JP2008285491A - Anticancer agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an anticancer agent effective for treating malignant tumors, especially ovarian cancer. <P>SOLUTION: The anticancer agent comprises a protein which is a variant of a diphtheria toxin and has activity of hindering an HB-EGF from binding to an EGF receptor and does not substantially have the toxicity of the diphtheria toxin, a protein which contains at least a receptor binding domain of the diphtheria toxin composed of part of the diphtheria toxin or a complex protein containing the protein or the like, has activity of hindering an HB-EGF from binding to an EGF receptor and does not substantially have the toxicity of the diphtheria toxin as an active ingredient. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to an anticancer agent that can be used for the treatment of various cancers including ovarian cancer.

Various treatments and drugs have been developed for malignant tumors, but there are still many cases where a sufficient therapeutic effect cannot be obtained yet. In particular, ovarian cancer is a malignant tumor that progresses quickly among gynecological malignant tumors and has a particularly poor prognosis.
The only treatment for ovarian cancer is currently chemotherapy centered on taxol. Although these chemotherapy have temporary effects, they often allow recurrence later, and new treatments have been developed. It has been demanded.

  HB-EGF is a cell growth factor of the EGF family, and is essential for the formation and regeneration processes of the body, and at the same time is known as a molecule related to the onset of vascular stenosis, arteriosclerosis, etc. (for example, non-patent literature) 1), this factor has never been targeted for the treatment of malignant tumors. This molecule is synthesized as a membrane-bound precursor (proHB-EGF) and is cleaved by proteases on the cell surface to produce secreted HB-EGF. The secretory type has a growth-promoting effect, but the membrane type has a growth-inhibiting effect, and it is thought that the secretory type and the membrane type are used properly to help form and maintain the tissue. .

  HB-EGF binds to and activates the EGF receptor (Her1) and the EGF receptor family Her4 (ErbB-4). However, the EGF receptor family (Her1, Her2, Her3, Her4) can form homodimers as well as all combinations of heterodimers, so that HB-EGF can activate all EGF receptor family molecules as a result. . HB-EGF is expressed in various tissues and is thought to work in a wide range of cells and tissues, but has been reported to promote the proliferation of fibroblasts, smooth muscle cells or keratinocytes. (For example, refer nonpatent literature 2).

  HB-EGF is synthesized as a membrane-bound precursor (proHB-EGF) as described above, but proHB-EGF has a signal sequence, a prosequence, a heparin-binding domain, an EGF-like domain, and a JACKSTA membrane from the N-terminus. It consists of a main, transmembrane domain, and cytoplasmic domain (Fig. 1). This proHB-EGF is cleaved by a protease (ectodomain shedding) at the portion indicated by an arrow in the figure and becomes a secreted form. ProHB-EGF ectodomain shedding is a pathway in which lysophosphatidic acid (LPA) or the like activates the Ras-Raf-MEK pathway via a G protein-coupled receptor, or a pathway in which phorbol esters activate PKC. It is proposed to be stimulated by (see, for example, Non-Patent Document 3).

  The secretory HB-EGF binds to the EGF receptor and promotes phosphorylation of the EGF receptor exists in the EGF-like domain (see, for example, Non-Patent Document 1).

  On the other hand, diphtheria toxin is a protein having a molecular weight of about 59,000 produced by diphtheria, but it is known that a membrane-bound precursor (proHB-EGF) of HB-EGF is used as a receptor (for example, non-patent literature). 4). Moreover, a mutant of diphtheria toxin such as CRM197 is known as an inhibitor of secretory HB-EGF (see, for example, Non-Patent Document 5). Database information on diphtheria toxin can be found in the gene EMBL; K01722, amino acid sequence SWISS-PROT; P00588, three-dimensional structure PDB; 1MDT or 1XDT. The gene for diphtheria toxin is encoded by phage lysogenized in the microbial cells.

  Diphtheria toxin is a simple protein consisting of 535 amino acids (the amino acid sequence of diphtheria toxin (SEQ ID NO: 1) and the base sequence of the gene encoding it (SEQ ID NO: 2) are shown in FIG. 2 and FIG. (Representing the sequence), it is divided into a fragment A part and a fragment B part that are separated by treatment with a reducing agent (FIG. 4), but according to the three-dimensional structure analysis, the fragment B part is further divided into two domains. Yes. The function of each domain is as follows. The catalytic domain corresponding to the fragment A portion (amino acid number 1 to 193 excluding the signal sequence) exhibits ADP ribosylation activity and the membrane corresponding to the N-terminal half of the fragment B portion. The transmembrane domain (amino acid numbers 194 to 378 excluding the signal sequence) has a property of forming a channel in the endosomal membrane, and a receptor-binding domain (signal sequence) corresponding to the C-terminal half of the fragment B portion. The amino acid numbers excluding 386 to 535) have the activity of binding to the cell surface diphtheria toxin receptor.

Fragment A (catalytic domain portion) of diphtheria toxin has an action of ADP-ribosylating EF-2 (peptide elongation factor 2) in the presence of NAD, thereby inhibiting protein synthesis. Therefore, for diphtheria toxin to be toxic, fragment A must enter the cytoplasm.
The mechanism by which fragment A enters the cytoplasm is that the receptor binding domain in fragment B binds to proHB-EGF, which is a receptor on the cell surface, so that diphtheria toxin is incorporated into endosomes by endocytosis and transmembranes within endosomes. The domain is inserted into the endosomal membrane, and finally fragment A passes through the endosomal membrane and is released into the cytoplasm, where it deactivates EF-2 (see, for example, Non-Patent Document 6).

  Both fragment A and fragment B are required for diphtheria toxin to exert toxicity. Thus, no matter which fragment is mutated, a protein that does not have the toxicity of diphtheria toxin is produced.

  Diphtheria toxin has been isolated from a detoxified mutant having a mutation in the catalytic domain, such as CRM197.

  On the other hand, the mutant of diphtheria toxin has an activity of inhibiting the binding between HB-EGF and the EGF receptor because diphtheria toxin binds to the EGF-like domain of secreted HB-EGF. It is the receptor-binding domain of diphtheria toxin that is involved in this binding. It has been reported that the 516th Lys and 530th Phe of diphtheria toxin are important for the binding of HB-EGF (see, for example, Non-Patent Document 7). In addition, the crystal structure of a complex comprising diphtheria toxin and the EGF domain of HB-EGF has been analyzed, and amino acids important for binding to HB-EGF between 381 and 535 have been reported (for example, non- (See Patent Document 8).

Thus, although it was recognized that the diphtheria toxin mutant binds to HB-EGF and inhibits the activity of HB-EGF, it has not been known that HB-EGF is a target for cancer treatment. Because of this and the complexity of the regulatory mechanism involving HB-EGF, no attempt has been made to use diphtheria toxin mutants in cancer therapeutics.
Eisuke Mekada et al. "Gene Medicine" Medical Do Co., Ltd., vol. 5 No. 2 2001, p. 131-134 Higashiyama, S .; et al. Cell Biol. (1993) 122, p. 933-940, Prezel, N .; et al Nature (1999) 402, p. 884-888 J. et al. G. Nagrich et al. Cell (1992) 69, p. 1051-1061 T.A. Mitamura et al J.M. Biol. Chem. (1995) 270, p. 1015 T.A. By Umata et al. Biol. Chem. (1998) 273, p. 8351 By Shen, HS et al. Biol. Chem. (1994) 269, p. 29077-29084 Gordon VL et al. Molecular Cell (1997) 1, p. 67-78

  An object of the present invention is to solve the above-described problems and achieve the following objects. That is, an object is to provide an anticancer agent effective for the treatment of malignant tumors, particularly ovarian cancer.

  The inventors have reexamined the function of secretory HB-EGF, which has been indispensable for the formation and regeneration processes of the body, and has been known as a protein related to the onset of vascular stenosis, arteriosclerosis, The present inventors have found that HB-EGF is involved in the growth and metastasis process of cancer cells. Based on this, the development of a new treatment method was advanced, and as a result, it was clarified that CRM197 having the ability to neutralize HB-EGF significantly suppresses the growth of human ovarian cancer-derived cells inoculated into nude mice. It came to.

That is, the means for solving the problems of the present invention are as follows.
<1> A protein of any one of the following (a), (b) and (c), which has an activity of inhibiting the binding of HB-EGF to the EGF receptor and substantially reduces the toxicity of diphtheria toxin. It is a cancer drug characterized by containing a protein that does not have as an active ingredient.
(A) a protein comprising a part of diphtheria toxin and comprising at least the receptor binding domain of the diphtheria toxin (b) an amino acid in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein of (a) Protein comprising sequence (c) Complex protein comprising any one of proteins (a) and (b) <2> The protein of any one of (a), (b) and (c) is a catalytic domain of diphtheria toxin It is an anticancer drug as described in <1> which does not have.
<3> The anticancer agent according to <2>, wherein the protein (c) is GST-DT.

  ADVANTAGE OF THE INVENTION According to this invention, the anticancer agent effective in the treatment of a malignant tumor, especially ovarian cancer can be provided.

  Based on the finding that HB-EGF has tumorigenicity in examining the function of HB-EGF, the inventors have known the inhibitory activity of HB-EGF on binding to the EGF receptor. We have demonstrated the anticancer effect of a detoxified mutant of diphtheria toxin.

The anticancer agent according to the first aspect of the present invention is a mutant of diphtheria toxin, which has an activity of inhibiting the binding between HB-EGF and the EGF receptor, and has substantially no toxicity of diphtheria toxin. It is characterized by containing as an active ingredient.
The mutant of diphtheria toxin represents a protein having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of diphtheria toxin, for example, one or several amino acids are deleted, substituted or substituted It is a protein consisting of an added amino acid sequence. In addition, the 25 signal sequences of diphtheria toxin may or may not be attached, and any sequence is included in the scope of the present invention.

  The anticancer drug of the second aspect of the present invention is a protein of any one of the following (a), (b) and (c), which has an activity of inhibiting the binding of HB-EGF to the EGF receptor and It contains a protein that does not substantially have the toxicity of diphtheria toxin as an active ingredient. (A) A protein comprising a part of diphtheria toxin and comprising at least a receptor binding domain of the diphtheria toxin. (B) A protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of the protein of (a). (C) A complex protein comprising the protein of any one of (a) and (b).

  The protein refers to a part of diphtheria toxin or a variant thereof, or a complex protein containing these proteins and retaining a receptor binding domain.

  Here, the toxicity of diphtheria toxin refers to the activity (ADP-ribosylation) of EF-2 (peptide elongation factor 2) held by fragment A by binding to a cell surface receptor and entering into the cell. It means that the protein synthesis function of cells is inhibited by ribosylation activity), which can be easily measured by the degree of protein synthesis inhibition. That is, it is a method in which a certain amount of diphtheria toxin is added to cultured cells and cultured for about 2 to 8 hours, followed by short-time cultivation in the presence of radioactive amino acids to measure the amount of radioactive amino acids incorporated into the protein.

Specifically, Vero cells (1 × 10 ⁵ cells) are inoculated on a 24-well plate and cultured in a CO ₂ incubator for 16 hours. After confirming that the cells have sufficiently adhered to the plate, each well is washed once with cold PBS (150 mM, NaCl-2.7 mM KCl-10 mM phosphate buffer, pH 7.2). At this time, the liquid should be taken in and out carefully so as not to peel off the cells. Add 0.5 ml of assay medium with serum. As the assay culture medium, a culture medium in which the concentration of leucine is reduced to about 1/10 from a normal medium is used. This is to increase the efficiency of incorporation of [ ³ H] leucine added later. However, since Ham's F12 medium has a low leucine content, it can be used as a culture medium for assay. Serum is added at the usual concentration.
Next, various concentrations of diphtheria toxin are added and cultured in a CO ₂ incubator for 2-5 hours. Add 10 μl of 3.7 MBq / ml [ ³ H] leucine and incubate for an additional hour.

  After discarding the culture and washing the wells once with PBS, the cells are lysed with 0.5 ml of 0.1 M NaOH and the solution is collected in a tube. Wash wells again with 0.5 ml of 0.1 N NaOH and collect the solution in the same tube.

  Add 0.5 ml of 20% trichloroacetic acid solution and stir well with a Vortex mixer. The resulting precipitate is trapped with a glass filter, and the filter is further washed with a 5% trichloroacetic acid solution.

  Finally, the filter is washed with 100% ethanol and dried.

  The filter is attached to a scintillator of toluene-PPO, and the radioactivity trapped on the filter is measured with a liquid scintillation counter. The value of the sample to which no diphtheria toxin was added is measured, and this is taken as 100%, and the value when the toxin is added is determined in%.

  A protein having substantially no toxicity of diphtheria toxin refers to a protein in which the toxicity of diphtheria toxin is detoxified or attenuated to a very low level. In the present invention, the toxicity of diphtheria toxin is a concentration of 1 ng / ml. In the above-mentioned Vero cell system, it means that there is no significant difference from the value of the sample to which diphtheria toxin was not added or the sample to which a diphtheria toxin variant not having a catalytic domain was added. The significant difference is preferably no significant difference at a significance level of 5% in the t test, more preferably no significant difference as a significance level of 1%, and even more preferably no significant difference as a significance level of 0.1%. .

However, among the present invention, CRM197 or even hitherto mutant toxicity of diphtheria toxin has been a no like DT52E148K, ^{10 10} of extremely weak toxicity (e.g. in CRM197 wildtype diphtheria toxin It is proved that about 1 / min) remains, and such a weakly toxic mutant is not excluded from the present invention. The toxicity level of diphtheria toxin is preferably the same as or lower than that of CRM197 from the viewpoint of eliminating side effects due to the toxicity of diphtheria toxin and improving safety. However, on the other hand, since the present invention suggests that the toxicity contributes to the effect of the anticancer agent, it is also preferable from the viewpoint of enhancing the anticancer effect that the toxicity is as low as that of CRM197. Therefore, the toxicity level of diphtheria toxin can be appropriately selected according to the formulation of the preparation.

  Toxicity of diphtheria toxin can be adjusted by mutating the catalytic domain essential for ADP-ribosylation of peptide elongation factor 2 or by deleting part or all of the catalytic domain .

  The function of the mutated catalytic domain can be accurately examined by directly measuring ADP ribosylation activity. The ADP ribosylation activity is determined by adding fragment A or a protein (mutated catalytic domain, etc.) whose ADP ribosylation activity is to be measured and NAD labeled with a radioisotope to EF-2 which has been separated and purified. It can be directly measured by ADP-ribosylating 2 and measuring the radioactivity incorporated into EF-2.

Specifically, the rabbit reticulocyte EF− obtained by the method described in the following literature (Moynihan, MR and Pappenheimer, AM Jr. Infect. Immun. (1981) 32, 575-582). In the two fractions, the final concentration of 20 mM Tris buffer (pH 7.8), 1 mM DTT (dithiothreitol), 0.1 to 1 μg / ml fragment A or 0.1 to 100 μg / ml ADP ribosylation activity was measured. The protein to be added is added, and [ ³² P] NAD (about 740 GBq / mmol) is added and mixed so that the final concentration is 370 KBq / ml, and the mixture is reacted at 37 ° C. for 10 minutes.

  The same volume of 10% -trichloroacetic acid solution is added to the reaction solution to precipitate the protein, the resulting precipitate is trapped with a glass filter, and the filter is further washed with 5% trichloroacetic acid solution.

  Finally, the filter is washed with 100% ethanol and dried.

  The filter is attached to a scintillator of toluene-PPO, and the radioactivity trapped on the filter is measured with a liquid scintillation counter.

  The measured radioactivity indicates the degree of ADP ribosylation activity, and the relative activity of the ADP ribosylation activity of the mutated protein is determined based on the radioactivity when using fragment A without the mutation. Can do.

In addition, the feature of having the activity of inhibiting the binding between secretory HB-EGF and the EGF receptor is that the inventors have examined in more detail based on domain information, a portion containing a receptor binding domain It was found that the amino acid sequence from the 378th to the 535th amino acid sequence should be included. That is, a gene in which GST (Glutathione-S-transferase) is fused with a sequence from 378th to 535th of diphtheria toxin is prepared and expressed in E. coli, and a fusion protein (GST-DT) having the above structure It was created. GST-DT inhibited the binding of ¹²⁵ I-labeled diphtheria toxin to HB-EGF in a concentration-dependent manner. From the degree of inhibition, it was found that GST-DT binds to HB-EGF with the same binding strength as diphtheria toxin. Therefore, it was found that what is necessary for the binding is the 378th to 535th, that is, the portion containing the receptor binding domain.

Here, whether or not it has the activity of inhibiting the binding between HB-EGF and the EGF receptor can be determined by the inhibition experiment on the binding of ¹²⁵ I-labeled diphtheria toxin to HB-EGF as described above.

  Therefore, a protein that has an activity of inhibiting the binding between HB-EGF and the EGF receptor and does not substantially have the toxicity of diphtheria toxin has a mutation in the catalytic domain while retaining the receptor binding domain. It can be obtained by preparing a diphtheria toxin mutant protein or a protein that is a part of diphtheria toxin that retains the receptor binding domain of diphtheria toxin and that lacks part or all of the catalytic domain.

  Examples of such mutants include CRM197, DT52E148K and GST-DT. They are substantially free of diphtheria toxin toxicity and inhibit the binding of HB-EGF to the EGF receptor. CRM197 is a mutant in which the 52nd Gly when counted excluding 25 signal sequences is mutated to Glu, and DT52E148K is the 148th Glu when counted without the signal sequence in addition to the mutation. Is a mutant mutated to Lys. GST-DT is a protein containing 378th to 535th when GST is counted excluding the signal sequence of diphtheria toxin. The amino acid sequence relating to CRM197 (the first 25 sequences represent a signal sequence) is represented by SEQ ID NO: 3, and the base sequence of the gene encoding it is represented by SEQ ID NO: 4. The amino acid sequence of GST-DT (SEQ ID NO: 5) and the base sequence of the gene encoding it (SEQ ID NO: 6) are shown in FIGS.

  Here, it has already been reported that CRM197 does not have the toxicity of diphtheria toxin, that is, has no ADP ribosylation activity (T. Uchida and AM Pappenheimer Jr. (1972) Science 175). , 901-903). In addition, it is known that a 148K mutant having a mutation in 148E has extremely weak activity (JT Barbieri and R. J. Collier (1987) Infect. Immun. 55, 1647-1651), 52E. DT52E148K, which is a double mutant obtained by further adding a mutation of 148K to CRM197, which is a mutant, is preferable as a safer mutant. Moreover, it was confirmed that the toxicity of these mutants was not significantly different from the value of the sample to which diphtheria toxin was not added even in the protein synthesis inhibition experiment. However, as described above, it is clear from the detailed analysis of the present invention that even these mutants still have extremely weak diphtheria toxin toxicity.

It is clear that GST-DT does not have the toxicity of diphtheria toxin because it completely lacks the catalytic domain.
(SEQ ID NO: 3)
MSRKLFASILIGALLGIGAPPSAHA GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILP GIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS
(SEQ ID NO: 4)
GTG AGCAGAAAACTGTTTGCGTCAATCTTAATAGGGGCGCTACTGGGGATAGGGGCCCCACCTTCAGCCCATGCAGGCGCTGATGATGTTGTTGATTCTTCTAAATCTTTTGTGATGGAAAACTTTTCTTCGTACCACGGGACTAAACCTGGTTATGTAGATTCCATTCAAAAAGGTATACAAAAGCCAAAATCTGGTACACAAGGAAATTATGACGATGATTGGAAAGAGTTTTATAGTACCGACAATAAATACGACGCTGCGGGATACTCTGTAGATAATGAAAACCCGCTCTCTGGAAAAGCTGGAGGCGTGGTCAAAGTGACGTATCCA GGACTGACGAAGGTTCTCGCACTAAAAGTGGATAATGCCGAAACTATTAAGAAAGAGTTAGGTTTAAGTCTCACTGAACCGTTGATGGAGCAAGTCGGAACGGAAGAGTTTATCAAAAGGTTCGGTGATGGTGCTTCGCGTGTAGTGCTCAGCCTTCCCTTCGCTGAGGGGAGTTCTAGCGTTGAATATATTAATAACTGGGAACAGGCGAAAGCGTTAAGCGTAGAACTTGAGATTAATTTTGAAACCCGTGGAAAACGTGGCCAAGATGCGATGTATGAGTATATGGCTCAAGCCTGTGCAGGAAATCGTGTCAGGCGATCAGTAGGTAGC CATTGTCATGCATAAATCTTGATTGGGATGTCATAAGGGATAAAACTAAGACAAAGATAGAGTCTTTGAAAGAGCATGGCCCTATCAAAAATAAAATGAGCGAAAGTCCCAATAAAACAGTATCTGAGGAAAAAGCTAAACAATACCTAGAAGAATTTCATCAAACGGCATTAGAGCATCCTGAATTGTCAGAACTTAAAACCGTTACTGGGACCAATCCTGTATTCGCTGGGGCTAACTATGCGGCGTGGGCAGTAAACGTTGCGCAAGTTATCGATAGCGAAACAGCTGATAATTTGGAAAAGACAACTGCTGCTCTTTCGATACTTCCT GTATCGGTAGCGTAATGGGCATTGCAGACGGTGCCGTTCACCACAATACAGAAGAGATAGTGGCACAATCAATAGCTTTATCGTCTTTAATGGTTGCTCAAGCTATTCCATTGGTAGGAGAGCTAGTTGATATTGGTTTCGCTGCATATAATTTTGTAGAGAGTATTATCAATTTATTTCAAGTAGTTCATAATTCGTATAATCGTCCCGCGTATTCTCCGGGGCATAAAACGCAACCATTTCTTCATGACGGGTATGCTGTCAGTTGGAACACTGTTGAAGATTCGATAATCCGAACTGGTTTTCAAGGGGAGAGTGGGCACGACATAAAAA TTACTGCTGAAAATACCCCGCTTCCAATCGCGGGTGTCCTACTACCGACTATTCCTGGAAAGCTGGACGTTAATAAGTCCAAGACTCATATTTCCGTAAATGGTCGGAAAATAAGGATGCGTTGCAGAGCTATAGACGGTGATGTAACTTTTTGTCGCCCTAAATCTCCTGTTTATGTTGGTAATGGTGTGCATGCGAATCTTCACGTGGCATTTCACAGAAGCAGCTCGGAGAAAATTCATTCTAATGAAATTTCGTCGGATTCCATAGGCGTTCTTGGGTACCAGAAAACAGTAGATCACACCAAGGTTAATTCTAAGCTATCGCTATTTT TGAAATCAAAAGC
TGA

  The fragment containing the receptor binding domain was synthesized by synthesizing the DNA sequence of the receptor binding domain by PCR using the gene (Pβ197) encoding CRM197 incorporated in the plasmid as a template, and using this as a GST fusion protein. Or histidine tag is inserted into the multicloning site of an expression vector (pGEX-3X, pQE-30), the resulting plasmid is incorporated into E. coli, and the gene encoded by the plasmid is synthesized in E. coli. Can be created.

  A mutant having a mutation in the catalytic domain can be prepared as follows. The CRM197 region is synthesized by PCR using the gene (Pβ197) encoding CRM197 incorporated in the plasmid as a template and the site to be mutated as a primer. The primer is synthesized and used by introducing a point mutation so as to have a mutation. The synthesized DNA can be prepared by incorporating it into a gene expression vector for E. coli (pET-22b), transducing E. coli, and expressing the mutant in E. coli.

The anticancer agent of the present invention can be used for the treatment of all malignant tumors such as breast cancer, prostate cancer, thyroid cancer, ovarian cancer, etc., but preferably can be used for malignant tumors expressing HB-EGF, particularly ovary. It can be suitably used for cancer.
The anticancer agent of the present invention has the activity of neutralizing the toxicity of the diphtheria toxin remaining in the mutant of the diphtheria toxin, and the mutant of the diphtheria toxin that does not suppress the binding of the mutant of the diphtheria toxin to cells Further comprising a monoclonal antibody,
It is also preferable that the diphtheria toxin mutant monoclonal antibody and the diphtheria toxin mutant form a complex.

  The mutant of the diphtheria toxin is attenuated by introducing the mutation, and substantially has no toxicity of the diphtheria toxin. As described above, it includes the case where toxicity remains. Therefore, a mutant of diphtheria toxin has an activity to neutralize the toxicity of diphtheria toxin remaining in the mutant of diphtheria toxin and does not inhibit binding of the mutant of diphtheria toxin to cells. The toxicity of diphtheria toxin can be further reduced by forming a complex.

  The diphtheria toxin mutant monoclonal antibody has an activity to neutralize the toxicity of the diphtheria toxin remaining in the diphtheria toxin mutant, and does not inhibit binding of the diphtheria toxin mutant to cells (ie, Monoclonal antibody that does not inhibit the binding activity to proHB-EGF can be prepared according to a conventional method as shown by the following document (Hayakawa S, J. Biol. Chem. 258, 4311-4317, 1983). . That is, among the clones that are producing antibodies against diphtheria toxin mutants, antibodies that have the activity of finally neutralizing the toxicity of diphtheria toxin but do not inhibit the binding of diphtheria toxin mutants to cells. You can create a clone that is created separately. Moreover, what has the said property with respect to the diphtheria toxin mutant to be used can also be selected from well-known diphtheria toxin monoclonal antibodies.

  The activity to neutralize the toxicity of diphtheria toxin is the colony formation that occurs when a complex of a diphtheria toxin mutant and the diphtheria toxin monoclonal antibody is added to Vero-H cells, and the diphtheria toxin mutant is added alone. Can be easily measured by seeing if inhibition of is inhibited.

On the other hand, the fact that the monoclonal antibody does not inhibit the binding of the diphtheria toxin mutant to the cells is due to the inhibition of the binding of ¹²⁵ I-labeled diphtheria toxin to HB-EGF and the IL-3 dependency described later. It can be examined by measuring the inhibitory action of HB-EGF on the proliferation activity of DER cell in which epidermal growth factor receptor gene is expressed in 32D cells (obtained from ATCC) showing proliferation ability.

  As a monoclonal antibody of a mutant of diphtheria toxin that has an activity to neutralize the toxicity of diphtheria toxin remaining in the mutant of diphtheria toxin and does not suppress binding of the mutant of diphtheria toxin to cells, for example, # 2 anti- DT mAb (patent biological deposit center deposit number: monoclonal antibody produced from a hybridoma of FERM P-19551).

  A complex with a mutant of diphtheria toxin can be prepared by mixing a mutant of diphtheria toxin and a monoclonal antibody at an appropriate ratio.

  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.

  The anticancer agent can be administered orally or parenterally (for example, intravenous, intramuscular, intraperitoneal, subcutaneous or intradermal injection, rectal administration, transmucosal administration, airway administration, etc.). . When applied to malignant tumors that are disseminated intraperitoneally, such as ovarian cancer, administration by intraperitoneal injection is preferable because it is directly delivered to cancer cells.

  Examples of the pharmaceutical composition suitable for oral administration include tablets, granules, capsules, powders, solutions, suspensions, syrups, etc. Examples of the pharmaceutical composition suitable for parenteral administration include , Injections, infusions, suppositories, transdermal absorption agents, and the like, but the dosage form is not limited thereto.

  There are no particular limitations on the type of formulation additive used in the production of the anticancer drug, and a person skilled in the art can appropriately select it. 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.

  Examples of pharmaceutical additives that can be used in the preparation of pharmaceutical preparations for oral administration include excipients such as glucose, lactose, D-mannitol, starch, or crystalline cellulose; carboxymethylcellulose, starch, carboxymethylcellulose calcium, etc. Disintegrating agents or disintegrating aids; binders such as hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, or gelatin; lubricants such as magnesium stearate or talc; coatings such as hydroxypropylmethylcellulose, sucrose, polyethylene glycol, or titanium oxide Agents: Bases such as petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat can be used.

Pharmaceutical additives that can be used for the preparation of pharmaceutical preparations for injection or infusion include aqueous solutions such as distilled water for injection, physiological saline, and propylene glycol, or solubilizers or solubilizers that can constitute dissolution-type injections when used Agents; tonicity agents such as glucose, sodium chloride, D-mannitol and glycerin; additives for preparation such as pH regulators such as inorganic acids, organic acids, inorganic bases and organic bases can be used.
The amount of the active ingredient contained in the anticancer agent of the present invention varies depending on the preparation form or administration route of the anticancer agent and cannot be generally defined, but is usually in the range of about 0.0001% to 70% in the final preparation. It can be selected and determined as appropriate.

  The anticancer agent of the present invention can be administered to mammals including humans.

  The dosage of the anticancer drug of the present invention should be appropriately increased or decreased according to conditions such as the patient's age, sex, weight, symptom, and administration route, but the amount of active ingredient per day for an adult is 1 kg of body weight. It is preferable that the range is about 1 μg to 30 mg per unit. The above-mentioned dose of medicine may be administered once a day or may be divided into several times. In addition, it may be administered once every several days to several weeks or once. Moreover, it can also administer with the component which suppresses a side effect, such as a steroid.

Hereinafter, although the Example of this invention is described, this invention is not limited to this Example at all.
Example 1
Production of CRM197 protein C7 (β197) lysogen stock (C7 (β197) available from ATCC (American Type Culture Collection) Bacteria collection (No. 39255) as C7 (β197) phage lysed diphtheria C7 (beta197) M1) ) And the late bacterial solution in the logarithmic growth phase is added to CY medium supplemented with 2% filtered maltose so that the initial OD590 is about 0.05. This OD corresponds to about 5 × 10 ⁷ cells / ml. The flask is placed on a rotary shaker at 200 revolutions per minute and incubated at 35 ° C. for 16 to 17 hours. When the OD becomes 10 to 15, the culture is terminated.

The CY medium is prepared as follows. That is, 10 g of casamino acid, 20 g of yeast extract, and 5 g of KH ₂ PO ₄ are dissolved in 1 l of distilled water. 2 ml of 50% CaCl ₂ . After adding 2H ₂ O, the pH is adjusted to 7.4. Filter after boiling. 2 ml Mueller and Miller solution II (22.5 g MgSO ₄ , 0.115 g β-alanine, 0.115 g nicotinic acid, 7.5 mg pimelic acid, 1 g CuSO ₄ .5H ₂ O, 1 g ZnSO ₄ .5H ₂ O, 1 g MnCl ₂ .4H ₂ O / 100 ml H ₂ O) and 1 ml Mueller and Miller solution III (20 g L-cystine, 20 ml concentrated hydrochloric acid / 100 ml H ₂ O) are added. A CY medium was obtained by dispensing 100 mL each and applying an autoclave.

  The CRM protein is purified as follows.

  The culture is centrifuged at 10,000g for 15 minutes. Add ammonium sulfate to the culture supernatant to a saturation state of 65%. Leave in an ice room for 24-48 hours. The precipitate is collected, dissolved in 0.02 M pH 7.2 Tris-HCl buffer, and dialyzed against the same solution.

  Centrifuge to remove insolubles, add to DE52 column and elute with a gradient of NaCl in 0.02 M pH 7.2 Tris-HCl buffer. CRM197 elutes when NaCl is 0.08M. The eluted liquid is saturated with ammonium sulfate to 65%. The precipitate is dissolved in 0.01 M Tris-HCl buffer and equilibrated again. Then, it is applied again to the DE52 column and precipitated again with ammonium sulfate. Subsequently, it is applied to a Sephacryl S-200 column and eluted with a solution of HEPES-NaOH, pH 7.2, 0.15 M NaCl. The eluted CRM197 is applied to a DeToxi gel to remove the LPS analog contained in the CRM197 sample and used in the experiment. The absorption of CRM197 at 280 nm corresponds to 1 OD of about 0.67 mg / ml.

(Example 2)
A tumorigenicity experiment was conducted using nude mice.

The ovarian cancer cell line SKOV-3 (available from ATCC) cultured in RPMI + 10% FBS was washed with EDTA / PBS (−) and recovered with 0.25% trypsin. After washing twice with RPMI + 10% FBS and twice with RPMI (without serum), 5 × 10 ⁶ cells were added to 250 μL of RPMI (with serum), and this was inoculated by subcutaneous injection on the back of nude mice.

  For a group of nude mice, administration of CRM197 was started 1 week after SKOV-3 cell inoculation, and intraperitoneally was administered 200 μg / day 5 times a week over 4 weeks (FIG. 7). In another group of nude mice, administration of CRM197 was started from 1 week after SKOV-3 cell inoculation, and 5 times a week at 100 μg / day was administered directly to the inoculated tumor over 4 weeks (FIG. 8). ). In addition, for another group of nude mice, administration of CRM197 was started 20 days after SKOV-3 cell inoculation, and 5 times a week was administered to the inoculated tumor at 100 μg / day for 3 weeks (FIG. 9). . In either case, nude mice that did not receive CRM197 were used as control experiments. The relationship between the administration time and the tumor volume is shown in FIGS. Here, the tumor volume was determined by measuring the major axis and minor axis of the formed tumor every 3 to 4 days, and the major axis x minor axis x minor axis x 1/2.

  From these results, it was found that in any case, administration of CRM197 suppressed tumor growth.

(Example 3)
It is a cell produced by transfecting the ovarian cancer cell line RMG-1 (registered with the Japan Collection of Research Bioresources Cell Bank), the ovarian cancer cell line OV47, and the ovarian cancer cell line SKOV-3 cells with the HB-EGF gene. SKOV-H was collected in the same manner as the SKOV-3 cells in Example 2, and inoculated by subcutaneous injection on the back of nude mice instead of SKOV-3 cells.

  Regardless of which cell was inoculated, 1 mg of CRM197 was administered intraperitoneally immediately after cell inoculation and one week later. In addition, nude mice that did not receive CRM197 were used as control experiments.

  The relationship between the administration time and the tumor volume is shown in FIGS. FIG. 10 shows the results for RMG-1 cells, FIG. 11 for OV47 cells, and FIG. 12 for nude mice inoculated with SKOV-H cells.

  From these results, it was found that in all cases, tumor growth was suppressed by administration of CRM197.

Example 4
CRM197 Cytotoxicity Test In the previous experiments, CRM197 Fragment A had no ADP-ribose transferase activity and was considered to be a completely non-toxic molecule. Since it is extremely important in the present invention to clarify whether it has toxicity as a toxin, the ADP ribose transferase activity of CRM197 was analyzed in more detail.

  First, in order to examine the cytotoxicity of CRM197, Vero cells (available from ATCC) and Vero-H cells in which HB-EGF was highly expressed in Vero cells (human HB-EGF cDNA manufactured by OriGene Technologies, Inc., pCDNA3.1 plasmid ( Cells cloned by Invitrogen) were transfected with cells prepared by transfection with various concentrations of CRM197 and cultured for 1 week, and the colony formation rate was examined.

FIG. 13 shows that Vero cells and Vero-H cells are seeded on a 6-well plate at a density of 300 cells / well, cultured for 10 hours, added with CRM197, cultured for 24 hours, and then cultured in a culture solution not containing CRM197 for 6 days. The colony formation rate when the number of colonies is counted is shown. FIG. 14 shows the colony formation rate when CRM197 was added to Vero cells and Vero-H cells and cultured for one week. As a result, Vero-H cells showed a decrease in the number of colonies when CRM197 was added and cultured for 24 hours with 1 μg / ml CRM197 and when cultured in the presence of CRM197 for one week with 100 ng / ml CRM197. confirmed. In Vero cells, no significant decrease in the number of colonies was observed under either condition. CRM197 was more toxic to Vero-H cells than Vero cells, suggesting that this toxicity was related to the diphtheria toxin receptor (proHB-EGF). In addition, when diphtheria toxin was allowed to act for 24 hours in place of CRM197 and then cultured for 6 days in a culture solution not containing diphtheria toxin, diphtheria toxin hardly observed the appearance of colonies at a concentration of about 1 fg / ml (toxin About 3% of the number of colonies obtained when not added). Therefore, it was found that the cytotoxicity of CRM197 measured by the colony formation method used here is 10 ¹⁰ or less that of diphtheria toxin, and the cytotoxicity of CRM197 is extremely weak.

(Example 5)
Protein Synthesis Inhibition Test by CRM197 The toxin action of diphtheria toxin is a protein synthesis inhibitory action based on ADP ribosylation of EF2. Therefore, in order to examine whether the toxicity of CRM197 is due to protein synthesis inhibition, the protein synthesis inhibition action by CRM197 was examined. CRM197 was exposed to Vero cells similar to those used in Example 4 and Vero-H cells for 36 hours, and the protein synthesis inhibitory ability was examined from the incorporation rate of [ ³ H] Leu into the protein. Specifically, Vero cells and Vero-H cells were seeded on a 24-well plate at a density of 1 × 10 ⁵ cells / ml, cultured for 16 hours, added with CRM197, and further cultured for 36 hours. Thereafter, [ ³ H] Leu was added and incubated for 1 hour, and then the amount of [ ³ H] Leu incorporated into the protein was measured with a liquid scintillation counter to determine the ability to inhibit protein synthesis.

  As a result, in Vero-H cells, inhibition of protein synthesis was observed with CRM197 at a concentration of 100 ng / ml or more. Under these conditions, almost no protein synthesis inhibitory effect was observed in Vero cells (FIG. 15).

(Example 6)
Cytotoxicity test of DT52E148K As in Example 4, the toxicity of DT52E148K, which is a mutant having two mutations in diphtheria toxin, and the recombinant protein GST-DT were measured by the colony formation method. As a result, DT52E148K was toxic to Vero-H cells, although it was weaker than CRM197 (FIG. 16). On the other hand, no cytotoxicity was observed in Vero cells (FIG. 17). Therefore, it was shown that these two mutations alone are insufficient to completely eliminate the toxicity. GST-DT did not show any toxicity against Vero cells or Vero-H cells.

(Example 7)
Resistance to CRM197 and DT52E148K of Vdtr cells In order to examine whether the cytotoxicity and protein synthesis inhibitory action of CRM197 and DT52E148K shown so far is inactivation of EF-2 by the ADP ribosylation activity of diphtheria toxin Diphtheria toxin resistant strain Vdtr cells were prepared. Specifically, the following documents (Moehring JM and Moehring TJ, Somat. Cell Genet. 5, 453-468, 1979;
Kohno K et al. Somat. Cell Genet. 11, 421-423, 1985), Vero cells were treated with EMS and then added with diphtheria toxin and cultured to obtain viable cells. Among these, cells that were resistant even when a high concentration of diphtheria toxin was added were selected to obtain Vdtr cells. Next, HB-EGF was highly expressed in the Vdtr cells, and Vdtr-4H cells were obtained (obtained by cloning the human-derived HB-EGF gene from OriGene Technologies, Inc. into pCDNA3.1 plasmid (Invitrogen)). , Cells created by transfection). FIG. 18 shows the protein synthesis inhibition rate when diphtheria toxin was added to Vdtr-4H cells, and FIG. 19 shows the results of cell-free ADP ribosylation assay in which fragment A was added to the cell lysate obtained from these cells. . In this cell, no ADP ribosylation of EF2 was observed even in the cell-free ADP ribosylation assay, indicating that diphtheria toxin resistance was caused by EF2 in this cell.

  In order to confirm whether the cytotoxicity of CRM197 is due to ADP ribosylation activity, the toxicity of CRM197 to Vdtr-4H cells in which HB-EGF was highly expressed in the Vdtr cells was examined. CRM197 was added to Vdtr-4H cells, and the toxicity was examined by the colony formation method as in Example 4. As a result, even when 100 μg / ml CRM197 was added, no toxicity was shown to Vdtr-4H cells (FIG. 20). Similarly, DT52E148K showed no toxicity at all (FIG. 20). From these results, it was revealed that the cytotoxicity and protein synthesis inhibition shown by CRM197 are due to the ADP ribosyltransferase activity of fragment A.

(Example 8)
ADP-ribosylation experiment of EF-2 in cell-free system The experimental results using Vdtr cells of Example 7 show that the cytotoxicity present in CRM197 and DT52E148K is due to residual ADP-ribosyltransferase activity. Yes. In order to further confirm this, an ADP ribosylation experiment was performed under Cell-free conditions. The following documents Gill, DM and Pappenheimer, AM Jr. J. et al. Biol. Chem. 246, 1492-1495, 1971), CRM197 or DT52E148K was added to EF-2 extracted from rabbit liver, and [ ³² P] NAD was added thereto and incubated at 37 ° C. for 10 minutes. The ADP ribosylation reaction was performed with cell-free. Thereafter, the amount of radioactivity was measured with a liquid scintillation counter. As a result, although it was very weak activity, CRM197 and DT52E148K were found to have the activity of ADP-ribosylating EF-2 (FIGS. 21 and 22). The upper right diagram in FIG. 21 shows the ADP ribosylation activity of CRM197 on an enlarged scale on the vertical axis. From this result, it was concluded that both CRM197 and DT52E148K have a slight activity to ADP-ribosylate EF2.

Example 9
Neutralization of cytotoxicity of CRM197 by diphtheria toxin monoclonal antibody # 2 anti-DT mAb (Diphtheria toxin monoclonal antibodies # 2)
When CRM197 is used as an inhibitor of HB-EGF growth activity, it is sometimes undesirable that CRM197 is weak but cytotoxic. Therefore, next, conditions for suppressing toxicity remaining in CRM197 were examined. Many monoclonal antibodies against diphtheria toxin have been isolated (Hayakawa S, J. Biol. Chem. 258, 4311-4317, 1983). Among these are antibodies that inhibit diphtheria toxin cytotoxicity but not diphtheria toxin binding to the receptor. Among these, it was found that # 2 anti-DT mAb neutralizes the toxicity of CRM197 as in the case of diphtheria toxin, but does not suppress the binding of CRM197 to HB-EGF.

  At the same time as CRM197, # 2 anti-DT mAb (patent biological deposit center accession number: monoclonal antibody produced from a hybridoma of FERM P-19551) was added to examine whether the toxicity of CRM197 was neutralized.

  The production of a monoclonal antibody against diphtheria toxin is shown by the following document (Hayakawa S, J. Biol. Chem. 258, 4311-4317, 1983), but briefly described as follows. The abdominal cavity of BALB / c mice was inoculated with 0.1 mg of formalin-treated diphtheria toxin together with Freund's adjuvant, and this was performed three times a week. Several days after the last inoculation, the spleen cells of this mouse were removed and fused with mouse myeloma cells SP2 / 0 cells. The cells after the fusion reaction were cultured in a HAT selection medium, and a clone producing an antibody against diphtheria toxin was isolated from the proliferating cells. Among clones making antibodies against diphtheria toxin, clones that finally produced antibodies that had the activity of neutralizing diphtheria toxin toxicity but did not inhibit diphtheria toxin binding to cells were isolated. .

  The CRM197 / # 2 anti-DT mAb complex was prepared by first mixing CRM197 (1 mg) and # 2 anti-DT mAb (10 mg) and incubating at 37 ° C. for 1 hour. This complex was added to Vero-H cells at various concentrations and cultured for 1 week, and the colony formation rate was examined. As a result, for example, CRM197 alone is killed by 100 ng / ml CRM197, but in the case of CRM197 / # 2 anti-DT mAb complex, colony formation is not suppressed at all even at a maximum amount of 10 μg / mL. 2 It was found that anti-DT mAb completely inhibited the cytotoxicity of CRM197 (FIG. 23).

(Example 10)
HB-EGF proliferative activity inhibitory action of CRM197 / # 2 anti-DT mAb complex HB-EGF proliferative activity inhibitory action of CRM197 / # 2 anti-DT mAb complex (action that inhibits proliferative activity of HB-EGF) did. An epidermal growth factor receptor gene (EGFR gene, manufactured by OriGene Technologies) was expressed in 32D cells (obtained from ATCC) showing proliferation ability in an IL-3 dependent manner to prepare a DER cell (EGFR gene was transformed into pCDNA3). .1 Cloned into plasmid (Invitrogen) and prepared by transfection). These cells grow in the absence of IL-3 due to the proliferative activity of HB-EGF. CRM197 and CRM197 / # 2 anti-DT mAb complex were added to the cells in the presence of HB-EGF. Cells were cultured for 48 hours and the number of expanded cells was determined by MTT assay. DER cell proliferated in the absence of CRM197 and CRM197 / # 2 anti-DT mAb complex, but in the presence of CRM197 and CRM197 / # 2 anti-DT mAb complex, DER cell proliferation was suppressed. (FIG. 24). When the growth activity inhibitory action of CRM197 and CRM197 / # 2 anti-DT mAb complex was compared, there was almost no difference between them and the same inhibitory effect was shown. That is, the CRM197 / # 2 anti-DT mAb complex has suppressed the cytotoxicity of CRM197, but has the same inhibitory activity as CRM197 alone with respect to the growth inhibitory action of HB-EGF. all right.

(Example 11)
Tumor suppressive effect by CRM197 / # 2 anti-DT mAb complex Each group of nude mice (3 individuals) was inoculated with SKOV-H cells, and CRM197 (1 mg / 1 individual / week) from 1 week after inoculation, Alternatively, administration of a CRM197 / # 2 anti-DT mAb complex (containing 1 mg of CRM197 / week) was started and administered intraperitoneally once a week for 4 weeks). As the CRM197 / # 2 anti-DT mAb complex, 1 mg of CRM197 and 8 mg of # 2 anti-DT mAb were incubated for 1 hour at room temperature. A nude mouse to which CRM197 was not administered was used as a control experiment. FIG. 25 shows the relationship between administration of CRM197 or CRM197 / # 2 anti-DT mAb complex and tumor volume. The tumor volume was measured in the same manner as in Example 2. This experiment revealed that the CRM197 / # 2 anti-DT mAb complex suppressed tumor growth, but the effect was weaker than that of CRM197 alone.

  Since the CRM197 / # 2 anti-DT mAb complex completely suppresses the weak cytotoxicity of CRM197, it can be expected that the CRM197 / # 2 anti-DT mAb complex is safer than using CRM197 alone. On the other hand, when CRM197 is used alone, the effect of suppressing tumor growth is stronger. This is considered to be a result of the addition of the weak cytotoxicity of CRM197 in addition to the HB-EGF growth activity inhibitory action of CRM197. Therefore, when using CRM197, it can be administered as a complex with # 2 anti-DT mAb when safety is more important, and CRM197 alone is also effective when effect is more important It is possible.

(Example 12)
Regarding DT52E148K and CRM197, the tumor suppressive effect was examined in the same manner as in Example 11 except that the administration was once a week for 3 weeks. FIG. 26 shows the relationship between DT52E148K or CRM197 and tumor volume. This experiment revealed that DT52E148K suppressed tumor growth, but its effect was weaker than CRM197. There are two mutations in the A fragment having ADP-ribose transferase activity, and the cytotoxicity is low (FIG. 16). This result regarding DT52E148K shows that in addition to the above-mentioned HB-EGF proliferation inhibitory activity of CRM197 in CRM197, This supports the presumption that the weak cytotoxicity of CRM197 has an effect on the suppression of tumor growth.

(Reference example)
A group of nude mice (each 10 mice) were inoculated with SKOV-3 cells or SKOV-H cells, respectively, and 1 week and 2 weeks after the inoculation, Taxol 40 mg / kg / week was administered intraperitoneally. . The results are shown in FIG. Taxol was found to be effective for SKOV-3 cells but less effective for SKOV-H cells. Since SKOV-H cells have the property of expressing a large amount of HB-EGF, it was found that taxol has a different mechanism of action from the anticancer agent of the present invention. Therefore, it is suggested that the anticancer agent of the present invention is effective in cases where taxol is not very effective.

  The present invention can be used for the production of anticancer agents effective for the treatment of various cancers including ovarian cancer.

FIG. 1 is a schematic diagram showing the structure of proHB-EGF. FIG. 2 shows the amino acid sequence and base sequence of diphtheria toxin. FIG. 3 shows the amino acid sequence and base sequence of diphtheria toxin (continuation of FIG. 2). FIG. 4 is a schematic diagram showing the domain structure of diphtheria toxin. FIG. 5 shows the amino acid sequence and base sequence of GST-DT. FIG. 6 shows the amino acid sequence and base sequence of GST-DT (continuation of FIG. 5). FIG. 7 is a graph showing the effect of CRM197 intraperitoneal injection on tumor growth in nude mice injected with SKOV-3 cells. FIG. 8 is a graph showing the effect on tumor growth by local injection of CRM197 in nude mice injected with SKOV-3 cells. FIG. 9 is a diagram showing the effect on tumor growth by local injection of CRM197 in nude mice injected with SKOV-3 cells. FIG. 10 is a graph showing the effect on the cancer growth rate after CRM197 injection in RMG-1 cell-injected nude mice. FIG. 11 is a graph showing the effect on cancer growth rate after CRM197 injection in OV47 cell-injected nude mice. FIG. 12 is a graph showing the effect on cancer growth rate after CRM197 injection in SKOV-H-injected nude mice. FIG. 13 is a graph showing toxicity to Vero cells and Vero-H cells when CRM197 is allowed to act for 24 hours. FIG. 14 is a graph showing toxicity to Vero cells and Vero-H cells when CRM197 is allowed to act for 1 week. FIG. 15 is a diagram showing protein synthesis inhibitory action by CRM197. FIG. 16 is a diagram showing the toxicity of CRM197, DT52E148K and GST-DT to Vero-H cells. FIG. 17 shows the toxicity of CRM197, DT52E148K and GST-DT to Vero cells. FIG. 18 is a diagram showing DT resistance of Vdtr-4H cells. FIG. 19 is a diagram showing an ADP-ribosylation reaction of EF-2 in Vdtr-4H cells and Vero-H cell lysate under cell-free conditions. FIG. 20 shows the resistance of Vdtr-4H cells to CRM197 and DT52E148K. FIG. 21 shows ADP-ribosylation of EF-2 by CRM197 and DT. FIG. 22 shows ADP-ribosylation of EF-2 by DT52E148K. FIG. 23 shows neutralization of CRM197 toxicity by # 2 anti-DT mAb. FIG. 24 is a diagram showing inhibition of HB-EGF cell proliferation activity by CRM197 / # 2 anti-DT mAb complex. FIG. 25 is a diagram showing the proliferation of cancer cells in nude mice by intraperitoneal administration of CRM197 and CRM197 / # 2 anti-DT mAb complex. FIG. 26 is a graph showing proliferation of cancer cells in nude mice by intraperitoneal administration of CRM197 and DT52E148K. FIG. 27 is a diagram showing the proliferation of cancer cells of Xenograft mice by administration of taxol.

Claims (3)

Any one of the following proteins (a), (b) and (c), which has an activity of inhibiting the binding of HB-EGF to the EGF receptor and substantially has no toxicity of diphtheria toxin An anticancer agent comprising a protein as an active ingredient.
(A) a protein comprising a part of diphtheria toxin and comprising at least the receptor binding domain of the diphtheria toxin (b) an amino acid in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein of (a) Protein consisting of sequence (c) Complex protein comprising any one of (a) and (b) The anticancer agent according to claim 1, wherein any one of the proteins (a), (b) and (c) does not have a catalytic domain of diphtheria toxin. The anticancer agent according to claim 2, wherein the protein of (c) is GST-DT.

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