JP2010501161A - Treatment or prevention of cancers that overexpress REG4 or KIAA0101 - Google Patents

Abstract

The invention features a method of inhibiting cancer cell growth by contacting a cell with a composition of siRNA that inhibits expression of REG4 or KIAA0101. Also included in the invention are methods of treating cancer. The invention also features products comprising nucleic acid sequences and vectors useful in the provided methods, and compositions containing them. The present invention also provides a method for inhibiting tumor cells such as, for example, pancreatic cancer cells, prostate cancer cells, breast cancer cells and bladder cancer cells, particularly pancreatic ductal adenocarcinoma (PDAC), by inhibiting the REG4 gene. To do. The invention also relates to a method of treating or preventing PDAC in a subject comprising administering to the subject a pharmaceutically effective amount of an antibody or fragment thereof that binds to a protein encoded by REG4. The invention also relates to a method for diagnosing chemo-radiotherapy resistance of cancer. The present invention also provides a therapeutic agent or therapeutic method for cancer using the polypeptide. The polypeptide of the present invention is composed of an amino acid sequence comprising an amino acid sequence consisting of a polypeptide comprising QKGIGEFF / SEQ ID NO: 21. The polypeptides of the present invention can be introduced into cancer cells by modifying the polypeptide with a transfection agent such as polyarginine.

Description

  This application claims the benefit of U.S. Provisional Patent Application No. 60 / 838,649 filed on August 18, 2006 and U.S. Provisional Patent Application No. 60 / 838,749 filed on August 18, 2006. The entire disclosure is hereby incorporated by reference for all purposes.

  The present invention relates to the field of biological science, and more particularly to the field of cancer research. In particular, the present invention relates to methods for treating and preventing cancers such as pancreatic cancer, prostate cancer, breast cancer and bladder cancer. In particular, the present invention relates to a composition comprising a nucleic acid capable of inhibiting the expression of genes encoding REG4 and KIAA0101. In certain embodiments, the compound is a small interfering RNA (siRNA) corresponding to the sequence of these genes.

  Alternatively, the present invention also treats pancreatic cancer, particularly pancreatic ductal adenocarcinoma (PDAC) in a subject, comprising administering to the subject a pharmaceutically effective amount of an antibody or fragment thereof that binds to a protein encoded by REG4. Or how to prevent. Furthermore, the present invention relates to a composition comprising a peptide that inhibits the interaction between PCNA and KIAA0101. In certain embodiments, the composition is a cell permeable dominant negative peptide that stores a PCNA binding motif (PIP box).

  Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the Western world and the worst mortality among common malignancies with a 5 year survival rate of only 4% ( DiMagno EP, et al. Gastroenterology 1999; 117: 1464-84., Wray CJ, et al. Gastroenterology 2005; 128: 1626-41.). In 2006, it was estimated that approximately 33,730 new cases were diagnosed with pancreatic cancer in the United States, 32,300 of which would die from the disease (Jemal A, et al. Cancer statistics, 2006. CA Cancer J Clin 2006; 56: 106-130.). Since the majority of PDAC patients are diagnosed at their advanced stage, there is currently no effective treatment to treat the disease. Several approaches that combine surgery with and without 5-FU or gemcitabine chemotherapy and radiation can improve the patient's quality of life (DiMagno EP, et al. Gastroenterology 1999; 117: 1464- 84., Wray CJ, et al. Gastroenterology 2005; 128: 1626-41.), But because of its highly progressive and chemoresistant nature, their treatment is beneficial for long-term survival in patients with PDAC. It has only a very limited effect.

  The very poor prognosis of PDAC arises for several reasons, including difficulty in detecting PDAC at an early stage (DiMagno EP, et al. Gastroenterology 1999; 117: 1464-84., Wray CJ, et al. Gastroenterology 2005; 128: 1626-41.). Despite improvements in diagnostic imaging techniques such as endoscopic ultrasound (EUS) and magnetic resonance cholangiopancreatography (MRCP) (DiMagno EP, et al. Gastroenterology 1999; 117: 1464-84., Wray CJ , et al. Gastroenterology 2005; 128: 1626-41.), many patients do not receive imaging techniques because there are no symptoms until the end of the course of the disease. Precise and convenient serum tests such as PSA (prostate specific antigen) for prostate cancer can easily detect early PDAC and can be applied for mass screening of PDAC. Early stage surgical resection of PDAC can provide a relatively favorable prognosis, with a 5-year survival of 50-60% (Wray CJ, et al. Gastroenterology 2005; 128: 1626-41.). Therefore, considering the biological progression of PDAC and resistance to chemotherapy, one of the realistic strategies to improve the prognosis of this deadly disease is to use a non-invasive serum test at an early stage. To screen for PDAC.

  Currently, CA19-9 is the only commercially available serum marker for PDAC, but it is far from the ideal tumor marker. The reason is that (i) only about 10-15% of individuals secrete CA19-9 due to the Lewis antigen status, (ii) it is not specific for pancreatic cancer and is elevated even in benign conditions, And (iii) it is usually in the normal range in early stage patients. (Sawabu N, et al. Pancreas 2004; 28: 263-7., Pleskow DK, et al. Ann Intern Med 1989; 110: 704-9.). Therefore, the establishment of screening methods through the development of new tumor markers that are more specific and more sensitive to PDAC is urgently needed.

  REG4 is a new member of the REG family (Hartupee JC, et al. Biochim Biophys Acta 2001; 1518: 287-93.) And has been reported as a tumor marker for PDAC. Molecules belonging to the REG (REGenerating islet-derived) family are secreted proteins that act in the tissue regeneration and inflammation in the digestive tract (Hartupee JC, et al. Biochim Biophys Acta 2001; 1518: 287-93., Watanabe T , et al. J Biol Chem 1990; 265: 7432-9., Uno M, et al. Adv Exp Med Biol 1992; 321: 61-6.). The expression level of this member has been reported to be up-regulated in some gastrointestinal cancers and to function as a trophic or anti-apoptotic factor in cancer (Unno M, et al. Adv Exp Med Biol 1992; 321: 61-6., Sekikawa A, et al. Gastroenterology 2005; 128: 642-53.).

  cDNA microarray technology has enabled practitioners to obtain a comprehensive profile of gene expression in normal and malignant cells and to compare gene expression in malignant and corresponding normal cells (Okabe et al., (2001 ) Cancer Res 61: 212937; Kitahara et al., (2001) Cancer Res 61: 3544-9; Lin et al., (2002) Oncogene 21: 4120-8; Hasegawa et al., (2002) Cancer Res 62: 7012-7). This technique allows the elucidation of the complex nature of cancer cells and helps understand the carcinogenic mechanism. Identification of genes that are deregulated in tumors will lead to a more precise and accurate diagnosis of individual cancers and the development of new therapeutic targets (Bienz and Clevers, (2000) Cell 103: 311-20).

  For example, in recent years, new approaches to cancer treatment using gene-specific siRNA have been tried in clinical studies (Bumcrot D et al., Nat Chem Biol 2006 Dec, 2 (12): 711-9). RNAi is considered to have already gained a position in the main technology base (Putral LN et al., Drug News Perspect 2006 Jul-Aug, 19 (6): 317-24; Frantz S, Nat Rev Drug Discov 2006 Jul, 5 (7): 528-9; Dykxhoorn DM et al., Gene Ther 2006 Mar, 13 (6): 541-52). Nevertheless, several challenges must be addressed before RNAi can be applied for clinical use. These challenges include the lack of RNA stability in vivo (Hall AH et al., Nucleic Acids Res 2004 Nov 15, 32 (20): 5991-6000, Print 2004; Amarzguioui M et al., Nucleic Acids Res 2003 Jan 15, 31 (2): 589-95), reduced drug toxicity (Frantz S, Nat Rev Drug Discov 2006 Jul, 5 (7): 528-9), delivery mode, accuracy of siRNA or shRNA used Sequence and cell type specificity. It is a well-known fact that there can be toxicities associated with silencing partially homologous genes or inducing universal gene suppression by activating inter-feron responses (Judge AD et al. Nat Biotechnol 2005 Apr, 23 (4): 457-62, Epub 2005 Mar 20; Jackson AL & Linsley PS, Trends Genet 2004 Nov, 20 (11): 521-4). Therefore, double-stranded molecules that target cancer-specific genes that do not have harmful side effects are necessary for the development of anticancer agents.

  Previously, we performed detailed and accurate analysis of pancreatic cancer expression profiles using laser microdissection and a broad genomic cDNA microarray of approximately 27,000 genes to purify the cancer cell population. (Nakamura et al., (2004) Oncogene, 23: 2385-400). Among the genes that we identified as being transactivated in pancreatic cancer cells (WO2004 / 031412), we focused on KIAA0101 as a target molecule for cancer treatment.

  PCNA (proliferating cell nuclear antigen) not only affects cell cycle progression through interaction with several cell cycle proteins, but is also important for DNA replication and repair (Prelich et al., (1987) Nature, 326: 471-5; Wyman and Botchan (1995) Curr Biol, 5: 334-7; Warbrick et al., (2000) Bioessays, 22: 997-1006). Its crystal structure is the same trimer protein in which PCNA is circular and functions as a clamping base necessary to mobilize DNA proteins involved in DNA synthesis or metabolism, such as DNA polymerase, DNA ligase, etc. (Krishna et al., (1994) Cell, 79: 1233-43). PCNA interacts with a number of DNA replication / repair enzymes, and some proteins have a conserved PCNA binding motif (PIP box, QXXL / I / MXXF / Y) to interact with PCNA (Jonsson et al. al., (1998) EMBO J, 17: 2412-25). PCNA overexpression is characteristic of cell proliferation, and clinical PCNA is useful as a general proliferation marker, especially in the prognosis of tumor development, similar to Ki67 / MIB-1 (Haitel et al., (1997) Am J Clin Pathol, 107: 22935).

  KIAA0101 was identified as p15PAF (PCNA-related factor) that binds to PCNA protein by yeast two-hybrid screening (Yu et al., (2001) Oncogene 20: 484-9) and was conserved to interact with PCNA Has a PIP box. However, its function has not been elucidated and the mode of cell proliferation or cancer progression by KIAA0101-PCNA interaction remains unclear (Yu et al., (2001) Oncogene, 20: 484-9; Simpson et al. (2006) Exp Cell Res. 312: 73-85).

  We are based on broad-genomic cDNA microarray analysis as well as RT-PCR and immunohistochemical analysis, and are new members of the REG family in cancer cells (eg, in PDAC cells), and / or The overexpression of KIAA0101 is reported here. In addition, the inventors have discovered that siRNA is used to reduce dramatic cell survival by knocking down endogenous REG4 and / or KIAA0101 expression in cancer (eg, PDAC) cell lines. Consistently, the addition of recombinant REG4 and / or KIAA0101 to the medium enhanced the growth of cancer (eg, PDAC cell lines in a dose-dependent manner. Monoclonal antibodies against REG4 and / or KIAA0101 have a growth-promoting effect. In combination, the growth of cancer (eg, PDAC cells) was significantly attenuated, indicating that REG4 and KIAA0101 are promising tumor markers for screening early stage cancers, including PDAC, And, for example, neutralization of REG4 and / or KIAA0101 with an antibody, inhibitory polypeptide or inhibitory polynucleotide (eg, siRNA) is abnormal (ie, abnormally high) REG4 and / or KIAA0101 overexpression and An effective prophylactic or therapeutic method is provided for cancers including PDAC caused by intracellular signal transduction.

  It is an object of the present invention to provide compounds useful for the treatment and / or prevention of cancer caused by abnormal (ie abnormally high) REG4 and / or KIAA0101 overexpression and / or intracellular signaling. Alternatively, it is an object of the present invention to provide methods and pharmaceutical compositions for treating or preventing cancer, such as pancreatic cancer, prostate cancer, breast cancer or bladder cancer, or both, using the compound. .

  The inventors confirm that suppression of REG4 or KIAA0101 expression using, for example, siRNA can achieve suppression of cancer growth. Accordingly, an object of the present invention provides a method for treating or preventing pancreatic cancer in a subject comprising administering to the subject a composition comprising a small interfering RNA (siRNA) that inhibits REG4 expression. That is. In further embodiments, the present invention is mediated by abnormal (ie, abnormally high) KIAA0101 overexpression and / or intracellular signaling comprising administering to said subject a composition comprising siRNA that inhibits expression of KIAA0101 For example, to provide or treat cancer in a subject, such as pancreatic cancer, prostate cancer, breast cancer or bladder cancer.

  The present invention also provides a pharmaceutical compound for treating or preventing pancreatic cancer comprising a pharmaceutically effective amount of a small interfering RNA (siRNA) that inhibits the expression of REG4 as an active ingredient and a pharmaceutically acceptable carrier. provide. The present invention further includes an abnormal (ie, abnormally high) KIAA0101 excess, eg, pancreatic cancer, prostate cancer, breast cancer or bladder cancer, comprising a pharmaceutically effective amount of siRNA that inhibits expression of KIAA0101 as an active ingredient Pharmaceutical compounds for treating or preventing cancer mediated by expression and / or intracellular signaling are provided.

  The invention further relates to the use of small interfering RNA (siRNA) that inhibits the expression of REG4 to produce a pharmaceutical composition for treating or preventing pancreatic cancer, and abnormal (ie, abnormally high) KIAA0101 Small molecule interference that inhibits the expression of KIAA0101 to produce a pharmaceutical composition for treating or preventing cancer (eg, pancreatic cancer, prostate cancer, breast cancer or bladder cancer) by overexpression and / or intracellular signaling Provide the use of RNA (siRNA). In certain embodiments, a preferred siRNA comprises the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 32 as a target sequence.

  The present invention relates to a double-stranded molecule comprising a sense strand and an antisense strand, the sense strand comprising a ribonucleotide sequence corresponding to the target sequence of SEQ ID NO: 5 or SEQ ID NO: 32, wherein the antisense strand is the sense strand A ribonucleotide sequence complementary to the strand, the sense strand and the antisense strand hybridize to each other to form the double-stranded molecule and when introduced into a cell expressing the REG4 gene or KIAA0101 gene, Double-stranded molecules inhibit the expression of the gene.

  In addition, REG4 has been identified to function as an autocrine or paracrine growth factor and mediated Akt signaling pathway. Furthermore, the inventors find that anti-REG4 antibodies neutralize the cell growth activity of REG4 in order to attenuate the proliferation of pancreatic cancer cells.

Accordingly, it is an object of the present invention to provide a method for treating or preventing pancreatic cancer in a subject comprising administering to the subject an anti-REG4 antibody or fragment thereof that neutralizes REG4 activity. The present invention also provides a composition comprising a pharmaceutically effective amount of an anti-REG4 antibody or fragment thereof that neutralizes the activity of REG4 as an active ingredient and a pharmaceutically acceptable carrier, that is, a pharmaceutical for treating or preventing pancreatic cancer A composition is provided. The present invention further provides the use of an anti-REG4 antibody or fragment thereof that neutralizes the activity of REG4 for the manufacture of a pharmaceutical composition for treating or preventing pancreatic cancer. In certain embodiments, the anti-REG4 antibody is a monoclonal antibody. In one embodiment, the anti-REG4 antibody comprises VH and VL chains, each VH and VL chain comprising CDR amino acid sequences designated CDR1, CDR2 and CDR3 separated by a framework amino acid sequence, each VH and The amino acid sequence of each CDR in the VL chain is as follows:
VH CDR1: SYWMH (SEQ ID NO: 20),
VH CDR2: NIYPGSGSTNYD (SEQ ID NO: 21),
VH CDR3: GGLWLRVDY (SEQ ID NO: 22),
VL CDR1: SASSSVSYMH (SEQ ID NO: 23),
VL CDR2: DTSKLAS (SEQ ID NO: 24), and
VL CDR3: QQWSSNPF (SEQ ID NO: 25).

The present invention also relates to a method for the treatment and / or prevention of pancreatic cancer comprising administering an antibody comprising each VH and VL chain, referred to as CDR1, CDR2 and CDR3 separated by framework amino acid sequences The amino acid sequence of each CDR in each VH and VL chain, including the CDR amino acid sequence, is as follows:
VH CDR1: SYWMH (SEQ ID NO: 20),
VH CDR2: NIYPGSGSTNYD (SEQ ID NO: 21),
VH CDR3: GGLWLRVDY (SEQ ID NO: 22),
VL CDR1: SASSSVSYMH (SEQ ID NO: 23),
VL CDR2: DTSKLAS (SEQ ID NO: 24) and
VL CDR3: QQWSSNPF (SEQ ID NO: 25).

  In addition, the present invention provides an inhibitory polypeptide comprising QKGIGEFF (SEQ ID NO: 46). In certain desirable embodiments, the amino acid sequence is VRPTPKWQKGIGEFFRLSPK (SEQ ID NO: 44) or TPKWQKGIGEFFRLSP (SEQ ID NO: 45). The present invention further provides pharmaceuticals or methods using these inhibitory polypeptides for the treatment and / or prevention of cancer.

  The present invention also encodes an inhibitory polypeptide comprising QKGIGEFF (SEQ ID NO: 46) (eg, at least VRPTPKWQKGIGEFFRLSPK (SEQ ID NO: 44), TPKWQKGIGEFFRLSP (SEQ ID NO: 45) amino acid sequence fragment) It relates to a method for the treatment and / or prevention of cancer comprising the step of administering an inhibitory polypeptide having a polynucleotide. Furthermore, the present invention relates to the use of a polypeptide of the invention or the use of a nucleotide encoding it in the manufacture of a pharmaceutical composition for the treatment and / or prevention of cancer.

  These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples. However, it should be understood that the foregoing summary of the invention and the following detailed description are preferred embodiments, and are not limited to the invention or any other embodiments of the invention.

REG4 mRNA and protein expression levels in PDAC cells. (A) REG4 and quantification in microdiverted normal pancreatic ductal epithelial cells (NPD) and microdissected PDAC cells (lanes 1-9) compared to normal vital organs including heart, lung, liver, kidney and brain RT-PCR analysis of TUBA as a control. In immunohistochemical experiments with (B, C, D) anti-REG4 antibodies, strong staining was observed in PDAC cells. Positive staining for REG4 was observed in cytoplasmic granules (B) and cytoplasmic membrane (C) suggesting REG4 secretion. In normal pancreatic tissue, acinar cells showed very slight staining, but not normal ductal epithelial cells and islet cells (D). Knockdown of REG4 expression by siRNA resulted in attenuation of pancreatic cancer cell growth. (A) The knockdown effect of the REG4 transcript was confirmed by semi-quantitative RT-PCR using cells transfected with siRNA expression vector (REG4-si2) and negative control vector (siEGFP) against REG4. . β2-MG was used to quantify RNA. EGFPsi did not show any effect on the level of REG4 transcript, whereas REG4-si2 showed a strong knockdown effect. (B, C) In transfection of REG4-si2 vector into SUIT-2, the number of viable cells measured by MTT assay (B) and colony formation number (C) does not show any knockdown effect on REG4 Compared to cells transfected with the vector, it caused a dramatic decrease. In the MTT assay (B), the bar graph is the average of absorbance from 3 experiments after 7 days of culture with Geneticin, the bar is the SD value, * is p <0.01 (Student t test) Show. The growth promoting effect of rhREG4 in PDAC cells. (A) Bioactive rhREG4 protein was produced by mammalian cells (FreeStyle ™ 293). rhREG4 was purified and analyzed by SDS-PAGE with Coomassie staining (left) and Western blot using antibodies specific for REG4 (right). (B) PK45P cells were incubated with 0, 0.1, 1 and 10 nM rhREG4 supplemented with 1% FBS. rhREG4 treatment promoted cell proliferation of PK45P cells in a dose-dependent manner. Data points represent the mean ratio of absorbance from 3 experiments compared to sample 0, bars represent SD values, * represents p <0.01 (Student t test). (C) Akt (Ser473) phosphorylation was dose-dependently enhanced by treatment of PK45P cells with 0, 0.1, 1 and 10 nM rhREG4. Phosphorylated Akt was detected by Western blot using an antibody specific for phosphorylated Akt (Ser473), and the blot was re-detected with an antibody against Akt to assess the total level of Akt. Anti-REG4 monoclonal antibody neutralization and growth inhibitory effect. (A) The binding ability of the anti-REG4 antibody was evaluated by Western blot with anti-REG4pAb using SUIT-2 medium using immunoprecipitation. Anti-REG4 mAb and pAb immunoprecipitated REG4 from SUIT-2 medium with high affinity. (B) Anti-REG4 mAb treatment stopped the growth promoting effect of rhREG4. PK45P was stimulated with 10 nM rhREG4 in the presence or absence of anti-REG4 mAb. The bar graph is the average ratio of absorbance from three experiments compared to the culture sample in (−) medium, the bar indicates the SD value, and * indicates p <0.01 (Student t test). (C) Effect of various concentrations of anti-REG4 mAb on the growth of SUIT-2 (REG4-positive) and MIAPaCa-2 (REG4-negative). Each cell line was incubated in the presence of various concentrations of anti-REG4 mAb. Anti-REG4 mAb treatment did not affect the cell growth of MIAPaCa-2 that did not express REG4 at all, but suppressed SUIT-2 cell growth in a dose-dependent manner. Data points are average ratios of absorbance from three experiments compared to culture samples in (−) medium, bars indicate SD values, * indicates p <0.01 (Student t test). (D) Anti-REG4 mAb treatment stopped phosphorylation of Akt stimulated by rhREG4. PK45P cells were treated with 10 nM rhREG4 in the presence or absence of anti-REG4 mAb. Phosphorylation of Akt was assessed by Western blot using an antibody specific for phosphorylated Akt (Ser473), and the blot was redetected with an antibody against Akt to assess the total level of Akt. 1, Non-stimulation: 2, 10 nM rhREG4: 3, 10 nM rhREG4 + anti-REG4 mAb. Anti-REG4 antibody treatment suppressed the growth of pancreatic cancer cells in vivo. (A) Tumor volume inoculated in nude mice was twice per week (300 μg / mouse ip.) Or control antibody (normal) during treatment with anti-REG4 antibody (34-1 mAb, n = 8) Mouse IgG, n = 9) were evaluated. Treatment of 34-1 mAb (filled circles and bold lines) resulted in a significant decrease in tumor volume compared to control IgG (P = 0.0598). (B) Tumors were weighed 30 days after initial treatment with anti-REG4 antibody (34-1 mAb) or control antibody. Treatment of 34-1 mAb (black box) resulted in a significant reduction in tumor weight compared to the control antibody (P = 0.0489). Overexpression of REG4 has been implicated in the resistance of pancreatic cancer to gamma radiation. (A) pCAGGSnHC-REG4-HA was transfected into a pancreatic cancer cell line PK45P that did not express REG4. After selection of geneticin resistant cells, the expression of recombinant REG4 in the cell line was confirmed by Western blotting. (B) After 48 hours of plain incubation, REG4 expression clones (C1-6, C2-6 and C10) or control clones (M1, M3 and M6) were 1, 5, using 60 cobalt sources. Irradiated with 10 or 30 Gy. After 48 hours, viable cells were measured using cell counts and the relative ratio of absorbance (each irradiation) / absorbance (non-irradiation) was evaluated. (C) After gamma irradiation of 0, 1 or 5 Gy, gamma-ray induced apoptosis was assessed by detecting the sub-G1 fraction using a flow cytometer. REG4 overexpression has been implicated in gemcitabine resistance in pancreatic cancer. (A) After 48 hours preincubation, REG4 expressing clones (C1-6, C2-6, C10) or control clones (M1, M3, M6) were treated with 0.1-100,000 nM gemcitabine for 48 hours . After incubation, viable cells were measured by counting cells and the relative ratio of absorbance (each treatment) / absorbance (non-treated control) was evaluated. (B) After 48 hours of 10 nM or 50 nM gemcitabine treatment, apoptosis was assessed by detecting the sub-G1 fraction using a flow cytometer. Immunostaining for REG4 in pancreatic adenocarcinoma specimens undergoing neo-CRT. (A), (B) Neoadjuvant chemoradiation therapy (neo-CRT) sensitive pancreatic adenocarcinoma specimens showed low or no expression of REG4. (C), (D) Neo-CRT insensitive pancreatic adenocarcinoma specimens showed strong expression of REG4. Results of KIAA0101 overexpression in pancreatic cancer cells. (A) Semi-quantitative RT-PCR confirmed that KIAA0101 expression was up-regulated in microdissected normal pancreatic duct cells and microdissected pancreatic cancer cells compared to normal pancreatic tissues . TUBA expression was used as a quantitative control. (B) Northern blot analysis showed no expression in vital organs (lane 7-11) including heart, lung, liver, kidney and brain, but in pancreatic cancer cell line (lane 1-6) Strong expression of was shown. (C) Immunohistochemical experiment using anti-KIAA0101 antibody. Acinar cells and normal ductal epithelium in normal pancreatic tissue showed no staining, but strong staining was observed in the nuclei of pancreatic cancer cells (arrowhead x400). Results of the effect of KIAA0101 knockdown by siRNAs on pancreatic cancer cell proliferation. (A) Two siRNA expression vectors (# 759si) specific for the KIAA0101 transcript and EGFP siRNA expression vector (EGFPsi) as a negative control were transfected into KLM-1 cells. The knockdown effect on the KIAA0101 transcript was confirmed by RT-PCR using β2MG expression as a quantitative control. EGFPsi did not show any effect at the KIAA0101 transcript level, but transfection with # 759si showed a strong knockdown effect. (B) Transfection of # 759si vector resulted in a dramatic reduction in the number of viable cells as measured by colony-number formation, compared to transfection using siRNA expression vectors that did not have a knockdown effect on KIAA0101. It was. (C) Transfection of # 759si vector resulted in a dramatic reduction in the number of viable cells as measured by MTT assay. ABS on the Y-axis in the MTT assay means the mean absorbance at 490 nm measured by a microplate reader and 630 nm as a reference. Results of exogenous overexpression of KIAA0101 resulting in promotion of cancer cell growth and transformation of NIH3T3. (A) Western blot analysis of 6 PK45P-derived cells (clone 1-6) constitutively expressing exogenous KIAA0101 and mock vectors (mock 1-4) transfected. Exogenous KIAA0101 expression induction was confirmed using an anti-HA tag antibody. ACTB was used as a loading control. (B) Proliferation measurement by MTT assay showed that 6 KIAA0101 clones (1-6, solid line) proliferated significantly more rapidly than 2 pseudo clones (1-4, dashed line). The X-axis and Y-axis indicate the relative growth rate calculated by the absorbance of the diameter by comparison with the number of days after sowing and the absorbance value on the first day as a control. Each mean value is plotted with an error bar representing standard error. All these experiments were performed in triplicate. (C) Three KIAA0101 overexpressing clones and three pseudoclones were established from NIH3T3 that did not express endogenous KIAA0101 mouse homologues. Three KIAA0101 overexpressing NIH3T3 clones (1-3) and pseudo-NIH3T3 clones were inoculated into the left and right flank of 8-week-old nude mice, respectively. Four weeks later, only KIAA0101 overexpressing NIH3T3 cells formed a tumor on the right side of nude mice. (D) Each tumor was immunostained with anti-KIAA0101 antibody and showed exogenous KIAA0101 expression. The KIAA0101 protein bound to several DNA replication proteins. (A) Immunoprecipitation fractions separated on an SDS-PAGE gel followed by silver staining show that some proteins were immunoprecipitated with KIAA0101 protein in cancer cell lysates compared to the control sample results. It was. Each differential band was analyzed by MALDI-TOF system after trypsin digestion in the gel, and they were identified as PCNA, POLD1 (polymerase δp125 subunit) and FEN1 (flap endonuclease-1). (B) These interactions were confirmed by immunoprecipitation experiments. All of these proteins are involved in DNA replication, and like KIAA0101, POLD1 and FEN1 also bind to PCNA. Inhibition of interaction between KIAA0101 and PCNA by cell-permeable dominant negative peptide. (A) Two dominant negative peptides (PIP20, PIP16) containing a PIP box ({q} KG {i} GE {ff} / SEQ ID NO: 21 shown in parentheses) and its conserved substituted alanine A mutant peptide (PIP20mt, PIP16mt) with a PIP box ({a} KG {a} GE {aa} / SEQ ID NO: 37 shown in parentheses) was designed and arginine to promote cell permeability ( R) Repeat was joined. (B) In in vitro experiments, immunoprecipitation confirmed the suppression of the interaction between PCNA and KIAA0101 by PIP20 treatment, but PIP20mt and scrambled peptide did not affect the interaction between PCNA and KIAA0101. (C) PIP20mut and scrambled peptide did not show cell growth suppression, but PIP20 treatment suppressed KIAA0101 cell growth in a dose dependent on KIAA0101 expression. On the other hand, PIP20 did not affect the growth of mouse normal cell line NIH3T3 cells that do not express the homologue of human KIAA0101. (D) Short PIP peptides (PIP16 and PIP16mt) were designed by deleting the N and C-terminal 4 residues in the retained PIP box motif, while PIP16 treatment strongly suppressed cancer cell growth, So PIP16 also affected the proliferation of NIH3T3.

Detailed description of preferred embodiments
Small interfering RNA :
The present invention is surprising that small interfering RNA (siRNA) selective for REG4 and / or KIAA0101 is effective in inhibiting cell proliferation of various cancer cells, including pancreatic cancer, prostate cancer, breast cancer and bladder cancer Based in part on the discovery. In particular, it was a surprising discovery that PDAC can be inhibited or suppressed by inhibition of the REG4 gene. The invention described herein is based in part on these discoveries.

  The present invention provides a method of inhibiting cell proliferation. Among the methods provided are methods comprising contacting a cell with a composition comprising a small interfering RNA (siRNA) that inhibits expression (eg, transcription or translation) of REG4 or KIAA0101. The invention also provides a method of inhibiting tumor cell growth in a subject. Such a method comprises administering to a subject a composition comprising a small interfering RNA (siRNA) that specifically hybridizes to a REG4 or KIAA0101 sequence. Another aspect of the invention provides a method of inhibiting the expression of the REG4 or KIAA0101 gene in cells of a biological sample.

  Gene expression may be inhibited by introducing a sufficient amount of a double-stranded ribonucleic acid (RNA) molecule into the cell to inhibit the expression of the REG4 or KIAA0101 gene. Another aspect of the invention relates to products comprising nucleic acid sequences and vectors, and compositions comprising them, for example, useful in the provided methods. Among the products provided are siRNA molecules that have the property of inhibiting expression of REG4 or KIAA0101 gene when introduced into a cell expressing REG4 gene or KIAA0101 gene. Among such molecules, the sense strand includes a sense and antisense strand, the sense strand includes a ribonucleotide sequence corresponding to the REG4 or KIAA0101 target sequence, and the antisense strand includes a ribonucleotide sequence complementary to the sense strand. There are molecules. The sense and antisense strands of the molecule hybridize to each other to form a double stranded molecule.

  As used herein, the term “organism” means any living organism that consists of at least one cell. The organism may be as simple as, for example, a eukaryotic single cell or as complex as a mammal including a human.

  As used herein, the term “biological sample” refers to an entire organism, or a tissue, cell, or component thereof (eg, but not limited to blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva). Bodily fluids including amniotic fluid, umbilical cord blood, urine, vaginal fluid, and semen). A “biological sample” further refers to a homogenate, lysate, extract, cell culture, or tissue culture, or fraction or portion thereof, prepared from an entire organism or cells, tissues, or components thereof. means. Finally, “biological sample” means a medium such as a nutrient medium or gel in which an organism has been grown, containing cellular components such as proteins or nucleic acid molecules.

  The invention features a method of inhibiting cell proliferation. Cell proliferation is inhibited by contacting the cells with a composition of REG4 or KIAA0101 small interfering RNA (siRNA). In addition, the cells are contacted with a transfection facilitating agent. The cells are provided in vitro, in vivo, or ex vivo. The subject is a mammal (eg, a human, non-human primate, mouse, rat, dog, cat, horse, or cow). The cell is a pancreatic duct cell. Alternatively, the cell is a tumor cell (ie, a cancer cell) such as a cancer cell or an adenocarcinoma cell. For example, the cells are pancreatic cancer cells, particularly pancreatic ductal adenocarcinoma cells, prostate cancer cells, breast cancer cells or bladder cancer cells. Inhibiting cell proliferation means that treated cells grow slower or have less viability than untreated cells. Cell proliferation is measured by proliferation assays well known in the art.

  The terms “polynucleotide” and “oligonucleotide” are used interchangeably herein and are represented by their generally accepted single letter codes unless otherwise indicated. The term refers to a nucleic acid (nucleotide) polymer in which one or more nucleic acids are linked by ester bonds. A polynucleotide or oligonucleotide can consist of DNA, RNA, or a combination thereof.

  As used herein, the term “duplex molecule” refers to, for example, small interfering RNA (siRNA, eg, double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and small interfering DNA. Means nucleic acid molecules that inhibit the expression of target genes including / RNA (siD / RNA, eg, DNA and RNA chimeric duplexes (dsD / RNA) or small hairpin DNA and RNA chimeras (shD / RNA)) .

  The term “siRNA” refers to a double-stranded RNA molecule that prevents translation of a target mRNA. Standard techniques for introducing siRNA into cells are used, including techniques where the template for transcription of RNA is DNA. The siRNA comprises a sense REG4 or KIAA0101 nucleic acid sequence, an antisense REG4 or KIAA0101 nucleic acid sequence, or both. The siRNA comprises two complementary molecules, or one transcript, for example, a hairpin that induces the production of microRNA (miRNA) in certain embodiments, sense and complementary antisense Constructed to have both sequences. The siRNA may be dsRNA or shRNA.

  As used herein, the term “dsRNA” refers to a construct of two RNA molecules that contain sequences that are complementary to each other and anneal to each other through complementary sequences to form a double-stranded RNA molecule. . Two strands of nucleotide sequences are RNA molecules having nucleotide sequences selected from non-coding regions of the target gene as well as “sense” or “antisense” RNA selected from sequences encoding the protein of the target gene sequence May also be included.

  As used herein, the term “shRNA” refers to an siRNA comprising a first region and a second region (ie, sense and antisense strands) that are complementary to each other and having a stem-loop structure. The degree of complementarity and orientation of the regions is sufficient for bases to pair between the regions, the first region and the second region are joined by a loop region, and the loop is a nucleotide (or nucleotide in the loop region) Resulting in a lack of base pairing between the analogues). The loop region of shRNA is a single-stranded region interposed between the sense strand and the antisense strand, and is sometimes referred to as “intervening single strand”.

  As used herein, the term “siD / RNA” refers to a double-stranded polynucleotide molecule composed of both RNA and DNA, including RNA and DNA hybrids and chimeras, that prevents translation of the target mRNA. To do. In this specification, a hybrid refers to a molecule in which a polynucleotide composed of DNA and a polynucleotide composed of RNA hybridize with each other to form a double-stranded molecule. Chimera, on the other hand, indicates that one or both of the strands that make up a double-stranded molecule can include RNA and DNA. Standard techniques for introducing siD / RNA into cells are used. The siD / RNA includes a CX sense nucleic acid sequence (also referred to as “sense strand”), a CX antisense nucleic acid sequence (also referred to as “antisense strand”), or both. siD / RNA may be constructed such that a single transcript has both sense and complementary antisense nucleic acid sequences derived from the target gene, eg, a hairpin. The siD / RNA may be either dsD / RNA or shD / RNA.

  As used herein, the term “dsD / RNA” refers to two molecules that contain sequences that are complementary to each other and anneal to each other through complementary sequences to form a double-stranded polynucleotide molecule. Means a construct. The two-strand nucleotide sequence has a nucleotide sequence selected from a non-coding region of the target gene as well as a “sense” or “antisense” polynucleotide sequence selected from the sequence encoding the protein of the target gene sequence. Polynucleotides may also be included. One or both of the two molecules that make up dsD / RNA are composed of both RNA and DNA (chimeric molecules), or else one of the molecules is composed of RNA and the other is DNA Constructed (hybrid duplex).

  As used herein, the term “shD / RNA” means siD / RNA comprising a first region and a second region (ie, sense strand and antisense strand) that are complementary to each other and having a stem-loop structure. The degree of complementarity and orientation of the regions is sufficient for bases to pair between the regions, the first region and the second region are joined by a loop region, and the loop is a nucleotide (or nucleotide in the loop region) Resulting in a lack of base pairing between the analogues). The loop region of shD / RNA is a single-stranded region interposed between the sense strand and the antisense strand, and is sometimes referred to as “intervening single strand”.

  This method is used, for example, to alter the expression of genes in cells that are abnormally upregulated in the expression of REG4 or KIAA0101, such as malignant transformation of cells. When siRNA binds to the REG4 or KIAA0101 transcript in the target cell, the production of REG4 or KIAA0101 by the cell is reduced. The length of the oligonucleotide is at least about 10 nucleotides and may be as long as a natural REG4 or KIAA0101 transcript. Preferably, the oligonucleotide is less than about 75, about 50, or about 25 nucleotides in length. Most preferably, the oligonucleotide is about 19 to about 25 nucleotides in length. Examples of REG4 or KIAA0101 siRNA oligonucleotides that inhibit REG4 or KIAA0101 expression in mammalian cells include target sequences, eg, oligonucleotides comprising nucleotides of SEQ ID NO: 5 or 32, respectively.

  Methods for designing double stranded RNA having the ability to inhibit gene expression in target cells are known. (See, eg, US Pat. No. 6,506,559, which is incorporated herein by reference in its entirety). For example, a computer program for designing siRNA is available from the Ambion website (www.ambion.com/techlib/misc/siRNA_finder.html). A computer program available from Ambion, Inc. selects nucleotide sequences for siRNA synthesis based on the following procedure.

siRNA target site selection
1. Scan downstream for AA dinucleotide sequences starting from the AUG start codon of the transcript. Record the occurrence of each AA and 3 ′ adjacent 19 nucleotides as potential siRNA target sites. Tuschl et al., Genes Dev 13 (24): 3191-7 (1999), is rich in regulatory protein binding sites in the 5 'and 3' untranslated regions (UTR) and the region near the start codon (within 75 bases). It is recommended not to design siRNAs because of the possibility. UTR binding proteins and / or translation initiation complexes may interfere with the binding of the siRNA endonuclease complex.
2. Compare potential target sites with appropriate genomic databases (human, mouse, rat, etc.) and exclude any target sequences that have significant homology with other coding sequences from consideration. It is proposed to use BLAST found on the NCBI server at www.ncbi.nlm.nih.gov/BLAST/.
3. Select a qualified target sequence for synthesis. To assess, it is typical to select several target sequences along the length of the gene.

  Also included in the present invention are isolated nucleic acid molecules comprising a target sequence, eg, a nucleotide sequence of SEQ ID NO: 5 or 32, or a nucleic acid molecule complementary to the nucleotide sequence of SEQ ID NOs: 5 and 32. As used herein, an “isolated nucleic acid” is a nucleic acid that has been removed from its original environment (eg, the natural environment if it is naturally occurring), and thus has been artificially altered from its natural state. It is a nucleic acid. In the present invention, isolated nucleic acids include DNA, RNA, and derivatives thereof. When the isolated nucleic acid is RNA or a derivative thereof, the base “t” is replaced with “u” in the nucleotide sequence.

  As used herein, the term “complementary” refers to Watson-Crick or Hoogsteen-type base pairing between nucleotide units of a nucleic acid molecule, and the term “binds” It means a physical or chemical interaction between a nucleic acid or compound, or a related nucleic acid or compound, or a combination thereof. If the polynucleotides contain modified nucleic acids and / or non-phosphodiester bonds, these polynucleotides can bind to each other in a similar manner. In general, complementary nucleic acid sequences hybridize under appropriate conditions to form a stable duplex with little or no mismatch. For the purposes of the present invention, two sequences with 5 or fewer mismatches are considered complementary. Furthermore, the sense and antisense strands of the isolated nucleotides of the present invention can form double stranded nucleotides or hairpin loop structures by hybridization.

  In preferred embodiments, such duplexes do not contain more than one mismatch for every 10 matches. In particularly preferred embodiments where the duplex strands are completely complementary, such duplexes do not contain mismatches. For REG4, the nucleic acid molecule is less than 1518 nucleotides in length, and for KIAA0101, the nucleic acid molecule is less than 1508 nucleotides in length. For example, the nucleic acid molecule is less than about 500, about 200, or about 75 nucleotides in length. Also included in the invention are vectors containing one or more of the nucleic acids described herein, and cells containing the vectors. The isolated nucleic acids of the invention are useful for siRAN against REG4 or KIAA0101, or DNA encoding siRNA. When the nucleic acid is used for siRNA or DNA encoding it, the sense strand is preferably longer than about 19 nucleotides, more preferably longer than 21 nucleotides.

  The double-stranded molecule of the present invention may comprise one or more modified nucleotides and / or non-phosphodiester bonds. Chemical modifications well known in the art can increase the stability, effectiveness, and / or cellular uptake of double-stranded molecules. Those skilled in the art are also aware of other types of chemical modifications that can be incorporated into the molecule (WO03 / 070744; WO2005 / 045037). In one embodiment, the modifications can be used to provide improved resistance to degradation or improved uptake. Examples of such modifications include phosphorothioate linkages, 2'-O-methyl ribonucleotides (especially on the sense strand of double-stranded molecules), 2'-deoxy-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal bases" (Universal base) "nucleotides, 5'-C-methyl nucleotides, and inverted deoxyabasic residue incorporation (US20060122137).

  In another embodiment, the modifications can be used to improve the stability of the double stranded molecule or to increase its targeting efficiency. Modifications include chemical cross-linking between two complementary strands of a double-stranded molecule, chemical modification of the 3 ′ or 5 ′ end of one strand of a double-stranded molecule, sugar modification, nucleobase modification and / or backbone modification, 2-fluoro modified ribonucleotides and 2'-deoxyribonucleotides (WO2004 / 029212) are included. In another embodiment, the modifications can be used to increase or decrease the affinity for complementary nucleotides in the target mRNA and / or in the complementary double-stranded molecular strand (WO2005 / 044976). For example, unmodified pyrimidine nucleotides can be replaced by 2-thio, 5-alkynyl, 5-methyl, or 5-propynyl pyrimidine. In addition, unmodified purines can be replaced by 7-deza, 7-alkyl, or 7-alkenyl-purines. In another embodiment, if the double-stranded molecule is a double-stranded molecule with a 3 ′ overhang, the 3 ′ terminal nucleotide overhanging nucleotide can be replaced by deoxyribonucleotides (Elbashir SM et al., Genes Dev 2001 Jan 15, 15 (2): 188-200). More specifically, it can be obtained from a public document such as US20060234970. The invention is not limited to these examples, and any known chemical modification can be made to the double-stranded molecule of the invention as long as the resulting molecule retains the ability to inhibit target gene expression. Can be used.

  Furthermore, the double-stranded molecule of the present invention may comprise both DNA and RNA, such as dsD / RNA or shD / RNA. Specifically, a hybrid polynucleotide of a DNA strand and an RNA strand or a DNA-RNA chimera polynucleotide exhibits increased stability. A mixture of DNA and RNA, that is, a hybrid double-stranded molecule consisting of a DNA strand (polynucleotide) and an RNA strand (polynucleotide), or a chimeric type containing both DNA and RNA in one or both of the single strand (polynucleotide) Double-stranded molecules and the like can also be formed to improve the stability of the double-stranded molecule. As long as the hybrid of DNA strand and RNA strand has an activity to inhibit the expression of the gene when introduced into a cell that expresses the target gene, the sense strand is DNA and the antisense strand is RNA or It may be a hybrid that is the opposite. Preferably, the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA. In addition, both the sense strand and the antisense strand are composed of DNA and RNA as long as the chimeric double-stranded molecule has an activity of inhibiting the expression of the gene when introduced into a cell that expresses the target gene. One of the sense strand and the antisense strand may be composed of DNA and RNA.

In order to improve the stability of a double-stranded molecule, the molecule preferably contains as much DNA as possible, whereas in order to induce inhibition of the expression of the target gene, the molecule must have sufficient inhibition of expression. It is required to be RNA within the range to be induced. As a preferred example of the chimeric double-stranded molecule, the upstream partial region of the double-stranded molecule (that is, the region adjacent to the target sequence inside the sense or antisense strand or its complementary sequence) is RNA. Preferably, the upstream partial region represents the 5 ′ side (5 ′ end) of the sense strand and the 3 ′ side (3 ′ end) of the antisense strand. That is, in a preferred embodiment, both the region adjacent to the 3 ′ end of the antisense strand, or the region adjacent to the 5 ′ end of the sense strand and the region adjacent to the 3 ′ end of the antisense strand consist of RNA. For example, the chimeric or hybrid double-stranded molecule of the present invention includes the following combinations.
Sense strand: 5 '-[DNA] -3'
3 '-(RNA)-[DNA] -5': antisense strand,
Sense strand: 5 '-(RNA)-[DNA] -3'
3 '-(RNA)-[DNA] -5': antisense strand and sense strand: 5 '-(RNA)-[DNA] -3'
3 '-(RNA) -5': antisense strand.
The upstream partial region is preferably a domain consisting of 9 to 13 nucleotides counted from the end of the target sequence inside the sense or antisense strand of the double-stranded molecule or the complementary sequence thereto. Furthermore, preferable examples of the above-described chimeric double-stranded molecule include a chain length of 19 to 21 nucleotides and a region at least in the upstream half of the polynucleotide (a 5 ′ region in the sense strand, 3 in the antisense strand). 'Side region' is RNA and the other half is DNA. In such a chimeric double-stranded molecule, the effect of inhibiting the expression of the target gene is much higher when the entire antisense strand is RNA (US20050004064).

  In the present invention, the double-stranded molecule may form a hairpin such as a short hairpin RNA (shRNA) and a short hairpin consisting of DNA and RNA (shD / RNA). shRNA or shD / RNA is a sequence of RNA or a mixture of RNA and DNA that produces a robust hairpin turn that can be used for silencing gene expression via RNA interference. An shRNA or shD / RNA contains a sense target sequence and an antisense target sequence on one single strand, and these sequences are separated by a loop sequence. In general, the hairpin structure is cleaved by cellular machinery to dsRNA or dsD / RNA, which subsequently binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA that matches the target sequence of dsRNA or dsD / RNA.

  The present invention is based in part on the discovery that the gene encoding REG4 is overexpressed in pancreatic ductal adenocarcinoma (PDACa) compared to non-cancerous pancreatic tissue, and the gene encoding KIAA0101 is non-cancerous tissue In comparison, it is based on the discovery that it is overexpressed in pancreatic cancer, prostate cancer cells, lung cancer cells and bladder cancer cells. The cDNA of REG4 (Genbank accession number: AY126670) is 1518 nucleotides long. On the other hand, the cDNA of KIAA0101 (Genbank accession number: NM — 014736) is 1508 nucleotides long. The nucleic acid and polypeptide sequences of REG4 are shown in SEQ ID NOs: 1 and 2, respectively, and in KIAA0101, they are shown in SEQ ID NOs: 39 and 40.

  The present invention discloses transfection of siRNA comprising SEQ ID NO: 5 resulting in growth inhibition of PDAC cell lines, and further transfection of siRNA comprising SEQ ID NO: 32 resulting in growth inhibition of pancreatic cancer cell lines.

A method of inhibiting cell proliferation .
The present invention relates to inhibiting cell proliferation, ie cancer cell proliferation, by inhibiting the expression of REG4 or KIAA0101. The expression of REG4 or KIAA0101 is inhibited by, for example, small interfering RNA (siRNA) that specifically targets the REG4 gene or KIAA0101 gene. The target of REG4 includes, for example, the nucleotide of SEQ ID NO: 5, and the target of KIAA0101 includes the nucleotide of SEQ ID NO: 32.

  The term “specifically inhibits” in the inhibitory polynucleotides and polypeptides herein means the ability of an agent or ligand to inhibit REG4 and / or KIAA0101 expression or biological function. . Specific inhibition typically involves expression of REG4 and / or KIAA0101 (eg transcription or translation) or measurable biological function (eg cell growth or proliferation, inhibition of apoptosis, intracellular from REG4 Signal, eg, EGF receptor / Akt / AP-1 signaling pathway for REG4, binding to PCNA or other intracellular protein for KIAA0101) is at least about 2 times background, preferably about 10 times, more preferably , About 100 times. Expression levels and / or biological function may be measured in a configuration comprising treated and untreated cells, cell populations before and after treatment. In certain embodiments, the expression or biological function of REG4 and / or KIAA0101 is completely inhibited. Typically, specific inhibition is a statistically significant decrease in REG4 / KIAA0101 expression or biological function using an appropriate statistical test (eg, p ≦ 0.05).

  In non-mammalian cells, double-stranded RNA (dsRNA) has been shown to have a powerful and specific silencing effect on gene expression, which is called RNA interference (RNAi) (Sharp PA. Genes Dev. 1999; 13 (2): 139-41.). dsRNA is processed into 20-23 nucleotide dsRNA called small interfering RNA (siRNA) by an enzyme containing RNase III motif. siRNAs, together with multicomponent nuclease complexes, specifically target complementary mRNAs (Hammond SM et. al., Nature. 2000; 404 (6775): 293-6; Hannon GJ. Nature. 2002; 418 ( 6894): 244-51.). In mammalian cells, siRNA composed of 20 or 21-mer dsRNA with 19 complementary nucleotides and 3 'terminal non-complementary thymidine or uridine dimer induces global changes in gene expression Without having been shown to have a gene-specific knockdown effect (Elbashir SM et al., Nature. 2001; 411 (6836): 494-8.).

  Cell proliferation is inhibited by contacting the cells with a composition comprising siRNA of REG4 or KIAA0101. The cells are further contacted with a transfection agent. Suitable transfection agents are known in the art. Inhibiting cell growth means that the cells grow at a slower rate or have reduced viability as compared to cells not exposed to the composition. Cell proliferation is measured by methods known in the art such as the MTT cell proliferation assay.

  REG4 or KIAA0101 siRNAs target a single target of the REG4 gene sequence or KIAA0101 gene sequence, respectively. Alternatively, siRNAs target multiple targets of REG4 or KIAA0101 gene sequences. For example, the composition comprises REG4 or KIAA0101 siRNA directed to two, three, four, five, or more target sequences of REG4 or KIAA0101. By REG4 or KIAA0101 target sequence is meant a nucleotide sequence that is identical to a portion of the REG4 gene or KIAA0101 gene (ie, a polynucleotide within the REG4 or KIAA0101 gene that is complementary to siRNA and equal in length). The target sequence can include the 5 ′ untranslated (UT) region, open reading frame (ORF), or 3 ′ untranslated region of the human REG4 or KIAA0101 gene. Alternatively, the siRNA is a nucleic acid sequence that is complementary to an upstream or downstream modulator of REG4 or KIAA0101 gene expression. Examples of upstream or downstream modulators include transcription factors that bind to the REG4 or KIAA0101 gene promoter, kinases or phosphatases that interact with REG4 or KIAA0101 polypeptides, REG4 or KIAA0101 promoters or enhancers.

  The REG4 or KIAA0101 siRNA that hybridizes to the target mRNA is encoded by the REG4 or KIAA0101 gene by binding to a single-stranded mRNA transcript in a linear fashion, thereby preventing translation and preventing protein expression Reduce or inhibit production of REG4 or KIAA0101 polypeptide products. Thus, the siRNA molecules of the invention can be defined by their ability to specifically hybridize with mRNA or cDNA from the REG4 or KIAA0101 gene under stringent conditions.

  In the present invention, the terms “hybridize” or “specifically hybridize” are used to refer to the ability of two nucleic acid molecules to hybridize under “stringent hybridization conditions”. The phrase “stringent hybridization conditions” refers to conditions, typically in complex nucleic acid mixtures, where a nucleic acid molecule hybridizes to its target sequence but does not detectably hybridize to other sequences. . Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Extensive guidance on nucleic acid hybridization can be found in Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10 ° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength, pH. Tm is the temperature (at a given ionic strength, pH, and nucleic acid concentration) at which 50% of the probe complementary to the target hybridizes with the target sequence at equilibrium (if the target sequence is present in excess) Tm occupies 50% of the probe in equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least twice background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions may be as follows: 50% formamide, 5x SSC, and 1% SDS, incubation at 42 ° C, or 5x SSC, 1% SDS, 65 Incubation at ° C and wash at 50 ° C in 0.2x SSC and 0.1% SDS.

  The siRNA of the invention is less than about 500, about 200, about 100, about 50, or about 25 nucleotides in length. Preferably, the siRNA is about 19 to about 25 nucleotides in length. An exemplary nucleic acid sequence for generating REG4 siRNA includes the nucleotide sequence of SEQ ID NO: 5 as the target sequence. Similarly, an exemplary nucleic acid sequence for generating KIAA0101 siRNA includes the nucleotide sequence of SEQ ID NO: 32 as the target sequence. Furthermore, in order to enhance the inhibitory activity of siRNA, nucleotide “u” can be added to the 3 ′ end of the antisense strand of the target sequence. The number of “u” added is at least about 2, generally from about 2 to about 10, and preferably from about 2 to about 5. The added “u” forms a single strand at the 3 ′ end of the antisense strand of the siRNA.

  The cell is any cell that expresses or overexpresses REG4 or KIAA0101. The cell is an epithelial cell such as a pancreatic duct cell. Alternatively, the cell is a tumor cell such as a carcinoma, adenocarcinoma, blastoma, leukemia, myeloma, or sarcoma. The cells are pancreatic cancer cells, particularly pancreatic ductal adenocarcinoma cells, prostate cancer cells, breast cancer cells and bladder cancer cells.

  REG4 or KIAA0101 siRNA is introduced directly into cells in a form that can bind to mRNA transcripts. Alternatively, DNA encoding REG4 or KIAA0101 siRNA is present in the vector.

  The vector can be, for example, an expression vector having a REG4 or KIAA0101 target sequence functionally linked control sequence adjacent to the REG4 or KIAA0101 sequence so that expression of both strands is possible (by transcription of the DNA molecule). It is generated by cloning (Lee, NS, et al., (2002) Nature Biotechnology 20: 500-5). An RNA molecule that is antisense to REG4 or KIAA0101 mRNA is transcribed by the first promoter (for example, the roux promoter sequence on the 3 ′ side of the cloned DNA), and is an RNA molecule that is the sense strand of the REG4 or KIAA0101 mRNA Is transcribed by a second promoter (eg, a roux promoter sequence 5 ′ of the cloned DNA). The sense strand and the antisense strand are hybridized in vivo to produce an siRNA construct for silencing the REG4 or KIAA0101 gene. Alternatively, two constructs are utilized to create the sense and antisense strands of the siRNA construct. The cloned REG4 or KIAA0101 may encode a construct with a secondary structure, such as a hairpin, where a single transcript has both sense and complementary antisense sequences from the target gene.

To form a hairpin loop structure, a loop sequence consisting of any nucleotide sequence can be placed between the sense and antisense sequences. Thus, the present invention also provides siRNA having the general formula 5 ′-[A]-[B]-[A ′]-3 ′, wherein [A] is mRNA or cDNA from REG4 or KIAA0101 Is a ribonucleotide sequence corresponding to a sequence that specifically hybridizes with. In a preferred embodiment, [A] is a ribonucleotide sequence corresponding to the sequence of nucleotides of SEQ ID NO: 5 or 32.
[B] is a ribonucleotide sequence consisting of 3 to 23 nucleotides, and
[A ′] is a ribonucleotide sequence consisting of the complementary sequence of [A].
Region [A] hybridizes with [A ′], and then a loop consisting of region [B] is formed.

The loop sequence may preferably be about 3 to about 23 nucleotides in length. The loop sequence can be selected, for example, from the group consisting of the following sequences (http://www.ambion.com/techlib/tb/tb_506.html). In addition, a loop sequence of 23 nucleotides also provides an active siRNA (Jacque, JM et al., (2002) Nature 418: 435-8.).
CCC, CCACC, or CCACACC: Jacque, JM et al., (2002) Nature, 418: 435-8.
UUCG: Lee, NS et al., (2002) Nature Biotechnology 20: 500-5; Fruscoloni, P. et al., (2003) Proc. Natl. Acad. Sci. USA 100 (4): 1639-44.
UUCAAGAGA: Dykxhoorn, DM et al., (2003) Nature Reviews Molecular Cell Biology 4: 457-67.

As an example, a preferred siRNA having the hairpin loop structure of the present invention is shown below. In the following structure, the loop sequence can be selected from the group consisting of CCC, UUCG, CCACC, CCACACC, and UUCAAGAGA. A preferred loop sequence is UUCAAGAGA (“ttcaagaga” in DNA).
5'-GACAGAAGGAAGAAACTCA- [B] -TGAGTTTCTTCCTTCTGTC-3 '(when the target sequence is SEQ ID NO: 5)

  The regulatory sequences adjacent to the REG4 or KIAA0101 sequences are the same or different so that their expression can be regulated independently or temporally or spatially. The siRNA is transcribed intracellularly by cloning the REG4 or KIAA0101 gene template into a vector containing, for example, an RNA polymerase III transcription unit derived from a nuclear small RNA (snRNA) U6 or a human H1 RNA promoter. In order to introduce the vector into the cell, a transfection facilitating agent can be used. FuGENE (Roche Diagnostices), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako Pure Chemical Industries) are useful as transfection promoters.

  Oligonucleotides of various parts of REG4 or KIAA0101 mRNA, and oligonucleotides complementary to those various parts, in tumor cells (e.g., pancreatic cell lines such as pancreatic cancer cell lines or pancreatic ductal adenocarcinoma (PDAC) cell lines). In use) was tested in vitro according to standard methods for the ability to reduce REG4 or KIAA0101 production. REG4 or KIAA0101 specific antibody or other detection of decreased REG4 or KIAA0101 gene product in cells contacted with the candidate siRNA composition compared to cells cultured in the absence of the candidate composition Detect using method. Sequences that reduce REG4 or KIAA0101 production in an in vitro cell assay or cell free assay are then tested for their inhibitory effect on cell proliferation. Sequences that inhibit cell growth in an in vitro cell assay are tested in vivo in rats or mice to confirm a decrease in REG4 or KIAA0101 production and a decrease in tumor cell growth in animals with malignant neoplasms.

How to treat malignant tumors
Patients with tumors characterized by REG4 or KIAA0101 overexpression are treated by administering siRNA of REG4 or KIAA0101, respectively. For example, siRNA therapy to inhibit REG4 or KIAA0101 expression in patients with or at risk of developing pancreatic cancer, particularly pancreatic ductal adenocarcinoma (PDAC), prostate cancer cells, breast cancer cells, and bladder cancer cells Is used. Such patients are identified by standard methods for specific tumor types. Pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC), prostate cancer cells, breast cancer cells, and bladder cancer cells are diagnosed by, for example, CT, MRI, ERCP, MRCP, computer tomography, or ultrasound. A treatment is effective if the treatment results in a clinical benefit such as a reduction in REG4 or KIAA0101 expression in the subject, or a reduction in tumor size, spread, or metastatic potential. “Effective” when the treatment is carried out prophylactically means that the treatment delays or prevents the formation of the tumor or prevents or reduces the clinical symptoms of the tumor. Efficacy is determined by any known method for diagnosing or treating a particular tumor type.

  siRNA therapy is performed by administering siRNA to a patient by standard vectors encoding the siRNA of the invention and / or gene delivery systems such as by delivery of synthetic siRNA molecules. Typically, synthetic siRNA molecules are chemically stabilized to prevent nuclease degradation in vivo. Methods for preparing chemically stabilized RNA molecules are well known in the art. Typically, such molecules comprise a backbone and nucleotides that are modified to prevent the action of ribonucleases. Other modifications are possible, for example cholesterol-binding siRNAs have shown improved pharmacological properties (Song et al. Nature Med. 9: 347-351 (2003)).

  Suitable gene delivery systems can include liposomes, receptor-mediated delivery systems, or viral vectors such as herpes viruses, retroviruses, adenoviruses, and adeno-associated viruses, among others. The therapeutic nucleic acid composition is formulated in a pharmaceutically acceptable carrier. The therapeutic composition may also include a gene delivery system as described above. A pharmaceutically acceptable carrier is a biologically compatible excipient suitable for administration to animals, such as saline. A therapeutically effective amount of a compound is an amount that can produce a medically desirable result in the treated animal, such as decreased production of REG4 or KIAA0101 gene product, cell proliferation, eg, decreased proliferation, or decreased tumor growth.

  Parenteral administration such as intravenous, subcutaneous, intramuscular, and intraperitoneal delivery routes can be used to deliver siRNA compositions of REG4 or KIAA0101. For the treatment of pancreas, prostate, breast, and bladder tumors, direct injection near the tissue or cancer is useful.

Dosage to any patient is many, including the patient's physique, body surface area, age, the specific nucleic acid to be administered, sex, time and route of administration, general health, and other drugs to be administered simultaneously Depends on the factors. When the nucleic acid is administered intravenously, the dose is about 10 ⁶ to 10 ²² copies of the nucleic acid molecule.

  Polynucleotides are administered by standard methods, such as injection into gaps in tissues such as muscle or skin, introduction into the circulation or body cavities, or inhalation or infusion. The polynucleotide is injected or delivered to an animal with a pharmaceutically acceptable liquid carrier, such as a water-soluble or partially water-soluble liquid carrier. The polynucleotide is associated with liposomes (eg, cationic or anionic liposomes). The polynucleotide contains genetic information necessary for expression by the target cell, such as a promoter.

Anti-REG4 antibodies and antigen binding proteins:
Antibody The inventor has shown that a monoclonal antibody against REG4 neutralized the proliferation promoting effect of REG4 and significantly attenuated PDAC cell growth. For example, it has been shown that treatment of diseases associated with cells expressing REG4, such as pancreatic cancer, is conveniently performed using antibodies that bind to REG4.

  The present invention relates to a pharmaceutical composition for treating or preventing cancer caused by abnormal overexpression of REG4 including pancreatic cancer. The composition comprises as active ingredients an antibody that binds to a pharmaceutically effective amount of a protein encoding REG4, or a fragment thereof, or a protein that binds to an antigen and a pharmaceutically acceptable carrier. The invention also relates to the use of an anti-REG4 antibody or a protein that binds to an antigen to produce a pharmaceutical composition for treating or preventing pancreatic cancer. The pharmaceutical composition of the present invention comprises an anti-REG4 antibody or a protein that binds to an antigen and a pharmaceutically acceptable carrier.

  An “isolated” or “purified” polypeptide is substantially free of cellular material such as carbohydrates, lipids, or other contaminating proteins from the cell or tissue source from which the protein is derived, or A polypeptide that is substantially free of chemical precursors or other chemicals when chemically synthesized. The term “substantially free of cellular material” includes preparations of a polypeptide in which the polypeptide is separated from cellular components of the cells from which the polypeptide is isolated or produced by recombinant techniques.

  Thus, a polypeptide that is substantially free of cellular material has less than about 30%, 20%, 10%, or 5% (by dry weight) of a heterologous protein (referred to herein as a “contaminating protein”). Polypeptide preparations are included. When the polypeptide is produced by recombinant techniques, the polypeptide is also preferably substantially free of media and is a polypeptide having a media that is less than about 20%, 10%, or 5% of the volume of the protein preparation. Contains preparations. When the polypeptide is produced by chemical synthesis, the polypeptide is preferably substantially free of chemical precursors or other chemicals, and is approximately 30%, 20%, 10%, 5% of the volume of the protein preparation ( Preparations of polypeptides with less than (by dry weight) chemical precursors or other chemicals involved in protein synthesis. The inclusion of an isolated or purified polypeptide by a particular protein preparation is, for example, simple after protein preparation sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis and gel coomassie brilliant blue staining. It can be shown by the appearance of a band. In preferred embodiments, the antibodies or fragments thereof of the invention are isolated or purified.

  An “isolated” or “purified” nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. An “isolated” or “purified” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or medium when chemically produced or is chemically synthesized. In some cases, it may be substantially free of chemical precursors or other chemicals. In a preferred embodiment, the nucleic acid molecule encoding the antibody of the present invention or fragment thereof is isolated or purified.

  “Antibodies” and “immunoglobulins” are glycoproteins having the same structural characteristics. Whereas antibodies exhibit binding specificity for specific antigens, immunoglobulins include both antibodies and other antibody-like molecules that are not defined for antigen specificity. The latter type of polypeptide is produced, for example, at low levels by the lymphatic system and at increased levels by myeloma.

  “Natural antibodies and immunoglobulins” are typically heterotetrameric glycoproteins of about 150,000 daltons that contain two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, but the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by a number of constant domains (CH). Each light chain has a variable domain (VL) at one end and a constant domain (CL) at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, The variable domain aligns with the variable domain of the heavy chain. Certain amino acid residues are thought to form an interface between the light and heavy chain variable domains (Chothia et al., (1985) J Mol Biol .; 186: 651-63; Novotny and Haber, (1985) Proc Natl Acad Sci USA .; 82: 4592-6).

  The term “variable” refers to the fact that certain portions of the variable domains differ significantly in sequence between antibodies, and are useful in the binding and specificity of each particular antibody for a particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. The variability is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions present in both the light chain variable domain and the heavy chain variable domain. The more highly conserved portions of variable domains are called the framework (FR). Each of the natural heavy and light chain variable domains contains four framework regions that are predominantly in a β-sheet configuration and are linked by three CDRs, which form a linking loop, In some cases it forms part of the β-sheet structure. The CDRs in each chain are held in close proximity to each other by the framework regions and contribute to the formation of the antigen binding site of the antibody with the CDRs from the other chain (Kabat et al., (1991) Sequences of Proteins of Immunological Interest , Fifth Edition, National Institute of Health, Bethesda, Md.). Constant domains are not directly involved in antibody binding to antigen, but exhibit various effector functions such as antibody involvement in antibody-dependent cellular toxicity.

  Papain digestion of the antibody produces two identical antigen binding fragments, called “Fab” fragments, each with a single antigen binding site, and the remaining “Fc” fragments. Pepsin treatment yields an F (ab ′) 2 fragment with two antigen binding sites. “Fv” is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of tight, non-covalent associations of one heavy chain variable domain and one light chain variable domain. In this arrangement, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Together, six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or one of the Fvs containing only three CDRs specific for the antigen, which has a lower affinity than the entire binding site) recognizes the antigen. Has the ability to bind.

  The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH-1) of the heavy chain. Fab ′ fragments differ from Fab fragments by the addition of several residues at the C-terminus of the heavy chain CH-1 domain including one or more cysteines from the antibody hinge region. Fab′-SH as used herein refers to Fab ′ in which the cysteine residue of the constant domain has a free thiol group. F (ab ′) 2 antibody fragments originally were produced as pairs of Fab ′ fragments which have hinge cysteines between the fragments. The chemical coupling of other antibody fragments is also known.

  The “light chain” of an antibody (immunoglobulin) from any vertebrate species is one of two distinct types, called κ (kappa) and λ (lambda), based on the amino acid sequence of its constant domain. Can be assigned to.

  Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to different classes. There are five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, some of which are in subclasses (isotypes), eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Further classification can be made. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

  The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population may be present in small amounts. Except for typical natural mutations. Monoclonal antibodies are very specific and are directed to a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies to different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to specificity, monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture and other immunoglobulins cannot be contaminated. Thus, the modifier “monoclonal” indicates the character of the antibody as obtained from a substantially homogeneous antibody population and should not be construed as requiring production of the antibody by any particular method. . For example, monoclonal antibodies used in accordance with the present invention may be prepared by the hybridoma method first described by Kohler and Milstein, (1975) Nature; 256: 495-7, or by the recombinant DNA method (Cabilly et al., (1984) Proc Natl Acad Sci USA.; 81: 3273-7).

  Monoclonal antibodies herein specifically include “chimeric” antibodies or immunoglobulins as long as they exhibit the desired biological activity, and in “chimeric” antibodies or immunoglobulins, a portion of the heavy and / or light chain. Is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, but the rest of the chain is from another species or another antibody class Or identical or homologous to the corresponding sequence in antibodies belonging to a subclass (Cabilly et al., Supra; Morrison et al., (1984) Proc Natl Acad Sci USA .; 81: 6851-5). Most typically, chimeric antibodies or immunoglobulins comprise human and murine antibody fragments, generally human constant regions and murine variable regions.

  “Humanized” forms of non-human (eg, murine) antibodies are special chimeric immunoglobulins, immunoglobulin chains, or fragments thereof derived from minimal sequences derived from non-human immunoglobulin. Such fragments also include Fv, Fab, Fab ′, F (ab ′) 2, or other antigen binding subsequences of the antibody. For the most part, humanized antibodies are non-human species, such as mice, rats, or rabbits, in which residues from the complementarity determining region (CDR) of the recipient have the desired specificity, affinity, and ability ( It is a human immunoglobulin (recipient antibody) that is replaced by residues from the CDRs of a donor antibody.

  In some cases, Fv framework residues of the human immunoglobulin may be replaced by corresponding non-human residues. In the present invention, several, two, or preferably one of the frameworks in the humanized antibody may be replaced by non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, a humanized antibody has a framework region in which all or substantially all of the CDR region corresponds to a CDR region of a non-human immunoglobulin, and all or substantially all of the framework region is a human immunoglobulin consensus sequence. Which may comprise substantially all of at least one, and typically two variable domains.

  Optionally, the humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details, see Jones et al., (1986) Nature; 321: 522-5; Riechmann et al., (1988) Nature; 332: 323-7; Presta, (1992) Curr Opin Struct Biol. 2: 593 Refer to -6.

  “Single-chain Fv” or “sFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single-chain polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allows the sFv to form the desired structure for antigen binding. Light and heavy chain polypeptides from naturally-aggregated but chemically separated antibody V regions into sFv molecules thought to fold into a three-dimensional structure substantially similar to the structure of the antigen binding site A number of methods have been described to identify chemical structures for conversion (US Pat. Nos. 5,091,513, 5,132,405, and 4,946,778; Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)).

Antigen binding proteins The present invention also includes antigen binding proteins or non-antibody binding proteins (eg, ligands) that specifically bind to REG4 or KIAA0101. Non-antibody ligands include antibody mimics using non-immunoglobulin protein backbones, including adnectins, avimers, single chain protein binding molecules and antibody-like binding peptidomimetics, as described in more detail below.

  Other compounds have been developed that target and bind to targets in a manner similar to antibodies. Certain of these “antibody mimics” use non-immunoglobulin protein scaffolds as alternative protein frameworks for antibody variable regions.

  For example, Ladner et al. (US Patent No. 5,260,203) is a single chain polypeptide binding molecule that has a binding specificity similar to the light and heavy chains of antibody variable regions that are aggregated but molecularly separated. Is described. Single chain binding molecules contain the antigen binding sites of both the heavy and light chain variable regions of an antibody linked by a peptide linker and are folded into a structure similar to a bipeptide antibody. Single chain binding molecules are superior to conventional antibodies in several respects, including molecular size, higher stability, and are more easily modified.

  Ku et al. (Proc. Natl. Acad. Sci. USA 92 (14): 6552-6556 (1995)) describe an alternative to antibodies based on cytochrome b562. Ku et al. (1995) created a library in which two loops of cytochrome b562 were randomized and selected for binding to bovine serum albumin.

  Lipovsek et al. (U.S. Patent Nos. 6,818,418 and 7,115,396) disclose an antibody mimic featuring a fibronectin or fibronectin-like protein backbone and at least one variable loop. As known as adenctin, these fibronectin-based antibody mimics exhibit many of the same properties as natural or processed antibodies, including high affinity and specificity for the target ligand. Any technique for evolving new or improved binding proteins can be used for these antibody mimetics.

  The structure of these fibronectin based antibody mimics is similar to the structure of the variable region of an IgG heavy chain. Therefore, these mimetics show similar antigen binding in nature and affinity as natural antibodies. Furthermore, these fibronectin based antibody mimics present certain advantages over antibodies and antibody fragments. For example, these antibody mimetics are stable under conditions that would normally destroy the antibody, as natural folding stability does not depend on disulfide binding. Furthermore, because the structure of these fibronectin-based antibody mimics is similar to that of IgG heavy chains, the process of loop randomization and shuffling is similar to the process of affinity maturation of antibodies in vivo. Can be performed in vitro.

  Beste et al. (Proc. Natl. Acad. Sci. USA 96 (5): 1898-1903 (1999)) discloses an antibody mimic (Anticalin®) based on the lipocalin backbone. Lipocalin is composed of a β-barrel with four hypervariable loops at the end of the protein. Beste (1999) induced random mutations in the loop and selected, for example, for binding to fluorescein. Three mutants showed specific binding to fluorescein, one of which showed binding similar to that of anti-fluorescein antibody. Further analysis revealed that all of the randomized positions are variable, indicating that Antiticalin® is suitable for use as an antibody surrogate.

  Anticalin® is a low-molecular-weight single-chain peptide, typically 160-180 residues, which reduces production costs, improves stability during storage, and reduces immune responses compared to antibodies. It has several advantages including:

  Hamilton et al. (U.S. Patent No. 5,770,380) discloses synthetic antibody mimics that use a strong non-peptide organic backbone of calixarene, which binds multiple variable peptide loops used as binding sites. All of the peptide loops protrude from each other on the same side geometrically from the calixarene. Because of this geometrical confirmation, all loops are available for binding, increasing the binding affinity for the ligand. However, compared to other antibody mimics, antibody mimics based on calixarenes are not composed solely of peptides and are therefore less susceptible to attack by protease enzymes. Being neither a backbone purely composed of peptides, DNA or RNA means that the antibody mimetic is relatively stable under extreme environmental conditions and has a long lifetime. Furthermore, antibody mimics based on calixarenes are relatively small and are unlikely to generate an immune response.

  Murali et al. (Cell. Mol. Biol. 49 (2): 209-216 (2003)) discusses a methodology for reducing antibodies to smaller peptidomimetics, “antibody-like peptidomimetics” (ABiP). I call it. It can also be used as an alternative to antibodies.

  Silverman et al. (Nat. Biotechnol. (2005), 23: 1556-1561) disclose a fusion protein that is a single-chain polypeptide containing multiple domains called “avimers”. Avimers were developed from human extracellular receptor domains by in vitro exon shuffling and phage display and are a class of binding proteins somewhat similar to antibodies in terms of affinity and specificity for various target molecules. It is. The resulting multidomain protein can contain multiple independent binding domains, which domains have improved affinity (sometimes sub-nanomolar) and specificity compared to single epitope binding proteins Can be shown. Further details regarding methods of construction and use of avimers are disclosed, for example, in US Patent Publication Nos. 20040175756, 20050048512, 20050053973, 20050089932, and 20050221384.

  In addition to non-immunoglobulin protein frameworks, antibody properties are also mimicked in compounds including RNA molecules and non-natural oligomers (eg, protease inhibitors, benzodiazepines, purine derivatives and β-turn mimetics) All are suitable for use in the present invention.

Neutralizing activity:
Presented are antibodies and non-antibody binding proteins that have the function of depriving pathogens of infectivity and toxin activity. Antibody-mediated neutralization can be accomplished by binding of the antigenic variable region to an antigen or may require complement intervention. For example, antiviral antibodies may require complement intervention to deprive the virus of infectivity. The Fc region is essential for complement involvement. Thus, such antibodies contain effector functions that require Fc for virus and cell neutralization.

  The present invention relates to the activity of REG4 that promotes the growth of cells, particularly cancer cells having the property of mediating abnormal high REG4 expression or intracellular signals, with anti-REG4 antibody and non-antibody binding protein specifically binding to REG4 Based in part on the finding to neutralize.

  The term "specifically binds" or "specific binding" or "added" or "adds" in the section of antibody or non-antibody binding protein refers to other agents that bind to REG4 expressed in cells or tissues Alternatively, it means a preferential association of a target epitope (eg, REG4) that binds or competes with a ligand and all or part of the drug or ligand. Of course, a particular degree of non-specific interaction can be distinguished through specific recognition of the antibody and non-target epitope. Nevertheless, specific binding can be distinguished as binding mediated through specific recognition of the target epitope. Typically, specific binding is stronger between an entity lacking the target epitope (eg, assay well or cell) and the binding antibody than between the entity having the target antibody (eg, assay well or cell) and the delivery molecule. Meeting. Specific binding is typically in the amount (per unit time) of agent or ligand that binds to a cell or tissue having a target epitope (ie REG4) compared to a cell or tissue lacking the target epitope. At least about a 2-fold increase over the background, preferably more than about 10-fold, and more preferably over a 100-fold increase. Specific binding between two entities generally means an affinity of at least 106M-1. An affinity of 108M-1 or higher is preferred. Specific binding can be determined for nucleic acids as well as protein drugs and ligands. Specific binding to a nucleic acid agent is determined using assays well known to those skilled in the art including, but not limited to, Northern blots, gel shift assays, and in situ hybridization. Specific binding to protein drugs and ligands includes, but is not limited to, gel electrophoresis, western blot, ELISA, flow cytometry and immunohistochemistry

The present invention also relates to a method for inhibiting cell growth of REG4-expressing cells, the method comprising the following steps:
1) contacting an anti-REG4 antibody or anti-REG4 non-antibody binding protein with a REG4-expressing cell; and
2) Neutralize cell growth activity of REG4.

  In the method or pharmaceutical composition of the present invention, REG4-expressing cells can be suppressed. For example, pancreatic cancer cells are preferred as the REG4-expressing cells of the present invention. Of these, pancreatic cancer or cells are preferred.

Cells and antibodies (or non-antibody binding proteins) can be contacted in vivo or in vitro. When targeting cancer cells in vivo as REG4-expressing cells, the method of the present invention is actually a method of treating or preventing cancer. Specifically, the present invention provides a method for treating cancer comprising the following steps:
1) administering an antibody or non-antibody binding protein that specifically binds to REG4 to a cancer patient; and
2) Inhibiting cancer cell proliferation using the function of an antibody or non-antibody binding protein that binds to REG4, where the function is to neutralize the cell proliferation activity of REG4.

  In the present invention, neutralizing the function of the anti-REG4 antibody or non-antibody binding protein means suppressing the activity of REG4 that promotes cell growth of REG4-expressing cells. For example, the inventors have confirmed that REG4 functions as an autocrine or paracrine growth factor and mediates the Akt signaling pathway. Thus, in a preferred embodiment of the invention, the anti-REG4 antibody or non-antibody binding protein blocks the REG4 autocrine / paracrine pathway and prevents subsequent Akt phosphorylation. The present inventors have also confirmed that the cell proliferation of REG4-expressing cells, particularly pancreatic cancer cells, is effectively suppressed using the neutralizing function. The present inventors further confirmed that REG4 is highly expressed in pancreatic cancer cells at a high rate. In addition, the expression level of REG4 in normal tissues is low. Considering this information together, a method for treating pancreatic cancer in which an anti-REG4 antibody is administered can be an effective method with little risk of side effects.

  However, the antibodies and non-antibody binding proteins of the present invention are not limited as long as they include a desired neutralizing function. Mutants, analogs or derivatives to the Fc site can be constructed, for example, by residue or sequence mutations, substitutions.

  Variant (or derivative) polypeptides include insertional variants. Here, the Fc amino acid sequence may be supplemented with one or more amino acid residues. The insertion can be located at one or both ends of the protein, or within the insertion region of the Fc amino acid. Insertion variants having additional residues at one or both ends include, for example, fusion proteins and proteins comprising amino acid tags or labels. For example, the Fc molecule can optionally include an N-terminal Met, particularly when the molecule is recombinantly expressed, for example, in bacterial cells such as E. coli.

  In Fc deficient mutants, one or more amino acid residues in the Fc polypeptide are removed. Deletions can be accomplished at one or both ends of the Fc polypeptide, or by removal of one or more residues within the Fc amino acid sequence. Deletion variants thus include all fragments of the Fc polypeptide sequence.

  In Fc deficient mutants, one or more amino acid residues of the Fc polypeptide are removed and replaced with alternative residues. In one embodiment, while substitutions are naturally conservative, the present invention allows substitutions that are non-conservative.

  Preferably, the parent polypeptide Fc region is a human Fc region, eg, a native sequence human Fc region human IgG1 (A and non-A allotype) or a human IgG3 Fc region. In one embodiment, variants with improved ADCC mediate ADCC substantially more effectively than the Fc region of antibodies with native sequence IgG1 or IgG3 and the antigen binding region of the variant. Preferably, the variant comprises or consists essentially of substitutions of two or three of the residues at positions 298, 333, and 334 of the Fc region. The residue numbering system in the immunoglobulin heavy chain is that of the EU index as in Kabat et al. (Supra), which is expressly incorporated herein by reference. More preferably, residues at positions 298, 333, and 334 are substituted (eg, with alanine residues). Furthermore, in order to create Fc region variants with improved ADCC activity, Fc region variants with improved binding affinity for FcγRIII, which are considered to be important FcRs for mediating ADCC, are generally produced. It is done. For example, to create such a variant, any one of amino acid positions 256, 290, 298, 312, 326, 330, 333, 334, 360, 378, or 430 in the parent Fc region or Further amino acid modifications (eg, insertions, deletions, or substitutions) may be introduced. Variants with improved binding affinity for FcγRIII may further have a reduced binding affinity for FcγRII, particularly a reduced affinity for FcγRIIb receptor inhibition.

  In any case, any variant amino acid insertion, deletion and / or substitution (1-50 amino acids, preferably 1-25 amino acids, more preferably 1-10 amino acids) is contemplated and within the scope of the present invention. Conservative amino acid substitutions are generally preferred.

Furthermore, the modification may be in the form of a modified amino acid such as a D-amino acid.
Accordingly, human-derived antibodies belonging to these classes are preferred in the present invention. Human antibodies can be obtained using antibody-producing cells obtained from humans or chimeric animals transplanted with human antibody genes (Ishida I, et al., (2002) Cloning and Stem Cells., 4:91 -102).

  Furthermore, the antibody Fc region can be joined to any variable region. Specifically, chimeric antibodies in which human constant regions are linked to variable regions of different animal species are known in the art. Alternatively, a human-human chimeric antibody can be obtained by binding an arbitrary constant region to a variable region derived from human. Furthermore, CDR grafting technology, which is a technology for substituting the complementarity determining region (CDR) corresponding to the variable region of a human antibody with the CDR of a heterologous antibody ("Immunoglobulin genes", Academic Press (London), pp260-274 Roguska MA, et al., (1994) Proc. Natl. Acad. Sci. USA., 91: 969-73).

  Replacement of the CDR replaces the binding specificity of the antibody. That is, a humanized antibody transplanted with a human REG4-binding antibody CDR recognizes human REG4. The transplanted antibody is also called a humanized antibody. Antibodies obtained in this manner and having an Fc region essential for effector function can be used as the antibody of the present invention regardless of the origin of the variable region. For example, an antibody comprising the Fc of human IgG is preferred in the present invention, even if the variable region comprises an amino acid sequence derived from another class or other species of immunoglobulin.

  Alternatively, antibody fragments lacking the Fc region are used as long as they have the desired neutralizing function. For example, in the present invention, Fv, Fab, Fab ', F (ab') 2 or other antigen-binding sequences of an antibody can be used as an antibody. In certain embodiments, an agent that enhances the neutralizing effect can be conjugated to an antibody or fragment thereof.

The VH and VL domains of the antibodies of the invention comprise three CDRs each designed as CDR1, CDR2 and CDR3 separated by a framework region. The amino acid sequence of CDR is not limited as long as the antibody specifically binds to REG4.
Preferred examples of CDR amino acid sequences are as follows:
VH CDR1: SYWMH (SEQ ID NO: 20),
VH CDR2: NIYPGSGSTNYD (SEQ ID NO: 21),
VH CDR3: GGLWLRVDY (SEQ ID NO: 22),
VL CDR1: SASSSVSYMH (SEQ ID NO: 23),
VL CDR2: DTSKLAS (SEQ ID NO: 24),
VL CDR3: QQWSSNPF (SEQ ID NO: 25).

  In a more preferred embodiment, VH comprises the amino acid sequence of SEQ ID NO: 18 and VL comprises the amino acid sequence of SEQ ID NO: 19. In certain embodiments, VH comprises an amino acid sequence having at least about 90%, 95%, 98% sequence identity over the entire length of SEQ ID NO: 18, and VL is at least about the entire length of SEQ ID NO: 19. Contains amino acid sequences with 90%, 95%, 98% sequence identity. Sequence identity can be measured using software defaults available in known techniques such as, for example, BLAST or ALIGN.

  In the present invention, the antibody may be a monoclonal antibody or a polyclonal antibody. Even when administered to humans, human polyclonal antibodies can be obtained using animals transplanted with the above human antibody genes. Alternatively, immunoglobulins constructed by genetic engineering techniques such as humanized antibodies, human-nonhuman chimeric antibodies, and human-human chimeric antibodies can also be used. Furthermore, a method for obtaining a human monoclonal antibody by cloning human antibody-producing cells is also known.

  In order to obtain the antibody of the present invention, a fragment containing REG4 or a partial peptide thereof can be used as an immunogen. In the present invention, REG4 may be derived from any species, preferably a mammal such as a human, mouse or rat, more preferably a human. The nucleotide and amino acid sequences of human REG4 are known in the art. The cDNA nucleotide sequence of REG4 is described in SEQ ID NO: 1, and the amino acid sequence encoded by the nucleotide sequence is described in SEQ ID NO: 2 (Genbank accession number AY126670). One skilled in the art can routinely isolate a gene containing the indicated nucleotide sequence and prepare fragments of the sequence as necessary to obtain a protein corresponding to the target amino acid sequence.

  For example, a gene encoding REG4 protein or a fragment thereof can be inserted into a known expression vector and used to transform host cells. The desired protein or fragment thereof can be recovered from the inside or outside of the host cell by any standard method and can also be used as an antigen. In addition, proteins, their lysates, and chemically synthesized proteins can be used as antigens. Furthermore, the cell itself that expresses the REG4 protein or a fragment thereof can be used as an immunogen.

  When a peptide fragment is used as the REG4 immunogen, it is preferable to select an amino acid sequence including a region predicted to be an extracellular region. The presence of the signal peptide is estimated to be 1-25 at the N-terminus of REG4. Therefore, for example, a region excluding the N-terminal signal peptide (25 amino acid residues) is preferable as an immunogen for obtaining the antibody of the present invention. Alternatively, an antibody that binds to the extracellular region of REG4 is preferred as the antibody of the present invention.

  Therefore, a preferable antibody in the present invention is an antibody having Fc essential for effector function and a variable region capable of binding to the extracellular REG4 region. For purposes of administration to humans, it is desirable to have an IgG Fc.

  Any mammal can be immunized with such an antigen. However, it is preferable to consider compatibility with the parent cell used in cell fusion. In general, rodents, rabbit eyes, or primates are preferred.

  Rodents include, for example, mice, rats, and hamsters. Rabbit eyes include, for example, rabbits. Primates include, for example, monkeys (Old World) monkeys such as cynomolgus monkeys (Macaca fascicularis), rhesus monkeys (Macaca mulatta), baboons, and chimpanzees.

  Methods for immunizing animals with antigens are well known in the art. Intraperitoneal or subcutaneous injection of antigen is a standard method of immunizing mammals. Specifically, the antigen can be diluted and suspended with an appropriate amount of phosphate buffered saline (PBS), physiological saline or the like. If desired, the antigen suspension can be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, emulsified and administered to a mammal. Following this, it is preferable to administer the antigen mixed with an appropriate amount of Freund's incomplete adjuvant several times every 4 to 21 days. Appropriate carriers can also be used for immunization. After immunization as described above, standard methods are to examine the serum for an increase to the desired antibody level.

  Polyclonal antibodies against the REG4 protein can be prepared from immunized mammals whose sera have been examined for an increase in the desired antibody. This is accomplished by collecting blood from these animals or by any conventional method of isolating serum from those blood. Polyclonal antibodies include serum containing polyclonal antibodies and fractions containing polyclonal antibodies isolated from serum. IgG and IgM can be prepared from a fraction that recognizes REG4 protein, for example, by using an affinity column to which REG4 protein is bound, and then further purifying this fraction on a protein A or protein G column. it can. In the present invention, the antiserum can also be used as a polyclonal antibody. Alternatively, purified IgG, IgM or the like can be used.

  To prepare monoclonal antibodies, immune cells may be collected from a mammal immunized with the antigen, examined for increased levels of the desired antibody in serum (as described above), and applied to cell fusion. The immune cells used for cell fusion are preferably obtained from the spleen. Other preferred parental cells that are fused to the immunogens described above include, for example, mammalian myeloma cells, and more preferably myeloma cells that have acquired the property of selecting fused cells with agents.

  The above immune cells and myeloma cells can be fused using a known method, for example, the method of Milstein et al. (Galfre, G. and Milstein, C., (1981) Methods. Enzymol .: 73, 3-46). it can.

  Hybridomas produced by cell fusion can be selected by culturing in a standard selection medium such as HAT medium (medium containing hypoxanthine, aminopterin, and thymidine). Cell culture in HAT medium is usually continued for a period of days to weeks, sufficient for killing all other cells (unfused cells) except the desired hybridoma. Standard limiting dilution is then performed to screen and clone hybridoma cells producing the desired antibody.

  Non-human animals can be immunized with the antigen to prepare the hybridoma in the above method. Furthermore, human lymphocytes derived from cells infected with EB virus or the like can be immunized in vitro using proteins, cells expressing the proteins, or suspensions thereof. The immunized lymphocytes may then be fused with unlimited-dividing human-derived myeloma cells (such as U266), thereby yielding a hybridoma that produces the desired human antibody capable of binding to the protein. (Publication Patent Publication (JP-A) Sho 63-17688).

  The obtained hybridoma is subsequently transplanted into the abdominal cavity of a mouse to extract ascites. The resulting monoclonal antibody can be purified using, for example, ammonium sulfate precipitation, protein A or protein G column, DEAE ion exchange chromatography, or an affinity column to which the protein of the present invention is bound. The antibody of the present invention can be used not only for purification and detection of the protein of the present invention but also as a candidate for agonists and antagonists of the protein of the present invention. These antibodies can also be applied to antibody therapy for diseases associated with the protein of the present invention. When the obtained antibody is administered to a human body (antibody therapy), a human antibody or a humanized antibody is preferable because of its low immunogenicity.

  For example, transgenic animals containing a repertoire of human antibody genes can be immunized with an antigen selected from proteins, protein-expressing cells, or suspensions thereof. Subsequently, antibody-producing cells are collected from the animal and fused with myeloma cells to obtain hybridomas, and anti-protein human antibodies can be prepared from these hybridomas (International Publication Nos. 92-03918, 94-94). No. 02602, 94-25585, 96-33735, and 96-34096).

  Alternatively, immune cells such as immunized lymphocytes that produce antibodies can be immortalized using oncogenes and used to prepare monoclonal antibodies.

  The monoclonal antibodies thus obtained can be prepared using genetic engineering methods (see, for example, Borrebaeck, C.A.K. and Larrick, J.W., (1990) Therapeutic Monoclonal Antibodies, MacMillan Publishers, UK). For example, recombinant antibodies can be obtained by cloning DNA encoding the antibody from immune cells such as immunized lymphocytes or hybridomas that produce the antibody, inserting these DNAs into appropriate vectors, and introducing them into host cells. Can be prepared. Recombinant antibodies prepared as described above are also contemplated in the present invention.

  Antibodies can be modified by conjugation with various molecules such as polyethylene glycol (PEG). Antibodies modified in this way can also be used in the present invention. The modified antibody can be obtained by chemically modifying the antibody. Such modification methods are conventional in the art. The antibody can also be modified with other proteins. Antibodies modified with protein molecules can be produced by genetic engineering. That is, a target protein can be expressed by fusion of an antibody gene and a gene encoding a modified protein molecule.

  Alternatively, the antibody may be a chimeric antibody containing a variable region derived from a non-human antibody and a constant region derived from a human antibody, or a complementarity determining region (CDR) derived from a non-human antibody, a framework region derived from a human antibody (FR ) And a humanized antibody containing a constant region. Such an antibody can be produced by a known technique.

  Standard sequences of molecular biology may be used to prepare DNA sequences encoding chimera and CDR graft products. The gene encoding the CDR of the antibody of interest can be prepared, for example, by synthesizing variable regions from the RNA of antibody-producing cells using the polymerase chain reaction (PCR) (eg, Larrick et al., “ Methods: a Companion to Methods in Enzymology ", vol. 2: page 106 (1991); Courtenay-Luck," Genetic Manipulation of Monoclonal Antibodies "in Monoclonal Antibodies: Production, Engineering and Clinical Application; Ritter et al. (Eds.) , page 166 (Cambridge University Press, 1995) and Ward et al., “Genetic Manipulation and Expression of Antibodies” in Monoclonal Antibodies: Principles and Applications; Birch et al. (eds.), page 137 (Wiley-Liss, Inc. , 1995)).

  DNA sequences encoding chimeric and CDR graft products can be synthesized completely or in part using oligonucleotide synthesis methods. Site-directed mutagenesis and polymerase chain reaction may be used as appropriate. For example, oligonucleotide-directed synthesis as described by Jones et al., (1986) Nature.; 321: 522-5 may be used. Alternatively, using existing variable region oligonucleotide-directed mutagenesis, for example, as described in Verhoeyen et al., (1988) Science .; 239: 1534-6 or Riechmann et al., (Supra). Also good. Also, for example, enzymes in oligonucleotides with gaps using T4 DNA polymerase as described by Queen et al., (1989) Proc Natl Acad Sci USA.; 86: 1002933; PCT Publication WO 90/07861 Target filling may be used.

  Any suitable host cell / vector system may be used for the expression of DNA sequences encoding CDR grafted heavy and light chains. Bacterial systems such as E. coli and other microbial systems are used for the expression of antibody fragments, in particular Fb and (Fab ') 2 fragments, in particular Fv fragments and single chain antibody fragments such as single chain Fvs Also good. For example, eukaryotic host cell expression systems such as mammals may be used, particularly for the production of larger CDR-grafted antibody products, including complete antibody molecules. Suitable mammalian host cells include CHO cells and myeloma cell lines or hybridoma cell lines.

  The antibody obtained as described above can be purified to homogeneity. For example, antibodies can be purified and separated according to common methods for purifying and separating proteins. For example, antibodies are appropriately selected and combined with column chromatography including but not limited to affinity chromatography, filtration, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing etc. Can be separated and isolated (Antibodies: A Laboratory Manual, Harlow and David, Lane (ed.), Cold Spring Harbor Laboratory, 1988).

  Protein A and protein G columns can be used as affinity columns. Exemplary protein A columns used include Hyper D, POROS, and Sepharose FF (Pharmacia).

  Exemplary chromatography (excluding affinity chromatography) includes ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (“Strategies for Protein Purification and Characterization: A Laboratory Course Manual "Daniel R. Marshak et al., (1996) Cold Spring Harbor Laboratory Press). Chromatography can be performed according to liquid phase chromatography procedures such as HPLC or FPLC.

  For example, antigen binding activity of the antibodies of the present invention using absorbance measurements, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and / or immunofluorescence. Can be measured. In ELISA, the antibody of the present invention is typically immobilized on a plate, the protein of the present invention is added to the plate, and then a sample containing the desired antibody such as a culture supernatant of a cell producing the antibody or a purified antibody Add. Next, a secondary antibody that recognizes the primary antibody and is tagged with an enzyme such as alkaline phosphatase is added and the plate is incubated. After washing, an enzyme substrate such as p-nitrophenyl phosphate is added to the plate and the absorbance is measured to evaluate the antigen binding activity of the sample. Protein fragments (such as C-terminal or N-terminal fragments) can be used as well as proteins. BIAcore (Pharmacia) can be used to evaluate the binding activity of the antibody.

  In addition, one or more anti-REG4 antibodies that inhibit the activity of REG4 are used for the methods and compositions of the invention. In the present invention, preferred anti-REG4 antibodies neutralize the activity of REG4 that promotes cell growth of pancreatic cancer. The neutralizing function of anti-REG4 antibodies can be assessed in vitro or in vivo. For example, the neutralizing function of an anti-REG4 antibody is evaluated by observing the effect of the antibody on the proliferation of REG4-expressing cells. Specifically, a neutralizing function is observed when the antibody suppresses cell proliferation to a detectable level when measured using a well-known method in this technical field. Alternatively, the function is also confirmed by suppression of the Akt signaling pathway (Sekikawa A, et al. Gastroenterology 2005; 128: 642-53., Bishnupuri KS, et al. Gastroenterology 2006; 130: 137-49.).

  In the present invention, anti-REG4 antibodies and non-antibody binding proteins can be administered as drugs to humans or other animals. In the present invention, non-human animals to which the antibody is administered can include mice, rats, guinea pigs, rabbits, chickens, cats, dogs, sheep, pigs, cows, monkeys, baboons, and chimpanzees. Antibodies and non-antibody binding proteins can be administered directly to a subject and can be formulated into dosage forms using known pharmaceutical preparation methods. For example, if desired, it can be administered parenterally in an injectable form, such as a sterile solution or suspension, using water or any other pharmaceutically acceptable liquid. For example, such compounds can be used in acceptable carriers or solvents, specifically sterile water, saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, solvents, Along with preservatives, binders and the like, it can be mixed into generally acceptable unit doses essential for pharmaceutical use.

  Other isotonic solutions including saline, glucose, and adjuvants (such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride) can be used as aqueous solutions for injection. They are also with suitable solubilizers such as alcohols, specifically ethanol and polyalcohols (e.g. propylene glycol and polyethylene glycol), and non-ionic surfactants (e.g. Polysorbate 80 (TM) or HCO-50). Can be used.

  Sesame oil or soybean oil can be used as an oily liquid and may include benzyl benzoate or benzyl alcohol as a solubilizer. Buffers (such as phosphate buffer and sodium acetate buffer), analgesics (such as procaine hydrochloride), stabilizers (such as benzyl alcohol and phenol), and antioxidants can be included in the formulation. The prepared injection solution can be filled into an appropriate ampoule.

  In the present invention, anti-REG4 antibody and non-antibody binding protein can be administered to a patient, for example, intraarterial, intravenous, transdermal, intranasal, transbronchial, topical, or intramuscular. Intravascular (intravenous) administration by infusion or injection is an example of a common method for systemic administration of antibodies to patients with pancreatic cancer. Furthermore, the method of inserting an intra-arterial catheter to the vicinity of the artery supplying nutrients to cancer cells and locally injecting an anticancer drug such as an antibody drug is effective as a local control treatment for metastatic lesions as well as the primary lesion of pancreatic cancer. .

  The dose and method of administration will vary depending on the weight and age of the patient and the method of administration, but can be routinely determined by one skilled in the art. Furthermore, DNA encoding the antibody can be inserted into a gene therapy vector and the vector administered for therapy. The dose and administration method vary depending on the weight, age, and condition of the patient, and those skilled in the art can appropriately select them. Usually, lower doses are administered first and increased sequentially until an effective effect is achieved without undesirable side effects.

  The anti-REG4 antibody and the non-antibody binding protein can be administered to a living body in an amount that can confirm the neutralizing function against REG4. For example, a typical dose of anti-REG4 antibody ranges from 0.1 mg to 250 mg / kg per day, although there are some differences depending on the symptoms. Usually, the dose per adult (body weight 60 kg) is 5 mg to 17.5 g / day, preferably 5 mg to 10 g / day, and more preferably 100 mg to 3 g / day. The administration schedule is 1 to 10 times at intervals of 2 to 10 days. For example, the course is observed after 3 to 6 administrations.

  Alternatively, an antibody, a nucleic acid sequence containing a sequence encoding a non-antibody binding protein, or a functional derivative thereof is administered to treat or prevent diseases associated with REG4-expressing cells, such as pancreatic cancer including PDAC, by gene therapy. . Gene therapy refers to a therapy performed by administering an expressed or expressible nucleic acid to a subject. In this aspect of the invention, the nucleic acid produces the antibody or antibody fragment that it encodes, which mediates a prophylactic or therapeutic effect.

  Any of the methods for gene therapy available in the art can be used according to the present invention. An exemplary method is described below.

  For a general review of gene therapy methods, see Goldspiel et al., (1993) Clin. Pharm .; 12: 488-505; Wu and Wu, (1991) Biotherapy .; 3: 87-95; Tolstoshev, ( 1993) Ann Rev Pharmacol Toxicol.; 32: 573-96; Mulligan, (1993) Science.; 260: 926-32; Morgan and Anderson, (1993) Ann Rev Biochem.; 62: 191-217; Clare Robinson Trends Biotechnol .; 11 (5): 155-215. Methods widely known and available in the field of recombinant DNA technology are Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

  In a preferred aspect, the composition of the invention comprises a nucleic acid encoding an antibody or non-antibody binding protein, the nucleic acid expressing an antibody or fragment thereof or chimeric protein or heavy or light chain in a suitable host. Is part of. In particular, such nucleic acids have a promoter operably linked to the antibody coding region, preferably a heterologous promoter, which promoter is inducible or constitutive and optionally tissue specific. In other specific embodiments, the antibody-encoding sequence and any other desired sequence are flanked by regions that promote homologous recombination at the desired site in the genome, thereby allowing the nucleic acid encoding the antibody to Nucleic acid molecules that cause intrachromosomal expression are used (Koller and Smithies, (1989) Proc Natl Acad Sci USA.; 86: 8932-5; Zijlstra et al., (1989) Nature.; 342: 435-8). In certain embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequence includes sequences encoding both the heavy and light chains of the antibody or fragments thereof.

  Delivery of the nucleic acid to the subject may be direct such that the subject is directly exposed to the nucleic acid or a vector carrying the nucleic acid, or the cells are first transformed in vitro with the nucleic acid and then transplanted into the subject. It may be indirect. These two approaches are known as in vivo or ex vivo gene therapy, respectively.

  In certain embodiments, the nucleic acid sequence can be administered directly in vivo, where the nucleic acid sequence is expressed to produce the encoded product. This can be accomplished by any of a variety of methods known in the art: for example, constructing nucleic acid sequences as part of a suitable nucleic acid expression vector so that they are intracellular. By administering them; for example, by infection with defective or attenuated retroviral vectors or other viral vectors (see US Pat. No. 4,980,286); or by direct injection of naked DNA; or by microparticle irradiation (eg, , Gene guns; by use of Biolistic, Dupont) or by coating with lipids or cell surface receptors or transfection agents; by encapsulation in liposomes, microparticles or microcapsules; or peptides known to enter the nucleus By administering the nucleic acid sequence in ligation to a receptor; Administering the nucleic acid sequence linked to a ligand that receives tosis (see, eg, Wu and Wu, (1987) J Biol Chem .; 262: 442932) Can be achieved for example).

  In other embodiments, a nucleic acid-ligand complex can be formed where the ligand comprises a fusogenic viral peptide that disrupts the endosome and allows the nucleic acid to avoid lysosomal degradation. In yet other embodiments, targeting specific receptors allows nucleic acids to be targeted in vivo for cell-specific uptake and expression (eg, PCT publications WO 92/06180, WO 92 / 22635, WO 92/20316, WO 93/14188, or WO 93/20221). Alternatively, nucleic acids can be introduced into cells and incorporated into host cell DNA for expression by homologous recombination (Koller and Smithies, (1989) Proc Natl Acad Sci USA.; 86: 8932-5; Zijlstra et al. al., (1989) Nature .; 342: 435-8).

  In a particular embodiment, viral vectors that contain nucleic acid sequences encoding the antibodies of the invention or fragments thereof are used. For example, retroviral vectors can be used (see Miller et al., (1993) Methods Enzymol .; 217: 581-99). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. A nucleic acid sequence encoding an antibody or non-antibody binding protein to be used for gene therapy is cloned into one or more vectors that facilitate delivery of the gene to the subject. Further details regarding retroviral vectors are described in Boesen et al., (1994) Biotherapy, which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to increase the resistance of stem cells to chemotherapy. ; 6: 291-302. Other references illustrating the use of retroviral vectors in gene therapy include: Clowes et al., (1994) J Clin Invest.; 93: 644-51; Kiem et al., ( 1994) Blood .; 83: 1467-73; Salmons and Gunzberg, (1993) Hum Gene Ther .; 4: 129-41; Grossman and Wilson, (1993) Curr Opin Genet Dev .; 3: 110-4.

  Another viral vector that can be used for gene therapy is adenovirus. Adenoviruses are particularly attractive vehicles for delivering genes to the respiratory epithelium. Adenoviruses originally infect respiratory epithelia where they cause mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage that they can infect non-dividing cells. Kozarsky and Wilson, (1993) Curr Opin Genet Dev .; 3: 499-503 provides a review of gene therapy based on adenovirus. Bout et al., (1994) Hum Gene Ther .; 5: 3-10 demonstrates the use of adenoviral vectors to transfer genes to the rhesus monkey airway epithelium. Other examples of the use of adenovirus in gene therapy are Rosenfeld et al., (1991) Science.; 252: 431-4; Rosenfeld et al., (1992) Cell.; 68: 143-55; Mastrangeli et al ., (1993) J Clin Invest.; 91: 225-34; PCT Publication WO 94/12649; Wang et al., (1995) Gene Ther.; 2: 775-83. In a preferred embodiment, adenoviral vectors are used.

  Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., (1993) Proc Soc Exp Biol Med .; 204: 289-300; US Pat. No. 5,436,146).

  Another approach for gene therapy involves transferring the gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer involves the transfer of a selectable marker to the cells. Subsequently, the cells are subjected to selection in order to isolate those cells that have taken up and are expressing the transferred gene. Subsequently, such cells can be delivered to the subject.

  In this embodiment, the nucleic acid is introduced into the cell prior to in vivo administration of the resulting recombinant cell. Such introduction includes transfection, electroporation, microinjection, infection with a viral vector or bacteriophage vector containing the nucleic acid sequence, cell fusion, gene transfer via chromosome, gene transfer via micronucleus, This can be done by any method known in the art, including but not limited to spheroplast fusion. Many techniques for introducing foreign genes into cells are known in the art (eg, Loeffler and Behr, (1993) Methods Enzymol .; 217: 599-618; Cotton et al., 1993, Methods Enzymol .; 217: 618-44; see Cline MJ. Pharmacol Ther. 1985; 29 (1): 69-92), used according to the present invention unless the necessary development and physiological function of the recipient cell is destroyed Can do. The procedure should include stable transfer of the nucleic acid into the cell so that the nucleic acid can be expressed by the cell, preferably inherited and expressed by its cell progeny.

  The resulting recombinant cells can be delivered to a subject by various methods known in the art. Recombinant blood cells (eg, hematopoietic stem cells or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.

  Cells into which nucleic acids can be introduced for gene therapy purposes include any desired cell type available, including epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; Blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes, etc .; various stem cells or progenitor cells, especially hematopoietic stem cells or progenitor cells such as bone marrow, Examples include, but are not limited to, those obtained from umbilical cord blood, peripheral blood, fetal liver and the like. In preferred embodiments, the cells used for gene therapy are autologous to the subject.

  In embodiments where recombinant cells are used for gene therapy, nucleic acid sequences encoding antibodies or fragments thereof can be introduced into cells such that they can be expressed by the cells or their progeny, followed by recombinant cells Administered in vivo for therapeutic effect. In certain embodiments, stem cells or progenitor cells are used. Any stem and / or progenitor cell that can be isolated and maintained in vitro could potentially be used according to this aspect of the invention (eg, PCT publication WO 94/08598; Temple and Anderson, (1992) Cell.; 71: 973-85; Rheinwald, (1980) Methods Cell Biol.; 21A: 229-54; Pittelkow and Scott, (1986) Mayo Clin Proc.; 61: 771-7).

  In certain embodiments, a nucleic acid introduced for gene therapy purposes is an inducible promoter operably linked to a coding region so that the expression of the nucleic acid can be controlled by controlling the presence or absence of an appropriate transcription inducer. Is included.

  Furthermore, the present invention relates to an immunogenic composition for inducing an antibody having a neutralizing function against REG4, comprising REG4 or an immunologically active REG4 fragment, or DNA or cells capable of expressing them as an active ingredient. Offer things. Alternatively, the present invention relates to the production of an immunogenic composition of REG4 or an immunologically active REG4 fragment, or a DNA or cell capable of expressing them, for inducing an antibody having a neutralizing function against REG4. Regarding use.

  In the present invention, neutralizing the activity of REG4 to promote cell proliferation in an autocrine / paracrine manner is accomplished by administration of an anti-REG4 antibody. Therefore, if the REG4 antibody can be induced in vivo, a therapeutic effect equivalent to that of antibody administration can be achieved. When administering an immunogenic composition comprising an antigen, the target antibody can be induced in vivo. Therefore, the immunogenic composition of the present invention is particularly useful in vaccine therapy against REG4-expressing cells. Therefore, the immunogenic composition of the present invention is useful, for example, as a vaccine composition for treating pancreatic cancer.

  The immunogenic composition of the present invention may comprise REG4 or an immunologically active REG4 fragment as an active ingredient. An immunologically active REG4 fragment means a fragment capable of inducing an anti-REG4 antibody that recognizes REG4, and includes a neutralizing function. Hereinafter, REG4 and immunologically active REG4 fragments are described as immunogenic proteins. Whether a given fragment induces a target antibody can be determined by actually immunizing the animal and confirming the activity of the induced antibody. Antibody induction and confirmation of its activity can be performed, for example, using the method described in the Examples.

  The immunogenic composition of the present invention may contain a pharmaceutically acceptable carrier in the same manner as the immunogenic protein which is an active ingredient. If desired, the composition can also be combined with an adjuvant. As an adjuvant, killed tuberculosis bacteria, diphtheria toxoid, saponin and the like can be used.

  Alternatively, DNAs that encode immunogenic proteins, or cells that hold those DNAs in an expressible state can be used as the immunogenic composition. A method of using DNA expressing a target antigen as an immunogen, so-called DNA vaccine, is well known. A DNA vaccine can be obtained by inserting DNA encoding REG4 or a fragment thereof into an appropriate expression vector.

  Retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, Sendai virus vectors and the like are examples of suitable vectors. Furthermore, it is also possible to directly introduce into the cell and express as a naked DNA a DNA operably linked to a DNA encoding an immunogenic protein downstream of the promoter. Naked DNA can be encapsulated in liposomes or viral envelope vectors and introduced into cells.

  As described above, the present invention provides a method for inducing an antibody having a neutralizing function against REG4, comprising administering REG4, immunologically active REG4 fragments, or DNA or cells capable of expressing them. I will provide a. By the method of the present invention, an antibody having a neutralizing function that suppresses proliferation of REG4-expressing cells such as pancreatic cancer is induced. As a result, a therapeutic effect on pancreatic cancer and the like can be obtained.

Dominant negative protein that inhibits KIAA0101:
The present invention relates to inhibitory polypeptides comprising QKGIGEFF. In some preferred embodiments, inhibitory polypeptides include QKGIGEFF (SEQ ID NO: 46); a polypeptide functionally equivalent to that polypeptide; or a polynucleotide encoding such a polypeptide, wherein The polypeptide lacks the biological function of the peptide consisting of SEQ ID NO: 40. The amino acid sequence shown in SEQ ID NO: 40 is disclosed in WO2004 / 31412. It is known that the growth of cancer cells can be controlled by inhibiting the expression of the amino acid sequence. However, it is a novel finding proved by the present inventors that a fragment containing a sequence having a specific mutation in the amino acid sequence inhibits the growth of cancer cells.

  The polypeptide comprising a selected amino acid sequence of the present invention may be of any length so long as the polypeptide inhibits cancer cell growth. Specifically, the length of the amino acid sequence is 8 to 70 residues, such as 8 to 50 residues, preferably 8 to 30 residues, more specifically 8 to 20 residues, even more specifically. May range from 8 to 16 residues. For example, the amino acid sequence VRPTPKWQKGIGEFFRLSPK / SEQ ID NO: 44 is preferred as the selected amino acid sequence. Accordingly, a polypeptide comprising or consisting of the amino acid sequence TPKWQKGIGEFFRLSP / SEQ ID NO: 45 is a preferred example of a polypeptide in the present invention. A polypeptide of the invention characterized by comprising the amino acid sequence QKGIGEFF / SEQ ID NO: 46 may also be meant as a “PIP binding motif (PIP box)”.

  The polypeptides of the present invention may comprise two or more “selected amino acid sequences”. Two or more “selected amino acid sequences” may be the same or different amino acid sequences. Furthermore, the “selected amino acid sequences” can be linked directly. Or some intervening arrangement | sequence may be arrange | positioned among them.

  The present invention further relates to polypeptides that are homologous (ie, have sequence identity) to the QKGIGEFF / SEQ ID NO: 46 polypeptide. In the present invention, a polypeptide homologous to the QKGIGEFF / SEQ ID NO: 46 polypeptide includes any mutation selected from the addition, deletion, substitution, and insertion of one or more amino acid residues, QKGIGEFF / SEQ ID NO: 46 is functionally equivalent to the polypeptide. The phrase “functionally equivalent to QKGIGEFF / SEQ ID NO: 46 polypeptide” means having a function of inhibiting the binding of KIAA0101 to PCNA. The QKGIGEFF / SEQ ID NO: 46 sequence preferably maintains the amino acid sequence that constitutes a functionally equivalent polypeptide to the QKGIGEFF / SEQ ID NO: 46 polypeptide. Therefore, the polypeptide functionally equivalent to the QKGIGEFF / SEQ ID NO: 46 peptide in the present invention preferably has an amino acid mutation at a site other than the QKGIGEFF / SEQ ID NO: 46 sequence. The amino acid sequence of the polypeptide functionally equivalent to the QKGIGEFF / SEQ ID NO: 46 peptide in the present invention is the QKGIGEFF / SEQ ID NO: 46 sequence, which is 60% or more than the “selected amino acid sequence”. Above, usually 70% or more, preferably 80% or more, more preferably 90% or more, or 95% or more and even more preferably 98% or more homology. Amino acid sequence homology can be determined using algorithms well known in the art, eg, BLAST or ALIGN set by default.

  Alternatively, the number of amino acids that can be mutated is not particularly limited as long as the activity of QKGIGEFF / SEQ ID NO: 46 peptide is maintained. Generally, at most about 50 amino acids, even more preferably at most about 30 amino acids, even more preferably at most about 10 amino acids, even more preferably at most about 3 amino acids. Similarly, the site of the mutation is not particularly limited as long as the mutation does not result in a loss of activity of the QKGIGEFF / SEQ ID NO: 46 peptide.

In one preferred embodiment, the activity of the QKGIGEFF / SEQ ID NO: 46 peptide includes apoptosis inducing effects in cells expressing KIAA0101, ie pancreatic cancer cells, prostate cancer cells, breast cancer cells, lung cancer cells, and bladder cancer cells. Apoptosis means cell death caused by the cell itself, sometimes called programmed cell death. In cells undergoing apoptosis, nuclear chromosome aggregation, nuclear fragmentation, or cytoplasmic condensation is observed. Methods for detecting apoptosis are well known. For example, TUNEL staining (terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling; Gavrieli et al., (1992) J. Cell Biol. 119: 493-501, Mori et al., (1994) Anat. & Embryol. 190 : 21-28) may confirm apoptosis. Alternatively, DNA ladder assays, annexin V staining, caspase assays, electron microscopy, or observation of conformational changes on the nucleus or cell membrane may be used to detect apoptosis. Any commercially available kit may be used to detect these behaviors in cells induced by apoptosis. For example, such apoptosis detection kits are commercially available from the following suppliers:
LabChem Inc.,
Promega,
BD Biosciences Pharmingen,
Calbiochem,
Takara Bio Company (CLONTECH Inc.),
CHEMICON InteRNAtional, Inc,
Medical & Biological Laboratories Co., Ltd., etc.

The polypeptide of the present invention can be chemically synthesized from any position based on the selected amino acid sequence. Methods used in normal peptide chemistry can be used for methods of synthesizing polypeptides. Specifically, these methods include those described in the following documents and Japanese patent publications:
Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol. 2, Academic Press Inc., New York, 1976;
Peputido gousei (Peptide Synthesis), Maruzen (Inc.), 1975;
Peputido gousei no kiso to jikken (Fundamental and Experimental Peptide Synthesis), Maruzen (Inc.), 1985;
Iyakuhin no kaihatsu (Development of Pharmaceuticals), Sequel, Vol. 14: Peputido gousei (Peptide Synthesis), Hirokawa Shoten, 1991;
International Patent Publication No. WO99 / 67288.

  The polypeptide of the present invention can also be synthesized by a known genetic engineering technique. An example of a genetic engineering technique is as follows. Specifically, a transformed cell is prepared by introducing DNA encoding a desired peptide into an appropriate host cell. The polypeptide of the present invention can be obtained by recovering the polypeptide produced by this transformed cell. Alternatively, the desired polypeptide can be synthesized in an in vitro translation system in which the elements necessary for protein synthesis are reconstituted in vitro.

When genetic engineering techniques are used, the polypeptide of the present invention can be expressed as a fusion protein with a peptide having a different amino acid sequence. A vector that expresses the desired fusion protein comprises ligating a polynucleotide encoding a polypeptide of the invention with a polynucleotide encoding a different peptide such that they are in the same reading frame, followed by It can be obtained by introducing the resulting nucleotide into an expression vector. The fusion protein is expressed by transforming an appropriate host with the resulting vector. Different peptides used to form the fusion protein include the following peptides:
FLAG (Hopp et al., (1988) BioTechnology 6, 1204-10),
6xHis, 10xHis consisting of 6 His (histidine) residues,
Influenza hemagglutinin (HA),
Human c-myc fragment,
VSV-GP fragment,
p18 HIV fragment,
T7-tag,
HSV-tag,
E-tag,
SV40T antigen fragment,
Lck-tag,
α-tubulin fragment,
B-tag,
Protein C fragment,
GST (glutathione-S-transferase),
HA (influenza hemagglutinin),
An immunoglobulin constant region,
β-galactosidase, and
MBP (maltose-binding protein).

  The polypeptide of the present invention can be obtained by treating the fusion protein thus produced with an appropriate protease and subsequently recovering the desired polypeptide. To purify the polypeptide, the fusion protein can be pre-captured by affinity chromatography that binds to the fusion protein, and the captured fusion protein can subsequently be treated with a protease. By protease treatment, the desired polypeptide is separated from the affinity chromatography, and the desired polypeptide with high purity is recovered.

  The polypeptides of the present invention include modified polypeptides. In the present invention, the term “modified” refers to binding to other substances, for example. Therefore, in the present invention, the polypeptide of the present invention may further contain other substances such as a cell membrane permeable substance. Other materials include organic compounds such as peptides, lipids, saccharides, and various natural or synthetic polymers. The polypeptide of the present invention may have any modification as long as it retains the desired activity of inhibiting the binding of KIAA0101 to PCNA. In certain embodiments, the inhibitory polypeptide can directly compete for binding of KIAA0101 to PCNA. Modifications can also confer additional functions to the polypeptides of the invention. Examples of additional functions include targetability, reachability, and stabilization.

  Preferred examples of the modification in the present invention include, for example, introduction of a cell membrane permeable substance. Usually, the intracellular structure is blocked from the outside by a cell membrane. For this reason, it is difficult to efficiently introduce extracellular substances into cells. Cell membrane permeability can be imparted to the polypeptide of the present invention by modifying the polypeptide with a cell membrane permeable substance. Consequently, by contacting the polypeptide of the present invention with a cell, the polypeptide can be delivered into the cell and allowed to act there.

“Cell membrane permeable substance” means a substance that can permeate the mammalian cell membrane and enter the cytoplasm. For example, certain liposomes fuse with the cell membrane and release its contents into the cell. On the other hand, certain polypeptides penetrate the cytoplasmic membrane of mammalian cells and enter the inside of the cell. As such a polypeptide having cell invasion activity, a cytoplasmic membrane or the like is preferable as a substance in the present invention. Specifically, the present invention provides the following general formula:
[R]-[D]
Wherein [R] represents a cell membrane permeable substance, and [D] represents a fragment sequence comprising QKGIGEFF / SEQ ID NO: 46. In the above general formula, [R] and [D] can be linked directly or indirectly through a linker. Peptides, compounds having multiple functional populations, etc. can be used as linkers. Specifically, an amino acid sequence containing -GGG- can be used as a linker. Alternatively, a cell membrane permeable substance and a polypeptide containing a selected sequence can be bound to the surface of the microparticle. [R] can be linked to any position of [D]. Specifically, [R] can be linked to the N-terminus or C-terminus of [D], or to the side chain of the amino acid constituting [D]. Furthermore, a plurality of [R] molecules can be linked to one [D] molecule. The [R] molecule can be introduced at a plurality of different positions in the [D] molecule. Alternatively, [D] can be modified with a plurality of [R] linked to each other.

For example, various natural or artificially synthesized polypeptides with cell membrane permeability have been reported (Joliot A. & Prochiantz A., Nat Cell Biol. 2004; 6: 189-96). All of these known cell membrane permeable substances can be used to modify polypeptides in the present invention. In the present invention, for example, any substance selected from the following group can be used as the cell-permeable substance.
Poly-arginine; Matsushita et al., (2003) J. Neurosci .; 21, 6000-7.
[Tat / RKKRRQRRR] (SEQ ID NO: 47) Frankel et al., (1988) Cell 55,1189-93.
Green & Loewenstein (1988) Cell 55, 1179-88.
[Penetratin / RQIKIWFQNRRMKWKK] (SEQ ID NO: 48)
Derossi et al., (1994) J. Biol. Chem. 269, 10444-50.
[Buforin II / TRSSRAGLQFPVGRVHRLLRK] (SEQ ID NO: 49)
Park et al., (2000) Proc. Natl Acad. Sci. USA 97, 8245-50.
[Transportan / GWTLNSAGYLLGKINLKALAALAKKIL] (SEQ ID NO: 50)
Pooga et al., (1998) FASEB J. 12, 67-77.
[MAP (model amphipathic peptide) / KLALKLALKALKAALKLA] (SEQ ID NO: 51)
Oehlke et al., (1998) Biochim. Biophys. Acta. 1414, 127-39.
[K-FGF / AAVALLPAVLLALLAP] (SEQ ID NO: 52)
Lin et al., (1995) J. Biol. Chem. 270, 14255-8.
[Ku70 / VPMLK] (SEQ ID NO: 53)
Sawada et al., (2003) Nature Cell Biol. 5, 352-7.
[Ku70 / PMLKE] (SEQ ID NO: 61)
Sawada et al., (2003) Nature Cell Biol. 5, 352-7.
[Prion / MANLGYWLLALFVTMWTDVGLCKKRPKP] (SEQ ID NO: 54)
Lundberg et al., (2002) Biochem. Biophys. Res. Commun. 299, 85-90.
[pVEC / LLIILRRRIRKQAHAHSK] (SEQ ID NO: 55)
Elmquist et al., (2001) Exp. Cell Res. 269, 237-44.
[Pep-1 / KETWWETWWTEWSQPKKKRKV] (SEQ ID NO: 56)
Morris et al., (2001) Nature Biotechnol. 19, 1173-6.
[SynB1 / RGGRLSYSRRRFSTSTGR] (SEQ ID NO: 57)
Rousselle et al., (2000) Mol. Pharmacol. 57, 679-86.
[Pep-7 / SDLWEMMMVSLACQY] (SEQ ID NO: 58)
Gao et al., (2002) Bioorg. Med. Chem. 10, 4057-65.
[HN-1 / TSPLNIHNGQKL] (SEQ ID NO: 59)
Hong & Clayman (2000) Cancer Res. 60, 6551-6

  In the present invention, the poly-arginine listed above as an example of a cell membrane permeable substance is composed of an arbitrary number of arginine residues. Specifically, for example, it is composed of 5 to 20 consecutive arginine residues. The preferred number of arginine residues is 11 (SEQ ID NO: 60).

Pharmaceutical composition comprising QKGIGEFF / SEQ ID NO: 46 The polypeptide of the invention inhibits the growth of cancer cells. Therefore, the present invention provides an agent for treating and / or preventing cancer, comprising, as an active ingredient, a polypeptide comprising QKGIGEFF / SEQ ID NO: 46, or a polynucleotide encoding the same. Alternatively, the present invention relates to a method for treating and / or preventing cancer comprising the step of administering a polypeptide of the present invention. Furthermore, the invention relates to the use of a polypeptide of the invention in the manufacture of a pharmaceutical composition for treating and / or preventing cancer. Cancers that can be treated or prevented by the present invention are not limited as long as the expression of KIAA0101 is upregulated in cancer cells. For example, the polypeptides of the present invention are useful for treating pancreatic cancer, lung cancer, prostate cancer, breast cancer, bladder cancer, kidney cancer or testicular tumor. Among them, pancreatic cancer is preferred as a target for treatment or prevention in the present invention.

  Alternatively, the inhibitory polypeptides of the present invention can be used to induce apoptosis of cancer cells. Therefore, the present invention provides an agent for inducing apoptosis of cells, which contains a polypeptide containing QKGIGEFF / SEQ ID NO: 46 or a polynucleotide encoding the same as an active ingredient. The agent for inducing apoptosis of the present invention may be used for treating cell proliferative diseases such as cancer. Cancers that can be treated or prevented by the present invention are not limited as long as the expression of KIAA0101 is upregulated in cancer cells. For example, the polypeptides of the present invention are useful for treating pancreatic cancer, prostate cancer, breast cancer, bladder cancer, lung cancer, kidney cancer or testicular tumor. Among them, pancreatic cancer is preferred as a target for treatment or prevention in the present invention. Alternatively, the present invention relates to a method for inducing apoptosis of a cell comprising administering a polypeptide of the present invention. Furthermore, the invention relates to the use of a polypeptide of the invention in the manufacture of a pharmaceutical composition for inducing apoptosis in cells.

  The inhibitory polypeptides of the present invention induce apoptosis in KIAA0101-expressing cells such as pancreatic cancer. Meanwhile, KIAA0101 expression is not observed in most normal tissues. In some normal tissues, the expression level of KIAA0101 is relatively low compared to cancer tissues. Therefore, the polypeptide of the present invention can induce apoptosis specifically in cancer cells.

  The polypeptides of the invention can be used to treat cancer or induce apoptosis in cells, including humans and other mammals such as mice, rats, guinea pigs, rabbits, cats, dogs, sheep, pigs, cows, monkeys, baboons. , And when administered as a medicinal agent prepared for chimpanzees, the isolated compound can be administered directly, or formulated as a suitable dosage form using known medicinal drug preparation methods You can also. For example, if necessary, the pharmaceutical agent can be administered orally as sugar-coated tablets, capsules, elixirs, and microcapsules, or as a sterile solution using water or any other pharmaceutically acceptable liquid or It can also be administered parenterally in an injectable dosage form that is a suspension. For example, in a unit dosage form necessary to produce a generally acceptable pharmaceutical agent, the compound is pharmaceutically acceptable carrier or solvent, specifically sterile water, saline, vegetable oil, emulsifier. , Suspensions, surfactants, stabilizers, flavoring agents, excipients, solvent agents, preservatives, and binders. Depending on the amount of active ingredient in these formulations, a suitable dose within the specified range can be determined.

  Examples of additives that may be mixed into tablets and capsules include binders such as gelatin, corn starch, tragacanth gum, and gum arabic; media such as crystalline cellulose; swelling such as corn starch, gelatin, and alginic acid Agents; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, or saccharin; and flavoring agents such as peppermint, winter green oil, or cherry. Where the unit dosage form is a capsule, the above ingredients can further include a liquid carrier such as oil. Sterile mixtures for injection can be formulated using a medium such as distilled water for injection in line with the practice of conventional pharmaceutical agents.

  As an aqueous solution for injection, physiological saline, glucose solution, and other isotonic solutions containing adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be used. They are combined with suitable solubilizers such as alcohols, specifically ethanol, and polyalcohols such as propylene glycol and polyethylene glycol, nonionic surfactants such as polysorbate 80 ™ and HCO-50, etc. Can be used.

  Sesame oil or soybean oil can be used as the oily liquid, and it can also be used in combination with benzyl benzoate or benzyl alcohol as a solubilizer. In addition, they can be formulated with buffers such as phosphate buffer and sodium acetate buffer; analgesics such as procaine hydrochloride; stabilizers such as benzyl alcohol and phenol; and antioxidants. The injection solution thus prepared is filled into a suitable ampoule.

  To administer the pharmaceutical compounds of the invention to a patient, for example, by intraarterial, intravenous or subcutaneous injection, and also by intranasal, transbronchial, intramuscular, or oral administration. Any method well known to those skilled in the art can be used. The dose and method of administration will vary depending on the weight and age of the patient and the method of administration. However, those skilled in the art can select them routinely. The DNA encoding the polypeptide of the present invention can be inserted into a vector for gene therapy, and the vector can be administered for therapy. The dose and administration method vary depending on the patient's weight, age, symptoms, and the like, and those skilled in the art can appropriately select them. For example, the dose of a compound that binds to the polypeptide of the present invention and modulates its activity, when orally administered to a healthy adult (body weight 60 kg), varies somewhat depending on the symptoms, but is about 0.1 mg to about 100 mg / day, preferably about 1.0 mg to about 50 mg / day, more preferably about 1.0 mg to about 20 mg / day.

  When the compound is administered parenterally in an injectable form to a healthy adult (body weight 60 kg), about 0.01 mg to about 30 mg, depending on the patient, target organ, symptoms, and method of administration It is convenient to inject intravenously a dose of about 0.1 mg to about 20 mg / day, more preferably about 0.1 mg to about 10 mg / day. Similarly, the compound can be administered to other animals in an amount converted from the dose at a body weight of 60 kg.

Pharmaceutical composition:
Accordingly, the present invention includes agents and methods useful for one or both of cancer prevention and treatment. These agents and methods include siRNA that inhibits the expression of REG4 or KIAA0101, an antibody that neutralizes the activity of REG4, a polypeptide comprising QKGIGEFF / SEQ ID NO: 46, a polypeptide functionally equivalent to that polypeptide, or A polynucleotide encoding these polypeptides of the present invention comprises at least one in an amount effective to achieve attenuation or arrest of disease cell proliferation. More specifically, in the specification of the present invention, a therapeutically effective amount means an amount effective to prevent progression of the subject to be treated or to ameliorate its existing symptoms.

  Individuals treated with the methods of the present invention include any individual suffering from cancer, including, for example, pancreatic cancer, prostate cancer, breast cancer and bladder cancer. Such individuals are, for example, vertebrates, such as mammals including humans, dogs, cats, horses, cows, or goats, or any other animal, especially a commercially important animal or domesticated animal. It is possible. For the purposes of the present invention, an increase in marker protein expression is at least 10%, preferably 15%, 20%, 25%, relative to the mean concentration of marker protein in normal cells for one or both marker proteins. Mean average cell marker protein concentration of 30%, 35%, 40%, 45%, 50%, 55%, or more.

  In the present specification, suitable pharmaceutical dosage forms include oral, rectal, nasal, topical (including oral and sublingual), vaginal, or parenteral (including intramuscular, subcutaneous and intravenous). Dosage forms suitable for administration or for administration by inhalation or inhalation are included. Preferably administration is intravenous. The dosage form is appropriately sealed by volume unit.

  Pharmaceutical dosage forms suitable for oral administration include capsules, cachets or tablets containing a predetermined amount of the active ingredient. Suitable dosage forms also include powders, granules, solubilizers, suspending agents and emulsifiers. The active ingredient is optionally administered as a bolus electuary or paste. Tablets and capsules for oral administration are, for example, bulking agents such as sugars including lactose, sucrose, mannitol or sorbitol, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose Conventional excipients such as cellulose preparations such as sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP), binders, lubricants, and / or humidifiers may be included. If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

  A tablet may be made by compression or molding, optionally with one or more pharmaceutical ingredients. A compressed tablet is a powdered or granulated active ingredient, optionally mixed with a binder, lubricant, inert diluent, lubricant, surfactant, or dispersant and compressed in a suitable device. Can be prepared. Molded tablets may be made by molding in a suitable apparatus a mixture of the powdered compound moistened with an inert diluent. The tablets can be coated according to methods well known in the art. Oral fluid preparations may be in the form of, for example, an aqueous or oily suspension, solution, emulsion, syrup, or elixir, or dried for reconstitution with water or other suitable solvent prior to use. It may be a product. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous solvents (which may include edible oils), pH adjusting agents, and / or preservatives. Tablets may optionally be formulated to provide slow release of the active ingredient, ie sustained release. Tablet packaging may contain one tablet taken once a month.

  Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions optionally including antioxidants, buffers, bacteriostatic agents, and solutions that render the formulation isotonic with the blood of the intended recipient; Also included are aqueous and non-aqueous sterile suspensions that may contain suspending agents and / or thickeners. The formulation can be placed in unit dose or multi-dose containers, such as sealed ampoules and vials, and a sterile liquid carrier (eg, saline or water for injection) can be added just prior to use. Can be stored under lyophilization conditions. Alternatively, the formulation can be for continuous infusion. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

  Formulations for rectal administration include suppositories containing standard carriers such as cocoa butter or polyethylene glycol. For topical administration to the oral cavity, for example, buccal or sublingual administration, sucrose and acacia flavor bases or lozenges containing the active ingredient in tragacanth, etc., and gelatin, glycerin, sucrose or acacia, etc. A base tablet containing an active ingredient in the base is included. When the active ingredient is administered nasally, a liquid spray, or a dispersible powder, or a droplet form can be used. The droplets can be formulated with an aqueous or non-aqueous base that also contains one or more dispersants, solubilizers, or suspending agents.

  For inhalation administration, the composition is suitably administered from an insufflator, nebulizer, pressurized container, or other convenient aerosol spray delivery means. The pressurized container contains a suitable propellant (dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas). In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that delivers a fixed amount.

  Alternatively, for administration by inhalation or inhalation, the composition may take the form of a dry powder composition, eg, a powder mixture consisting of the active ingredient and a suitable powder base such as lactose or starch. The powder composition can be in unit dosage form, eg in capsules, cartridges, gelatin, or blister packs, which can be administered with an inhaler or aspirator.

  Other formulations include implantable devices and adhesive patches that release therapeutic agents.

  If desired, the above-described formulations adapted for sustained release of the active ingredient can be used. The pharmaceutical composition may also contain other active ingredients such as antibacterial agents, immunosuppressive agents, or preservatives.

  In addition to the ingredients specifically mentioned above, it should be understood that the formulations of the present invention may include other agents well known in the art for the type of formulation. For example, formulations suitable for oral administration can include flavoring agents.

  For example, depending on the route of administration, the concentration of the test compound administered, or whether the treatment uses a protein, a nucleic acid encoding the test compound, or a drug containing cells capable of secreting the test compound as an active ingredient It will be apparent to those skilled in the art that things may be more preferred.

  The pharmaceutical compositions of the invention are manufactured in a manner known per se, for example by conventional mixing, dissolving, granulating, dragee forming, grinding, emulsifying, encapsulating, entrapping or lyophilizing processes. Yes. Proper formulation is dependent upon the route of administration chosen.

Dosage and Schedule Determination of effective dosage ranges for the agents of the present invention is well within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein. Effective doses for test compound treatment can be estimated initially from cell culture assays and / or animal models. For example, a dose in an animal model can be calculated to reach a circulating concentration range that includes an IC50 determined by cell culture (a dose at which 50% of cells exhibit the desired effect). In addition, the toxicity and therapeutic efficacy of the test compound can be determined by, for example, cell culture or determination of LD50 (a dose that is lethal to 50% of the population) and ED50 (a dose that is therapeutically effective for 50% of the population) It can be determined by standard pharmaceutical procedures in laboratory animals. The dose ratio between toxic and therapeutic effects is the therapeutic index (ie, the ratio of LD50 to ED50). Compounds that exhibit high therapeutic indices are preferred. Data obtained from these cell culture assays and animal studies can be used in defining the dosage range for use in humans. The dosage of such compounds will fall within the range of circulating concentrations that include the ED50 with little or no toxicity. The dose may vary within this range depending on the dosage form used and the route of administration utilized. The exact dosage form, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See, for example, Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p1. Dosage amount and interval may be adjusted individually to provide plasma levels of the active test compound which are sufficient to maintain the desired effects.

  In particular, for each of the conditions described above, the composition (eg, polypeptide or organic compound) can be administered orally or by injection at a dose of about 0.1 to about 250 mg / kg / day. The adult dose range is generally about 5 mg to about 17.5 g / day, preferably about 5 mg to about 10 g / day, and most preferably about 100 mg to about 3 g / day. Tablets or other unit dosage forms provided in individual units are suitably unit doses that show efficacy in multiple doses of the same unit dose, for example about 5 mg to about 500 mg, usually about Includes amounts containing 100 mg to about 500 mg.

  The dose used will depend on several factors, including the age and sex of the subject, the exact disease to be treated, and its severity. The route of administration may also vary depending on the condition and severity. In any event, taking into account the factors discussed above, an appropriate and optimal dosage can be routinely calculated by those skilled in the art.

  Usually an effective or effective amount of one or more REG4 or KIAA0101 inhibitors is first administered a low dose or small amount of REG4 and / or KIAA0101 inhibitor and then abnormal REG4 and / or KIAA0101 overexpression or Increase and / or need for sequentially administered doses or doses until the desired effect of inhibiting or preventing intracellular signal-mediated cancer is observed in the formulated subject with minimal or no toxic side effects Depending on the addition of a second REG4 and / or KIAA0101 inhibitor. Suitable methods for determining the appropriate dose and schedule for administering the pharmaceutical composition of the present invention include, for example, Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., Brunton, et al., Eds., McGraw -Hill (2006), and Remington: The Science and Practice of Pharmacy, 21st Ed., University of the Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins (2005), both incorporated herein by reference. It is done.

Gene therapy
SiRNA that inhibits the expression of REG4 or KIAA0101, an antibody that neutralizes REG4 activity, and a cell-permeable dominant negative peptide that inhibits the interaction between KIAA0101 / PCNA can be used to treat cancer patients with gene therapy Delivery. Alternatively, a neutralizing antibody or a polypeptide comprising QKGIGEFF / SEQ ID NO: 46 of the present invention is used as a peptide that directly changes the activity between REG4 or KIAA0101 / PCNA. In certain aspects, gene therapy embodiments include nucleic acid sequences that encode the appropriate identified peptides of the present invention. In a preferred embodiment, the nucleic acid sequence includes regulatory elements necessary for expression of the peptide in the target cell. The nucleic acid can be prepared for stable insertion into the genome of the target cell (see, eg, description of the homologous recombination cassette vector of Thomas, KR and Capecchi, MR (1987) Cell 51: 503).

  Delivery of the nucleic acid to the patient may be direct (in which case the patient is directly exposed to the nucleic acid or nucleic acid-containing vector) or may be indirect (in which case the cell is first transformed in vitro with the nucleic acid. To be transplanted to the patient). These two approaches are known as in vivo or ex vivo gene therapy, respectively.

  For a general review of gene therapy methods, see Goldspiel et al., (1993) Clinical Pharmacy 12: 488-505; Wu and Wu, (1991) Biotherapy 3: 87-95; Tolstoshev, (1993) Ann. Rev. Pharmacol. Toxicol. 33: 573-596; Mulligan, (1993) Science 260: 926-932; and Morgan and Anderson, (1993) Ann. Rev. Biochem. 62: 191-217; May, (1993) TIBTECH 11 ( 5): Please refer to 155-115. Methods commonly known in the field of recombinant DNA technology that can be used are Ausubel et al. (Eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, Gene Transfer and Expression. , A Laboratory Manual, Stockton Press, NY.

Diagnosis of cancer chemo-radiotherapy resistance The term “diagnose” is meant to include prediction and likelihood analysis. The method is intended to be used clinically in decision making concerning therapeutic interventions including cancer therapeutic intervention, diagnostic criteria such as disease stage, and disease monitoring and follow-up.

Diagnosis Method for Cancer Chemo-Radiotherapy Resistance The expression of sREG4 was found to be specifically elevated in surgical samples in patients with pancreatic cancer resistant to chemo-radiotherapy. Thus, the REG4 gene identified here, as well as its transcription and translation products, has found diagnostic utility as a marker for cancer chemo-radiotherapy resistance, and the REG4 gene in cell samples By measuring expression, one can diagnose cancer chemo-radiotherapy resistance.

Specifically, the present invention provides the following methods for diagnosing chemo-radiotherapy resistance of cancer by determining the expression level of REG4 in a subject:
[1] A method for diagnosing chemo-radiotherapy resistance of a cancer in a subject comprising measuring the expression level of the REG4 gene in a biological sample from the subject, the expression of the gene compared to the normal subject level A method wherein increased levels indicate that the subject is suffering from a chemo-radiation resistant risk or a chemo-radiation resistant cancer.
[2] The method of [1], wherein the expression level is at least 10% greater than the normal control level.
[3] The method of [1], wherein the expression level is measured by a method selected from the following group:
(a) detection of mRNA of said gene;
(b) detecting the protein encoded by the gene, and
(c) detecting the biological activity of the protein encoded by the gene.
[4] The method according to [3], which is measured by detecting hybridization of a probe to a gene transcript of the gene.
[5] The method according to [4], wherein the hybridization step is performed on a DNA array.
[6] The method of [3], wherein the expression level is measured by detecting binding of an antibody to a protein encoded by the REG4 gene as the gene expression level.
[7] The method of [1], wherein the biological sample comprises a biopsy, sputum or blood.
[8] The method of [1], wherein the biological sample derived from the subject contains epithelial cells.
[9] The method of [1], wherein the biological sample derived from the subject contains cancer cells.
[10] The method of [1], wherein the biological sample derived from the subject contains cancer epithelial cells.
[11] The method of [1], wherein the cancer is pancreatic cancer.

  The subject diagnosed by this method is preferably a mammal. Exemplary mammals include, but are not limited to, for example, humans, non-human primates, mice, rats, dogs, cats, horses and cows.

  It is preferred to collect a biological sample from the subject being diagnosed to make the diagnosis. Any biological material can be used as a biological sample for determination as long as it contains the transcriptional or translational product of REG4 of interest. Biological samples include, but are not limited to, body tissue and body fluids such as blood, sputum, and urine. Preferably, the biological sample comprises a cell population comprising epithelial cells, more preferably cancer epithelial cells, or epithelial cells from tissue suspected of being cancerous. Further, if necessary, the cells may be purified from the obtained body tissue and fluid and then used as a biological sample.

  According to the present invention, the expression level of the REG4 gene is determined in a subject-derived biological sample. Expression levels can be determined at the transcription (nucleic acid) product level using methods known in the art. For example, mRNA of the REG4 gene may be quantified using a probe by a hybridization method (for example, Northern hybridization). Detection may be performed on a chip or array. Use of the array is preferred for detecting the expression level of multiple genes (eg, various cancer specific genes) including the REG4 gene of the present invention. One skilled in the art can prepare such probes using the sequence information of the REG4 gene (SEQ ID NO: 1; Genbank accession number AY126670). For example, cDNA of REG4 gene may be used as a probe. If necessary, the probe may be labeled with a suitable label, such as a dye and isotope, and the expression level of the gene can be detected as the intensity of the hybridized label.

  Furthermore, the transcription product of the REG4 gene can be quantified using a primer by a detection method based on amplification (for example, RT-PCR). Such primers can also be prepared based on the available sequence information of the gene. For example, the primers (SEQ ID NOs: 3 and 4) used in the examples can be used for detection by RT-PCR or Northern blot, but the invention is not limited thereto. Specifically, the probe or primer used for the method of the present invention hybridizes to the mRNA of the REG4 gene under stringent conditions, moderately stringent conditions, or low stringent conditions. . As used herein, the phrase “stringent conditions” is shown in the section “Small interfering RNA”.

  Alternatively, translation products can be detected for the diagnosis of the present invention. For example, the amount of REG4 protein may be determined. Methods for determining the amount of a protein as a translation product include immunoassay methods that use an antibody that specifically recognizes the protein. The antibody may be monoclonal or polyclonal. In addition, any fragment or variant of the antibody (e.g., chimeric antibody, scFv, Fab, F (ab ') 2, Fv, etc.) can be detected as long as the fragment retains the ability to bind to REG4 protein. May be used for Methods for preparing these types of antibodies for protein detection are well known in the art, and any method can be used in the present invention to prepare such antibodies and their equivalents. .

  As another method for detecting the expression level of the REG4 gene based on its translation product, the intensity of staining may be observed through immunohistochemical analysis using an antibody against the REG4 protein. That is, when strong staining is observed, it indicates an increase in the presence of protein and at the same time a high expression level of the REG4 gene. Furthermore, translation products can also be detected based on their biological activity.

  The expression level of the REG4 gene in the biological sample is increased or decreased by, for example, 10%, 25%, or 50% from the control level of the REG4 gene; or 1.1 times, 1.5 times, 2.0 times, 5.0 times If it rises above, above 10.0 times, or more, it can be considered as rising.

  The control level can be determined at the same time as the test biological sample by using a sample previously collected and stored from a subject with known disease state (cancerous or non-cancerous). Alternatively, the control level may be determined by statistical methods based on results obtained by analyzing a predetermined expression level of the REG4 gene in a sample from a subject with known disease status . Furthermore, the control level may be a database of expression patterns from previously tested cells. Furthermore, according to aspects of the present invention, the expression level of the REG4 gene in a biological sample may be compared to a plurality of control levels determined from a plurality of reference samples. It is preferred to use a control level determined from a reference sample derived from a tissue type similar to that of the patient-derived biological sample. Furthermore, it is preferable to use a standard value for the expression level of the REG4 gene in a population whose disease state is known. The standard value may be obtained by any method known in the art. For example, an average value ± 2S.D. Or a range of average values ± 3S.D. May be used as the standard value.

  In the context of the present invention, a control level determined from a biological sample that has been found not to be cancerous is referred to as a “normal control level”. On the other hand, if the control level is determined from a chemo-radiation resistant biological sample, this is referred to as the “chemo-radiation resistant control level”.

  If the expression level of the REG4 gene is elevated compared to the normal control level or is similar to the chemo-radiation resistant control level, the subject is suffering from chemo-radiation resistant cancer, Or it can be diagnosed as being at risk of chemo-radiation resistance.

  The difference between the expression level of the test biological sample and the control level is the expression level of a control nucleic acid, such as a housekeeping gene, whose expression level is known not to vary depending on the cancerous or non-cancerous state of the cell. It can be standardized. Exemplary control genes include, but are not limited to, β-actin, glyceraldehyde 3-phosphate dehydrogenase, and ribosomal protein P1.

  Aspects of the invention are described in the following examples, which are not intended to limit the scope of the invention described in the claims.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although similar or equivalent methods and materials described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.

  The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1
1． General method
Serum samples of clinical samples before and after surgery (3 or 4 weeks after therapeutic resection) were obtained from 7 patients who had undergone therapeutic resection of pancreatic ductal adenocarcinoma with informed consent. Normal paraffin-embedded tissue sections of PDAC were also obtained from surgical specimens resected at the same center. A tissue microarray sample in which 31 PDAC tissues and 2 endocrine tumor tissues were spotted on both the primary and secondary sides was obtained from ISU ABXIS (Seoul, Korea).

Cell line
PDAC cell lines, PK45P and SUIT-2 were provided by Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Japan) and Kyushu Medical Center (Fukuoka, Japan). MIAPaCa-2 was purchased from the American Type Culture Collection (ATCC, Rockville, MD). These cell lines contain RPMI 1640 (Sigma-Aldrich, St. Louis, MO) (PK45P) containing 10% fetal bovine serum (Cansera InteRNAtional, Ontario, Canada) and 1% antibiotic / antibacterial solution (Sigma-Aldrich). And for SUIT-2) and Dulbecco's Modified Eagle's Medium (Sigma-Aldrich) (for MIAPaCa-2). Cells were maintained at 37 ° C. in 5% CO ₂ and humidified air. FreeStyle ™ 293 cells (Invitrogen, Carlsbad, CA) were suspended in FreeStyle ™ 293 Expression Medium (Invitrogen) and orbitally shaken at 125 rpm using an Erlenmeyer flask (Corning, NY). Rotated on machine and allowed to grow. Cells were maintained at 37 ° C. in 8% CO ₂ and humidified air. Pancreatic cancer cell lines MIAPaCa-2 and Panc-1 grown in Dulbecco's Modified Eagle's Medium or RPMI1640 (Sigma-Aldrich, St. Louis, MO), and the normal rodent cell line NIH3T3 are American Type Culture Collection (ATCC, Rockville, MD). Pancreatic cancer cell lines PK-59, KLM-1, PK45P and PK-1 were provided by Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Japan) and stored at RPMI1640 (Sigma-Aldrich). Both media were supplemented with 10% fetal calf serum (Cansera InteRNAtional, Ontario, Canada) and 1% antibiotic / antibacterial solution (Sigma-Aldrich). Cells were maintained at 37 ° C. in 5% CO ₂ and humidified air.

2． Semi-quantitative RT-PCR for REG4
Purification of PDAC cells, pancreatic cancer cells and normal pancreatic ductal epithelial cells has been previously described (Nakamura T, et al. Oncogene 2004; 23: 2385-400.). Purified cell populations and RNA from normal human heart, lung, liver, kidney, brain, pancreas (BD Biosciences, Palo Alto, Calif.) And normal pancreatic duct cells were double T7-based in vitro transcription (Epicentre Technologies, Madison) , WI) followed by synthesis into single stranded cDNA. Total RNA from human pancreatic cancer cell lines was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's recommendations. The extracted RNA was treated with deoxyribonuclease I (Roche, Mannheim, Germany) and reverse transcribed into single stranded cDNA using Superscript II reverse transcriptase (Invitrogen) and oligo (dT) primers. We prepared appropriate dilutions of single-stranded cDNA with α-tubulin (TUBA) as a quantitative control for PCR amplification.

Primer sequence is
For TUBA
5'-AAGGATTATGAGGAGGTTGGTGT-3 '(SEQ ID NO: 9) and
5'-CTTGGGTCTGTAACAAAGCATTC-3 '(SEQ ID NO: 10);
For REG4,
5′-CCAATTGCTATGGTTACTTCAGG-3 ′ (SEQ ID NO: 3) and
5′-GAAAAACAAGCAGGAGTTGAGTG-3 ′ (SEQ ID NO: 4),
For KIAA0101 (NM_014736, the amino acid sequence is represented by SEQ ID NO: 40 encoded by the nucleic acid represented by SEQ ID NO: 39)
5′-AGCTTTGTTGAACAGGCATTT-3 ′ (SEQ ID NO: 26) and
5′-GGCAGCAGTACAACAATCTAAGC-3 ′ (SEQ ID NO: 27),
For β2MG
5'-CACCCCCACTGAAAAAGAGA-3 '(SEQ ID NO: 28) and
5′-TACCTGTGGAGCAAGGTGC-3 ′ (SEQ ID NO: 29).

  All reactions include initial denaturation at 94 ° C for 2 minutes followed by 94 ° C for 30 seconds, 23 cycles (for TUBA) or 28 cycles (for REG4), and 72 ° C for 1 minute, or 95 ° C After a 5 minute initial denaturation step, perform 30 cycles of 95 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 30 seconds (β2MG), or 28 cycles (for KIAA0101) GeneAmp PCR system 9700 (PE Applied Biosystems , Foster, CA).

3． Antibody against REG4
An expression vector for His-tagged full-length human REG4 was transfected into 293T cells, and recombinant REG4 (REG4-His) was purified from the culture solution using a TALON purification kit (Clontech, San Diego, Calif.). REG4-His protein was prepared for injection by emulsifying the antigen solution with Freund's complete adjuvant. Anti-REG4 polyclonal antibody (pAb) was prepared using rabbit (Medical and Biological Laboratories, Nagoya, Japan) against REG4-His protein, and immune serum was purified by affinity column according to standard protocol. Mouse monoclonal antibody (mAb) was made by inoculating BALB / C mice with REG4-His with Freund's complete adjuvant. Lymphocytes were fused with myeloma cells P3U1, and single clone hybrid cells were generated and evaluated by standard techniques.

Four. Recombinant protein for antibody and KIAA0101 A cDNA fragment encoding the full-length KIAA0101 (111 amino acids, NP_055551) (SEQ ID NO: 15) was generated using KOD-Plus polymerase (TOYOBO) and the pET21 vector (Novagen ) And cloned. Recombinant KIAA0101 protein fused at the COOH end with a polyhistidine tag was expressed in E. coli, BL21 codon plus (Stratagene) and purified using Ni-NTA resin (QIAGEN) under natural conditions according to the supplier's protocol . Repurification was performed using a High Performance Liquid Chromatography AKTA explorer (Amersham) equipped with MonoS HR 5/5 (Amersham). The protein was immunized in rabbits and immune serum was purified according to basic methods using an affinity column packed with Affi-Gel 10 activated affinity media (Bio-Rad) conjugated with recombinant KIAA0101 protein. The affinity-purified anti-KIAA0101 polyclonal antibody was used for detection of KIAA0101 protein.

Five. Immunohistochemical staining of REG4 Sections were autoclaved and deparaffinized in citrate buffer, pH 6.0 for 15 minutes at 108 ° C. Endogenous peroxidase activity was inactivated by incubation for 30 minutes in 0.33% hydrogen peroxide diluted in methanol. After incubation with fetal bovine serum for blocking, sections were incubated with anti-REG4 pAb (1: 1000) for 1 hour at room temperature. After washing with PBS, immunodetection was performed using peroxidase labeled anti-mouse immunoglobulin (Envision kit, Dako Cytomation, Carpinteria, CA). Finally, the reaction was matured with 3,3'-diaminobenzidine (Dako) and the cells were counterstained with hematoxylin.

  With global genomic cDNA microarray analysis, we identified dozens of genes that were overexpressed in PDAC cells (Hartupee JC, et al. Biochim Biophys Acta 2001; 1518: 287-93). Among them, we focused on REG4 that we confirmed overexpression in 7 out of 9 microdisected PDAC cell populations investigated by RT-PCR (FIG. 1A). . In PDAC cells, its mRNA levels were clearly higher than normal pancreas and vital organs including heart, lung, kidney and brain.

  Immunohistochemical analysis with pAb against REG4 in other series of PDAC tissues showed a strong signal on REG4 goblet cell-like vesicles (Figure 1B) or on the cell surface of cancer cells (Figure 1C), Normal pancreatic acinar cells (FIG. 1D) showed slight staining of REG4 and no signal in duct cells and islet cells. Adult vital organs including heart, lung, kidney and brain showed no staining (data not shown). In addition, tissue microarrays in another series of 31 PDAC tissues showed high levels of REG4 expression in 14 of 31 PDACs, and 35 (55%) of all 64 PDACs with anti-REG4 antibodies Showed positive staining. Well-differentiated PDAC (G1) showed more positive REG4 staining than undifferentiated (G2, G3 and G4) PDAC (p = 0.0001, chi-square test).

6． Establishment of cells that stably express REG4
To create a REG4 expression vector, the entire coding sequence of REG4 cDNA was amplified by PCR using the following primer pair at the restriction enzyme site;
5′-CGGAATTCATGGCTTCCAGAAGCATGC-3 ′ (SEQ ID NO: 63) and
5'-ATAAGAATGCGGCCGCTGGTCGGTACTTGCACAGG-3 '(SEQ ID NO: 64),
These primers contain EcoRI and NotI restriction sites indicated by the first and second underline. The product was inserted into the EcoRI and NotI sites of pCAGGSnHC to express the HA tag protein. The plasmid was transfected into REG4 negative PDAC cell line, PK45P cell using FuGENE6 (Roche) according to the method recommended by the manufacturer. Cell populations were selected using 0.5 mg / ml Geneticin (Invitrogen) and clonal PK45P cells were subcloned by limiting dilution. To assess the level of HA-tagged REG4 expression, several clones were isolated from 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA, 0.1% protease. Recovered with lysis buffer containing inhibitor cocktail III (Calbiochem, San Diego, CA). The sample was centrifuged to remove the precipitate. The amount of supernatant protein was determined by the Bradford method. The 10 mg portion was subjected to 15% SDS-PAGE and detected by Western blotting using an anti-HA antibody (3F10) (Roche). The amount of each protein was normalized by anti-β-actin antibody (Sigma-Aldrich). Three clones that constitutively expressed REG4 were established (C1-6, C2-6, C10). Control PK45P cells transfected with empty pCAGGSnHC-HA vector were also established (M1, M3, M6).

7． Gamma and gemcitabine resistance assays
Each clone (C1-6, C2-6, C10) expressing 3,000 cells of REG4 or control clone (M1, M3, M6) is seeded in each well of a 96-well culture dish and contains 10% FBS Incubated in medium. After 48 hours of preincubation, the cells were treated with 1, 5, 10, or 30 Gy of gamma irradiation using a 60 cobalt source, or 48 to 0.1 to 100,000 nM gemcitabine. After 48 hours, viable cells were measured using Cell counting kit-8 (DOJINDO). To assess percent inhibition values, the relative ratio of absorbance (each treatment) / absorbance (untreated control) was calculated. For FACS analysis, 48,000 cells / well were seeded in 6-well plates and after 48 hours preincubation, the cells were irradiated with 0, 1 or 5 Gy gamma irradiation using a 60 cobalt source, or 48 hours 10 nM or Treated with 50 nM gemcitabine. 48 hours later, cells were collected, washed with PBS, fixed with cold 70% ethanol, stained with propidium iodide (10 mg / ml) and ribonuclease A (100 mg / ml), and FACSCalibur ™ Flow Cytometry System Cell cycle analysis was performed by (BD) analysis. The percentage of aneuploid cells was calculated by cell cycle analysis software (BD CELLQuest ™).

8． Immunohistochemical staining for KIAA0101 Normal sections of pancreatic cancer tissue were obtained from surgical specimens resected under appropriate informed consent. Normal pancreas sections were purchased from Biochain (Hayward, CA). Sections were deparaffinized by autoclaving at 108 ° C. in Dako Cytomation Target Retrieval Solution High pH (Dako, Carpinteria, Calif.) For 15 minutes. After blocking of endogenous peroxidase and protein, sections were incubated with anti-KIAA0101 antibody (1: 200 dilution) for 30 minutes at room temperature. After washing with PBS, immunodetection was performed with peroxidase-labeled anti-rabbit immunoglobulin (Envision kit, Dako). Finally, the reaction was matured with 3,3′-diaminobenzidine (Dako) and counterstained with hematoxylin.

9． Northern Blot Analysis We extracted total RNA from several pancreatic cancer cell lines using Trizol reagent (Invitrogen, Carlsbad, Calif.) And performed Northern blot analysis. After treatment with deoxyribonuclease I (Nippon Gene, Osaka, Japan), mRNA was purified with Micro-FastTrack (Invitrogen) according to the manufacturer's protocol. Similar to mRNA isolated from normal human adult heart, lung, liver, kidney, brain and pancreas (BD Biosciences, Palo Alto, CA), 1 μg of each mRNA from pancreatic cancer cell lines is separated on a 1% denatured agarose gel. , Transferred to nylon membrane. A 702 bp probe specific for KIAA0101 was prepared by PCR using the following primer set:
Forward, 5′-AGCTTTGTTGAACAGGCATTT-3 ′ (SEQ ID NO: 1), and
Reverse, 5'-GGCAGCAGTACAACAATCTAAGC-3 '(SEQ ID NO: 2).

  Hybridization using a random-primed α32 P-dCTP labeled probe was performed according to the instructions of the Megaprime DNA labeling system (Amersham Biosciences, Buckinghamshire, UK). Prehybridization, hybridization and washing were performed according to the supplier's recommendations. Blots were performed by autoradiography for 10 days at −80 ° C. using intensifying screens.

10. Small interfering RNA (siRNA) expression constructs and transfection for KIAA0101 knockdown To knockdown the expression of endogenous KIAA0101 in pancreatic cancer cells, we have previously described: A psiU6BX3.0 vector for short hairpin RNA expression was used for the target gene (Taniuchi et al., (2005) Cancer Res, 65: 105-12). The target sequence of the synthetic oligonucleotide for KIAA0101 siRNA was as follows:
# 759si; 5'-GCCATATTGTCACTCCTTCTA-3 '(SEQ ID NO: 7) and
EGFPsi; 5′-GAAGCAGCACGACTTCTTC-3 ′ (SEQ ID NO: 8) (negative control).

  Pancreatic cancer cell line that highly expressed KIAA0101, KLM-1 and MIA-PaCa2 were seeded on 10cm plate, and 8μg of plasmid designed to express siRNA against KIAA0101 was used according to the manufacturer's instructions using FuGENE6 (Roche) And transfected.

  Cells were selected for 5 days with Geneticin (Sigma-Aldrich) 0.5 mg / ml (for KLM-1) or 0.8 mg / ml (for MIAPaCa-2) and harvested to analyze the knockdown effect of KIAA0101 expression . For colony formation assay, transfectants expressing siRNAs were grown for 14 days in medium containing geneticin. After fixation with 4% paraformaldehyde, transformed cells were stained with Giemsa solution to assess colony formation. Cell viability was quantified using Cell counting kit-8 (DOJINDO, Kumamoto, Japan). After 14 days in culture in geneticin-containing medium, the solution was added at a final concentration of 10%. After culturing at 37 ° C. for 3 hours, the absorbance at 450 nm was measured using a Microplate Reader 550 (Bio-Rad, Hercules, Calif.).

11． Establishment and growth of exogenous KIAA0101-expressing cells in vitro and in vivo
KIAA0101 cDNA was prepared by PCR amplification using a forward primer containing a Kozak sequence and a NotI linker and a reverse primer containing a NotI linker. The PCR product was inserted into the NotI site of mammalian expression vector, pCAGGS / HA for expression of HA tag protein. Add FuGENE6 (Roche) according to the manufacturer's protocol to pCAGGS-KIAA0101-HA or empty pCAGGS / HA pseudovector, PK45P that slightly expresses KIAA0101, and NIH3T3 cells that hardly express detectable KIAA0101 homolog (NP_080791) Used to transfect.

Next, geneticin resistant clones were selected in medium containing geneticin 0.9 mg / ml for NIH3T3 and 0.5 mg / ml for PK45P. The expression of exogenous KIAA0101 in each clone was confirmed by Western blot analysis using anti-FLAG tag antibody (Sigma-Aldrich) and anti-β-actin antibody (Sigma-Aldrich). For the proliferation assay, 7,500 cells each of KIAA0101 expression clone (PK45P-KIAA0101) or control clone (PK45P-Mock) were seeded in each well of a 24-well culture dish and incubated in medium containing 10% FBS. Cell viability was quantified by MTT assay. The experiment was repeated at least 3 times. For in vivo transformation, one stable clone of NIH3T3-KIAA0101 and one NIH3T3-Mock 5 × 10 ⁶ cells were inoculated into the left and right flank of an 8 week nude mouse and 4 weeks later Tumors were collected. Each tumor was weighed and immunostained with anti-KIAA0101 antibody.

12． Immunoprecipitation and mass spectrometry analysis for KIAA0101 association complex
In order to identify proteins that bound to the KIAA0101 protein, the inventors performed immunoprecipitation experiments using anti-KIAA0101 antibodies. Pancreatic cancer cell lines KLM-1 and PK59 overexpressing KIAA0101 are lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% NP-40, Protease Inhibitor Cocktail Set III (Calbiochem, San Diego, CA)) Dissolved in. Equal amounts of total protein were incubated with 2 μg anti-KIAA0101 polyclonal antibody or rabbit IgG (Santa Cruz Biotechnologies, Santa Cruz, Calif.) For 1 hour at 40 ° C. The immune complex was incubated with protein G Sepharose (protein G Sepharose, South San Francisco, Calif.) For 1 hour and washed with lysis buffer.

  Coprecipitated proteins were separated by 5-20% gradient SDS-PAGE and stained with a silver staining kit (Wako, Osaka, Japan). A band different from that of the precipitated protein and the control IgG with the anti-KIAA0101 polyclonal antibody was excised, digested in gel with trypsin, and peptided using an AXIMA-CFR MALDI-TOF mass spectrometer (Shimadzu Corporation, Tsukuba, Japan). Analyzed by mass fingerprint. Peptide mass was searched with 10 ppm mass accuracy, and protein database searches were performed using the database optimization program IntelliMarque (Shimadzu). Binding proteins identified by this method were confirmed by immunoprecipitation using anti-KIAA0101, anti-PCNA antibody (Santa-Cruz), anti-POLD1 antibody (Santa-Cruz), and anti-FEN1 antibody (Santa-Cruz).

13. Cell penetrating peptide treatment and KIAA0101-PCNA interaction In order to inhibit the interaction between KIAA0101 and PCNA in a dominant negative manner, we have used arginine (R) repeat cell penetrating peptide (Noguchi et al., (2004) Nat Med., 10: 305-9), a PIP box motif peptide of KIAA0101 was designed. PIP20 was RRRRRRRRRRRGGG-VRPTPKW {qKGiGEff} RLSPK (SEQ ID NO: 9) (PIP box motif is shown in parentheses and conserved residues are shown in lower case). PIP20mut replaced the conserved residue of the PIP box motif with alanine: RRRRRRRRRRRGGG-VRPTPKW {aKGaGEaa} RLSPK (SEQ ID NO: 10). A scrambled peptide was also designed as a negative control; RRRRRRRRRRRGGG-IFKQWPRGETKPRVLSPKGF (SEQ ID NO: 11). In addition, the inventors also designed shorter peptides containing the PIP box motif: PIP16; RRRRRRRRRRRGGG-TPKW {qKGiGEff} RLSP (SEQ ID NO: 12), PIP16mut; RRRRRRRRRRRGGG-TPKW {aKGaGEaa} RLSP (sequence Number: 13). They were synthesized by Sigma-Aldrich and purified by HPLC to a grade of over 95%. The cancer cell line KLM-1 expressing KIAA0101 and the normal mouse cell line NIH3T3 not expressing the mouse KIAA0101 homolog were treated with a series of concentrations of these cell-penetrating peptides (5 μM, 10 μM and 20 μM). On days 1 and 3, cells were contacted with the respective peptides, and on day 5, viable cell numbers were assessed by the MTT assay described above.

Example 2
1． siRNA expression vector and colony formation assay / MTT assay
To knock down the expression of endogenous REG4 in PDAC cells, we have previously described (Shimokawa T, et al. Cancer Res 2003; 63: 6116-20.) The psiU6BX3.0 vector was used for short hairpin RNA expression against the target gene.

The target sequence of the synthetic oligonucleotide for siRNA for REG4 is as follows:
REG4-si2; 5′-GACAGAAGGAAGAAACTCA-3 ′ (SEQ ID NO: 5) and
EGFPsi; 5′-GAAGCAGCACGACTTCTTC-3 ′ (SEQ ID NO: 6) (negative control).

PDAC cell lines SUIT-2 (REG4 positive) and MIAPaCa-2 (REG4 negative) are seeded onto 6-well plates and expressed siRNA (10 μg) using FuGENE6 (Roche, Basel, Switzerland) according to manufacturer's instructions Plasmids designed to be transfected. siRNA expressing plasmids were prepared by cloning double stranded oligonucleotides. The paired oligonucleotide sequences are as follows:
5'-CACCGACAGAAGGAAGAAACTCATTCAAGAGATGAGTTTCTTCCTTCTGTC-3 '(SEQ ID NO: 11) and
5′-AAAACTGTCTTCCTTCTTTGAGTAAGTTCTCTACTCAAAGAAGGAAGACAG-3 ′ (SEQ ID NO: 12): for si2.

  Cells are selected for 7 days at Geneticin (Sigma-Aldrich) 0.9 mg / ml (for SUIT-2) or 0.8 mg / ml (for MIAPaCa-2), and then to analyze the knockdown effect on REG4 expression It was recovered. For colony formation assay, siRNA expression transfectants were grown for 7 days in geneticin-containing medium. After methanol fixation, transformed cells were stained with 0.1% crystal violet solution to evaluate colony formation. In the MTT assay, cell viability was quantified using Cell counting kit-8 (DOJINDO, Kumamoto, Japan). After 7 days of culture in Geneticin-containing medium, the solution was added at a final concentration of 10%. After 1.5 hours of incubation at 37 ° C., the absorbance was measured using a Microplate Reader 550 (Bio-Rad, Hercules, Calif.) At 490 nm and 630 nm as a reference.

  In order to investigate the role of REG4 overexpression in PDAC cell growth, we constructed several expression vectors designed to express REG4 specific siRNA and expressed endogenous REG4. They were transfected into the PDAC cell line SUIT-2, which is expressed at high levels. Of the three plasmids we tested in SUIT-2 cells, REG4-si2 showed a significant knockdown effect on the endogenous REG4 transcript (Figure 2A), and this transfection was performed using the MTT assay (Figure The decrease in the number of colonies (FIG. 2C) was confirmed in the same manner as this, as confirmed by the decrease in living cells measured by 2B). At this time, transfection of other plasmids (negative control siEGFP) did not affect the knockdown effect on REG4 expression and the proliferation of SUIT-2. On the other hand, REG4-si2 did not affect the cell viability of MIAPaCa-2 that does not express REG4, thus eliminating the possibility of the “non-specific” effect of REG4-si2 (data not shown). The growth suppression effect of this siRNA expression vector (REG4-si2) correlated well with the gene silencing effect, and these data indicated an important role for REG4 in pancreatic cancer cell survival and / or proliferation.

Example 3
1． Production of biologically active recombinant human REG4
To create a biologically active form of REG4, the entire coding sequence of REG4 cDNA was amplified by PCR using a primer pair with a restriction enzyme site;
5'-CG {GAATTC} ATGGCTTCCAGAAGCATGC-3 '(forward), (SEQ ID NO: 7) and
5′-ATAAGAAT {GCGGCCGC} TGGTCGGTACTTGCACAGG-3 ′ (reverse), (SEQ ID NO: 8), which contain EcoRI and NotI restriction sites, respectively, shown in parentheses. The product was inserted into the EcoRI and NotI sites of pCAGGS to express the HA tag protein. FreeStyle ™ 293 cells were seeded at 1.5 × 10 ⁵ cells / ml in 30 ml medium. The REG4-HA / pCAGGS vector was transfected into the cells using FuGene 6 according to the instructions. The medium was collected after 48 hours and recombinant human REG4 (rhREG4) was purified with HA agarose (Sigma-Aldrich).

2． Immunoprecipitation
SUIT-2 cells cultured in 10 cm dishes were washed and further cultured in serum-free medium for 2 days. After centrifugation at 10,000 xg for 15 minutes at 4 ° C, the supernatant was treated with Protein G Sepharose (Zymed Laboratories, San Francisco, CA) for 1 hour at 4 ° C. The pretreated supernatant was then added to a mixture of protein G sepharose preincubated with anti-REG4 mAb. Incubation was performed by gentle rotation for 4 hours at 4 ° C. after two washes. The bound protein was separated and eluted by 15% SDS-PAGE. After electrophoretic separation, proteins were transferred to nitrocellulose membrane (Amersham) and examined with anti-REG4 pAb. Protein bands were visualized by a chemiluminescent detection system (ECL, Amersham).

3． Akt phosphorylation To assess the level of phosphorylated Akt, PK45P cells were treated for 6 hours in the presence or absence of 0, 0.1, 1 or 10 nM rhREG4 and 100 μg / ml anti-REG4 mAb. After treatment, the cells are washed with cold PBS, 50 mM HEPES, pH 7.5, 200 mM NaCl, 2.5 mM EDTA, 2.5 mM EGTA, 10 mM NaF, 1 mM Na3VO4, 0.5% Triton X- Recovery was performed with a lysis buffer containing 100, 0.5 mM 1,4-dithiothreitol and 0.1% protease inhibitor cocktail III (Calbiochem, San Diego, Calif.). The sample was centrifuged to remove the precipitate. The amount of protein in the supernatant was measured by the Bradford method. The 20 μg portion was subjected to 10% SDS-PAGE and detected by Western blotting using an anti-pSer473 Akt antibody (Abcam, Cambridge, Mass.). The total amount of Akt protein was assessed by anti-Akt antibody (Santa Cruz Biotechnology, CA).

  To study the effect of secreted REG4 on pancreatic cancer cell growth, we generated a biologically active rhREG4 protein by using the mammalian system (FreeStyle ™ 293-F) (FIG. 3A) and reduced REG4 low Cell proliferation assays were performed by treating PK45P cells exhibiting expression with several concentrations of rhREG4 (0-10 nM). FIG. 4B shows that the presence of REG4 protein in the medium clearly promotes cell growth in a dose-dependent manner. This indicates that secreted REG4 has a function of promoting cell proliferation in an autocrine / paracrine manner outside the cell. One downstream target of the REG family has been reported to be the Akt signaling pathway (Sekikawa A, et al. Gastroenterology 2005; 128: 642-53., Bishnupuri KS, et al. Gastroenterology 2006; 130: 137- 49.). To investigate whether rhREG4 can activate the Akt signaling pathway in PDAC cells, PK45P cells were incubated in the presence of a series of doses of rhREG4 and phosphorylated Akt was converted to serine 473 specific phosphorylation. Detection was performed by Western blot analysis using oxidized Akt antibody. rhREG4 treatment clearly showed increased phosphorylation of Akt (FIG. 3C), whereas the total expression level of Akt was not changed by treatment with rhREG4.

  These data indicated that REG4 stimulates cell proliferation via the Akt signaling pathway in PDAC cells.

  To assess the therapeutic predisposition of anti-REG4 mAb, we performed a cell proliferation assay by treating PDAC cells with anti-REG4 mAb. First, the present inventors confirmed the binding affinity of several anti-REG4 mAbs by immunoprecipitation using a cell culture medium. FIG. 4A shows that one anti-REG4 mAb (34-1) binds with high affinity to endogenous REG4 protein in SUIT-2 medium. A neutralization assay using PK45P showed that anti-REG4 mAb clone 34-1 completely stopped the growth-promoting effect of rhREG4 treatment, whereas the control antibody did not show neutralizing activity (FIG. 4B). . Proliferation assays using SUIT-2 cells expressing high levels of endogenous REG4 showed that anti-REG4 mAb treatment inhibited SUIT-2 cell proliferation in a dose-dependent manner (FIG. 4C). On the other hand, anti-REG4 mAb did not affect the cell proliferation of MIAPaCa-2 that does not express REG4.

  In addition, the inventors also examined the effect on Akt phosphorylation by treating PK45P cells with rhREG4 and anti-REG4 mAb, and anti-REG4 mAb treatment was induced by rhREG4-treated PDAC. Akt phosphorylation of cells was suppressed (FIG. 4D). This showed that anti-REG4 mAb can inhibit the Akt signaling pathway in PDAC cells by neutralizing secreted REG4 and blocking the autocrine / paracrine pathway. Therefore, these data suggested that anti-REG4 antibodies have neutralizing activity against REG4 promoted cell proliferation.

To assess the therapeutic potential of anti-REG4 mAb, we performed a tumor growth assay by treating mice inoculated with PDAC cells with anti-REG4 mAb. 5 × 10 ⁶ cells of REG4-expressing PDAC cell line SUIT-2 were inoculated into the flank of 8-week-old nude mice, and the major axis (L) and minor axis (S) of the tumor were measured twice a week. Tumor volume was calculated by L × L × S × 0.52. Antibody treatment with anti-REG4 mouse mAb 34-1 (300 μg / mouse ip) was initiated when the tumor volume reached 100-200 mm 3. The antibody was treated intraperitoneally twice per week and, as a control, non-specific mouse IgG (Acris) was treated on the same schedule. As shown in FIG. 5A, tumor growth (P = 0.0598) was suppressed by 34-1 mAb treatment. On day 30, tumors were collected and weighed. 34-1 mAb treatment significantly suppressed tumor weight (Figure 5B, P = 0.0489).

Example 4
1． Expression of REG4 in chemo-radiation resistant PDAC in vitro
To investigate how REG4 expression is involved in the resistance of PDAC to chemo-radiotherapy, we generated three clones that constitutively express REG4 from REG4-negative PDAC cells and were exposed to gamma irradiation or gemcitabine in vitro. These clones were processed. Expression of exogenous REG4 in C1-6, C2-6 and C10 clones and non-expression in pseudo clones were confirmed by Western blot analysis (FIG. 6A). Compared to non-irradiation, gamma irradiation suppressed cell viability of REG4-expressing cells and mock cells. However, as shown in FIG. 6B, REG4-expressing cells have low sensitivity to gamma irradiation, and the survival rate of REG4-expressing cells (C1-6, C10, C2-6) after 30 Gy gamma irradiation is non-irradiated. In comparison, it was suppressed to 60%, but the survival rate of pseudo cells was suppressed to less than 40% (P <0.001). FACS analysis after gamma irradiation showed that 1-Gly gamma irradiation induced a pseudo-G1 population (apoptotic cells) to 28.7%, but only 10.73% of REG4-expressing cells (Fig. 6C). Furthermore, 5-Gly gamma irradiation induced pseudocell sub-G1 population (apoptotic cells) to 46.47%, but only 24.68% in REG4-expressing cells (FIG. 6C). These findings indicate that REG4 expression in PK45P cells strongly contributes to resistance to gamma irradiation.

  REG4 positive or negative cells were then treated with a range of concentrations of gemcitabine and their viability assessed. As shown in FIG. 7A, REG4-expressing cells (C1-6, C10, C2-6) were less sensitive to gemcitabine compared to pseudo cells. The IC50 of gemcitabine for REG4-expressing cells was about 100 nM for C1-6 and C10 (FIG. 6A), and about 50 nM for C2-6 with less REG4 expression than C1-6 and C10, but the IC50 for pseudocells Was about 30 nM. But this difference is not dominant. Furthermore, FACS analysis showed that REG4-expressing cells tended to show fewer apoptotic cells after treatment with 50 nM gemcitabine, but was not statistically significant (FIG. 7B). High concentration treatment with gemcitabine showed no significant difference in apoptotic cells. These findings indicate that the expression of REG4 in PK45P cells may have some contribution to resistance to gemcitabine, but that contribution is not very strong. Therefore, in PDAC cells, REG4 expression may contribute to radiation resistance more than gemcitabine-based chemotherapy.

2． REG4 expression in neoadjuvant chemo-radiation resistant patients
To evaluate the association between REG4 expression and treatment resistance in pancreatic cancer, surgical specimens from patients who received neoadjuvant chemo-radiotherapy treatment (neo-CRT) were investigated. They were treated with 3D confocal radiation therapy (total 50 Gy) and in parallel with gemcitabine (1 g / m2 / week for 3 weeks) followed by 8 weeks of follow-up. Nineteen surgical specimens were immunostained with anti-REG4 antibody and evaluated for REG4 expression and pathological response to neo-CRT.

  Of the 19 neo-CRT specimens, 9 showed a histological response to neo-CRT, but 10 did not. Regarding REG4 expression, 2 out of 9 responders showed REG4 expression in tumor cells (FIGS. 8A and B), while 7 out of 10 non-responders showed REG4 expression. (FIGS. 8C and D). This REG4 expression was significantly associated with the response to neo-CRT (chi-square test, P = 0.0028). This means that the expression of REG4 allows cancer cells to survive under cytotoxic treatments such as chemotherapy and radiation therapy.

Example 5
Overexpression of KIAA0101 in pancreatic cancer cells Among the genes overexpressed in pancreatic cancer cells by wide-area genomic cDNA microarray analysis (Nakamura T., Oncogene, 23: 2385-400, 2004.), the present inventors I focused on KIAA0101. Our microarray data showed overexpression of KIAA0101 in all beneficial cases of pancreatic cancer cells examined by the inventors (14 of 14 beneficial cases showed more than 10-fold in expression signal) The overexpression was confirmed by RT-PCR in 8 of the 9 microdisected pancreatic cancer cell populations examined (FIG. 9A). Northern blot analysis using the KIAA0101 cDNA fragment as a probe identified an approximately 1.2 kb highly expressed transcript in all cancer cell lines examined by the inventors;
Expression was not observed in any vital organ including lung, heart, liver and kidney (FIG. 9B). Immunohistochemical analysis using a polyclonal antibody against KIAA0101 showed a strong signal at the nucleus of pancreatic cancer cells in all pancreatic cancer tissue sections of 5 patients (FIG. 9C). Staining was not observed in normal pancreatic epithelium and acinar cells (FIG. 9C) and important normal organs including lung, heart, liver and kidney.

Example 6
Effect of KIAA0101 knockdown by siRNAs on the growth of pancreatic cancer cells To investigate the biological importance of KIAA0101 overexpression in cancer cells and its potential as a molecular target for cancer therapy, we have determined that the KIAA0101 mRNA sequence Several siRNA expression vectors specific for, were constructed and transfected into KLM-1 and MIAPaCa-2, which endogenously express KIAA0101 mRNA at high levels. When the # 759si construct by the present inventors was used, the knockdown effect was confirmed by RT-PCR (FIG. 10A). Compared to EGFPsi, which has no apparent knockdown effect, the colony formation assay using KLM-1 (Figure 10B) and MTT assay (Figure 10C) dramatically increased the number of cells transfected with # 759si. It was clarified to decrease. Similar effects were obtained in the MIA-PaCa2 cell line. These findings indicated that KIAA0101 plays an important role in cancer cell proliferation, and suppression of KIAA0101 function has potential as a novel molecular target for cancer therapy.

Example 7
Exogenous overexpression of KIAA0101 promoted cancer cell growth and transformed NIH3T3.To further explore the potential tumorigenic properties of KIAA0101, we constitutively expressed exogenous KIAA0101. Several clones of PK45P-derived cell line, PK45P-KIAA0101, to be expressed were established. We also prepared control PK45P cells transfected with a mock vector (PK45P-Mock) and compared their growth rates. Western blot analysis confirmed the expression of exogenous KIAA0101 in 6 clones (FIG. 11A). Growth curves measured in the MTT assay show that 6 clones of PK45P-KIAA0101 (solid line), 4 PK45P-pseudo clones (dashed line, FIG. 11B) proliferate significantly rapidly, and KIAA0101 expression Have been shown to promote the growth of cancer cells.

  Of the cell lines examined by the inventors, only the normal mouse NIH3T3 cell line did not show expression of the KIAA0101 homolog. Therefore, the present inventors forced NIH3T3 to express KIAA0101 and examined whether overexpression of KIAA0101 transforms NIH3T3 in vivo. As shown in FIG. 11C, three clones of NIH3T3-KIAA0101 formed tumors on the right side of nude mice, but NIH3T3-sham did not form on the left side, and immunohistochemical analysis of these tumors. Staining showed positive staining for KIAA0101 in the nuclei of tumor cells (FIG. 11D). These results implied that KIAA0101 has the ability to transform normal cells into tumor cells.

Example 8
Identification of PCNA, POLD1, and FEN1 as a complex of KIAA0101 protein To further investigate the biological function of KIAA0101, the present inventors used immunization with a polyclonal antibody against KIAA0101 to identify the complex of KIAA0101 protein. Sedimentation was performed. Immunoprecipitation fractions separated on SDS-PAGE gels and stained with silver showed that some proteins immunoprecipitated with KIAA0101 protein from cancer cell lysates compared to the control sample results ( Figure 12A). Each protein was analyzed by MALDI-TOF system after in-gel trypsin digestion; identified as PCNA, POLD1 (polymerase δ125 subunit) and FEN1 (flap endonuclease-1). These interactions were confirmed by the immunoprecipitation experiment shown in FIG. 12B. All of these proteins are involved in DNA replication / repair, and POLD1 and FEN1 also bind to PCNA, similar to KIAA0101 (Jonsson et al., (1998) EMBO J. Apr 15; 17 (8): 2412 -25, Zhang et al., (1999) J Biol Chem. Sep 17; 274 (38): 26647-53; Bruning & Shamoo (2004) Structure. Dec; 12 (12): 2209-19.) Suggests that KIAA0101 is involved in DNA replication through interaction with PCNA.

Example 9
Inhibition of interaction between KIAA0101 and PCNA by cell-permeable dominant negative peptide
In order to inhibit the interaction between KIAA0101 and PCNA via the PIP box motif of KIAA0101, we designed a dominant negative peptide containing this PIP box, and in order to facilitate cell permeability, arginine ( R) Repeats were bound (FIG. 13A). In an in vitro study, we confirmed suppression of the interaction between PCNA and KIAA0101 by immunoprecipitation. In the presence of PIP20, PCNA did not immunoprecipitate with KIAA0101, but PIP20mut (FIG. 13A) and scrambled (FIG. 13B) in which the conserved residues were replaced by alanine did not affect the interaction. Next, the present inventors evaluated whether these peptides inhibit cancer cell proliferation by treating cancer cells and normal fibroblast NIH3T3 cells with these peptides. Mouse KIAA0101 (NP_080791) was highly homologous to humans, and the target region of the dominant negative peptide in human KIAA0101 was 100% identical to that of mouse KIAA0101. FIG. 13C demonstrated that PIP20 inhibited cancer cell growth in a dose-dependent manner, but not PIP20mut and scrambled peptides. On the other hand, PIP20 did not affect the growth of mouse normal cell line NIH3T3 cells that do not express the homologue of human KIAA0101. These findings indicated that PIP20 specifically inhibits cell proliferation by targeting KIAA0101.

  Next, the inventors designed a short PIP peptide (PIP16 and PIP16mut shown in FIG. 13A) by deleting several residues in the region leading to the N-terminus and C-terminus with a maintained PIP box motif. Cancer cell lines and NIH3T3 cells were treated. Treatment with PIP16 strongly suppressed cancer cell growth, but PIP16 also affected the growth of NIH3T3, and its growth inhibitory effect was lost by substituting the conserved residues of the PIP box motif with alanine, similar to PIP20. (Fig. 13D). These findings suggested that the effect of PIP16 is not specific to KIAA0101, and that PIP16 affects the interaction between PCNA and other DNA replication proteins.

Discussion The present invention has shown its potential as a molecular target for PDAC therapy. To invent the role or function of secreted REG4 in the development or progression of pancreatic cancer, we knocked down endogenous REG4 expression with siRNA in PDAC cell lines and exposed cancer cells with recombinant REG4 . Findings from this experiment indicated that REG4 functions as an autocrine / paracrine growth factor and is transmitted to the Akt signaling pathway via an unknown receptor. However, how REG4 transmits the Akt signaling pathway is unknown and is a problem that must be investigated in further studies in addition to identifying the REG4 receptor (Kobayashi S, et al. J Biol Chem 2000; 275 : 10723-6.).

  Recent studies have reported that REG4 and other family members, REG1, can function as anti-apoptotic factors for colorectal or gastric cancer via the Akt pathway (Sekikawa A, et al. Gastroenterology 2005; 128 : 642-53., Bishnupuri KS, et al. Gastroenterology 2006; 130: 137-49.). Therefore, in this study, we evaluated that the activation pathway by secretory REG4 and the Akt signal pathway are downstream pathways of REG family signals involved in cancer growth or anti-apoptosis. The REG family is thought to be expressed in the tissue regeneration process or tissue damage or play an important role in tissue regeneration (Unno M, et al. Adv Exp Med Biol 1992; 321: 61-6., Zhang YW, et al. World J Gastroenterol 2003; 9: 2635-41.). Given the activation of the Akt signaling pathway by REG4 and other REG members, they may be related to the sensitivity of cancer to chemotherapy or radiotherapy, and the effects of REG4 expression and chemo-radiotherapy in vitro or in vivo It is interesting to investigate the relationship between.

  Monoclonal antibodies specific for REG4 neutralize secreted REG4 in the culture medium in vitro, and treatment of these neutralizing antibodies blocks REG4's autocrine / paracrine pathway and subsequent Akt phosphorylation It is noteworthy to significantly suppress PDAC cell proliferation by protecting against. These findings indicate the feasibility of neutralizing antibody therapy targeting REG4. Bevacizumab, a humanized monoclonal antibody against VEGF, is currently approved in combination with intravenous 5-fluorouracil-containing therapy for first-line treatment of metastatic colorectal cancer. In addition to anti-angiogenic factor antibodies, for example HGF (Burgess T, et al. Cancer Res. 2006; 66: 1721-9.) And IL-6, (Trikha M, et al. Clin Cancer Res 2003; 9: 4653 -65.) Antibodies against blood ligands are under investigation as anti-cancer agents, and REG4 target neutralizing antibody therapy is also offered as a novel treatment for other cancers that express PDAC and REG4 Also good.

  In conclusion, we have shown the promising feasibility of REG4 as a serodiagnostic marker for PDAC and a molecular target for PDAC treatment and targeted REG4 along with other screening or other anti-cancer therapies Combining new methods may improve the disastrous prognosis of current PDACs.

  Furthermore, the present inventors have identified KIAA0101 as one of the genes overexpressed in pancreatic cancer cells. Information and several reports of microarray analysis in various human cancers of the present inventors (Peiwen et al., (2001) Oncogene, 20: 484-9; Mizutani et al., (2005) Cancer, 103: 1785-90 ), At the RNA level, many other cancer cells also overexpress KIAA0101, whose expression is widely observed in highly proliferating cells. Immunohistochemical analysis using anti-KIAA0101 antibody showed that KIAA0101 is highly expressed in normal intestinal mucosal crypts and lymph node germinal centers where cancer cells and proliferating cells are present. The expression of KIAA0101 is dependent on the cell cycle, and the expression is highest in the S phase where DNA replication is most active (data not shown), indicating that the expression of KIAA0101 is associated with cell proliferation. It means strongly to be involved. In fact, knockdown of KIAA0101 by siRNA in the present invention suppressed cell proliferation of cancer cells, and in a previous report, KIA0101 was identified as a PCNA binding protein by the yeast two-hybrid system (Yu et al. , (2001) Oncogene, 20: 484-9).

  PCNA serves as a clamp base that slides along the DNA template and is an auxiliary protein essential for DNA replication and DNA repair processes that interact with DNA synthesis or metabolic enzymes (Wyman and Botchan, (1995) Curr Biol., 5: 334-7; Warbrick, (2000) Bioessays, 22: 997-1006; Krishna et al., (1994) Cell, 79: 1233-43). We also bind KIAA0101 directly to PCNA through its conserved PIP box motif, KIAA0101 regulates PCNA function by binding to PCNA, or other PCNA such as p21. Shown to compete with binding partners. (Waga et al., (1994) Nature 369: 574-8; Chen et al., (1996) Nucl Acid Res 24: 1727-33; Kontopidis et al., (2005) PNAS, 102: 1871-6). In the present invention, the inventors focused on the motif of this PIP box, and in order to specifically inhibit the interaction between PCNA and KIAA0101, cell-permeable arginine repeat (Noguchi et al., (2004) Nat A dominant negative peptide linked to Med., 10: 305-9) was designed. PIP20mut did not inhibit cancer cell growth, but PIP20 inhibited cancer cell growth in a dose-dependent manner, and PIP20 did not affect the proliferation of NIH3T3 that does not express KIAA0101. This suggests its high specificity.

  Interestingly, we designed PIP16 and PIP16, shorter peptides that strongly inhibit cancer cell growth than PIP20, so that some residues in the adjacent region of PIP20 were retained while retaining the PIP box. Deficient. However, it seemed to lose its specificity for KIAA0101-PCNA interaction. PCNA interacts with many proteins via the PIP box motif (Wyman and Botchan, (1995) Curr Biol., 5: 334-7; Warbrick, (2000) Bioessays, 22: 195-1006; Krishna et al., (1994) Cell, 79: 1233-43; Chen et al., (1996) Nucl Acid Res 24: 1727-33; Kontopidis et al. (2005) PNAS, 102: 1871-6), PIP16 It plays an important role in PCNA and cell proliferation, making it possible to inhibit the interaction between numerous other widely expressed proteins. Adjacent residues of PIP20 may be important for specific suppression between KIAA0101 and PCNA. Similarly, p21 PIP box-derived peptides that interact with PCNA enable the suppression and killing of cancer cells, and inhibition of PCNA-dependent DNA replication is a promising approach to cancer therapy (Chen et al ., (1996) Nucl Acid Res 24: 1727-33; Kontopidis et al. (2005) PNAS, 102: 1871-6).

  Intracellular protein-protein interactions constitute a major control point in many signal transduction pathways, but reflect a broad, shallow, hydrophobic protein interface that is targeted by small molecules. Has proved to be difficult (Walensky et al., (2004) Science, 305: 1446-70). Peptides are attractive candidates for stable or unstable protein-protein interactions, but their effectiveness as reagents in vivo is due to loss of secondary structure, sensitivity to proteolysis and penetration into untreated cells. Due to the difficulty, it is very flawed. However, recent reports on peptide modifications have shown their feasibility as pharmaceutical targets (Walensky et al., (2004) Science, 305: 1446-70) and the targeted protein-protein interaction Protein-protein that has more appeal to drug development and offers promising feasibility for drug development through further structural analysis (Kontopidis et al., (2005) PNAS, 102: 1871-6) or DDS improvement Peptides that inhibit the interaction may be provided.

Industrial applicability:
The inventors have shown that cell growth is suppressed by small interfering RNA (siRNA) that specifically targets the REG4 gene or KIAA0101 gene. Therefore, this novel siRNA is useful as a target for the development of anticancer drugs. For example, agents that block REG4 expression or inhibit its activity find therapeutic use as anti-cancer agents, particularly anti-cancer agents for the treatment of pancreatic cancer such as pancreatic ductal adenocarcinoma (PDAC). Similarly, agents that block KIAA0101 expression or inhibit its activity find therapeutic use as anti-cancer agents for the treatment of pancreatic cancer, prostate cancer, breast cancer and bladder cancer.

  The inventors also showed that a monoclonal antibody against REG4 neutralized its growth promoting effect and significantly attenuated PDAC cell proliferation. Therefore, for example, treatment of diseases involving REG4-expressing cells such as pancreatic cancer is appropriately performed using an antibody that binds to REG4.

  The inventors have also shown that KIAA0101 interacts with PCNA and that inhibition of that interaction leads to inhibition of cancer cell growth. Therefore, agents that inhibit the binding between the interaction of PCNA and KIAA0101 and that inhibit its activity have therapeutic uses as anticancer agents.

  The present invention thus provides novel polypeptides and other compounds useful for treating or preventing cancer. The polypeptide of the present invention consists of an amino acid sequence comprising QKGIGEFF / SEQ ID NO: 46. The polypeptide of the present invention can be administered to suppress the growth of cancer cells or to induce apoptosis. The polypeptide of the present invention is expected to show a cell growth inhibitory effect against various cancers. In particular, it was confirmed that the polypeptide of the present invention has an effect of inhibiting cell growth of pancreatic cancer.

  Pancreatic cancer is an important cancer where it is still desirable to provide an effective method of treatment. Thus, the present invention is important in that it provides an effective method for treating and / or preventing pancreatic cancer.

  While the invention has been described and described in detail with reference to specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention. It is.

Claims (76)

  A method of treating or preventing pancreatic cancer in a subject, comprising administering to the subject a composition comprising at least one small interfering RNA (siRNA) that inhibits the expression of REG4 or KIAA0101.   2. The method of claim 1, wherein the siRNA comprises a sense strand nucleic acid sequence and an antisense strand nucleic acid sequence that specifically hybridize to a sequence derived from REG4 or KIAA0101.   2. The method of claim 1, wherein the cancer to be treated is selected from the group consisting of pancreatic cancer, prostate cancer, breast cancer and bladder cancer.   4. The method of claim 3, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).   2. The method of claim 1, wherein the siRNA inhibits REG4 expression and the cancer being treated is PDAC.   2. The method of claim 1, wherein the siRNA inhibits KIAA0101 expression and the cancer being treated is pancreatic cancer, prostate cancer, breast cancer and bladder cancer.   3. The method of claim 2, wherein the siRNA comprises a ribonucleotide sequence corresponding to the sequence of SEQ ID NO: 5 or SEQ ID NO: 32 as a target sequence.   The siRNA has a general formula 5 ′-[A]-[B]-[A ′]-3 ′, wherein [A] is a nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 32. The corresponding ribonucleotide sequence, [B] is a ribonucleotide loop sequence consisting of 3 to 23 nucleotides, and [A '] is a ribonucleotide sequence consisting of the complementary sequence of [A]. 7. The method according to 7.   A double-stranded molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises a ribonucleotide sequence corresponding to the target sequence of SEQ ID NO: 5 or SEQ ID NO: 32, wherein the antisense strand is attached to the sense strand; Comprising a ribonucleotide sequence that is complementary, the sense strand and the antisense strand hybridize to each other to form the double-stranded molecule, and the double-stranded molecule expresses a REG4 or KIAA0101 gene A double-stranded molecule that inhibits expression of the gene when introduced into a living cell.   10. The double-stranded molecule of claim 9, wherein the target sequence comprises at least about 10 consecutive nucleotides from the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 39.   11. The double-stranded molecule of claim 10, wherein the target sequence comprises from about 19 to about 25 contiguous nucleotides derived from the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 39.   12. The double-stranded molecule of claim 11, wherein the double-stranded molecule is a single ribonucleotide transcript comprising a sense strand and an antisense strand linked via a single-stranded ribonucleotide sequence.   11. The double-stranded molecule of claim 10, wherein the double-stranded molecule is an oligonucleotide length of less than about 100 nucleotides.   14. The double-stranded molecule of claim 13, wherein the double-stranded molecule is an oligonucleotide length of less than about 75 nucleotides.   15. The double stranded molecule of claim 14, wherein the double stranded molecule is an oligonucleotide length of less than about 50 nucleotides.   16. The double stranded molecule of claim 15, wherein the double stranded molecule is an oligonucleotide length of less than about 25 nucleotides.   17. The double stranded polynucleotide of claim 16, wherein the double stranded molecule is an oligonucleotide of between about 19 and about 25 nucleotides in length.   A vector encoding the double-stranded molecule according to claim 10.   19. The vector of claim 18, wherein the vector encodes a transcript having secondary structure and comprises a sense strand and an antisense strand.   20. The vector of claim 19, wherein the transcript further comprises a single stranded ribonucleotide sequence linking the sense strand and the antisense strand.   A vector comprising a polynucleotide comprising a combination of a sense strand nucleic acid and an antisense strand nucleic acid, wherein the sense strand nucleic acid comprises the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 32, and the antisense strand nucleic acid comprises: A vector consisting of a sequence complementary to the sense strand.   The polynucleotide has the general formula 5 ′-[A]-[B]-[A ′]-3 ′, wherein [A] is the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 32. 22. The vector of claim 21, wherein [B] is a nucleotide sequence consisting of 3 to 23 nucleotides and [A ′] is a complementary nucleotide sequence of [A].   A pharmaceutical composition for treating or preventing pancreatic cancer, comprising a pharmaceutically effective amount of a small interfering RNA (siRNA) that inhibits the expression of REG4 as an active ingredient, and a pharmaceutically acceptable carrier.   24. The pharmaceutical composition of claim 23, wherein the siRNA comprises the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 32 as the target sequence.   siRNA has the general formula 5 ′-[A]-[B]-[A ′]-3 ′, where [A] is a ribonucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO: 5 25. The composition of claim 24, wherein [B] is a ribonucleic sequence consisting of 3 to 23 nucleotides, and [A ′] is a complementary ribonucleotide sequence of [A].   For treating or preventing pancreatic cancer, prostate cancer, breast cancer and bladder cancer, comprising a pharmaceutically effective amount of a small interfering RNA (siRNA) that inhibits expression of KIAA0101 as an active ingredient, and a pharmaceutically acceptable carrier Pharmaceutical composition.   27. The pharmaceutical composition of claim 26, wherein the siRNA comprises the nucleotide sequence of SEQ ID NO: 32 as the target sequence.   The siRNA has the general formula 5 ′-[A]-[B]-[A ′]-3 ′, wherein [A] is a ribonucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO: 32 28. The composition of claim 27, wherein [B] is a ribonucleotide sequence consisting of 3 to 23 nucleotides and [A ′] is a complementary ribonucleotide sequence of [A].   Use of a small interfering RNA (siRNA) that inhibits the expression of REG4 for the manufacture of a pharmaceutical composition for treating or preventing pancreatic cancer.   30. Use according to claim 29, wherein the siRNA comprises the nucleotide sequence of SEQ ID NO: 5 as the target sequence.   Use of small interfering RNA (siRNA) that inhibits expression of KIAA0101 for the manufacture of a pharmaceutical composition for treating or preventing pancreatic cancer, prostate cancer, breast cancer and bladder cancer.   32. Use according to claim 31, wherein the siRNA comprises the nucleotide sequence of SEQ ID NO: 32 as the target sequence.   A method for treating or preventing pancreatic cancer in a subject, comprising administering to the subject an anti-REG4 antibody or fragment thereof that neutralizes REG4 activity.   34. The method of claim 33, wherein the activity of REG4 to be neutralized is an activity that promotes cell proliferation of pancreatic cancer in an autocrine / paracrine manner.   34. The method of claim 33, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).   34. The method of claim 33, wherein the anti-REG4 antibody is a monoclonal antibody. The antibody comprises a VH and a VL chain, each VH and VL chain comprising a CDR amino acid sequence called CDR1, CDR2 and CDR3 separated by a framework amino acid sequence, of each CDR within each VH and VL chain 34. The method of claim 33, wherein the amino acid sequence is selected from the group consisting of:
VH CDR1: SYWMH (SEQ ID NO: 20),
VH CDR2: NIYPGSGSTNYD (SEQ ID NO: 21),
VH CDR3: GGLWLRVDY (SEQ ID NO: 22),
VL CDR1: SASSSVSYMH (SEQ ID NO: 23),
VL CDR2: DTSKLAS (SEQ ID NO: 24), and
VL CDR3: QQWSSNPF (SEQ ID NO: 25).   38. The method of claim 36, wherein the human VH comprises the amino acid sequence of SEQ ID NO: 18 and the human VL comprises the amino acid sequence of SEQ ID NO: 19.   A pharmaceutical composition for treating or preventing pancreatic cancer, comprising, as an active ingredient, a pharmaceutically effective amount of an anti-REG4 antibody or fragment thereof that neutralizes the activity of REG4, and a pharmaceutically acceptable carrier.   40. The pharmaceutical composition of claim 39, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).   40. The pharmaceutical composition of claim 39, wherein the anti-REG4 antibody is a monoclonal antibody. The antibody comprises a VH and a VL chain, each VH and VL chain comprising a CDR amino acid sequence called CDR1, CDR2 and CDR3 separated by a framework amino acid sequence, of each CDR within each VH and VL chain 40. The pharmaceutical composition of claim 39, wherein the amino acid sequence is selected from the group consisting of:
VH CDR1: SYWMH (SEQ ID NO: 20),
VH CDR2: NIYPGSGSTNYD (SEQ ID NO: 21),
VH CDR3: GGLWLRVDY (SEQ ID NO: 22),
VL CDR1: SASSSVSYMH (SEQ ID NO: 23),
VL CDR2: DTSKLAS (SEQ ID NO: 24), and
VL CDR3: QQWSSNPF (SEQ ID NO: 25).   43. The composition of claim 42, wherein the human VH comprises the amino acid sequence of SEQ ID NO: 18 and the human VL comprises the amino acid sequence of SEQ ID NO: 19. An amino acid sequence of each CDR in each VH and VL chain, comprising CDR amino acid sequences called CDR1, CDR2 and CDR3, each comprising a VH and VL chain, each VH and VL chain separated by a framework amino acid sequence Where the antibody is:
VH CDR1: SYWMH (SEQ ID NO: 20),
VH CDR2: NIYPGSGSTNYD (SEQ ID NO: 21),
VH CDR3: GGLWLRVDY (SEQ ID NO: 22),
VL CDR1: SASSSVSYMH (SEQ ID NO: 23),
VL CDR2: DTSKLAS (SEQ ID NO: 24), and
VL CDR3: QQWSSNPF (SEQ ID NO: 25).   45. The antibody of claim 44, wherein the human VH comprises the amino acid sequence of SEQ ID NO: 18 and the human VL comprises the amino acid sequence of SEQ ID NO: 19.   45. An isolated polynucleotide encoding the antibody of claim 44.   45. A vector comprising a polynucleotide encoding the antibody of claim 44.   45. An isolated host cell comprising a vector comprising a polynucleotide encoding the antibody of claim 44.   49. Producing an antibody comprising culturing the host cell of claim 48 and obtaining the antibody from the cultured host cell to express the polynucleotide.   45. A pharmaceutical composition for treating or preventing pancreatic cancer, comprising a polynucleotide encoding the antibody of claim 44 or a vector comprising the same.   Use of an antibody that binds to a protein encoded by REG4, or a fragment thereof, for the manufacture of a pharmaceutical composition for the treatment or prevention of pancreatic cancer. The antibody comprises a VH and a VL chain, each VH and VL chain comprising a CDR amino acid sequence called CDR1, CDR2 and CDR3 separated by a framework amino acid sequence, and each CDR within each VH and VL chain 52. Use according to claim 51, wherein the amino acid sequence of is:
VH CDR1: SYWMH (SEQ ID NO: 20),
VH CDR2: NIYPGSGSTNYD (SEQ ID NO: 21),
VH CDR3: GGLWLRVDY (SEQ ID NO: 22),
VL CDR1: SASSSVSYMH (SEQ ID NO: 23),
VL CDR2: DTSKLAS (SEQ ID NO: 24), and
VL CDR3: QQWSSNPF (SEQ ID NO: 25).   Either or both of the treatment and prevention of cancer comprising, as an active ingredient, a polypeptide comprising QKGIGEFF / SEQ ID NO: 46, a polypeptide functionally equivalent to the polypeptide, or a polynucleotide encoding the polypeptide An agent for reducing the biological function of the peptide consisting of SEQ ID NO: 40.   54. The agent of claim 53, wherein the biological function is cell proliferation activity.   54. The agent of claim 53, wherein the polypeptide consists of 8 to 30 residues.   54. The agent of claim 53, wherein the polypeptide comprises the amino acid sequence VRPTPKWQKGIGEFFRLSPK / SEQ ID NO: 44 or TPKWQKGIGEFFRLSP / SEQ ID NO: 45.   54. The agent of claim 53, wherein the polypeptide consists of the amino acid sequence VRPTPKWQKGIGEFFRLSPK / SEQ ID NO: 44 or TPKWQKGIGEFFRLSP / SEQ ID NO: 45.   54. The agent according to claim 53, wherein the active ingredient is a polypeptide, and the polypeptide is modified with a cell membrane permeable substance.   The polypeptide has the following general formula: [R]-[D], wherein [R] represents a cell membrane permeable substance, and [D] is a fragment sequence comprising QKGIGEFF / SEQ ID NO: 46 59. The agent of claim 58, wherein the polypeptide comprises: or an amino acid sequence of a polypeptide functionally equivalent to the polypeptide, wherein the polypeptide lacks the biological function of the peptide consisting of SEQ ID NO: 40. 59. The agent of claim 58, wherein the cell membrane permeable substance is any one selected from the group consisting of:
Polyarginine;
TAt / RKKRRQRRR / SEQ ID NO: 47;
Penetratin / RQIKIWFQNRRMKWKK / SEQ ID NO: 48;
Buforin II / TRSSRAGLQFPVGRVHRLLRK / SEQ ID NO: 49;
Transportan / GWTLNSAGYLLGKINLKALAALAKKIL / SEQ ID NO: 50;
MAP (amphiphilic peptide) / KLALKLALKALKAALKLA / SEQ ID NO: 51;
K-FGF / AAVALLPAVLLALLAP / SEQ ID NO: 52;
Ku70 / VPMLK / SEQ ID NO: 53 or PMLKE / SEQ ID NO: 61;
PRION / MANLGYWLLALFVTMWTDVGLCKKRPKP / SEQ ID NO: 54;
pVEC / LLIILRRRIRKQAHAHSK / SEQ ID NO: 55;
Pep-1 / KETWWETWWTEWSQPKKKRKV / SEQ ID NO: 56;
SynB1 / RGGRLSYSRRRFSTSTGR / SEQ ID NO: 57;
Pep-7 / SDLWEMMMVSLACQY / SEQ ID NO: 58; and
HN-1 / TSPLNIHNGQKL / SEQ ID NO: 59.   61. The agent of claim 60, wherein the polyarginine is Arg 11 (SEQ ID NO: 60).   54. The agent according to claim 53, wherein the cancer is any one selected from the group consisting of pancreatic cancer, prostate cancer, breast cancer and bladder cancer.   Either or both of the treatment and prevention of cancer comprising administering a polypeptide comprising QKGIGEFF / SEQ ID NO: 46, a polypeptide functionally equivalent to the polypeptide, or a polynucleotide encoding these polypeptides A method wherein the polypeptide lacks the biological function of the peptide consisting of SEQ ID NO: 40.   A polypeptide comprising QKGIGEFF / SEQ ID NO: 46, a polypeptide functionally equivalent to the polypeptide, or a polypeptide thereof in the manufacture of a pharmaceutical composition for either or both of cancer treatment and prevention Use of an encoding polynucleotide, wherein the polypeptide lacks the biological function of the peptide consisting of SEQ ID NO: 40.   A pharmaceutical composition comprising a polypeptide comprising QKGIGEFF / SEQ ID NO: 46, or a polypeptide functionally equivalent to said polypeptide, and a pharmaceutically acceptable carrier, wherein the polypeptide comprises SEQ ID NO: 40 A composition that lacks the biological function of a peptide comprising:   A polypeptide comprising QKGIGEFF / SEQ ID NO: 46, or an amino acid sequence of a polypeptide functionally equivalent to the polypeptide, wherein the polypeptide lacks the biological function of the peptide consisting of SEQ ID NO: 40 peptide.   68. The polypeptide of claim 66, wherein the biological function is cell proliferation activity.   68. The polypeptide of claim 66, wherein the polypeptide consists of 8 to 30 residues.   68. The polypeptide of claim 66, wherein the polypeptide comprises the amino acid sequence VRPTPKWQKGIGEFFRLSPK / SEQ ID NO: 44 or TPKWQKGIGEFFRLSP / SEQ ID NO: 45.   68. The polypeptide of claim 66, wherein the polypeptide consists of the amino acid sequence VRPTPKWQKGIGEFFRLSPK / SEQ ID NO: 44 or TPKWQKGIGEFFRLSP / SEQ ID NO: 45.   68. The polypeptide of claim 66, wherein the polypeptide is modified with a cell membrane permeable substance.   A polypeptide having the following general formula: [R]-[D], wherein [R] represents a cell membrane permeable substance, and [D] is a fragment sequence comprising QKGIGEFF / SEQ ID NO: 46 72 or an amino acid sequence of a polypeptide functionally equivalent to a polypeptide comprising the fragment sequence, wherein the polypeptide lacks the biological function of the peptide consisting of SEQ ID NO: 40. The described polypeptide. The polypeptide according to claim 72, wherein the cell membrane permeable substance is any one selected from the group consisting of:
Polyarginine;
Tat / RKKRRQRRR / SEQ ID NO: 47;
Penetratin / RQIKIWFQNRRMKWKK / SEQ ID NO: 48;
Buforin II / TRSSRAGLQFPVGRVHRLLRK / SEQ ID NO: 49;
Transportan / GWTLNSAGYLLGKINLKALAALAKKIL / SEQ ID NO: 50;
MAP (amphiphilic peptide) / KLALKLALKALKAALKLA / SEQ ID NO: 51;
K-FGF / AAVALLPAVLLALLAP / SEQ ID NO: 52;
Ku70 / VPMLK / SEQ ID NO: 53;
Ku70 / PMLKE / SEQ ID NO: 61;
PRION / MANLGYWLLALFVTMWTDVGLCKKRPKP / SEQ ID NO: 54;
pVEC / LLIILRRRIRKQAHAHSK / SEQ ID NO: 55;
Pep-1 / KETWWETWWTEWSQPKKKRKV / SEQ ID NO: 56;
SynB1 / RGGRLSYSRRRFSTSTGR / SEQ ID NO: 57;
Pep-7 / SDLWEMMMVSLACQY / SEQ ID NO: 58; and
HN-1 / TSPLNIHNGQKL / SEQ ID NO: 59.   74. The polypeptide of claim 73, wherein the polyarginine is Arg 11 (RRRRRRRRRRR / SEQ ID NO: 60). A method of diagnosing chemo-radiotherapy resistance in a subject comprising the following steps:
(A) detecting the expression level of the REG4 gene in a biological sample derived from a patient;
(B) comparing the detected expression level with a control level; and
(C) if the detected expression level is increased relative to a normal control level, the subject is suffering from chemo-radiation resistant cancer or the risk of chemo-radiation resistant The stage of determining that there is.   76. The method of claim 75, wherein the cancer is pancreatic cancer.

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