibody). SKBR3 cells strongly expressed both CD239 and Her2. (B) Results of cell proliferation assay with mouse anti-CD239 monoclonal antibody. Mouse anti-CD239 monoclonal antibodies (87202, 87207, BRIC221) conjugated with diphtheria toxin and Herceptin inhibited cell growth of the breast cancer cell line (SKBR3). * <0.01 The isolation of phage antibodies that bind to CD239 is shown. (A) Biopanning of phage antibodies that bind to CD239. Using a phage antibody library derived from the peripheral blood of a human hepatocellular carcinoma patient, three rounds of biopanning were performed on the immobilized solubilized CD239 recombinant protein. Bound phage antibody was detected by ELISA. In the third round, phages that bind to CD239 were enriched. (B) Cloning of anti-CD239 phage antibody. Thirteen types of phage clones were isolated. Wild shows the control which added the original phage which does not have a scFv library, and None shows the control which does not add a phage. The amino acid sequence of scFv in the cloned anti-CD239 phage antibody is shown. When DNA was extracted from each phage clone and the amino acid sequence of scFv was clarified from the base sequence, it was divided into three groups (groups ac). The sequence of group a including clone C7 used for production of scFv-Fc binding to CD239 is shown. As a result of sequence alignment, the amino acid sequences of scFv obtained from the clones of group a were very similar. Different amino acids are boxed. The amino acid sequence of scFv in the cloned anti-CD239 phage antibody is shown. The sequences of groups b and c are shown. All scFvs obtained from the four clones of group b had the same amino acid sequence. The structure of C7-Fc and its expression in cells are shown. (A) Schematic diagram of scFv-Fc. (B) Examination of a signal sequence effective for secretion of C7-Fc. The signal peptide sequences of mouse IgG κ chain (SEQ ID NO: 33), human laminin γ2 chain (SEQ ID NO: 34), human Lu / B-CAM (SEQ ID NO: 35), and human α-dystroglycan (SEQ ID NO: 36) were used. (C) Comparison of secretion efficiency by each signal peptide. The secretion efficiency of C7-Fc fused with the signal peptide of human laminin γ2 chain was superior to other signal peptides. There was no difference between cells. 2 shows the purification of the anti-CD239 recombinant antibody (C7-Fc) and the binding affinity with the CD239 protein. (A) SDS-PAGE of purified anti-CD239 recombinant antibody (C7-FC). C7-FC purified by protein A sepharose from the culture supernatant was separated by electrophoresis under 10% gel, reducing conditions. Fc is a control recombinant protein. (B) ELISA of purified anti-CD239 recombinant antibody (C7-FC). It shows that C7-Fc binds to solubilized CD239 recombinant protein. (C) C7-Fc sensorgram. (D) Affinity analysis of C7-Fc, 87202, BRIC221 for solubilized CD239 recombinant protein. The binding of anti-CD239 recombinant antibody (C7-Fc) to CD239 on the cell surface is shown. Using CD239 forced expression cells (CD239 (Lu) / HT1080F) and control cells (control / HT1080F), the binding activity of C239-Fc and CD239 expressed on the cell surface was evaluated by flow cytometry. C7-Fc specifically recognized cell surface CD239. The cell growth inhibitory activity with respect to the breast cancer cell line which highly expresses CD239 of the anti-CD239 recombinant antibody (C7-Fc + DT) couple | bonded with the diphtheria toxin is shown. (A) Expression of CD239 in breast cancer cell lines (SKBR3, BT474). Analysis was performed by flow cytometry using C7-Fc and Fc. SKBR3 cells and BT474 cells expressed CD239 strongly. (B) Cell growth inhibitory activity of C7-Fc conjugated with diphtheria toxin. Both SKBR3 cells and BT474 cells were inhibited from cell growth, but SKBR3 with strong CD239 expression was more effectively inhibited. * <0.01 The cell growth inhibitory activity of the anti-CD239 recombinant antibody (C7-Fc + DT) which combined diphtheria toxin with respect to the breast cancer cell line which forcedly expressed CD239 is shown. (A) Expression of CD239 in MCF7 cells and CD239 (Lu) / MCF7 cells in which CD239 is forcibly expressed. Analysis was performed by flow cytometry using C7-Fc and Fc. CD239 expression in MCF7 cells is lower than that in SKBR3 cells and BT474 cells, etc., but it was confirmed that CD239 (Lu) / MCF7 cells that were forced to express expressed at the same level as SKBR3 cells and BT474 cells. It was. (B) Cell growth inhibitory activity of C7-Fc conjugated with diphtheria toxin. Although growth suppression of MCF7 cells was not observed, CD239 (Lu) / MCF7 cells were inhibited by C7-Fc and diphtheria toxin combination. This result indicates that C7-Fc to which diphtheria toxin is bound specifically acts on cells that highly express CD239 in terms of cell growth inhibitory action. * <0.01 2 shows the cell growth inhibitory activity of C7-Fc conjugated with diphtheria toxin in vitro and in vivo. (A) In vitro cytotoxic activity of C7-Fc combined with diphtheria toxin against CD239 forced expression cells (CD239 (Lu) / HT1080F) and control cells (control / HT1080F) transplanted into nude mice. Injury activity was shown against CD239 forced expression cells (CD239 (Lu) / HT1080F). * <0.01 (b) Administration of C7-Fc conjugated with diphtheria toxin to tumor-bearing mice. CD239 forced expression cells (CD239 (Lu) / HT1080F) and control cells (control / HT1080F) were transplanted into nude mice to prepare tumor-bearing mice. IT was administered to the tumor mass. There was no difference in the size of the tumor mass before administration, but a significant difference was observed on the 7th day after administration. * <0.01

Hereinafter, the present invention will be described in more detail.
The present inventors have found that CD239 is strongly expressed in human breast cancer tissue and breast cancer cell line SKBR3 as shown in detail in the following Examples (FIGS. 1-1, 1-2, 2a).

  A mouse anti-CD239 monoclonal antibody was examined for its effect on CD239-expressing breast cancer cells. The antibody alone has no cytotoxic activity, but the mouse anti-CD239 monoclonal antibody bound with diphtheria toxin lacking the cell membrane binding domain is intracellular. It was found that diphtheria toxin showed an effect and was effective as a cancer cell growth inhibitor (FIG. 2b).

Next, the present inventors have used human single-chain Fv expressing genes encoding antibody heavy chain variable region (VH chain or VH) and antibody light chain variable region (VL chain or VL) from the peripheral blood of hepatocellular carcinoma patients. A phage antibody that binds to human CD239 was cloned from the phage display library (FIGS. 3a and b), and the nucleotide sequence and amino acid sequence of the gene encoding the anti-human CD239scFv antibody molecule (antibody fragment) were clarified (FIG. 4). -1 and 4-2). Furthermore, a recombinant antibody (C7-Fc) was prepared by fusing this scFv antibody and human IgG ₁ Fc (FIGS. 5, 6, and 7).

  Similar to the findings obtained with the mouse monoclonal antibody, the recombinant antibody C7-Fc alone did not show cell growth inhibitory activity, but C7-Fc bound to diphtheria toxin was internalized in the cell and diphtheria toxin was Was found to be effective as an anticancer agent (FIG. 8). This effect was not shown for cells with weak CD239 expression, but was found to show cell growth inhibitory activity only for cells with high expression (FIG. 9). C7-Fc conjugated with diphtheria toxin showed cell growth inhibitory activity not only in vitro but also in vivo (FIG. 10).

  In one embodiment, the present invention provides an anticancer agent characterized in that it is a complex of a polypeptide capable of specifically recognizing human CD239 (ie, luteran blood group antigen) and a cytotoxic drug. .

More specifically, examples of the polypeptide capable of specifically recognizing human CD239 include antibodies capable of specifically binding to human CD239, particularly IgG antibodies, or antigen-binding fragments and antigen-binding derivatives thereof. . For example, a polypeptide is a complete antibody that specifically binds to human CD239, Fab, Fab ′, F (ab ′) ₂ fragment, or a heavy chain variable region (VH) and a light chain variable region (VL) linked by a linker. Single-chain antibody fragments (scFv fragments) and the like. By using a single chain, for example, compared to the case of a complete antibody or the like, the operation for producing a recombinant antibody can be saved, and the production efficiency with a phage or the like can be increased. Although scFv is widely recognized as an antibody fragment in this field, strictly speaking, since it is a synthetic polypeptide in which variable regions are linked by a linker, it is considered appropriate to describe it as an antigen-binding derivative. Polypeptides that can be used in the present invention include these fragments and derivatives, and derivatives that can be understood by those skilled in the art within a range that does not affect antigen binding properties, such as modifications to facilitate antibody purification and enhance stability. Also included are the applied derivatives. In the present specification, the antigen-binding fragment and the antigen-binding derivative are sometimes referred to as “antibody” for convenience.

  Further, for example, a polypeptide that can specifically recognize and bind to human CD239 can be obtained by screening using a peptide library. Furthermore, for example, a polypeptide of laminin containing laminin α5 chain or a fragment thereof can also be suitably used as the polypeptide of the present invention.

  The binding between the polypeptide and human CD239 can be confirmed, for example, in vitro using an enzyme-linked immunosorbent assay (ELISA), a BIAcore binding assay, or the like. Alternatively, the presence of CD239 in the biological sample can be confirmed using a polypeptide labeled with fluorescence or the like.

When scFv fragments are used, the linker may be any linker that is commonly used in the art, and is not particularly limited. For example, 5 to 25, preferably 10 to 20 amino acid residues. (GGGS) _n , (GGGGS) _n (n represents an integer of 1 to 4), or a GS linker consisting of the amino acid sequence represented by SEQ ID NO: 14 can be suitably used. . In addition, in order to form a complex with a drug, for example, an Fc region of an antibody heavy chain constant region can be linked to the above polypeptide. By binding to Fc, stabilization in blood and extension of half-life, induction of antibody-dependent cellular cytotoxicity (ADCC) can also be expected.

  The antibody is preferably a humanized antibody or a human antibody, and particularly preferably a human antibody in consideration of use as an anticancer agent or a marker or carrier for humans. Therefore, a polypeptide that can be preferably used in the present invention is an antibody comprising a framework region (FR) of a human antibody and a complementarity determining region (CDR) that can provide high binding affinity for CD239 ( Or an antigen-binding fragment or antigen-binding derivative thereof).

In the present specification, the above-mentioned “high binding affinity” is not particularly limited, but is 10 ⁻⁸ M or less, preferably 10 ⁻⁹ M or less, more preferably 10 ^{− in} binding to CD239. It can have a KD value of ¹⁰ M or less. Therefore, a polypeptide capable of specifically recognizing human CD239 is characterized by having the above binding affinity for human CD239.

As will be demonstrated in the following examples, the present inventors have identified a polypeptide capable of specifically recognizing human CD239 as a more specific example of the present invention.
VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1,
VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3,
And VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4,
VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6
It has been found that it can include. Antibodies having these CDR combinations correspond to the antibody group described as group a in the following Examples.

The antibody group included in the above group a has heavy chain and light chain all having the same CDR sequence, and the FR1 to FR4 framework region sequences are very similar and have similar functions. obtain. Therefore, polypeptides that can be used in the present invention are:
An antibody heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having 5 amino acid additions, substitutions and deletions to the amino acid sequence;
And an antibody light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having addition, substitution and deletion of 5 amino acids or less to the amino acid sequence.

As another example of the present invention, a polypeptide capable of specifically recognizing human CD239 is:
VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 19,
VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 20 and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 21
May be included. Antibodies having these CDR combinations correspond to the antibody group described as group b in the following Examples.

In the antibody group included in the group b, all heavy chains have the same CDR sequence, the framework region sequences are very similar, and can have the same function. Therefore, polypeptides that can be used in the present invention are:
It may contain an antibody heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence having 5 amino acid additions, substitutions and deletions with respect to the amino acid sequence. Group b polypeptides contain little light chain-derived sequence, but those skilled in the art will understand that they can bind antigen even in the absence of light chain (Trends in Biotechnology, 21 484-490 (2003)).

As yet another example of the present invention, a polypeptide capable of specifically recognizing human CD239 is:
VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 24,
VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 25, and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 26,
VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 27,
VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 28, and VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 29,
May be included. Antibodies having these CDR combinations correspond to the antibody group described as group c in the following Examples.

The antibody group included in the above-mentioned group c has heavy chain and light chain that all have the same CDR sequence, and the FR1 to FR4 framework region sequences are very similar and have similar functions. obtain. Therefore, polypeptides that can be used in the present invention are:
An antibody heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence having 5 amino acid additions, substitutions and deletions to the amino acid sequence;
And an antibody light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 31 or an amino acid sequence having addition, substitution and deletion of 5 or less amino acids to the amino acid sequence.

  When the polypeptide having the above sequence characteristic is an scFv fragment linked by a linker, for example, the polypeptide capable of specifically recognizing human CD239 is selected from the group consisting of SEQ ID NOs: 15-18, 23 and 32. It may consist of an amino acid sequence selected or an amino acid sequence having 90% or more sequence identity to the amino acid sequence.

  As will be understood by those skilled in the art, the polypeptide that can be used in the present invention may be any polypeptide that can specifically recognize CD239, and the position of the epitope recognized by CD239 is not particularly limited. For example, the antibody groups shown in groups a to c above can recognize different epitopes of CD239. Similarly, in the following examples, mouse antibodies that have been confirmed to specifically bind to CD239 and commercially available anti-CD239 antibodies can also recognize different epitopes, but specifically bind to CD239 to form a complex. As long as it is internalized in cancer cells together with the prepared drug, it can be suitably used in the present invention.

  Therefore, in the present invention, a commercially available anti-CD239 antibody or the like may be used as the polypeptide, or a polypeptide having the desired binding specificity can be synthesized based on the above sequence information. Furthermore, a polynucleotide encoding these polypeptides can be introduced into a suitable host cell and expressed to obtain a recombinant protein. In this case, the polynucleotide may be DNA or RNA, and the means for introduction into the host cell may be appropriately selected from those used in the art. As a vector for introducing a polynucleotide into a host cell, a virus vector, a plasmid vector, a phage vector, or the like can be appropriately used. Examples of host cells that can be used include bacteria such as E. coli, yeast, insect cells, animal cells, and the like.

  For example, as described in the following examples, a DNA encoding a polypeptide used in the present invention (C7-Fc in the examples) is mixed with a signal sequence, a poly A sequence, a regulatory sequence such as a promoter sequence, and a selection marker. A polypeptide capable of specifically recognizing human CD239 can be obtained as a recombinant protein by incorporating it into a vector containing it, introducing it into an appropriate host cell and culturing.

  In an embodiment in which a polypeptide capable of specifically recognizing human CD239 is obtained as a recombinant protein, a preferred polypeptide is the polypeptide specified by the above SEQ ID NO. That is, the present invention also provides a recombinant protein having the above amino acid sequence that can specifically recognize human CD239.

The recombinant protein of the present invention includes, for example, the following (a) to (i):
(A) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1,
VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3,
And VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4,
VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6
An amino acid sequence comprising
(B) an antibody heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having 5 amino acid additions, substitutions and deletions to the amino acid sequence;
And an amino acid sequence comprising an antibody light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having addition, substitution and deletion of 5 or less amino acids to the amino acid sequence,
(C) an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 18, or an amino acid sequence having 90% or more sequence identity to the amino acid sequence,
(D) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 19,
VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 20 and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 21
An amino acid sequence comprising
(E) an amino acid sequence comprising the antibody heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 22, or an amino acid sequence having additions, substitutions and deletions of 5 or less amino acids to the amino acid sequence;
(F) the amino acid sequence represented by SEQ ID NO: 23, or an amino acid sequence having 90% or more sequence identity to the amino acid sequence,
(G) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 24,
VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 25, and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 26,
VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 27,
VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 28, and VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 29,
An amino acid sequence comprising
(H) an antibody heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence having 5 amino acid additions, substitutions and deletions with respect to the amino acid sequence;
And an amino acid sequence comprising an antibody light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having additions, substitutions and deletions of 5 or less amino acids to the amino acid sequence, and (i) SEQ ID NO: 32, or an amino acid sequence selected from amino acid sequences having 90% or more sequence identity to the amino acid sequence.

  The recombinant protein of the present invention can be further expressed by linking with a signal sequence derived from IgGκ chain, laminin γ2 chain, Lu / B-CAM, α-dystroglycan and the like. Preferred signal sequences include, but are not limited to, mouse IgG kappa chain (SEQ ID NO: 33), human laminin γ2 chain (SEQ ID NO: 34), human Lu / B-CAM (SEQ ID NO: 35), human α-dystroglycan (sequence No. 36), more preferably human laminin γ2 chain (SEQ ID NO: 34).

  In order to obtain the above-described polypeptide as a recombinant protein, the present invention also provides a polynucleotide encoding the recombinant protein disclosed in the present invention.

  The present invention further provides an expression vector containing the above-mentioned polynucleotide and a host cell containing the vector.

  The present invention further provides a method for producing the recombinant protein of the present invention, which comprises culturing the above host cell and recovering the expressed protein.

Drugs that bind to polypeptides that can specifically recognize human CD239 to form complexes include proteins such as toxins that can be cytotoxic to cells, especially cancer cells, antimitotic drugs, alkylation Agents, antibiotics, antimetabolites, apoptotic agents, alkaloid agents, taxoid agents, radioisotopes and the like. Examples of the toxin include diphtheria toxin, saporin toxin, Pseudomonas aeruginosa toxin, abrin toxin, ricin A toxin, tumor necrosis factor, and interferon-γ. The drug is selected from anti-mitotic agents, alkylating agents, antibiotics, antimetabolites, apoptotic agents, alkaloid agents, taxoid agents, and combinations thereof. Examples of the radioisotope include ⁹⁰ Y, ¹³¹ I and the like. A drug that can be particularly preferably used in the present invention is diphtheria toxin.

  The method for linking the polypeptide and the drug is not limited, but it is preferable that the polypeptide is bound by, for example, a covalent bond. For example, the drug may be added to the polypeptide via a linker. The linker may be any substance that can form a connecting part between a drug and a polypeptide, such as a peptide, a fatty acid, and a fatty acid ester. The linker may be inserted between the drug and the polypeptide as a spacer for not inhibiting the three-dimensional structure of the polypeptide or as a cleavable linking moiety containing a protease recognition site.

  For example, when diphtheria toxin is used as a drug, in order to exert toxicity only to the target cell, for example, the cell binding domain of natural diphtheria toxin is deleted, and instead, an IgG binding molecule such as Protein G To form a complex with the polypeptide having the above Fc region (for example, Biochem. Biophys. Res. Commun., 2014, Vol. 454, pp , See 600-603).

  The anticancer agent of the present invention can act specifically on cancer cells expressing CD239. Examples of cancer cells include breast cancer, skin cancer, liver cancer, colon cancer, ovarian cancer and the like. The anticancer agent of the present invention has a growth inhibitory effect particularly on breast cancer.

  Although the anticancer agent of the present invention can be used alone, it can be used in combination with anticancer agents and anticancer treatments having different mechanisms.

  The present invention also provides a pharmaceutical composition containing the anticancer agent of the present invention described above alone or in combination with other active ingredients.

  The anticancer agent or pharmaceutical composition of the present invention can be administered locally or systemically, and the dosage form is not limited. For example, in the case of breast cancer treatment, it is administered by injection or infusion to the affected area or the vicinity of the affected area. It is preferable. In addition to the anticancer agent of the present invention and other active ingredients, the pharmaceutical composition can contain carriers, excipients, buffers, stabilizers and the like that are commonly used in the art depending on the dosage form. . The dose of the anticancer agent of the present invention varies depending on the patient's body weight, age, severity of disease, etc., and is not particularly limited. For example, the dose is 0.001 to 10 mg / kg body weight per day. It can be administered once to several times, every 2 days, every 3 days, every week, every 2 weeks, every month, every 2 months, and every 3 months.

In another embodiment, the present invention further provides a marker for detecting a CD239-expressing cancer cell comprising a polypeptide capable of specifically recognizing human CD239. The marker of the present invention can be used alone or in combination with other markers, for example, for detection of breast cancer that highly expresses CD239. For example, the markers of the present invention can detect the presence of CD239 in biological samples such as blood and biopsy samples. Alternatively, the marker of the present invention can be injected into the body of a subject suspected of having a cancer that expresses CD239 to detect the presence of cells that express CD239. When used as a marker, the binding between CD239 and the polypeptide can be detected by means commonly used in the art. The polypeptide can also be labeled with a radioactive substance such as Cu ⁶⁴ , In ¹¹¹ , and I ¹³¹ .

  The present invention further provides, in another embodiment, a carrier for delivering an agent that is cytotoxic to CD239-expressing cancer cells, comprising a polypeptide that can specifically recognize human CD239. The carrier of the present invention can be an anti-CD239 antibody internalized with CD239 as an antigen. Such carriers can be a platform for antibody-drug conjugates.

  Also in these embodiments, the polypeptide capable of specifically recognizing human CD239 is, for example, as described above, an antibody capable of specifically binding to human CD239, particularly an IgG antibody, or an antigen-binding fragment thereof and antigen binding. And may be a recombinant protein as described above.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[Example 1: Expression of CD239 and Her2 in breast cancer patient tissue]
Mouse anti-CD239 monoclonal antibody (87207, R & D Systems, Minneapolis, MN) and rabbit anti-Her2 monoclonal antibody (29D8, Cell Signaling Technology, Danvers, MA) against breast cancer tissue (BioChain, Newark, CA, patient numbers 1-34) ) Was used for fluorescent immunostaining.

  First, the tissue sections were blocked with 1% BSA / PBS (-) containing 10% goat serum for 10 minutes. A solution of mouse anti-CD239 monoclonal antibody (10 μg / ml) and rabbit anti-Her2 monoclonal antibody (diluted 400 times) was added to the blocked sections, and incubated at room temperature for 1 hour. After 1 hour, the solutions were removed, anti-mouse IgG antibody labeled with Alexa Fluor 488 and anti-rabbit IgG antibody labeled with Alexa Fluor 594 were added and further incubated for 1 hour. (Keyence, Osaka, Japan) was observed (Fig. 1-1 (a)).

  FIG. 1-2 (b) shows the results of classifying strong expression as ++, intermediate expression as ++, weak expression as +, and non-expressing as − for each tissue. In addition, the expression of CD239 and Her2 in each tissue were compared (FIG. 1-2 (c)). As a result, it was revealed that breast cancer tissue that strongly expresses Her2 also has strong CD239 expression. On the other hand, some breast cancer tissues that strongly express CD239 were not accompanied by Her2 expression.

  This indicates that CD239 may be a new therapeutic target molecule in Her2-negative breast cancer. It has also been shown that it can be a new marker for CD239-expressing cancer cells.

[Example 2: Expression of CD239 and Her2 in breast cancer cell lines]
The expression of CD239 and Her2 in the breast cancer cell line SKBR3 (purchased from ATCC) was analyzed using a flow cytometer (FIG. 2 (a)). SKBR3 cells were detached with a cell dissociation buffer (Life Technologies, Carlsbad, Calif.) And suspended in 0.1% BSA / 1 mM EDTA / PBS (−) to 1 × 10 ⁷ cells / ml. The solution was dispensed to 100 μl / tube, mouse anti-CD239 monoclonal antibody (87207) and trastuzumab (Herceptin: Chugai, Tokyo, Japan) were added to 10 μg / ml and reacted on ice for 1 hour. Next, the cells were washed with 0.1% BSA / 1 mM EDTA / PBS (−), and reacted with anti-mouse IgG antibody and anti-human IgG antibody labeled with Alexa488 for 1 hour, respectively. After further washing, analysis was performed using a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA).

  As a result, it was clarified that the expression of CD239 was very strong in SKBR3 cells as well as Her2.

[Example 3: Analysis of breast cancer cell growth inhibitory activity by mouse anti-CD239 monoclonal antibody conjugated with diphtheria toxin]
As the diphtheria toxin (DT3C) to be bound to the mouse anti-CD239 monoclonal antibody, one containing a transmembrane domain, a toxin domain, and an IgG binding region derived from protein G (Biochem. Biophys. Res. Commun., 2014, Vol. 454) was used. Pp. 600-603).

  SKBR3 cells were used for the cell proliferation assay. For cell culture, MacCoy'5A medium supplemented with low IgG fetal calf serum (Life Technologies, Carlsbad, CA) was used. Cells are seeded in 96-well plates, and after 1 day of culture, mouse anti-CD239 monoclonal antibody (87202, 87207: R & D Systems, Minneapolis, MN; BRIC221: Serotec, Oxford, UK) or equimolar to a final concentration of 15 nM An antibody conjugated with diphtheria toxin (DT3C) was added. The cells were cultured for 4 days, and the number of viable and proliferating cells was quantified by MTT (Dojin, Kumamoto, Japan). The produced formazan was dissolved and the absorbance at 570 nm was measured.

  As a result, the single mouse anti-CD239 monoclonal antibody did not affect the proliferation of SKBR3 cells (FIG. 2 (b)), but when diphtheria toxin (DT3C) was bound to the mouse anti-CD239 monoclonal antibody, cell proliferation was increased. Inhibited. This indicates that internalization occurs in which CD239 on the cell surface is taken up into the cell.

[Example 4: Construction of phage antibody library from peripheral blood of hepatocellular carcinoma patient and screening of anti-CD239 phage antibody]
The construction of the phage library is described in J. Org. D. With reference to the method reported by Marks et al. (J. Mol. Biol., 1991, Vol. 222, pp. 581-597), the peripheral blood of four hepatocellular carcinoma patients was constructed as a starting material. The library had a diversity of 1.85 × 10 ⁸ clones.

  The phages that bind to CD239 were concentrated by biopanning (FIG. 3 (a)). In an ELISA plate (Maxisorp, ThermoFisher Scientific, Waltham, MA), a solubilized CD239 recombinant protein (J. Biol. Chem., 2002, Vol. 277, pp. 44864-44869) at a concentration of 0.5 μg / ml, Immobilization was performed overnight at 4 ° C. The blocking buffer was 0.5% BSA / PBS (-), and blocking was performed for 2 hours at room temperature. After blocking, washing was performed only once with a blocking buffer, and thereafter with a washing buffer (0.05% Tween20, PBS (−)).

Phages containing the scFv library (1.0 × 10 ¹⁰ (cfu)) were suspended in 0.5% BSA / PBS (−), added to wells immobilized with BSA, and nonspecific adsorption was performed twice for 30 minutes. Thereafter, the solubilized CD239 recombinant protein was transferred to a well in which it was immobilized, and reacted at room temperature for 1 hour. The bound phage was washed 5 times with a washing buffer, eluted with glycine-HCl (pH 2.0), and neutralized with Tris-HCl (pH 9.1). The eluted phage is E. coli. The cells were infected with E. coli TG-1, seeded on 2TYAG plates (2TY medium, 1.5% agar, 2% glucose, 100 μg / ml ampicillin), and cultured at 30 ° C. overnight. After culturing, the cells were further grown in 2TY medium, M13KO7 helper phage was added at MOI = 5, and the mixture was allowed to stand at 37 ° C. for 30 minutes for infection. After centrifugation at 1000 × g for 10 minutes, the pellet was suspended in 2TYAK (2TY medium, 100 μg / ml ampicillin, 50 μg / ml kanamycin) and cultured overnight at 37 ° C. After culturing, phages were prepared by mixing 0.2 volumes of 2.5 M NaCl / 20% PEG 6000 with the supernatant collected by centrifugation at 1000 × g for 10 minutes, and used for the next round.

  In biopanning using solubilized CD239 recombinant protein, three rounds of selection were performed. As the blocking buffer, 0.5% BSA / PBS (−) was used in the first and second rounds, and 5% skim milk / PBS (−) was used in the third round. The solubilized CD239 recombinant protein was immobilized on an ELISA plate at a concentration of 0.5 μg / ml in the first round and at a concentration of 0.25 μg / ml in the second and third rounds. Washing after the phage reaction to the target antigen was increased by 5 times per round. In the third round, IgA was used as an absorbed antigen.

  The phages concentrated by biopanning were infected with E. coli to form colonies. The formed colonies were randomly picked and cultured overnight at 37 ° C. in 2TYAG (2TY medium, 2% glucose, 100 μg / ml ampicillin). A part of the culture was mixed with 2TYAG and cultured at 37 ° C. for 1.5 hours, and M13KO7 helper phage was added at MOI = 5 to infect for 30 minutes at 37 ° C. After centrifugation at 1000 × g for 10 minutes, the pellet was suspended in 2TYAK (2TY medium, 100 μg / ml ampicillin, 50 μg / ml kanamycin) and grown overnight at 37 ° C. After centrifugation at 2150 × g for 10 minutes at 4 ° C., the supernatant was collected and mixed with 0.2 volumes of 2.5 M NaCl / 20% PEG 6000 to isolate clonal phage.

The isolated clonal phage was subjected to ELISA to confirm its specificity for CD239 (FIG. 3 (b)). The solubilized CD239 recombinant protein was immobilized on an ELISA plate at 4 ° C. overnight or at room temperature for 2 hours. Blocking was performed with 0.5% BSA / PBS (-) for 2 hours at room temperature. After blocking, it was washed only once with a blocking buffer and then with a washing buffer. Each phage was added to each well at about 1 × 10 ⁹ (cfu) and allowed to react for 1 hour at room temperature. To detect bound phage, a complex of Anti-Filamentous Phages M13 fd F1 antibody [B62-FE2] (biotin) (Abcam, Cambridge, UK) and peroxidase-labeled streptavidin (VECTOR, Burlingame, CA) was added to 0.5% BSA. A solution diluted with / PBS (-) was used. The diluted complex was reacted with the phage for 1 hour at room temperature, developed with TMB solution (CALBIOCHEM, San Diego, CA) for 30 minutes, and stopped with 1N HCl. Absorbance at 450 nm was measured with a microplate reader (BIO-RAD, Hercules, CA).

  The scFv genes of 14 types of isolated phage clones were determined using BigDye Terminator Cycle Sequencing Kit (Life Technologies, Carlsbad, CA) (FIGS. 4-1 and 4-2). As a result of determining the amino acid sequence from the analyzed base sequence and aligning with VH and VL, it was revealed that the amino acid sequence was roughly divided into three groups (groups a to c). Table 1 below shows the scFv contained in each phage clone and the amino acid sequence of its partial region, arranged by SEQ ID NO.

  As shown in Table 1, multiple types of scFv having the same CDR sequence could be obtained from different clones. This may indicate that the clones from which they are derived are similar or that mutations were introduced at the stage of library construction, but only sequences that can bind to CD239 with high specificity were selected. Is.

  The sequence of the entire variable region of scFv obtained from different clones was also very similar. In group a, three amino acid sequences (SEQ ID NOs: 7 to 9) were found in the heavy chain variable region (VH), and four amino acid sequences (SEQ ID NOs: 10 to 13) were found in the light chain variable region (VL). However, there were only 2 or 3 amino acid differences in each framework region.

[Example 5: Expression and purification of anti-CD239scFv fused with human IgG ₁ Fc]
DNA of anti-CD239 phage antibody was extracted according to a conventional method, and DNA encoding scFv (clone C7) as a template was amplified by PCR. Anti-CD239scFv-Fc (C7-Fc, FIG. 5 (a)) takes the form of an antibody that does not exist in the living body, and there is a possibility that it is not secreted efficiently from cultured cells. Therefore, in order to efficiently secrete C7-Fc from cultured cells, a signal peptide was selected. For the signal peptide, the sequences of mouse IgG kappa chain (SEQ ID NO: 33), human laminin γ2 chain (SEQ ID NO: 34), human Lu / B-CAM (SEQ ID NO: 35), human α-dystroglycan (SEQ ID NO: 36) were used. (FIG. 5 (b)). In order to fuse each signal peptide to scFv, a primer spanning two DNA fragments was designed, and PCR was performed again using each signal peptide and scFv DNA as a template. At that time, an appropriate restriction enzyme site should be provided so that the expression vector (J. Biol. Chem., 2007, Vol. 282, pp. 14853-14860) incorporating a sequence encoding human IgG ₁ Fc is matched with the frame. Designed at both ends of the DNA fragment, a zeocin resistance gene was also incorporated for selection.

A plasmid expressing anti-CD239scFv (C7-Fc) fused with human IgG ₁ Fc was introduced into HEK293 cells or CHO cells using Lipofectamin 2000 (Life Technologies, Carlsbad, Calif.). Comparison of expression by ELISA revealed that the signal peptide of human laminin γ2 chain effectively secreted C7-Fc in all cells (FIG. 5 (c)).

  HEK293 cells expressing C7-Fc were selected by Zeocin (Life Technologies, Carlsbad, CA) and used for mass culture. In a state where cells expressing C7-Fc were confluent, serum-free culture was performed for 5 days to prepare a culture supernatant containing C7-Fc. The culture supernatant was subjected to centrifugation at 1000 rpm for 5 minutes to remove cells, and further centrifuged at 10,000 rpm for 10 minutes to remove cell debris and the like. Next, the culture supernatant containing C7-Fc was added with ammonium sulfate to 80% and concentrated by salting out. The concentrated culture supernatant was dialyzed against PBS (−). After dialysis, centrifugation was performed at 10,000 rpm for 10 minutes to remove insoluble components. Next, the supernatant containing C7-Fc was added to a column packed with protein A-Sepharose (GE Healthcare, Piscataway, NJ) and allowed to bind for 1 hour while rotating at 4 ° C. The bound C7-Fc was washed three times with 10 column volumes of PBS (−), eluted with glycine-HCl (pH 2.5), and neutralized with Tris-HCl (pH 8.8). Each fraction was dialyzed against PBS (-) and the concentration was measured by protein quantification. Furthermore, the purity of the purified C7-Fc was confirmed by SDS-PAGE (FIG. 6 (a)).

[Example 6: Binding of purified anti-CD239 recombinant antibody (C7-Fc) to antigen]
Soluble CD239 recombinant protein was immobilized on an ELISA plate at 4 ° C. overnight. After blocking with 1% BSA / PBS (-), add anti-CD239 recombinant antibody (C7-Fc) diluted with washing buffer (0.05% Tween20, PBS (-)) and let stand at room temperature for 1 hour did. After washing with a washing buffer, biotin-labeled anti-human IgG Fc antibody (Jackson ImmunoResearch, West Grove, PA) was added and left at room temperature for 1 hour. Further, after washing with a washing buffer, peroxidase-labeled streptavidin was added and left at room temperature for 1 hour. After washing with washing buffer, o-phenylenediamine dihydrochloride (SIGMA, St Louis, MO) is dissolved in 50 mM phosphate-citrate buffer to a concentration of 0.4 mg / ml, and H ₂ O ₂ is added immediately before use. The color was added to 0.012%, and color was allowed to develop for 30 minutes at room temperature. Absorbance at 450 nm was measured using Multiskan Go (Thermo Fisher Scientific, Vantaa, Finland).

  As a result, it was revealed that the anti-CD239 recombinant antibody (C7-Fc) reacts with the soluble CD239 recombinant protein (FIG. 6 (b)).

  The binding dissociation constant between the anti-CD239 recombinant antibody (C7-Fc) and the soluble CD239 recombinant protein was measured by Biacore T100 system (GE Healthcare) (FIG. 6 (c)). An HBS-EP buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Tween 20) was used as a running buffer at 25 ° C. and a flow rate of 50 μl / min. A soluble CD239 recombinant protein was immobilized on a sensor chip CM5 (GE Healthcare) by amino coupling so as to be 100 response units (RU). The anti-CD239 recombinant antibody (C7-Fc) was diluted at different concentrations using HBS-EP buffer, reacted at a low concentration for 2 minutes, and measured. The regeneration of the sensor chip was performed with glycine-HCl (pH 3.0) for 5 seconds, and then with glycine-HCl (pH 2.2) for 5 seconds. BIA evaluation was used for data analysis.

As a result, the binding dissociation constant with the soluble CD239 recombinant protein was 2.5 × 10 ⁻⁹ M, which was found to be equivalent to the mouse anti-lutelan monoclonal antibody, which is a full-length antibody (FIG. 6 ( d)).

[Example 7: Analysis of binding of anti-CD239 recombinant antibody (C7-Fc) to CD239 on cells by flow cytometer]
The binding activity of the purified anti-CD239 recombinant antibody (C7-Fc) to CD239 on the cell surface was analyzed by a flow cytometer. As the cells, human fibrosarcoma-derived HT1080 cells (purchased from Human Science Research Resource Bank (HSRRB, Osaka, Japan)) that did not express CD239 were used. DNA encoding the Lu form of CD239 (NCBI Accession No: NP — 005572.2) was introduced into HT1080 cells using the Flp-In system (Life Technologies, Carlsbad, Calif.) (J. Biol. Chem., 2013, Vol. 288, pp. 30990-31001). Control cells were designated as control / HT1080F cells, and cells expressing CD239 were designated as CD239 (Lu) / HT1080F cells. The cells were detached with a cell dissociation buffer (Life Technologies, Carlsbad, Calif.) And suspended in 0.1% BSA / 1 mM EDTA / PBS (−) so as to be 1 × 10 ⁷ cells / ml. The solution was dispensed to 100 μl / tube, purified anti-CD239 recombinant antibody (C7-Fc) and control recombinant protein (Fc) were added to 10 μg / ml, and the mixture was allowed to react on ice for 1 hour. Next, the cells were washed with 0.1% BSA / 1 mM EDTA / PBS (−), and reacted with an anti-human IgG antibody labeled with Alexa488 for 1 hour. After further washing, analysis was performed using a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA).

  As a result, as shown in FIG. 7, the anti-CD239 recombinant antibody (C7-Fc) strongly binds to CD239 / HT1080F cells expressing CD239 and does not bind to control / HT1080F as control cells. It was clarified that it specifically binds to CD239 expressed in E. coli.

[Example 8: Growth inhibitory activity of diphtheria toxin-conjugated anti-CD239 recombinant antibody (C7-Fc + DT) against a breast cancer cell line that highly expresses CD239]
The expression of CD239 in breast cancer-derived SKBR3 cells and BT474 cells (ATCC, Manassas, VA) was analyzed using a flow cytometer. Cells were prepared as described in Example 7, using anti-CD239 recombinant antibody (C7-Fc) and control recombinant protein (Fc).

  As a result, it was clarified that the expression of CD239 in BT474 cells was similarly strong with a slightly weaker level than that of SKBR3 cells (FIG. 8 (a)).

  In the proliferation assay of SKBR3 cells, as described in Example 3, MacCoy'5A medium supplemented with low IgG fetal calf serum (Life Technologies, Carlsbad, CA) was used. In addition, the proliferation assay of BT474 cells was performed by adding low IgG fetal calf serum to DMEM / F12 medium, and the others were performed in the same manner as described above.

  As a result, the single anti-CD239 recombinant antibody (C7-Fc) did not affect the proliferation of either SKBR3 cells or BT474 cells, but it inhibited proliferation when diphtheria toxin (DT3C) was bound. (FIG. 8 (b)). This indicates that internalization takes place in the cell after the anti-CD239 recombinant antibody binds to CD239 on the cell surface.

[Example 9: Cell growth inhibitory activity of diphtheria toxin-conjugated anti-CD239 recombinant antibody (C7-Fc + DT) against a breast cancer cell line in which CD239 is forcibly expressed]
The expression of CD239 in breast cancer-derived MCF7 cells (purchased from Human Science Research Resource Bank (HSRRB, Osaka, Japan)) was analyzed with a flow cytometer. Cells were prepared as described in Example 7, using anti-CD239 recombinant antibody (C7-Fc) and control recombinant protein (Fc). CD239 expression in MCF7 was found to be weaker than SKBR3 and BT474 (FIG. 9 (a)).

  MCF7 cell proliferation assays were performed using DMEM medium plus low IgG fetal calf serum, and the others were performed as described above. As a result, inhibition of cell proliferation was not observed with the anti-CD239 recombinant antibody (C7-Fc + DT) conjugated with diphtheria toxin (DT3C) (FIG. 9 (b)). This result suggested that the anti-CD239 recombinant antibody (C7-Fc + DT) conjugated with diphtheria toxin (DT3C) had no effect on cells with weak CD239 expression. In order to verify that the growth inhibition of the anti-CD239 recombinant antibody (C7-Fc + DT) conjugated with diphtheria toxin (DT3C) depends on the expression level of CD239, cells in which CD239 was forcibly expressed in MCF7 were prepared. DNA encoding the Lu form of CD239 (NCBI Accession No: NP — 005572.2) was amplified by PCR. The obtained DNA fragment was inserted into pIRESpuro3 (Clontech, Mountain View, CA) according to a conventional method to construct an expression vector. Using Lipofectamin2000 (Life Technologies, Carlsbad, Calif.), Plasmids were introduced into MCF7 cells, and expression cells were selected and cloned with 0.5 μg / ml puromycin (Wako, Osaka, Japan).

  As a result of analyzing the expression of CD239 in the obtained CD239 (Lu) / MCF7 cells using a flow cytometer, a clone that strongly expresses CD239 was obtained (FIG. 9 (a)). Proliferation assays were performed using the CD239 (Lu) / MCF7 cells.

  As a result, cell growth inhibition was not seen with MCF7, but growth inhibition was seen with CD239 (Lu) / MCF7 cells (FIG. 9 (b)). This indicates that strong CD239 expression is necessary for the anti-CD239 recombinant antibody (C7-Fc + DT) conjugated with diphtheria toxin (DT3C) to exhibit cell growth inhibitory activity.

[Example 10: In vitro and in vivo cell growth inhibitory activity of anti-CD239 recombinant antibody (C7-Fc + DT) conjugated with diphtheria toxin]
Confirmed in vitro cell growth inhibitory activity of anti-CD239 recombinant antibody (C7-Fc + DT) conjugated with diphtheria toxin (DT3C) against CD239 forced expression cells (CD239 (Lu) / HT1080F) and control cells (Control / HT1080F) did. Cell proliferation assays were performed using DMEM medium plus low IgG fetal calf serum and the others were performed as described in Example 8.

  As a result, the anti-CD239 recombinant antibody (C7-Fc + DT) conjugated with diphtheria toxin (DT3C) does not show cell growth inhibition with respect to control cells (control / HT1080F), but CD239 forced expression cells (CD239 ( Cell proliferation was inhibited against Lu) / HT1080F) (FIG. 10 (a)).

CD239 forced expression cells (CD239 (Lu) / HT1080F) and control cells (control / HT1080F) were transplanted into nude mice (KSN / slc, 7-11 weeks, purchased from Hamamatsu, Japan). Each cell is detached with 0.05% trypsin / EDTA, suspended in PBS (-) to make 6.66x10 ⁷ cells / ml, and then dispensed to make 5x10 ⁶ cells / 75 μl / tube. Until left on ice. Immediately before use, it was mixed with 75 μl of Cultrex Basement Membrane Extract (Trevigen, Gaithersburg, MD), and this was transplanted subcutaneously with a 25G needle to gel. On the 10th day after transplantation, the size of the tumor mass was measured, the volume of the tumor mass was determined by (width) ² x length x 0.5, and anti-CD239 recombinant antibody (C7-) conjugated with diphtheria toxin (DT3C) Fc + DT) was administered. Before administration, there was no significant difference in the tumor mass size of CD239 forced expression cells (CD239 (Lu) / HT1080F) and control cells (control / HT1080F) (FIG. 10 (b), left panel). Diphtheria toxin (DT3C) in an equimolar amount was bound to 5 μg of anti-CD239 recombinant antibody (C7-Fc), and prepared with PBS (−) so as to be 20 μl. The administration method was intratumoral injection (IT) administered directly to the tumor mass.

  On the 7th day after administration, the size of the tumor mass was measured. As a result, the anti-CD239 recombinant antibody (C7-Fc + DT) conjugated with diphtheria toxin (DT3C) was found to be CD239 forced expression cells (CD239 (Lu) / HT1080F) The tumor mass was significantly reduced (FIG. 10 (b), right panel). The results of this tumor-bearing mouse showed that CD239 was internalized in tumor cells even in vivo, and anti-CD239 recombinant antibody (C7-Fc + DT) conjugated with diphtheria toxin (DT3C) was effective as an anticancer agent .

  The anticancer agent of the present invention can suppress the growth of cancer cells that highly express CD239. The polypeptide provided by the present invention can be used as a marker for CD239-expressing cancer cells, as a carrier for delivering drugs to cancer cells, and as a platform for searching for drugs and linkers of ADCs. There is a utility that can be.

Claims (16)

  An anticancer agent, which is a complex of a polypeptide capable of specifically recognizing human CD239 and a cytotoxic drug.   The anticancer agent according to claim 1, wherein the polypeptide is an antibody that specifically binds to human CD239, or an antigen-binding fragment or antigen-binding derivative thereof. The polypeptide is the following (a) to (i):
(A) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1,
VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3,
And VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4,
VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6
An antibody or antigen-binding fragment or antigen-binding derivative thereof,
(B) an antibody heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having 5 amino acid additions, substitutions and deletions to the amino acid sequence;
And an antibody comprising an antibody light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having 5 amino acid additions, substitutions and deletions to the amino acid sequence, or an antigen-binding fragment or antigen thereof Binding derivatives,
(C) an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 18, or a polypeptide consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence,
(D) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 19,
VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 20 and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 21
An antibody or antigen-binding fragment or antigen-binding derivative thereof,
(E) An antibody or antigen-binding fragment thereof comprising an antibody heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22, or an amino acid sequence having 5 amino acid additions, substitutions and deletions with respect to the amino acid sequence Or an antigen-binding derivative,
(F) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 23 or an amino acid sequence having 90% or more sequence identity to the amino acid sequence,
(G) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 24,
VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 25, and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 26,
VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 27,
VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 28, and VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 29,
An antibody or antigen-binding fragment or antigen-binding derivative thereof,
(H) an antibody heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence having 5 amino acid additions, substitutions and deletions with respect to the amino acid sequence;
And an antibody comprising an antibody light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having addition, substitution and deletion of 5 or less amino acids to the amino acid sequence, or an antigen-binding fragment or antigen thereof The anticancer agent according to claim 2, selected from a binding derivative and (i) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 32 or an amino acid sequence having 90% or more sequence identity to the amino acid sequence. .   The anticancer agent according to any one of claims 1 to 3, wherein the drug is diphtheria toxin.   The anticancer agent according to any one of claims 1 to 4, which has a growth inhibitory effect on breast cancer.   The pharmaceutical composition containing the anticancer agent of any one of Claims 1-5. The following (a) to (i):
(A) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1,
VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3,
And VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4,
VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6
An amino acid sequence comprising
(B) an antibody heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having 5 amino acid additions, substitutions and deletions to the amino acid sequence;
And an amino acid sequence comprising an antibody light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having addition, substitution and deletion of 5 or less amino acids to the amino acid sequence,
(C) an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 18, or an amino acid sequence having 90% or more sequence identity to the amino acid sequence,
(D) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 19,
VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 20 and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 21
An amino acid sequence comprising
(E) an amino acid sequence comprising the antibody heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 22, or an amino acid sequence having additions, substitutions and deletions of 5 or less amino acids to the amino acid sequence;
(F) the amino acid sequence represented by SEQ ID NO: 23, or an amino acid sequence having 90% or more sequence identity to the amino acid sequence,
(G) VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 24,
VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 25, and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 26,
VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 27,
VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 28, and VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 29,
An amino acid sequence comprising
(H) an antibody heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence having 5 amino acid additions, substitutions and deletions with respect to the amino acid sequence;
And an amino acid sequence comprising an antibody light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having additions, substitutions and deletions of 5 or less amino acids to the amino acid sequence, and (i) SEQ ID NO: A recombinant protein having an amino acid sequence selected from the amino acid sequence shown by 32 or an amino acid sequence having 90% or more sequence identity to the amino acid sequence.   Furthermore, the recombinant protein of Claim 7 containing the signal peptide which consists of an amino acid sequence selected from sequence number 33-36.   A polynucleotide encoding the recombinant protein according to claim 7 or 8.   An expression vector comprising the polynucleotide according to claim 9.   A host cell comprising the expression vector according to claim 10.   A method for producing a recombinant protein according to claim 7 or 8, comprising culturing the host cell according to claim 11 and recovering the expressed protein.   A marker for detecting a CD239-expressing cancer cell, comprising a polypeptide capable of specifically recognizing human CD239.   14. The marker according to claim 13, wherein the polypeptide is the recombinant protein according to claim 7.   A carrier for delivering an agent having cytotoxicity against CD239-expressing cancer cells, comprising a polypeptide capable of specifically recognizing human CD239.   The carrier according to claim 15, wherein the polypeptide is the recombinant protein according to claim 7.

Images