JP2010229038A - Carcinostatic agent against triple-negative breast cancer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a therapeutic agent for effectively treating a triple-negative breast cancer that is negative for expression of an estrogen receptor, expression of a progesterone receptor and expression of HER2. <P>SOLUTION: Employment of CRM197 or a variant thereof as an effective ingredient of a carcinostatic agent against the triple-negative breast cancer achieves effective treatment of the triple-negative breast cancer which has not been solved by conventional techniques. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an anticancer agent capable of effectively treating triple negative breast cancer exhibiting estrogen receptor expression negative, progesterone receptor expression negative, and HER2 expression negative.

  Breast cancer is a malignant tumor whose incidence is acceleratingly increasing, including its younger age, and the number of deaths due to breast cancer among middle-aged women occupies the first place in Japan and developed countries. To date, many therapeutic drugs such as adriamycin, taxol and other anti-cancer drugs, anti-estrogen preparations, and herceptin targeting HER2 (human epidermal growth factor receptor type 2) have been developed for breast cancer and have a certain effect. ing.

  However, in triple negative breast cancer (hereinafter sometimes simply referred to as TNBC) having the characteristics of estrogen receptor expression negative, progesterone receptor expression negative, and HER2 expression negative, which accounts for 10 to 15% of breast cancers, With the above-mentioned therapeutic agents, an effective improvement effect is not recognized, an effective treatment method has not yet been established, and the prognosis is extremely poor (see, for example, Non-Patent Documents 1-3). To date, therapeutic effects have been clinically tested against TNBC using anti-EGFR (epidermal growth factor receptor) antibodies and low molecular weight compounds that act on EGFR, but no clear efficacy has been demonstrated. . Furthermore, research using TNBC cell lines (BT-549 cell line, MDA-MB-231 cell line: Rahman M et al., Breast Cancer Res. Treat., 2008 doi: 10.1007 / s10549-008-9924-5) However, the search for effective therapeutic drugs has not progressed at all.

  Thus, TNBC cannot be treated with conventional therapeutic agents for breast cancer, and an effective treatment method for TNBC has not been established.

Preu CM et al., Nature, 406: 747-752, 2000 Sotiriou C et al., Proc. Natl. Acad. Sci. USA., 100: 10393-10398, 2003 (Sotiriou C et al., J. Natl. Cancer Inst., 98: 262-272, 2006)

  An object of the present invention is to solve the above-described problems of the prior art. That is, an object of the present invention is to provide a therapeutic agent for TNBC for which no effective therapeutic method has been found.

  The present inventors analyzed TNBC in detail, and confirmed that the expression level of HB-EGF (Heparin-binding EGF-like Growth Factor) in TNBC was significantly lower than that of breast cancer other than TNBC. Thus, it is generally very difficult to predict that an HB-EGF inhibitor is effective for TNBC with a low HB-EGF expression level. However, the present inventors have surprisingly found that CRM197 or a variant thereof is effective for the treatment of TNBC. The present invention has been completed by further studies based on this finding.

That is, this invention provides the invention of the aspect hung up below.
Item 1. An anticancer agent for triple negative breast cancer showing estrogen receptor expression negative, progesterone receptor expression negative, and HER2 expression negative, comprising CRM197 or a variant thereof as an active ingredient.
Item 2. Item 2. The anticancer agent according to Item 1, wherein the active ingredient is CRM197 or DT52E148K.
Item 3. Use of CRM197 or a variant thereof for the production of an anti-cancer agent for triple negative breast cancer showing estrogen receptor expression negative, progesterone receptor expression negative and HER2 expression negative.
Item 4. A method of treating triple negative breast cancer, comprising administering a therapeutically effective amount of CRM197 or a variant thereof to a patient with triple negative breast cancer that exhibits negative estrogen receptor expression, negative progesterone receptor expression, and negative HER2 expression.

  The anticancer agent of the present invention has an excellent antitumor effect against TNBC, and can greatly improve the prognosis of TNBC. In addition, an effective therapeutic method has not been established for TNBC, and the present invention will provide the world's first innovative cancer drug that enables effective treatment of TNBC.

In the reference test example 1, it is a figure which shows the result of having measured the expression level of HB-EGF in breast cancer (TNBC9 cases, 36 cases other than TNBC). In the reference test example 1, it is a figure which shows the result of having measured the expression level of the EGFR ligand (HB-EGF, amphiregulin, TGF (alpha), and EGF) in the breast cancer of 9 cases of TNBC. In Reference Test Example 1, expression levels of EGFR ligands (HB-EGF, amphiregulin, TGFα, Epiregulin, Betacellulin, EGF, and Epigen) in breast cancer cells (BT-549 strain and MDA-MB-231 strain) exhibiting the characteristics of TNBC It is a figure which shows the result of having measured. In Example 2, it is a figure which shows the result of having evaluated the apoptosis-inducing effect exerted on the breast cancer cells (BT-549 strain and MDA-MB-231 strain) which show the characteristic of TNBC by CRM197. In Example 3, it is a figure which shows the result of having evaluated the antitumor effect with respect to the tumor formed with the breast cancer cell which shows the characteristic of TNBC by administration of CRM197.

  The anticancer agent of the present invention is an antitumor agent against TNBC showing estrogen receptor expression negative, progesterone receptor expression negative, and HER2 expression negative, and is characterized by containing CRM197 or a variant thereof as an active ingredient. The present invention is described in detail below.

  The anticancer agent of the present invention contains CRM197 or a variant thereof as an active ingredient.

  CRM197 used in the present invention is a diphtheria toxin mutant in which diphtheria toxin is attenuated by mutating the catalytic domain while retaining the receptor binding domain of diphtheria toxin, and has 25 amino acids in the amino acid sequence of diphtheria toxin. This is known as a mutant in which the 52nd Gly is counted as Glu when counted except for the signal sequence. The amino acid sequence of CRM197 (the first 25 sequences represent a signal sequence) is shown in SEQ ID NO: 1. In the present specification, the amino acid sequence number in CRM197 is numbered with the amino acid at position 26 (Gly) as the number 1 except the signal sequence (1st to 25th) in the amino acid sequence of SEQ ID NO: 1. .

  Moreover, as a variant of CRM197 used in the present invention, one in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of CRM197 is exemplified. In CRM197, Glu, which is the 52nd amino acid when counted excluding 25 signal sequences, contributes to the attenuation of diphtheria toxin. The 52nd amino acid (Glu) when counting except the signal sequence is used without mutation. In the variant of CRM197 used in the present invention, the number of amino acid substitutions, deletions, insertions and / or additions is not particularly limited, but is usually 1 to 10, preferably 1 to 5, more preferably Examples are 1 to 3, particularly preferably 1 to 2. In addition, in the mutant of CRM197 used in the present invention, the amino acid substitution, deletion, insertion and / or addition sites are not particularly limited, and the catalytic domain (the amino acid sequence from the 1st to the 193rd) Any one or more of the receptor binding domain (the 378th to 535th amino acid sequence portion), the transmembrane domain (194th to 377th amino acid sequence portion) existing between the catalytic domain and the receptor binding domain A site.

  The CRM197 mutant used in the present invention is preferably one that can inhibit the binding between HB-EGF and the EGF receptor by binding to HB-EGF, more preferably the receptor binding domain of diphtheria toxin is mutated. Examples thereof include DT52E148K as a suitable example. DT52E148K is a mutant in which the 148th Glu in the CRM197 amino acid sequence, counted without the signal sequence, is mutated to Lys.

  The anticancer agent of the present invention is applied to TNBC showing estrogen receptor expression negative, progesterone receptor expression negative, and HER2 expression negative. That is, the therapeutic agent of the present invention is used for the treatment of TNBC in mammals including humans, particularly for the treatment of TNBC in humans.

Whether a breast cancer is TNBC is determined by measuring whether or not the above three proteins are expressed by immunohistochemistry on breast cancer cells collected from a breast cancer patient. Specifically, a breast cancer that is determined not to express any estrogen receptor, progesterone receptor, or HER2 by the following measurement is TNBC to which the anticancer agent of the present invention is applied.
<Measurement of estrogen receptor expression>
Estrogen receptor expression in breast cancer cells is determined using the Allred scoring system. Specifically, using an anti-human estrogen receptor monoclonal antibody (eg, clone 1D5, DAKO, Glostrup), the estrogen receptor of breast cancer cells is stained, and the average percentage of stained cells and the degree of staining are determined according to the following criteria. To score. Subsequently, the score of the ratio of the stained cells and the average score of the degree of staining are summed, and a case where the total score is less than 3 is negative, and a case where it is 3 or more is determined as positive. In addition, human-derived ductal epithelial cells are used as a positive staining positive control (positive control having a degree of staining score of 3).

<Measurement of progesterone receptor expression>
Progesterone receptor expression in breast cancer cells is determined using the Allred scoring system. Specifically, an anti-human progesterone receptor monoclonal antibody (eg, clone PgR636, DAKO, Glostrup) is used to stain the progesterone receptor of breast cancer cells, and the average percentage of stained cells and the degree of staining are determined according to the following criteria. To score. Subsequently, the score of the ratio of the stained cells and the average score of the degree of staining are summed, and a case where the total score is less than 3 is negative, and a case where it is 3 or more is determined as positive. Human-derived cervical cells are used as positive control for strong staining (positive control with a degree of staining score of 3).

<Measurement of HER2 expression>
For HER2 expression in breast cancer cells, in the immunostaining using anti-HER2 antibody, the degree of staining is scored according to the following criteria, and a score of 0 or 1+ is negative, a score of 2+ or 3+ is determined as positive To do. More specifically, it is determined that 2+ and 3+ are positive and 0 and 1+ are negative according to the score according to HercepTest kit scoring guidelines (DAKO, Glostrup).

  The anticancer agent of the present invention can be formulated as the active ingredient as it is or in combination with a pharmaceutically acceptable pharmaceutical carrier or additive.

  The dosage form of the anticancer agent of the present invention is not particularly limited, and may be either oral administration or parenteral administration. Examples of parenteral administration include intravenous, intramuscular, intraperitoneal, subcutaneous or intradermal injection, rectal administration, transmucosal administration, and airway administration. Of these dosage forms, preferably intravenous, intraperitoneal, or subcutaneous.

  When the anticancer agent of the present invention is prepared into an oral dosage form, examples of the dosage form include tablets, granules, capsules, powders, solutions, suspensions, syrups and the like. In addition, when the anticancer agent of the present invention is prepared in a parenteral dosage form, examples of the dosage form include injections, infusions, suppositories, and transdermal absorption agents. However, the dosage form of the anticancer agent of the present invention is not limited to these.

  The types of pharmaceutical carriers and additives used for the anticancer agent of the present invention are not particularly limited, and can be appropriately selected by those skilled in the art. For example, excipients, disintegrants or disintegration aids, binders, lubricants, coating agents, bases, solubilizers or solubilizers, dispersants, suspending agents, emulsifiers, buffers, antioxidants, antiseptics Agents, isotonic agents, pH adjusters, solubilizers, stabilizers and the like can be used, and the individual specific components used for these purposes are well known to those skilled in the art.

  Specifically, when the anticancer agent of the present invention is prepared in an oral dosage form, the pharmaceutical carrier and additive used include, for example, glucose, lactose, D-mannitol, starch, crystalline cellulose and the like. Forming agents; disintegrating agents or disintegrating aids such as carboxymethylcellulose, starch, or carboxymethylcellulose calcium; binders such as hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, gelatin; lubricants such as magnesium stearate or talc; hydroxy Coating agents such as propylmethylcellulose, sucrose, polyethylene glycol or titanium oxide; bases such as petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, hard fat and the like can be mentioned.

  In addition, when the anticancer agent of the present invention is prepared as an injection or an infusion, the pharmaceutical carrier and additive used may be, for example, an aqueous solution such as distilled water for injection, physiological saline, or propylene glycol. Solubilizers or solubilizers that can constitute a time-dissolving injection; isotonic agents such as glucose, sodium chloride, D-mannitol, glycerin; pH regulators such as inorganic acids, organic acids, inorganic bases, or organic bases Is mentioned.

  The compounding amount of the above active ingredient in the anticancer agent of the present invention is appropriately set based on the dosage, formulation form, administration route, etc. described later, but is usually appropriately within the range of about 0.0001 to 70% by weight in the anticancer agent. Just choose.

  The dose of the anticancer agent of the present invention is appropriately set according to the age, sex, weight, symptom, administration route, etc. of the patient. Usually, the dose of the active ingredient per day for an adult is from 1 μg / kg body weight. A range of about 50 mg is desirable. The above-mentioned dose of medicine may be administered once a day or may be divided into several times.

  Also, the administration schedule of the anticancer agent of the present invention is appropriately set according to the age, sex, weight, symptom, administration route, etc. of the patient. For example, it may be administered once a week for 6 to 8 weeks, every other day for 2 to 3 weeks, or daily for 10 to 14 days.

  In addition, the anticancer agent of the present invention may be administered in combination with known anticancer agents such as taxol, taxotere, 5-FU, cisplatin, carboplatin, adriamycin, camptotensin and the like, if necessary.

  EXAMPLES Hereinafter, although an Example etc. are given and this invention is demonstrated, this invention is not limited to these Examples.

Reference test example 1
For 45 breast cancer tissues collected from breast cancer patients with informed consent, whether or not it is TNBC was measured according to the TNBC criteria described above, 9 of the 45 cases were TNBC, and 36 were TNBC It wasn't. About these breast cancer tissues and breast cancer cells (BT-549 strain (ATCC Number: HTB-122) and MDA-MB-231 strain (ATCC Number: HTB-26)) showing characteristics of TNBC, EGFR ligand (HB-EGF , Amphiregulin, TGFα, Epiregulin, Betacellulin, EGF, and Epigen) were measured. Specifically, RNA extraction was performed on the breast cancer tissue and breast cancer cells of TNBC using TRIzol, followed by cDNA synthesis using SuperScript II reverse transcriptase (Invitrogen Corp.). Using the cDNA thus obtained, the expression level of the EGFR ligand was measured by Real Time PCR method (Applied Biosystems) using TaqMan probe. The expression level of EGFR ligand was analyzed using GAPDH as an endogenous control (mRNA Expression Index = number of copies of each EGFR ligand mRNA / number of copies of GAPDH mRNA × 10000).

  Fig. 1 shows the expression levels of HB-EGF in 45 breast cancer tissues. Fig. 2 shows the expression levels of EGFR ligands (HB-EGF, amphiregulin, TGFα, and EGF) in 9 breast cancer tissues that are TNBC. The results and the results of measuring the expression level of HB-EGF for breast cancer cells (BT-549 strain and MDA-MB-231 strain) showing the characteristics of TNBC are shown in FIG.

  As a result, it was confirmed that the expression level of HB-EGF in TNBC was significantly lower than that of breast cancer other than TNBC (see FIG. 1). Moreover, it became clear that the expression level of EGFR ligands other than HB-EGF in TNBC is even smaller than that of HB-EGF (see FIG. 2). Furthermore, in breast cancer cells (BT-549 strain and MDA-MB-231 strain) showing the characteristics of TNBC, it was confirmed that the expression level of EGFR ligands other than HB-EGF was smaller than that of HB-EGF (Fig. 3).

  From the results of this study, TNBC has a unique feature that the expression level of HB-EGF itself is small, and in TNBC, the expression level of EGFR ligands other than HB-EGF is much smaller than the expression level of HB-EGF. It has been elucidated.

Example 1: Evaluation of apoptosis-inducing effect of breast cancer cells showing characteristics of TNBC
Breast cancer cells (BT-549 strain and MDA-MB-231 strain) showing the characteristics of TNBC were cultured in a culture dish using RPMI-1640 medium containing 10% fetal bovine serum, and then adhered to the culture dish Was removed with trypsin and washed with RPMI-1640 containing 10% by volume fetal calf serum to collect breast cancer cells. Subsequently, 6 ml of RPMI-164 (serum-free medium) was placed in a 6 cm culture dish coated with polylysine, and 1 × 10 ⁶ breast cancer cells collected as described above were seeded thereon. Then, after leaving still at 37 ° C. and 5% CO ₂ to completely adhere the cells to the culture dish, CRM197 (produced by Osaka University Microbial Disease Research Group; final concentration 100 nM), anti-EGFR monoclonal antibody (clone LA1, Upstate Biotechnologies (Lake Placid, NY); final concentration 100 nM) and Herceptin (Chugai pharmaceutical Co, Tokyo; final concentration 1 μM) were added, and the cells were cultured at 37 ° C., 5% CO ₂ for 48 hours.

  Induction of apoptosis in each breast cancer cell after culture was evaluated by the TUNEL method (MEBSTAIN Apoptosis Kit Direct (MBL code no. 8445)). Specifically, each breast cancer cell after the above culture was collected, washed once with RPMI-1640 containing 10% by volume fetal bovine serum, and further washed twice with PBS containing 0.2% by volume fetal bovine serum albumin. It was. The breast cancer cells thus washed were allowed to stand in PBS containing 4% by volume paraformaldehyde for 30 minutes at 4 ° C., and then washed twice with PBS containing 0.2% by volume fetal bovine serum albumin. Thereafter, an ethanol aqueous solution (containing 70% ethanol) is added to the collected cell residue, and after sufficient stirring, left at 20 ° C. for 30 minutes, and then with PBS containing 0.2% fetal bovine serum albumin. Washed twice. 30 μl of TdT reaction solution (containing FITC-dUTP and TdT) was added to the cells thus fixed, and after sufficient stirring, the cells were reacted at 37 ° C. for 1 hour. Since the cells in which apoptosis was induced by the reaction were specifically labeled with FITC, the FITC-labeled cells were detected by flow cytometry (Becton Dickinson, FACScalibur).

  The obtained results are shown in FIG. From these results, CRM197 was able to induce apoptosis in breast cancer cells showing the characteristics of TNBC, but anti-EGFR monoclonal antibody and Herceptin, which are conventionally used as a therapeutic agent for breast cancer, are different from breast cancer cells showing the characteristics of TNBC. Thus, apoptosis could not be induced.

  The results of this study revealed that CRM197 is effective as a therapeutic agent for TNBC.

Example 2: Evaluation of antitumor effect on TNBC
After breast cancer cells (MDA-MB-231 strain) 5 × 10 ⁶ cells indicating characteristics of TNBC were housed were seeded subcutaneously into nude mice (BALB / c-nu / nu ), of 100 mm ³ or more in tumor volume From the time when formation was observed (one week after seeding of breast cancer cells), CRM197 was administered into the abdominal cavity of nude mice at 50 mg / kg for 10 consecutive days, and reared for 8 weeks after seeding of breast cancer cells. Tumor volume in nude mice was measured weekly. As a control, the test was performed under the same conditions as described above except that physiological saline was administered instead of CRM197. In this test, the CRM197 administration group and the control group were carried out with n = 10, and the tumor volume was determined by major axis × minor axis × minor axis × 1/2.

  The obtained results are shown in FIG. From these results, it was confirmed that administration of CRM197 disappeared tumor cells formed by breast cancer cells exhibiting the characteristics of TNBC, and CRM197 was effective in treating TNBC.

Claims (2)

  An anticancer agent for triple negative breast cancer showing estrogen receptor expression negative, progesterone receptor expression negative, and HER2 expression negative, comprising CRM197 or a variant thereof as an active ingredient.   The anticancer agent according to claim 1, wherein the active ingredient is CRM197 or DT52E148K.

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