Muc-1 cytoplasmic domain peptides as inhibitors of cancer

[0001] This invention relates to the regulation of cell growth, and in particular the regulation of tumor cell growth. In particular, MUC1 peptides derived from a specific region in the MUC1 cytoplasmic domain have been shown to inhibit oligomerization and translocation of MUC1 to the nucleus, causing inhibition and even death of MUC1-expressing cancer cells.

2. Related Art [0002] Mucins are highly O-glycosylated proteins that are expressed mainly in epithelial cells. Secreted and membrane-bound mucins form a physical barrier that protects the epithelial cell boundaries from damage caused by toxins, microorganisms, and other forms of stress that occur when in contact with the external environment. Transmembrane mucin 1 (MUC1) can also signal to the inside of the cell via its cytoplasmic domain. MUC1 does not show sequence similarity with other membrane-associated mucins, except for the presence of the protein domain from sea urchin-centrokinase-agrin (SEA) sperm (Duraisamy et al., 2006). In this respect, MUC1 is translated as a single polypeptide and then self-cleaved in the SEA domain (Macao, 2006).

[0003] The N-terminal MUC1 subunit (MUC1-N) contains a variable number of tandem repeats with a high proportion of serine and threonine residues that are modified by O-glycosylation (Siddiqui, 1988). MUC1-N extends beyond the cell glycocalyx and is bound to the cell surface by non-covalent binding to transmembrane

MUC1 C-terminal subunit (MUC1-C) (Merlo, 1989). MUC1-C consists of a 58 amino acid extracellular domain, a 28 amino acid transmembrane domain, and a 72 amino acid cytoplasmic domain that interacts with various signal molecules (Kufe, 2008). MUC1-N excretion to the protective physical barrier leaves MUC1-C on the cell surface as a potential receptor for transduction of intracellular signals, for growth and survival (Ramasamy et al., 2007; Ahmad et al., 2007).

[0004] Available evidence indicates that human cancers utilize the effect of MUC1 in contributing to oncogenicity. In this context, along with transformation and loss of polarization, MUC1 is expressed at high levels across the entire cell surface in breast and other epithelial tissue cancers (Kufe, 1984). Other work has shown that overexpression of MUC1 induces anchor-independent growth and oncogenicity (Li et al., 2003a; Raina et al., 2004; Ren et al., 2004; Wei et al., 2005), at least partly by stabilizing β-catenin (Huang et al., 2005). In addition, according to the function in the survival of normal epithelial cells, MUC1 overexpression confers cancer cells resistance to stress-induced apoptosis (Ren et al., 2004; Yin and Kufe, 2003; Yin et al., 2004; Yin et al., 2007).

[0005] Loss of apical membrane restriction allows complexation with epidermal growth factor receptor (EGFR) and co-activation of EGFR-mediated signaling (Li et al., 2001; Ramasamy et al., 2007). overexpression

MUC1 in cancer cells is also associated with the accumulation of MUC1-C in cy2 tozole and the targeting of this subunit (Li et al., 2003b; Li et al., 2003c) and mitochondria (Ren et al., 2004; Ren; et al., 2006). Importantly, MUC1-C oligomerization is necessary to target it to the nucleus and interact with various effectors (Leng et al., 2007). For example, the cytoplasmic domain MUC1-C (MUC1-CD) acts as a substrate for c-Src (Li et al., 2001), c-Abl (Raina et al., 2006), Cδ protein kinase (Ren et al., 2000 ) and 3β glycogen synthase kinase (Li et al., 1998) and directly interactions with the effector molecule of the Wnt pathway, β-catenin (Yamamoto et al., 1997; Huang et al., 2005) and with the p53 tumor suppressor (Wei et al. ., 2005). Thus, although oligomerization appears to be important, there is no direct evidence that interfering with MUC1 oligomer formation would have beneficial effects on cancer cells, much less on how this could be achieved.

SUMMARY OF THE INVENTION [0006] Thus, according to a first embodiment of the present invention, an MUC1 peptide consisting of at least 6 consecutive MUC1 residues and no more than 20 consecutive MUC1 residues is provided, and comprising a CQCRRK sequence, with an amino terminal cysteine

CQCRRK is obstructed at its NH2 terminus by at least one amino acid residue that does not necessarily correspond to the native MUC-1 transmembrane sequence for use in inhibiting MUC1-positive tumor cell in a subject. In other words, the first embodiment of the present invention provides the use of MUC1 peptide, as defined above, in the manufacture of a medicament for inhibiting MUC1 positive tumor cell in a subject. The peptide for use in the first embodiment of the present invention may contain at least 7 consecutive MUC1 residues, at least 8 consecutive MUC1 residues, and this sequence may more specifically comprise CQCRRK (SEQ ID NO: 4) or CQCRRKN ​​(SEQ ID NO: 53). The peptide for use in the first embodiment of the present invention may contain no more than 10 consecutive residues, 11 consecutive residues, 12 consecutive residues, 13 consecutive residues, 14 consecutive residues, 15 consecutive residues, 16 consecutive residues, 17 consecutive residues, 18 consecutive residues or 19 subsequent MUC1 residues. The peptide for use in the first embodiment of the present invention may be fused to a cell targeting domain, such as poly-DR, poly-DP or poly-DK. The peptide for use in the first embodiment of the present invention may contain L-amino acids alone, D-amino acids alone, or a mixture of L- and D-amino acids.

[0007] The MUC1-positive tumor cell that is inhibited according to the first embodiment of the present invention may be a cancer cell, leukemia cell or myeloma cell, such as a prostate or breast cancer cell. For example, the peptide may be used to inhibit MUC1-positive tumor cell in a subject by administering the peptide to the subject intravenously, intraarterially, intratumorally, subcutaneously or intraperitoneally, or by local, regional, systemic or continuous administration. Inhibition may include causing growth arrest of this cancer cell, apoptosis of that cancer cell, and / or necrosis of the tumor tissue comprising the cancer cell. The subject may be a human.

[0008] The subject of the first aspect of the present invention may be a recipient of a second cancer therapy. The second anti-cancer therapy may be surgery, chemotherapy, radiation therapy, hormone therapy, toxin therapy, immunotherapy and cryotherapy. Second anti-cancer therapy may be administered before this peptide, after that peptide or at the same time as this peptide. The subject of the first embodiment of the present invention may be evaluated for MUC1 expression in a tumor cell prior to administration of this peptide and / or the effect of this peptide on MUC1 expression in the tumor of that subject may be assessed.

[0009] The peptide can be used to inhibit MUC1 positive tumor cells in a subject, according to the first embodiment of the present invention, by administering this peptide at a dose of 0.1-500 mg / kg / day or 10-100 mg / kg / day. The peptide may be administered daily, for example for 7 days, 2 weeks, 3 weeks, 4 weeks, a month, weeks, 8 weeks, two months, 12 weeks or 3 months. The peptide may be administered weekly, for example for 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks or 12 weeks.

[0010] In a second embodiment of the present invention, there is provided a pharmaceutical composition comprising: (a) MUC1 peptide consisting of at least 6 consecutive MUC1 residues and no more than 20 consecutive MUC1 residues, and comprising the CQCRRK sequence, wherein the amino terminus cysteine ​​CQCRRK is obstructed at its end, NH2, by at least one amino acid residue that need not correspond to the native MUC1 transmembrane sequence, and (b) a pharmaceutically acceptable carrier, buffer or diluent. The peptide may contain at least 7 or 8 consecutive MUC1 residues. A peptide may contain no more than 10 consecutive residues, 11 consecutive residues, 12 consecutive residues, 13 consecutive residues, 14 consecutive residues, 15 consecutive residues, 16 consecutive residues, 17 consecutive residues, 18 consecutive residues or 19 consecutive MUC1 residues. The peptide can be linked to a cell targeting domain, such as poly-DR, polyD-P or poly-DK. The peptide may be at least 8 residues long, and at least two non-contiguous residues form a bridge through their side chains. The bridge may include a linker, chemically modified side chains or a hydrocarbon clamp. Linkers may contain modifications that stabilize the alpha helical structure of this peptide. The buffer may contain β-mercaptoethanol, glutathione or ascorbic acid, or another reducing agent that maintains the peptide in a monomeric state.

[0011] In a third embodiment of the present invention there is provided an MUC1 peptide consisting of at least 6 consecutive MUC1 residues and no more than 20 consecutive MUC1 residues, and comprising the CQCRRK sequence, wherein the amino terminal cysteine ​​CQCRRK is obstructed at its NH2 end by at least one amino acid residue that does not necessarily correspond to the native MUC1 transmembrane sequence. The peptide may contain at least or 8 consecutive MUC1 residues. A peptide may contain no more than 10 consecutive residues, 11 consecutive residues, 12 consecutive residues, 13 consecutive residues, 14 consecutive residues, 15 consecutive residues, 16 consecutive residues, 17 consecutive residues, 18 consecutive residues or 19 consecutive MUC1 residues. The peptide can be linked to a cell targeting domain, such as poly-DR, poly-DP or poly-DK.

[0012] Also provided, in a fourth embodiment of the present invention, the MUC1 peptide according to the third embodiment of the present invention or the pharmaceutical composition according to the second embodiment of the present invention for use in medicine.

[0013] In a fifth embodiment of the present invention, there is provided a method of inhibiting in vitro oligomerization and transport of MUC1 to the nucleus in a cell, comprising contacting the cell expressing MUC1 with a MUC1 peptide composed of at least 6 consecutive MUC1 residues and no more than 20 consecutive MUC1 residues, and comprising the CQCRRK sequence, wherein the amino terminus cysteine ​​CQCRRK is obscured at its NH2 terminus by at least one amino acid residue, which does not necessarily correspond to the native MUC1 transmembrane sequence. The peptide may contain at least 7 consecutive MUC1 residues, at least 8 consecutive MUC1 residues, and the sequence may in particular comprise CQCRRK or CQCRRKN. A peptide may contain no more than 10 consecutive residues, 11 consecutive residues, 12 consecutive residues, 13 consecutive residues, 14 consecutive residues, 15 consecutive residues, 16 consecutive residues, 17 consecutive residues, 18 consecutive residues or 19 consecutive MUC1 residues. The peptide can be linked to a cell targeting domain, such as poly-DR, poly-DP or poly-DK. A peptide may contain only L-amino acids, only D-amino acids or a mixture of L- and D-amino acids.

[0014] A MUC1-expressing cell that is inhibited according to the fifth embodiment of the present invention may be a cancer cell, such as a cancer cell, leukemia cell, or myeloma cell, such as a prostate or breast cancer cell.

[0015] A sixth embodiment of the present invention provides an MUC1 peptide consisting of at least 6 consecutive MUC1 residues and no more than 20 consecutive MUC1 residues, and comprising the sequence CQCRRK or KRRQCQ, wherein all amino acid residues of this peptide are D-amino acids. The peptide may comprise the sequence KRRCQC (SEQ ID NO: 49).

[0016] The tumor cell may be, e.g., a breast cancer cell, lung cancer, colon cancer, pancreatic cancer, kidney cancer, stomach cancer, liver cancer, bone cancer, hematological cancer, neural tissue cancer, melanoma, ovarian cancer, testicular cancer, cancer prostate, cervical cancer, vaginal cancer or bladder cancer.

[0017] Advantageous aspects of the above embodiments of the present invention may include killing tumor cells. This may include, before, after or at the same time as performing the above-described uses of the present invention, exposing the subject to one or more additional therapies. These therapies can be, for example, one or more forms of ionizing radiation and / or one or more chemotherapeutic agents. The one or more chemotherapeutic agents may be, for example, cisplatin, carboplatin, procarbazine, mechloretamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunorubicin, bleomycinomycinomycinomycinomycinomycinomycinomycinomycinomycin , tamoxifen, taxol, transplatin, 5-fluorouracil, vincristine, vinblastine, methotrexate or an analog of any of the above. Hormone therapy, immunotherapy, toxin therapy, cryotherapy and surgery are also considered as combination therapies.

[0018] It is contemplated that any of the uses, methods or compositions described herein may be performed with respect to any of the other uses, methods or compositions described herein.

[0019] The use of the singular in connection with the term "comprising" in the claims and / or description may include "one" element, but is also consistent with the meaning of "one or more", "at least one" and "one or more" than one. " The word "about" means plus / minus 5% of the indicated number.

[0020] Other objects, features and advantages of the present invention will be apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples indicating specific embodiments of the invention are given by way of illustration only.

BRIEF DESCRIPTION OF THE FIGURES [0021] The following drawings form part of the present description and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description.

FIG. 1A-D. MUC1 / CQC peptide blocks MUC1 oligomerization. (FIG. 1A). A schematic representation of the MUC1-C subunit and the 72 amino acid sequence of MUC1-CD is shown. The 15 N-terminal amino acids (shaded sequence) of MUC1 / CQC and mutated MUC1 / AQA peptides were synthesized using the poly-dArg transduction domain. (FIG. 1B) His-MUC1-CD (1.4 mg / ml) was immobilized on a BIAcore sensor chip. MUC1 / CQC was injected over the chip at 10 μΜ. Raw binding data were analyzed using BIAevaluation version 3.0 software and fitted to Langmuir 1: 1 binding model. (FIG. 1C) Purified His-MUC1-CD (1.5 mg / ml) were incubated in PBS, 200 μΜ MUC1 / CQC or 200 μΜ MUC1 / AQA for 1 h at room temperature. Proteins were separated on a non-reducing SDS-polyacrylamide gel and analyzed by anti-MUCI-C immunoblotting. (FIG. 1D) 293 cells were transiently transfected to express the empty vector or GFP-MUC1-CD and Flag-MUCI-CD. 48 h after transfection, cells were treated with 5 μΜ MUC1 / CQC or MUC1 / AQA for 3 days. Cells were then harvested for immunoblotting with anti-MUC 1 -C (left panel). Whole cell lysates were also precipitated from anti-Fl ag and pellets were tested by immunoblotting with indicated antibodies (right panels).

FIG. 2A-C. MUC1 / CQC peptide blocks MUC1-C nuclear localization. (FIG. 2A), ZR-75-1 cells were incubated with 5 μM FITC-labeled MUC1 / CQC peptide for the indicated time, and then analyzed by flow cytometry. The mean fluorescence index (MFI) is shown on each panel. (FIG. 2B-C) ZR-75-1 cells (FIG. 2B) and MCF-7 (FIG. 2C) were incubated in the presence of 5 μM MUC1 / CQC or MUC1 / AQA peptide for 3 days. Whole cell lysates (WCL) (left panels) and cell nuclei lysates (right panels) were immunoblotted with the indicated antibodies.

FIG. 3A-D. MUC1 / CQC peptide induces S phase arrest and necrosis. ZR-75-1 (FIGS. 2A-B) and MCF-7 (FIGS. 2C-D) cells were treated with 5 μM MUC1 / CQC or MUC1 / AQA for 3 and 4 days. Cells were fixed and analyzed for cell cycle distribution by flow cytometry (FIGS. 2A and 2C). The percentage of cells in the G1, S and G2 / M phase are shown on panels. Cells were also stained with propidium iodide and analyzed by flow cytometry for necrosis (FIGS. 2B and 2D). The percentage of necrotic cells is shown on panels.

FIG. 4A-E. MUC1 / CQC selectivity for breast cancer cells expressing MUC1. (FIG. 4A) ZR-75-1 cells were stably infected with empty lentivirus (vector) or MUC1 expressing siRNA. Infected cell lysates were immunoblotted with the indicated antibodies. (FIG. 4B) ZR-75-1 / vector cells were not treated (diamonds), and ZR-751 / vector cells (squares) and ZR-75-1 / MUC1siRNA cells (triangles) were treated with 5 μM MUC1 / CQC peptide for the indicated time. The number of viable cells was determined by exclusion method using trypan blue. (FIG. 4C) 293 cells were untreated (diamonds) and treated with 5 μM MUC1 / CQC peptide (squares) or MUC1 / AQA (triangles) for the indicated time. The number of viable cells was determined by exclusion method using trypan blue. (FIG. 4D) MCF-10A cells were left untreated (left panel) and treated with 5 μM MUC1 / CQC (middle panel) or MUC1 / AQA (right panel). After 3 days, the cells were analyzed for cell cycle distribution. (FIG. 4E) MCF-10A cells were untreated (diamonds) and treated with 5 μΜ MUC1 / CQC (squares) or MUC1 / AQA (triangles) for the indicated time. The number of viable cells was determined by exclusion method using trypan blue.

FIG. 5A-C. MUC1 / CQC peptide blocks the growth of ZR-75-1 breast cancer xenografts. (FIG. 5A) Female Balb-c nu / nu mice aged four to six weeks were implanted with 17-e-estradiol implants. After 24 h, ZR-75-1 breast cancer cells (embedded in Matrigel) were injected subcutaneously in the flank. When the tumors were ~ 150 mm in size³, mice were matched in pairs and injected intraperitoneally with PBS (vehicle control; filled squares), 50 mg / kg MUC1 / AQA peptide (peptide control, empty squares) or 10 mg / kg MUC1 / CQC peptide (filled triangles) daily for 21 days. Another group was treated with 50 mg / kg MUC1 / CQC peptide daily for 6 days (empty triangles). Mice were weighed twice a week and tumor measurements were carried out every 4 days. (FIGS. 5B and 5C). On day 24 (asterisk) tumors taken from the control group and the 50 mg / kg / day treated group x 6 days were stained with H&E (FIG. 5B) and anti-MUC1 antibody (FIG. 5C).

FIG. 6A-B. Prolonged effect of MUC1 / CQC peptide on ZR-75-1 tumors. (FIG. 6A) Mice were injected with ZR-75-1 cells as described in the legend of FIG. 5A. When the tumors were ~ 275 mm³ the mice were paired into groups and injected intraperitoneally with PBS (vehicle control; empty squares) or 30 mg / kg MUC1 / CQC peptide (peptide control, filled squares) daily for 21 days. Control mice were sacrificed on day 32 when tumors reached ~ 1200 mm³. Treated mice were monitored until day 52, when tumors were harvested for H&E staining (FIG. 6B).

FIG. 7. The 7-mer MUC1 inhibits prostate cancer. DU145 prostate cancer cells were treated with 5 μΜ short CQC peptides (7-mers) or 5 μΜ long CQC peptides (15-mers) for 4 days. Cell growth was measured by MTT test. Data indicate the percentage inhibition of growth compared to untreated cells (control).

FIG. 8. Sequences of clamped MUC1-CD peptides.

FIG. 9A. Effect of clamped MUC1-CD peptide on the growth of H1650 non-small cell lung cancer cells. To assess sensitivity to MUC1 inhibition, NSCLC H1650 cells were treated with 1 and 5 μΜ clamped MUC1 CQC peptide (GO-200-1B) for 7 days. Treatment of H1650 5 μΜ GO-200-1B cells was associated with significant growth inhibition followed by a decrease in cell number.

FIG. 9B. Effect of GO-200-2B on cell proliferation. The H-1975 non-small cell lung cancer cell line was cultured in DMEM with the addition of 10% heat inactivated fetal bovine serum and 100 units / ml penicillin, 100 μg / ml streptomycin and 2 mmol / L L-glutamine. Cells were re-seeded one day prior to surgery. Cells were treated with 5 μΜ GO-200-2B for 3 days and cell viability was determined by trypan blue exclusion.

FIG. 10. Effect of various YUCI-CP peptides with CQC region on the growth of hormone-dependent breast cancer cells. To determine if exposure to different Y1U peptides (TC'D containing the CQC region affect growth, breast cancer cells

ZR-75-1 was treated with 5 μΜ of different peptides for 4 days and monitored for cell proliferation. Indeed, substantial growth inhibition was observed compared to cells that were left untreated.

FIG. 11. Effect of various MUC1-CD peptides with CQC region on the growth of non-small cell lung cancer cells. A549 non-small cell lung cancer cells were treated with 5 μΜ GO-203, GO-203-2 or GO-203cyc for 7 days. The number of viable cells on day 7 was determined by trypan blue exclusion and the percentage inhibition of growth was calculated by comparing the cell growth for untreated cells.

FIG. 12. Effect of various MUC1-CD peptides with the CQC region on the growth of H1975 non-small cell lung cancer cells. H1975 non-small cell lung cancer cells were treated with 5 μΜ of different MUC1-CD peptides with the CQC region for 6 days. The number of viable cells on day 6 was determined by trypan blue exclusion. The results indicate that treatment of H1975 5 μΜ different peptides was associated with significant growth inhibition.

FIG. 13. Effect of various MUC1-CD peptides with CQC region on triple negative breast cancer cell growth. MDA-MB-231 triple-negative breast cancer cells were treated with 5 μΜ of different MUC1-CD peptides with the CQC region for 6 days. The number of viable cells on day 6 was determined by trypan blue exclusion. The results show that treatment of MDAMB-231 cells with different peptides was associated with significant growth inhibition.

FIG. 14. Effect of shorter GO-203 peptides on ZR-75-1 breast cancer cell proliferation. Human ZR-75-1 breast cancer cells were cultured in RPMI1640 with the addition of 10% heat inactivated fetal bovine serum, 100 units / ml penicillin, 100 μβ / ml streptomycin. Cells were treated with different peptides at a concentration of 5 μΜ daily for four days, and cell viability was determined by trypan blue exclusion. In contrast to GO-210, treatment of human breast cancer cells ZR-75-1 5 μΜ GO-203 (SEQ ID NO: 53), GO-207 (SEQ ID NO: 4), GO-208 (SEQ ID NO: 50 ) and GO-209 (SEQ ID NO: 54) every day for 4 days was associated with significant growth inhibition.

FIG. 15A-D. The effect of GO-203 in combination with various anti-cancer drugs. ZR-75-1 cells were exposed to the indicated concentrations of cisplatin (FIG. 15A), doxorubicin (FIG. 15B), rh-TNF-α (FIG. 15C) and taxol (FIG. 15D) alone or in combination with GO-203. The treatment was sequential for cisplatin, doxorubicin and taxol, the cells exposed to these agents for 72 h and then treated with 5 μΜ GO-203 for 72 h. In studies using rh-TNF, cells were simultaneously exposed to different concentrations of rh-TNF alone and in combination with 5 μΜ GO-203 for 72 h. The MTS test was used to determine cell survival.

FIG. 16. GO-203 acts additively or synergistically with anti-cancer agents. The graphs show association indicators for different levels of influence (fraction with influence). The level of influence was obtained by treating the cells with the indicated combinations of anticancer drugs and GO-203.

DESCRIPTION OF SAMPLE CHARACTERS

I. The present invention [0022] MUC1 has been extensively studied by the inventors and others for its role in cancer. As discussed above, human MUC1 is a heterodimeric glycoprotein translated as a single polypeptide that is cleaved into N- and C-terminal subunits in the endoplasmic reticulum (Ligtenberg et al., 1992; Macao et al., 2006; Levitin et al., 2005). The abnormal overexpression of MUC1 found in most human cancers (Kufe et al., 1984) leads to anchor-independent growth and oncogenicity (Li et al., 2003a; Huang et al., 2003; Schroeder et al., 2004; Huang et al., 2005). Other studies have shown that MUC1 overexpression confers resistance to apoptosis induced by oxidative stress and genotoxic anticancer agents (Yin and Kufe, 2003; Ren et al., 2004; Raina et al., 2004; Yin et al., 2004; Raina et al. ., 2006; Yin et al., 2007).

[0023] The family of bound and secreted mucins works by providing a protective barrier on the surface of the epithelial cell. When the epithelial layer is damaged, the closing rims between adjacent cells are broken and a loss of polarization occurs, as the cells initiate a hegulin-induced repair program (Vermeer et al., 2003). MUC1-N is excreted from the cell surface (Abe and Kufe, 1989), leaving MUC1-C to act as a transmitter of stress signals from the environment to the interior of the cell. To this end, MUC1-C forms complexes on the cell surface with members of the ErbB and MUC1-C receptor family is directed to the nucleus in response to stimulation heregulin (Li et al., 2001; Li et al., 2003c). MUC1-C also acts in the integration of the ErbB receptor and Wnt signaling pathways through direct interactions between the MUC1 cytoplasmic domain (CD) and members of the catenin family (Huang et al., 2005; Li et al., 2003c; Yamamoto et al., 1997; Li et al., 1998; Li et al., 2001; Li i Kufe, 2001). Other studies have shown that MUC1-CD is phosphorylated by 3β glycogen synthase kinase, c-Src, Cδ and c-Abl protein kinase (Raina et al., 2006; Li et al., 1998; Li et al., 2001; Ren et al., 2002).

[0024] The mechanisms responsible for targeting MUC1-C to the nucleus are unclear. Proteins containing the classic nuclear localization signal (NLS) are imported into the nucleus by initial binding to importin α and then, in turn, to importin β (Weis, 2003). The α / β load-importin complex attaches to the nuclear pores by binding to eoporins and is transported through the pores in a GTPase-dependent mechanism. Classic NLSs are one-part with a single 4-5 basic amino acid moiety or two-part with two basic amino acid moieties separated by 10-12 amino acid linkers. MUC1-CD contains an RRK motif that does not correspond to the prototype one-piece NLS (Hodel et al., 2002). However, some proteins containing non-classical NLS are transported through the nuclear pores by direct binding to importin β (Kau et al., 2004). Importin β binds to several nucleoporins (Ryan and Wente, 2000), including Nup62, which is located on both sides, cytoplasmic and nucleoplasmic, of nuclear pore complexes (Percipall et al., 1997). Other studies have shown that β-catenin is imported into the nucleus in a mechanism independent of importin and nucleoporin (Suh and Gumbiner, 2003).

[0025] In 2006, the inventors reported that MUC1 is imported into the nucleus by a mechanism involving binding to Nup62. They also showed that MUC1 forms oligomers using the CQC motif in the cytoplasmic domain of MUC1, and that MUC1 oligomerization is necessary for import into the nucleus. Here, they expanded their work to include further understanding of the role that the CQC motif plays in creating oligomers. They also showed that short peptides corresponding to this region can interfere with the formation of MUC1 oligomers, preventing cancer cells from being transported to the nucleus. These peptides are capable of inhibiting the growth of cancer cells as well as inducing apoptosis in such cells and even tumor necrosis. These and other aspects of the invention are described in detail below.

II. MUC1

A. Structure [0026] Y1LC1 is a mucin type glycoprotein that is expressed at the peak of normal epithelial secretory cells (Kufe et al., 1984). YUC1 forms a heterodimer after synthesis as a single polypeptide and cleavage of the precursor into two subunits in the endoplasmic reticulum (Ligtenberg et al., 1992). This fission can occur through an autocatalytic process (Levitan et al., 2005). The N-terminal ΜϋΠ> 250 kDa subunit (ΜΙΕ N-ter, ΜυΟ-N) contains a variable number of 20-amino acid tandem repeats that are inaccurate with strongly conservative changes and are modified by O-linked glycans (Gendler et al., 1988; Siddiqui et al., 1988). Μυϋ1-Ν is bound to the cell surface by dimerization with ~ 23 kDa C-terminal subunit (ΜυΟ C-ter, ΜυΟ-C), which includes the 58-amino acid extracellular region, the 28-amino acid transmembrane domain and the 72-amino acid cytoplasmic domain ( CD, SEQ ID NO: 1) (Ylerlo et al., 1989). The human ΜυΟ sequence is shown below:

GSVWQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFS AQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCOCRRKNYGQLDIFPAR DTYHPMSEYPTYHTHGRYVPPSSSNSPYSSPSS

The bold sequence indicates CD, and some are underlined by the oligomer inhibitory peptide (SEQ ID NO: 3) described in the examples.

[0027] After transformation of a healthy epithelium into cancers, ΜυΟ is abnormally overexpressed in the cytosol and on the entire cell membrane (Kufe et al., 1984; Perey et al., 1992). Cell membrane bound ΜυΟ is directed to endosomes by clocytin-mediated endocytosis (Kinlough et al., 2004). In addition, ΜυΟ-C, but not Μυθ-N, is directed to the nucleus (Baldus et al., 2004; Huang et al., 2003; Li et al., 2003a; Li et al., 2003b; Li et al., 2003c; Wei et al., 2005; Wen et al., 2003) and mitochondria (Ren et al., 2004).

B. Function [0028] ΜυΟ interacts with members of the ErbB receptor family (Li et al., 2001b; Li et al., 2003c; Schroeder et al., 2001) and with the effector Wnt, β-catenin (Yamamoto et al., 1997) . The epidermal growth factor receptor and c-Src phosphorylate the cytoplasmic domain ΜυΟ (cytυΟ-CD) at Y-46, and thereby increase the binding of ΜυΟ and β-catenin (Li et al., 2001a; Li et al., 2001b). Binding of ΜυΟ and β-catenin is also regulated by 3β glycogen synthase kinase and Cδ protein kinase (Li et al., 1998; Ren et al., 2002). ΜυΟ exhibits co-localization with β-catenin in the nucleus (Baldus et al., 2004; Li et al., 2003a; Li et al., 2003c; Wen et al., 2003) and coactivation of transcription of target genes for Wnt (Huang et al. , 2003). Other studies have shown that Μϋί1 also binds directly to p53 and regulates transcription of p53 target genes (Wei et al., 2005). It is worth stressing that overexpression of ΜυΟ is sufficient to induce anchorage-independent growth and oncogenicity (Huang et al., 2003; Li et al., 2003b; Ren et al., 2002; Schroeder et al., 2004).

[0029] Most mitochondrial proteins are encoded in the nucleus and imported into mitochondria by translocation complexes in the outer and inner mitochondrial membrane. Certain mitochondrial proteins contain N-terminal mitochondrial targeting sequences and interact with Tom20 in the outer mitochondrial membrane (Truscott et al., 2003). Other mitochondrial proteins contain internal targeting sequences and interact with the Tom70 receptor (Truscott et al., 2003). Recent studies have shown that mitochondrial proteins without internal targeting sequences are delivered to Tom70 by the HSP70 and HSP90 complex (Young et al., 2003).

III. MUC1 peptides

A. Structure [0030] The present invention contemplates the design, production and use of various MUC1 peptides. The structural features of these peptides are as follows. First, these peptides contain no more than 20 consecutive MUC1 residues. Thus, the term "peptide containing no more than 20 consecutive residues", even when it includes the term "comprising", cannot be understood to include a greater number of consecutive MUC1 residues. Secondly, these peptides contain the CQCRRK motif. Thus, the peptides will contain at least these six consecutive residues from the MUC1-C domain. Third, these peptides contain at least one amino acid residue attached to the NH2 terminal side of the first C residue in the CQCRRK motif, such that the first C residue is "obstructed" by the at least one amino acid attached thereto. This residue may be native to MUC1 (i.e. from the transmembrane domain), it can be randomly selected (any of the 20 naturally occurring amino acids or their analogs), or it can be part of another peptide sequence (e.g., purification tag sequence, stabilizing sequence or cell targeting domain).

[0031] Typically, the peptides will be composed of 50 or fewer residues, once again containing no more than 20 consecutive MUC1 residues. The total length can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or residues. The following peptide length ranges are considered: 6-50 residues, 7-50 residues, 7-25 residues, 6-20 residues, 7-20 residues and 7-15 residues. The number of consecutive MUC1 residues may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. The following ranges of consecutive residues 6-20, 7-20 are considered residues and 6-15 residues or 7-15 residues.

[0032] In the present invention, amino acids of the L configuration, amino acids of the D configuration or a mixture thereof can be used. Although L-amino acids account for the vast majority of the amino acids found in proteins, D-amino acids are found in some proteins produced by exotic marine organisms, such as cone snails. They are also common components of bacterial peptidoglycan cell walls. D-serine can act as a neurotransmitter in the brain. The L and D convention for determining amino acid configuration does not refer to the optical activity of the amino acid itself, but rather to the optical activity of the glycerol aldehyde isomer from which this amino acid can be theoretically synthesized (D-glycerol aldehyde is clockwise, Lglycerol aldehyde is left-handed).

[0033] One form of the "only-D" peptide is a retro-inverso peptide. Retroinverso modification of naturally occurring polypeptides involves a synthetic combination of amino acids with an α-carbon stereochemistry opposite to the stereochemistry of the corresponding L-amino acids, i.e. D-amino acids, in reverse order to the native peptide sequence. Thus, the retro-inverso analog has inverted ends and inverted peptide bonding direction (NH-CO instead of CO-NH) while maintaining approximately the side chain topology as in the native peptide sequence. See U.S. Patent No. 6,261,569, incorporated herein by reference.

[0034] As mentioned above, the present invention contemplates the fusion or conjugation of a cell targeting domain (also called a cell targeting vector or cell transduction domain). Such domains are well known in the art and are usually characterized as short amphipathic or cationic peptides and peptide derivatives, often containing many lysine and arginine residues (Fischer, 2007).

Particularly interesting are the poly-D-Arg and poly-D-Lys sequences (e.g., right-handed, eight-residue lengths), while others are shown in Table 1 below.

TABLE 1

<td>CDD / CTD PEPTIDES</td><td>SEQ ID NO</td>

<td>QAATATRGRSAASRPTERPRAPARSASRPRRPVE</td><td> 5</td>

<td>RQIKIWFQNRRMKWKK</td><td> 6</td>

<td>RRMKWKK</td><td> 7</td>

<td>RRWRRWWRRWWRRWRR</td><td> 8</td>

<td>RGGRLSYSRRRFSTSTGR</td><td> 9</td>

<td>YGRKKRRQRRR</td><td> 10</td>

<td>RKKRRQRRR</td><td> 11</td>

<td>YARAAARQARA</td><td> 12</td>

<td>RRRRRRRR</td><td> 13</td>

<td>kkkkkkkk</td><td> 14</td>

<td>GWTLNSAGYLLGKINLKALAALAKXIL</td><td> 15</td>

<td>LLILLRRRIRKQANAHSK</td><td> 16</td>

<td>SRRHHCRSKAKRSRHH</td><td> 17</td>

<td>NRARRNRRRVR</td><td> 18</td>

<td>RQLRIAGRRLRGRSR</td><td> 19</td>

<td>KLIKGRTPIKFGK</td><td> 20</td>

<td>RRIPNRRPRR</td><td> 21</td>

<td>KLALKLALKALKAALKLA</td><td> 22</td>

KLAKLAKKLAKLAK

PEPTIDES CDD / CTD SEQ ID NO

GALFLGFLGAAGSTNGAWSQPKKKRKV 24

KETWWETWWTEWSQPKKKRKV 25

GALFLGWLGAAGSTMGAKKKRKV 26

MGLGLHLLVLAAALQGAKSKRKV 27

AAVALLPAVLLALLAPAAANYKKPKL 28

MANLGYWLLALFVTMWTDVGLCKKRPKP 29

LGTYTQDFNKFHTFPQTAIGVGAP 30

DPKGDPKGVTVTVTVTVTGKGDPXPD 31

PPPPPPPPPPPPPP 32

VRLPPPVRLPPPVRLPPP 33

PRPLPPPRPG 34

SVRRRPRPPYLPRPRPPPFFPPRLPPRIPP 35

TRSSRAGLQFPVGRVHRLLRK 36

GIGKFLHSAKKFGKAFVGEIMNS 37

KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK 38

ALWMTLLKKVLKAAAKAALNAVLVGANA 39

GIGAVLKVLTTGLPALISWIKRKRQQ 40

INLKALAALAKKIL 41

GFFALIPKIISSPLPKTLLSAVGSALGGSGGQE 42

LAKWALKQGFAKLKS 43

SMAQDIISTIGDLVKWIIQTVNXFTKK 44

LLGDFFRKSKEKIGKEFKRIVQRIKQRIKDFLANLVPR- 45

TES

LKKLLKKLLKKLLKKLLKKL 46

KLKLKLKLKLKLKLKLKL 47

PAWRKAFRWAWRMLKKAA [0035] As also mentioned above, peptides modified for in vivo use are considered by attaching, at the amino and / or carboxy terminus, of a blocking agent to facilitate peptide survival in vivo. This may be useful in situations where the peptide ends tend to degrade by proteases prior to cell uptake. Such blocking agents may include, but are not limited to, additional linked or unbound peptide sequences that can be attached to the residue at the amino and / or carboxy terminus of the peptide to be administered. These agents can be added by chemical reaction during peptide synthesis or by recombinant DNA techniques using methods known in the art. Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art may be attached to residues at the amino and / or carboxyl terminus.

B. Synthesis [0036] Preparation of peptides using solid phase synthesis techniques (Merrifield, 1963) will be preferred. Other techniques for peptide synthesis are well known to those skilled in the art (Bodanszky et al., 1976; Peptide Synthesis, 1985; Solid Phase Peptide Synthelia, 1984). Suitable protecting groups for use in such syntheses can be found in the above texts, as well as in Protective Groups in Organic Chemistry, 1973. These synthetic methods include sequentially attaching one or more amino acid residues or appropriately protected amino acid residues to the growing peptide chain. Typically, the amino or carboxyl group of the first amino acid residue is protected by a suitable selectable deprotecting group. Another selectively removable protecting group is used for amino acids containing a reactive side group, such as lysine.

[0037] Taking solid phase synthesis as an example, the protected amino acid or its derivative is attached to an inert solid support via its unprotected carboxy or amino group. The amino or carboxy protecting group is then selectively removed and mixed with the next amino acid in the sequence having the properly protected complementary (amino or carboxy) group and reacted with the residue already attached to the solid support. Then the amino or carboxyl protecting group is removed from this newly added amino acid residue, and then another (properly protected) amino acid is added, and so on. After all of the desired amino acids have been combined into the correct sequence, all remaining terminal and side protecting groups (and solid support) are removed sequentially or simultaneously to form the final peptide. The peptides of the invention are preferably free of benzylated or methylbenzylated amino acids. Such protecting group moieties may be used during the synthesis, but are removed before use of the peptides. Additional reactions, as described elsewhere, may be necessary to create intramolecular connections to maintain conformation.

[0038] In addition to the 20 standard amino acids that can be used, there are a huge number of "non-standard" amino acids. Two of them can be determined by the genetic code, but they are rather rare in proteins. Selenocysteine ​​is introduced into certain proteins through the UGA codon, which is normally a stop codon. Pyrolysin is used by some methanogenic archaea in enzymes that they use to produce methane. It is coded by the UAG codon. Examples of non-standard amino acids that are not found in proteins include lanthionine, 2-aminoisobutyric acid, dehydroalanine, and the gam14 ma-aminobutyric acid neurotransmitter. Non-standard amino acids often occur as intermediates in the metabolic pathways of standard amino acids - for example, ornithine and citrulline are found in the urea cycle, which is part of the catabolism of amino acids. Custom amino acids are usually created by modifying standard amino acids. For example, homocysteine ​​is formed by the transsulfuration pathway or by demethylation of methionine via the intermediate metabolite Sadenosyl-methionine, and hydroxyproline is formed by post-translational modification of proline.

C. Linkers [0039] Linkers and cross-linking agents can be used to fuse MUC1 peptides with other protein sequences. Bifunctional cross-linking reagents are widely used for a variety of purposes, including for the production of affinity matrices, modification and stabilization of various structures, identification of ligand and receptor binding sites, and structural studies. Homobifunctional reagents that have two identical functional groups have been shown to be very effective in causing cross-linking between the same and different macromolecules or subunits of the macromolecule, and linking polypeptide ligands to their specific binding sites. Heterobifunctional reagents contain two different functional groups. By using different reactivity of two different functional groups, crosslinking can be controlled both selectively and sequentially. These bifunctional cross-linking reagents can be divided by the specificity of their functional groups, e.g., groups specific for amino, sulfhydryl, guanidine, indole or carboxyl groups. Of these, reagents targeting free amino groups have become particularly popular because of their commercial availability, ease of synthesis, and mildness of the reaction conditions under which they can be used. Most heterobifunctional cross-linking reagents contain a group that reacts with a primary amino group and a group that reacts with a thiol group.

[0040] In another example, heterobifunctional cross-linking reagents and methods of using cross-linking reagents are described in US Pat. No. 5,889,155, specifically incorporated herein by reference in its entirety. These cross-linking reagents combine a nucleophilic hydrazide residue with an electrophilic maleimide residue, allowing coupling, in one example, of aldehydes to free thiol groups. The cross-linking reagent can be modified to cross-link different functional groups and is therefore useful for cross-linking polypeptides. In cases where a specific peptide does not contain in its native sequence a residue susceptible to the effect of a given cross-linking reagent, conservative amino acid changes in the primary sequence may be used by genetic or synthetic methods.

[0041] Another use of linkers in the context of peptides as therapeutic agents is the so-called "Stapled Peptide" technique from Aileron Therapeutics. The general approach to peptide "clamping" is that two key residues within the peptide are modified by attaching linkers to amino acid side chains. After synthesis, the linkers are joined by means of a catalyst, thus creating a physical bridge limiting the peptide to its natural α-helix shape. In addition to helping to maintain the native structure needed to interact with the target molecule, this conformation also provides resistance to peptidase activity as well as cell membrane penetration properties. US Patent Nos. 7192713 and 7183059 describing this technology are incorporated herein by reference. See also Schafmeister et al., Journal of the American Chemical Society, 2000. 122 (24): pp. 5891-5892.

D. Design, variants and analogs [0042] The present invention focuses on peptides containing the CQCRRK sequence. After identifying this key structure in the formation of MUC1 oligomers, the inventors also consider the possibility of using CQCRRK sequence variants. For example, certain unnatural amino acids that meet the structural limitations of the CQC sequence can be substituted without loss, and maybe even to the benefit of biological function. In addition, the present inventors also consider formulating structurally similar compounds that mimic key parts of the peptide or polypeptides of the present invention. Such compounds, which may be referred to as peptidomimetics, can be used in the same manner as the peptides of the invention, and therefore also their functional counterparts.

[0043] Certain mimetics that mimic elements of the secondary and tertiary structure of proteins are described in Johnson et al. (1993). The rationale for using peptide mimetics is that the protein peptide backbone exists primarily to orient the amino acid side chains in such a way as to facilitate intermolecular interactions, such as between an antibody and / or an antigen. The peptidomimetic is therefore designed to allow intermolecular interactions similar to a natural molecule.

[0044] Methods for making specific structures have been disclosed in the art. For example, α-helix mimetics are disclosed in US Patent Nos. 5,456,128; 5710245; 5840833; and 5,851,184. Methods for producing conformationally limited β-bends and β-protuberances are described, for example, in US Patent Nos. 5,440013; 5618914; and 5670155. Other types of mimetic turns include reverse turns and γ-turns. Reverse bend mimetics are disclosed in U.S. Patent Nos. 5,475,085 and 5,929,237, and γ-bend mimetics are described in U.S. Patent Nos. 5,672,681 and 5,674,976.

[0045] As used herein, the term "molecular modeling" means quantitative and / or qualitative analysis of structure and function for physical protein-protein interactions based on information on three-dimensional structure and models of protein-protein interaction. This includes standard numerical-based molecular dynamics and energy minimization models, interactive computer graphic models, modified molecular mechanics models, distance geometry, and other structure-based constraint models. Molecular modeling is usually performed using a computer and can be further optimized using known methods. Computer programs that use X-ray crystallography data are particularly useful for designing such compounds. Programs such as RasMol, for example, can be used to create three-dimensional models. Computer programs such as INSIGHT (Accelrys, Burlington, MA), GRASP (Anthony Nicholls, Columbia University), Dock (Molecular Design Institute, University of California in San Francisco) and Auto-Dock (Accelrys) allow further manipulation and allow the introduction of new structure. These methods may include the additional step of sending the 3-D compound structure model to the output device. In addition, data on the 3-D structure of candidate compounds can be compared with a computer database, for example, of 3-D structures.

[0046] Compounds of the invention may also be interactively designed based on structural information from compounds described herein using other structure-based design / modeling techniques (see, e.g., Jackson, 1997; Jones et al., 1996). Candidate compounds can then be tested in standard tests known to those skilled in the art. Exemplary tests are described in this description.

[0047] The structure of 3-D biological macromolecules (e.g., proteins, nucinic acids, carbohydrates and lipids) can be determined on the basis of data obtained using various methods. These methodologies that are most effectively used to assess the 3-D structure of a protein include: (a) X-ray crystallography; (B) nuclear magnetic resonance (NMR) spectroscopy; (C) analysis of physical distance restrictions created between specific places on the macromolecule e.g. intramolecular chemical cross-linking between residues in a protein (e.g., PCT / US00 / 14667, the disclosure of which is incorporated herein by reference in its entirety) and (d) molecular modeling methods based on knowledge of the primary structure of the protein of interest, e.g. homology modeling techniques, threading algorithms, or ab initio structural modeling using computer programs such as MONSSTER (Modeling Of New Structures from Secondary and Tertiary Restraints) (see, e.g., International Application No. PCT / US99 / 11913, the disclosure of which is incorporated herein by reference all by reference). In accordance with the present invention, other molecular modeling techniques (e.g. Cohen et al., 1990; Navia et al., 1992, the disclosures of which are incorporated herein in their entirety by reference.) All these methods obtain data that can be subject to computer analysis. Other spectroscopic methods that may also be useful in the method of the invention but currently do not provide structural details of biomolecules at the atomic level include circular dichroism and fluorescence and ultraviolet / visible light absorption spectroscopy. X-ray crystallography is the preferred method of analysis. A description of this procedure and NMR spectroscopy is provided below.

[0048] X-ray crystallography. X-ray crystallography is based on X-ray diffraction with a characteristic wavelength on electron clouds surrounding the atomic nucleus in the crystal of the molecule or molecule of interest. The technique uses crystals of purified biological macromolecules or complexes of molecules (but they often contain components in the form of solvents, cofactors, substrates or other ligands) to determine the atoms that make up a specific biological macromolecule with atomic resolution. The condition for unraveling the 3-D structure by X-ray crystallography is a high order of the crystal that will strongly bend the X-rays. In this method, the X-ray beam is directed at a regular, repeating matrix of many identical molecules so that the X-rays are diffracted on the matrix according to a pattern from which the structure of a single molecule can be obtained. Highly ordered crystals of, for example, globular protein molecules are large, spherical or ellipsoidal objects with irregular surfaces. The crystals contain large channels between individual molecules. These channels, which usually occupy more than half the volume of the crystal, are filled with disordered solvent particles, and the protein particles are in contact in only a few small regions. This is one of the reasons why protein structures in crystals are usually the same as those in solution.

[0049] Methods for obtaining interesting proteins are described below. The formation of crystals depends on many different parameters, including pH, temperature, concentration of the biological macromolecule, type of solvent and precipitation reagent, as well as the presence of added ions or protein ligands. Many routine crystallization experiments may be needed to screen all these parameters for a combination that will give a crystal suitable for X-ray diffraction analysis. Crystallization machines can automate and speed up the work of setting up a large number of crystallization experiments in a reproducible manner (see, e.g., US Pat. No. 5,790,421, the disclosure of which is incorporated herein by reference in its entirety).

[0050] Crystallization of the polypeptide occurs in a solution in which the concentration of the polypeptide exceeds the maximum solubility (ie, the solution of the polypeptide is supersaturated). Such solutions can be brought back into equilibrium by reducing the concentration of the polypeptide, preferably by precipitating the crystals of the polypeptide. It is often possible to induce crystallization of polypeptides from supersaturated solutions by the addition of agents that change the surface charge of the polypeptide or interfere with the interaction between the polypeptide and a large amount of water by promoting interactions that lead to crystallization.

[0051] Crystallization is generally carried out between 4 ° C and 20 ° C. Substances known as "precipitation reagents" are often used to reduce the solubility of a polypeptide in a concentrated solution by forming an energetically unfavorable, precipitating, depleted layer around the polypeptide molecules (Weber, 1991). In addition to the precipitation reagents, other materials are sometimes added to the polypeptide crystallization solution. These include buffers for adjusting the pH of the solution and salts to reduce the solubility of the polypeptide. Various precipitation reagents are known in the art and include the following: ethanol, 3-ethyl-2-4-pentanediol and many polyglycols, such as polyethylene glycol (PEG). Precipitation solutions may contain, for example, 13-24% PEG 4000, 5-41% ammonium sulfate and 1.0-1.5 M sodium chloride and a pH in the range 5.0-7.5. Other additives may include 0.1 M Hepes, 2-4% butanol, 20-100 mM sodium acetate, 50-70 mM citric acid, 120-130 mM sodium phosphate, 1 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM dithiothreitol (DTT) ). These agents are prepared in buffers and added in various combinations to the crystallization buffer. Crystallized proteins can be modified, e.g., by phosphorylation or by the use of a phosphate mimetic (e.g., tungstate, cacodylate or sulfate).

[0052] Commonly used methods for crystallization of polypeptides include the following techniques: batch, hanging drop, embryo initiation and dialysis. In each of these methods, it is important to induce continuous crystallization after nucleation by maintaining the supersaturated solution. In the batch method, the polypeptide is mixed with precipitation reagents until supersaturation is achieved and the vessel is sealed and set aside until crystals appear. In the dialysis method, the polypeptide is held in a sealed dialysis membrane which is placed in a solution containing the precipitation reagent. Membrane equilibration increases the concentration of polypeptide and precipitation reagent, which causes the polypeptide to achieve supersaturation.

[0053] In a preferred hanging drop technique (McPherson, 1976), the initial polypeptide mixture is formed by adding a precipitation reagent to the concentrated polypeptide solution. The polypeptide and precipitation reagent concentrations are such that in the initial form the polypeptide does not crystallize. A small drop of this mixture is placed on the slide, which is inverted and suspended over the second solution tank. Then the system closes tightly. Typically, the second solution contains a higher concentration of precipitation reagent or other dehydrating agent. The difference in precipitation reagent concentrations means that the protein solution has a higher vapor pressure than the second solution. Because the system containing these two solutions is sealed, equilibrium is established and water from the polypeptide mixture passes into the second solution. This balance increases the concentration of the polypeptide and precipitation reagent in the polypeptide solution. At a critical concentration of the polypeptide and precipitation reagent, a polypeptide crystal may form.

[0054] In another crystallization method, a nucleation site is introduced into the concentrated polypeptide solution. Generally, a concentrated polypeptide solution is prepared and a seeding polypeptide crystal is introduced into this solution. If the levels of the polypeptide and any precipitation reagents are normal, the seed crystal will provide a nucleation site around which a larger crystal will form.

[0055] Yet another crystallization method is an electrocrystallization method that uses dipole moments of protein macromolecules that self-assemble into a Helmholtz layer near the electrode (see, e.g., US Patent No. 5,597,557, the disclosure of which is incorporated herein by reference in its entirety) .

[0056] Some proteins may be resistant to crystallization. However, many techniques are available to those skilled in the art to induce crystallization. For example, removal of flexible polypeptide segments from the amino or carboxy terminus of a protein may facilitate the production of crystalline protein samples. Removal of such sections can be accomplished using molecular biology techniques or by treating the protein with proteases such as trypsin, chymotrypsin or subtilisin.

[0057] In diffraction experiments, a narrow and parallel x-ray beam is obtained from an x-ray source and directed to the crystal to form bent beams. Incident primary beams cause damage to both macromolecules and solvent molecules. The crystal is therefore cooled (e.g. to a temperature between -220 ° C and -50 ° C) to extend its lifetime. The primary beam must hit the crystal from many directions to create all possible diffraction spots, so the crystal is rotated in the beam during the experiment. Diffraction spots are recorded on a film or electronic detector. The exposed film must be digitized and quantified in the scanning device, while electronic detectors provide the detected signals directly to the computer. Electronic area detectors significantly reduce the time required to collect and measure diffraction data. Each bent beam, which is recorded as a spot on a film or detector plate, is determined by three properties: the amplitude, which is measured by the intensity of the spot; wavelength, which is determined by the x-ray source; and the phase that is lost in x-ray experiments. All three properties are needed for all deflected beams to determine the position of the atoms causing the deflection of the beam. One way of determining the phase is called multiple isomorphic substitution (MIR), which requires the introduction of exogenous X-ray scatters (e.g. heavy atoms, such as metal atoms) into a crystal unit cell. For a more detailed description of the MIR, see U.S. Patent No. 6093573 (column 15), the disclosure of which is incorporated herein by reference in its entirety.

[0058] Atomic coordinates refer to Cartesian coordinates (positions x, y and z) obtained from mathematical equations involving Fourier synthesis for data derived from patterns obtained by diffraction of a monochromatic X-ray beam on atoms (scattering centers) of biological biomass in crystalline form. Diffraction data is used to calculate electron density maps of repeating units in a crystal (elemental cells). Electron density maps are used to determine the position (atomic coordinates) of individual atoms in a crystal unit cell. Absolute atomic coordinate values ​​convey information about the spatial relationships between atoms, because the absolute values ​​assigned to atomic coordinates can be changed by rotary and / or progressive movement along the x, y and / or z axis, together or separately, while maintaining the same relative spatial relationship between atoms. Thus, the biological macromolecule is considered (e.g. protein) whose set of absolute atomic coordinates can be rotatably or progressively adjusted to coincide with a set of predetermined values ​​based on analysis of another sample, has the same atomic coordinates as those obtained from another sample.

[0059] Further details regarding X-ray crystallography can be obtained from simultaneously considered US Application No. 2005/0015232, US Patent No. 6093573 and International Applications No. PCT / US99 / 18441, PCT / US99 / 11913 and PCT / US00 / 03745. The disclosures of all these patent documents are incorporated herein by reference in their entirety.

[0060] NMR spectroscopy. While X-ray crystallography requires individual crystals of the macromolecule of interest, WIR measurements are carried out in solution under physiological conditions. However, the structures obtained using the WIR method are not as detailed as those obtained from crystallographic studies.

[0061] Although the use of WIR spectroscopy has until recently been limited to the resolution of 3-D structures of relatively small molecules (e.g., proteins composed of 100-150 amino acid residues), recent advances include isotope labeling of the molecule of interest and spectroscopy optimized for transverse relaxation measurements (CARE) enabled the extension of this methodology to analyze much larger molecules, e.g. proteins with a molecular weight of 110 kDa (Wider, 2000).

[0062] W1R uses radio frequency radiation to study the environment of magnetic atomic nuclei in a homogeneous magnetic field pulsed with a specific radio frequency. Impulses interfere with the nuclear magnetization of those atoms whose nuclei have a non-zero spin. Transient time domain signals are detected when the system returns to equilibrium. Fourier transformation of the transient signal into the frequency domain gives a one-dimensional WIR spectrum. The peaks in these spectra represent chemical shifts of various active nuclei. The chemical shift of an atom depends on its local electronic environment. Two-dimensional NMR experiments can provide information about the proximity of various atoms in the structure and in three-dimensional space. Protein structures can be determined by carrying out several two (and sometimes 3- or 4-) dimensional WIR experiments and using the information obtained as limitations in a set of protein folding simulations.

[0063] More information on WIR spectroscopy, including detailed descriptions of how raw data obtained from a WIR experiment can be used to determine the 3-D structure of a macromolecule, can be found in: Protein WIR Spectroscopy, Principles and Practice, (1996); Gronenborn et al. (1990); and Wider (2000), supra, disclosures of which are incorporated herein in their entirety by reference.

[0064] Of interest are also peptidomimetic compounds that are designed based on the amino acid sequences of the compounds of the invention that are peptides. Peptidomimetic compounds are synthetic compounds with a three-dimensional conformational "motif" that is essentially the same as the three-dimensional conformation of a selected peptide. The peptide motif provides the peptidomimetic compound with the ability to inhibit Μϋί1 oligomerization. Domimetic peptides may have additional properties that increase their utility in vivo, such as increased cellular permeability and a longer biological half-life. Peptidomimetics usually have a skeleton that is partially or completely non-peptidic but contains side groups that are identical to the side groups of amino acid residues found in the peptide on which the peptidomimetic is based. Several types of chemical bonds, e.g. ester, thioester, thioamide, retroamide, reduced carbonyl, dimethylene and ketomethylene bonds are known in the art as generally useful peptide bond substitutes in the construction of protease resistant peptidomimetics.

IV. therapies

A. Pharmaceutical preparations and routes of administration [0065] Where clinical applications are contemplated, it will be necessary to formulate the pharmaceutical compositions in a form suitable for their intended use. Generally, this will mean making compositions that are substantially free of pyrogens as well as other impurities that may be harmful to humans or animals.

[0066] It will usually be desirable to use appropriate salts and buffers to confer stability to delivery vectors and to allow uptake by target cells. Buffers will also be used when recombinant cells are introduced into the patient. The aqueous compositions of the present invention contain an effective amount of the cell vector, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions are also referred to as inoculum. The expression "pharmaceutically or pharmacologically acceptable" refers to molecules and compositions that do not cause adverse, allergic or other adverse reactions when administered to an animal or human. The term "pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion agents, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except when any standard medium or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Additional active ingredients may also be included in the composition.

[0067] The active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be by any common route provided that the target tissue is available for this route. Such routes of administration include oral, nasal, buccal, rectal, vaginal or local. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions will usually be administered in the form of the pharmaceutically acceptable compositions described above. Of particular interest is direct administration to the tumor, tumor perfusion or local or regional administration to the tumor, for example to the local or regional vascular system or lymphatic system, or in place of the resected tumor.

[0068] These active compounds may also be administered parenterally or intraperitoneally.

Solutions of the active compounds in the form of the free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

[0069] Pharmaceutical forms suitable for use by injection include sterile aqueous solutions or dispersions and sterile powders for preparing sterile injectable solutions or dispersions immediately prior to use. In all cases, the form must be sterile and must be liquid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be accomplished by a variety of antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be achieved by using absorption retarding agents in the compositions, e.g., aluminum monostearate and gelatin.

[0070] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount into a suitable solvent with the various other ingredients listed above, if necessary followed by sterilization by filtration. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle that contains a basic dispersion medium and other required ingredients from those mentioned above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred production methods are vacuum drying and freeze drying techniques in which the active ingredient powder is prepared with any additional desired ingredient from a solution previously sterilized by filtration.

[0071] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except when any standard medium or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Additional active ingredients may also be included in the composition.

[0072] For oral administration, the polypeptides of the present invention may be incorporated with excipients and used in the form of non-swallowed mouthwashes and dentifrices. A mouthwash can be prepared by incorporating the active ingredient in the required amount into a suitable solvent, such as sodium borate solution (Dobell's solution). Alternatively, the active ingredient may be included in an antiseptic rinse containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including gels, pastes, powders and suspensions. The active ingredient may be added in a therapeutically effective amount to the toothpaste, which may contain water, binders, abrasives, flavors, foaming agents and desiccants.

[0073] The compositions of the present invention may be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (prepared with free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic and the like . Salts formed with free carboxyl groups may also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides, and organic bases, such as isopropylamine, trimethylamine, histidine, procaine and the like.

[0074] After formulation, the solutions will be administered in a manner compatible with the dosed formulation and in a quantity that is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as injectable solutions, drug-releasing capsules, and the like. For parenteral administration, for example in aqueous solution, the solution should be buffered appropriately when necessary and the liquid diluent should first be made isotonic with a sufficient amount of saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those skilled in the art in light of the present disclosure. For example, one dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected at the proposed infusion site (see for example 'Remington's Pharmaceutical Sciences', 15th edition, pages 1035-1038 and 1570-1580) . Some changes in dosage will definitely be made depending on the condition of the patient being treated. The person responsible for administering the medicine will in each case determine the appropriate dose for the individual. In addition, for administration to humans, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biologics standards.

B. Types of Tumors and Individuals [0075] Tumor cells for which medical applications and in vitro methods of the present invention can be utilized include essentially any cell that expresses MUC1, and more specifically that which overexpresses MUC1. A suitable cancer cell may be a breast cancer cell, lung cancer, colon cancer, pancreatic cancer, kidney cancer, stomach cancer, liver cancer, bone cancer, hematological cancer (e.g. leukemia or lymphoma), nervous tissue cancer, melanoma, ovarian cancer, testicular cancer, prostate cancer, cervical cancer, vaginal cancer or bladder cancer. In addition, the medical applications of the invention can be used for a wide range of species, e.g. humans, non-human primates (e.g. apes, baboons or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs , hamsters, rats and mice.

C. Medical uses [0076] Peptides or analogs that inhibit the formation of MUC1 oligomers are generally useful in anti-cancer therapeutic or prophylactic agents. They may be administered to mammalian subjects (e.g., human patients with breast cancer), alone or in combination with other drugs and / or radiation therapy. These compounds can also be administered to individuals who are genetically and / or environmentally (due to, for example physiological and / or environmental factors) susceptible to cancer, e.g., individuals with a family history of cancer (e.g., breast cancer), subjects with chronic inflammation or chronic stress, or individuals who are exposed to natural or unnatural environmental carcinogens (e.g. excessive exposure to sunlight, industrial carcinogens or tobacco smoke).

[0077] Where the uses of the present invention are used in subjects with cancer, prior to administration of the compound, the tumor may optionally be examined for MUC1 expression (expression of MUC1 protein or MUC1 mRNA) by methods known in the art. In this way, individuals can be identified as having cancer expressing or overexpressing MUC1. Such tests can be carried out in vitro on tumor cells obtained from an individual. Alternatively, in vivo imaging techniques may be used, for example using MUC1-specific radiolabeled antibodies. In addition, body fluids (e.g. blood or urine) from individuals with cancer can be tested for elevated levels of MUC1 protein or MUC1 protein fragments.

[0078] The required dose depends on the chosen route of administration; the nature of the preparation; the nature of the patient's disease; the size, weight, surface area, age and sex of the individual; other drugs administered; and evaluation of the attending physician. Suitable doses are in the range of 0.0001 mg / kg - 100 mg / kg. Significant differences in the required dose are to be expected due to the variety of compounds available and the varying efficacy of different routes of administration. For example, oral administration would be expected to require a higher dosage than intravenous injection. Differences in these dosage levels can be adjusted using standard empirical optimization procedures as is well known in the art. Administration can be single or multiple (e.g., 2, 3, 4, 5, 6, 8, 10, 20, 50, 100, 150 or more times). Capulation of the polypeptide in a suitable delivery vehicle (e.g. polymer microparticles or implantation devices) can increase the efficiency of administration, especially when administered orally.

V. Combination Therapies [0079] Resistance of tumor cells to DNA damaging agents is a major problem in clinical oncology. One of the goals of current cancer research is to find ways to improve the effectiveness of chemo- and radiotherapy. One way is to combine such traditional therapies with gene therapy. In the context of the present invention, similar use is contemplated for MUC1 peptide therapy in combination with chemotherapeutic, radiotherapeutic or immunotherapist intervention.

[0080] To kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis, or reverse or otherwise reduce the malignant phenotype of cancer cells using medical applications, in vitro methods and compositions of the present invention, the target cell is generally exposed on MUC1 peptide and at least one other therapy. These therapies will be administered in a combined amount effective to kill or inhibit cell proliferation. This process may involve exposing cells to agents / therapies at the same time. This can be accomplished by contacting the cell with a single composition or pharmacological preparation that contains both agents, or by contacting the cell with two separate compositions or two separate preparations at the same time, one composition containing the MUC1 peptide and the other containing the agent .

[0081] Alternatively, administration of MUC1 may be before or after other therapy at intervals of several minutes to weeks. In embodiments in which the other treatment and the MUC1 peptide are administered separately to the cell, it should generally be ensured that a significant period of time does not pass between the times of each administration, so that these therapies will still be able to exert a beneficial combined effect on the cell. In such cases, exposing the cell to both factors is considered 12-12 hours apart, about 6-12 hours apart, or only about 12 hours late. In some situations, it may be desirable to significantly extend the treatment period; however, with a few days (2, 3, 4, 5, 6 or 7) up to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) a break between the respective administrations.

[0082] It is also conceivable that more than one administration of MUC1 peptide or other therapy will be desired. Various combinations can be used, wherein "A" means MUC1 peptide and "B" means second therapy, as shown below:

A / B / AB / A / BB / B / AA / A / BB / A / AA / B / BB / B / B / AB / B / A / B

A / A / B / BA / B / A / BA / B / B / AB / B / A / AB / A / B / AB / A / A / BB / B / B / A

A / A / AB B / A / A / AA / B / A / AA / A / B / AA / B / B / BB / A / B / BB / B / A / B

Other connections are also being considered. Again, to achieve cell killing, both therapies are delivered to the cell in a combined amount effective to kill the cell.

[0083] Agents or agents suitable for use in combination therapy include any chemical compound or method of treatment that causes DNA damage when applied to a cell. Such means and factors include radiation and waves that cause DNA damage, such as γ radiation, X-rays, UV radiation, microwaves, electron emissions and the like. Various chemical compounds, also described as "chemotherapeutics" or "genotoxic agents", are intended for use in the combination treatments disclosed herein. In the treatment of cancer according to the invention, the tumor cells will be contacted with the agent, outside the expression construct. This can be achieved by limited irradiation of the tumor site using radiation such as X-rays, UV light, γ rays or even microwaves. Alternatively, the tumor cells can be contacted with the agent by administering to the subject a therapeutically effective amount of the pharmaceutical composition.

[0084] Various classes of chemotherapeutic agents are contemplated for use in combination with the peptides of the present invention. For example, selective estrogen receptor antagonists ("SERMs") such as Tamoxifen, 4-hydroxy tamoxifen (Afimoxyfen), Falsodex, Raloxifene, Bazedoxifene, Klomifene, Femarelle, Lasofoxyfene, Ormeloxyfen and Toremifene.

[0085] Chemotherapeutic agents contemplated for use include, e.g., camptothecin, actinomycin-D, mitomycin C. The invention also includes the use of a combination of one or more DNA damaging agents, both based on radiation and real compounds such as the use of rays X-ray with cisplatin or the use of cisplatin with etoposide. The agent may be prepared and used as a combined therapeutic composition or kit when combined with the MUC1 peptide as described above.

[0086] Heat shock protein 90 is a regulatory protein found in many eukaryotic cells. HSP90 inhibitors have been shown to be useful in the treatment of cancer. Such inhibitors include Geldanamycin, 17- (allylamino) -17-demetoxygeldan-mycin, PU-H71 and Rifabutin.

[0087] Agents that directly crosslink DNA or form addition compounds are also contemplated. Agents such as cisplatin and other DNA alkylating agents may be used. Cisplatin has been widely used to treat cancer, with effective doses used in clinical applications of 20 mg / m² for 5 days, once every three weeks in total in three throws. Cisplatin is not absorbed orally and must therefore be delivered by intravenous, subcutaneous, tumor or intraperitoneal injection.

[0088] Agents that damage DNA also include compounds that interfere with DNA replication, mitosis, and chromosome segregation. Such chemotherapeutic compounds include Adriamycin, also known as Doxorubicin, Etoposide, Verapamil, Podophyllotoxin and the like. These compounds, widely used in clinical situations for the treatment of cancer, are administered as intravenous bolus injections in doses ranging from 25-75 mg / m² at 21-day intervals for doxorubicin up to 35-50 mg / m² for etoposide intravenously or orally in an amount equal to twice the intravenous dose. Microtubule inhibitors such as taxanes are also contemplated. These molecules are diterpenes produced by plants of the genus Taxus and include paclitaxel and docetaxel.

[0089] Epidermal growth factor receptor inhibitors, such as Iressa, mTOR, which is the target of rapamycin in mammals, also known as FK506 binding protein, 12-protein associated with rapamycin 1 (FRAP1) is a serine / threonine protein kinase that regulates cell growth. cell proliferation, cell motility, cell survival, protein synthesis and transcription. Rapamycin and its analogues ("rapalogs") are therefore contemplated for use in combination cancer therapy in accordance with the present invention.

[0090] Another possible combination therapy using the peptides claimed herein is TNF-α (tumor necrosis factor alpha), a cytokine involved in systemic inflammation, and a member of the cytokine group that stimulates the acute phase response. The main function of TNF is to regulate the cells of the immune system. TNF is also able to induce apoptotic cell death, trigger inflammation, and inhibit oncogenesis and viral replication.

[0091] Agents that interfere with the synthesis of precursors and nucleic acid subunits and their accuracy also lead to DNA damage. As such, many nucleic acid precursors have been developed. Particularly useful are agents that have undergone extensive testing and that are readily available. As such, agents such as 5-fluorouracil (5-FU) are preferentially used by tumor tissue, making this agent particularly useful in targeting cancer cells. Although it is quite toxic, 5-FU can be used in a wide variety of carriers, including topically, but intravenous administration at doses ranging from 3 to 15 mg / kg / day is commonly used.

[0092] Other factors that cause DNA damage and are widely used include those commonly known as γ-rays, x-rays, and / or targeted delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents such as microwaves and UV radiation are also contemplated. Most likely, all of these factors cause a wide range of DNA damage, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. Dosage ranges for x-rays include daily doses of 50 to 200 x-rays for extended periods (3 to 4 weeks 26 days) to single doses of 2,000 to 6,000 x-rays. Dosage ranges for radioisotopes vary greatly and depend on the isotope's half-life, strength and type of radiation emitted and its absorption by tumor cells.

[0093] A person skilled in the art may refer to "Remington's Pharmaceutical Sciences" 15th edition, chapter 33, in particular pages 624-652. Some changes in dosage will definitely be made depending on the condition of the patient being treated. The person responsible for administering the medicine will in each case determine the appropriate dose for the individual. In addition, for administration to humans, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biologics standards.

[0094] The inventors suggest that local or regional delivery of MUC1 peptides to cancer patients will be very effective for the treatment of clinical disease. Similarly, chemo- or radiotherapy can be directed to a specific affected area in the patient's body. Alternatively, regional or systemic delivery of the expression construct and / or agent may be appropriate in some cases, for example when extensive metastases are present.

[0095] In addition to combining MUC1 therapy with chemo- and radiotherapy, combination with immunotherapy, hormone therapy, toxin therapy and surgery is also contemplated. In particular, targeted therapies such as Avastin, Erbitux, Gleevec, Herceptin and Rituxan can be used.

[0096] It should also be emphasized that any of the above therapies can itself be used in the treatment of cancer.

VI. Examples [0097] The following examples are included to show specific embodiments of the invention. Those skilled in the art will recognize that the techniques disclosed in the following examples represent techniques developed by the inventor so that they work well in practicing the invention, and therefore may be considered specific methods of practicing it. However, those skilled in the art should be aware in the light of the present disclosure that many changes can be made to the specific forms that have been disclosed and still obtain the same or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1 - Materials and methods [0098] Cell culture. Human breast cancer cell lines ZR-75-1, ZR-751 / we, ZR-75-1 / MUC1siRNA (Ren et al., 2004) were cultured in RPMI1640 medium supplemented with 10% heat inactivated fetal bovine serum (HI-FBS) , 100 U / ml penicillin and 100 μg / ml streptomycin (Invitrogen) in a humidified incubator at 37 ° C and 5% CO2. MCF-7 human breast cancer cells and 293 cells were cultured in Dulbecco's modified Eagle's medium with 10% HI-FBS, antibiotics and 2 mM L-glutamine. MCF-10A human breast epithelial cells were cultured in growth medium for breast epithelial cells (MEGM; Lonza). Cells were treated with MUC1 / CQC or MUC1 / AQA peptides synthesized by MIT Biopolymer Laboratory, Cambridge, MA. Viability was determined by the exclusion method using trypan blue.

[0099] Immunoprecipitation and analysis by Immunoblotting. Whole cell and cell nuclei lysates were prepared as described (Leng et al., 2007).

Soluble proteins were immunoprecipitated using an anti-Flag antibody (Sigma, St. Louis, MO). Immunoprecipitates and soluble proteins were analyzed by immunoblotting using anti-Hi s antibodies (Cell Signaling Technology,

Danvers, MA), anti-GFP (Millipore, Danvers, MA), anti-Flag, anti-MUC1-C (Ab1;

NeoMarkers, Fremont, CA), anti-L-B (EMD, La Jolla, CA) or anti-e-actin (Sigma). Reactivity was detected using horseradish peroxidase and chemiluminescence secondary antibodies.

[0100] Cell transfection. 293 cells were transfected with vectors expressing GFP, GFP-MUC1-CD or Flag-MUC1-CD in the presence of lipofectamine as described (Leng et al., 2007).

[0101] Peptide uptake. Cells were incubated with FITC-labeled MUC1 / CQC peptide (MIT Biopolymer Laboratory), washed with cold PBS, fixed with 1% paraformaldehyde / PBS and analyzed for fluorescence by flow cytometry.

[0102] Analysis of cell cycle distribution, apoptosis and necrosis. Cells were harvested, washed with PBS, fixed with 80% ethanol and incubated in PBS containing 40 μg / ml RNAse and 40 μg / ml propidium iodide for 30 min at 37 ° C. Cell cycle distribution was assessed by flow cytometry. Sub-G1 DNA content was assessed by propidium iodide staining of ethanol-fixed and permeabilized citrate buffer cells and monitoring by flow cytometry as described (Yin et al., 2007). To assess necrosis, cells were incubated with 1 μg / ml propidium iodide / PBS for 5 minutes at room temperature and then monitored by flow cytometry as described (Yin et al., 2007).

[0103] Human breast cancer xenograft model. Female Balb-c nu / nu mice (Charles River Laboratories, Wilmington, MA) 4-6 weeks old weighing 18-22 grams, were implanted subcutaneously with 17-e-estradiol (0.72 mg; Innovative Research, Sarasota, FL ) using a trocar. After 24 hours, 1x10⁷ ZR75-1 cells embedded in Matri-Gel (BD Biosciences) were injected subcutaneously in the flank. When tumors were detectable at ~ 150 mm³ (Cohort 1) or 275 mm³ (Cohort 2), the mice were matched in pairs to the experimental and control groups. Each group contained 5 mice, each marked on the ear and followed throughout the study. The first dose was given during pairing to groups (day 1). Phosphate buffered saline (carrier), MUC1 / CQC peptide and MUC1 / AQA peptide were administered daily by intraperitoneal injection. Mice were weighed twice a week and tumor measurements were carried out using a caliper every 4 days. Tumor volume (V) was calculated using the formula V = W2 x L / 2, where W and L are smaller and larger in diameter, respectively. After killing, mice were perfused with saline followed by phosphate buffered formalin by intracardial administration. Tumors were excised, fixed by immersion for 4 h, dehydrated in a series of ethanol concentrations and processed for routine embedding in paraffin. Tumors were assessed by H&amp;E staining and anti-MUC 1 immunoperoxidase staining as described (Kufe, 1984).

[0104] Drugs and cytokines. Cisdiaminodichloroplatin (II), Doxorubicin (Adriamycin), Taxol (Paclitaxel) were purchased from Sigma (St. Louis, MO). rh-TNF-alpha was purchased from Promega (Madison, WI). Peptide GO-203 was synthesized by Anaspec Inc.

[0105] In vitro cytotoxicity tests and combinations. Cells were plated in a 96-well flat-bottom microtiter plate (Fi sher) at 1000 cells per well for 6-day experiments or 3000 cells per well for 3-day experiment. Cells were then cultured for 24 h. Antineoplastic agents and GO-203 were diluted to the concentrations indicated and added to the cells. GO-203 (5 μmol / L) was added every 24 h for 72 h. Cell viability was determined by adding MTS reagent to the cells and reading absorbance at 490 nm on a microplate reader.

[0106] Data analysis. IC50 values ​​for all anti-cancer drugs were determined by non-linear regression analysis using Graphpad Prism (GraphPad Software, San Diego CA). The CombiTool computer program (version 2.001, IMB Jena Biocomputing Group) was used to calculate the association rate for the dose range in the presence of 5 μmol / l GO-203.

EXAMPLE 2 - Results [0107] Effect of MUC1 / CQC peptide on MUC1 oligomer formation. The MUC1 cytoplasmic domain (MUC1-CD) contains the CQC motif, which is necessary for oligomer formation and nuclear localization (Leng et al., 2007). To determine if a small molecule could be designed to block oligomerization, the inventors synthesized a peptide derived from the N-terminal MUC1-CD region that contains the CQC motif (MUC1 / CQC peptide; FIG. 1A). The poly D-arginine transduction domain was included in the synthesis to facilitate entry of the peptide into cells (Fischer, 2007) (FIG. 1A). As a control, a similar peptide in which the CQC motif was changed to AQA (MUC1 / AQA peptide; FIG. 1A) was synthesized. To assess the binding properties of MUC1-CD peptide, the inventors immobilized tagged His-MUC1-CD on a chip with a BIAcore sensor. The MUC1 / CQC peptide bound to His-MUC1-CD with a dissociation constant (Kd) of 30 nM (FIG. 1B), which is similar to the results obtained for MUC1-CD oligomers (Leng et al., 2007). In contrast, no apparent MUC1 / AQA peptide binding occurred (data not shown). Purified tagged His MUC1-CD forms oligomers, detected by polyacrylamide gel electrophoresis (FIG. 1C). Incubation of His-MUC1CD with MUC1 / CQC peptide significantly reduced oligomer formation and caused monomer growth (FIG. 1C). In addition, incubation with MUC1 / AQA peptide had little or no effect (FIG. 1C). To assess the effect on MUC1 oligomerization in vivo, 293 cells were transfected with GFP-MUC1-CD and Flag-MUC1-CD expressing vectors (FIG. 1D, left). GFP-MUC1-CD and Flag-MUC1-CD complexes were detectable by co-precipitation of lysates from cells not exposed to peptide (FIG. 1D, right). Consistent with the in vitro results, incubation of transfected 293 cells with MUC1 / CQC peptide was associated with impaired interaction between Flag-MUC1-CD and GFP-MUC1-CD (FIG. 1D, right). In addition, the MUC1 / AQA peptide had no visible effect (FIG. 1D, right). These results indicate that the MUC1 / CQC peptide binds to MUC1-CD and blocks the formation of MUC1-CD oligomers in vitro and in cells.

[0108] MUC1 / CQC peptide blocks MUC1-C targeting to the nucleus. ZR-75-1 and MCF-7 human breast cancer cells overexpress endogenous MUC1 and are therefore potential models for assessing the effect of MUC1 / CQC peptide (Ramasamy et al., 2007). To assess uptake, ZR-75-1 cells were incubated with 5 μM FITC-MUC1 / CQC peptide (FIG. 2A). After 2 h, flow cytometry analysis of the cells showed a significant increase in fluorescence intensity with an average (MFI) of 145 (FIG. 2A). Further increase in MFI was found after 6 and 24 h (FIG. 2A). MUC1-C oligomerization is necessary for its import into the nucleus (Leng et al., 2007). Treatment of ZR-75-1 cells with MUC1 / CQC or MUC1 / AQA peptide had no effect on MUC1-C levels in the cell (FIG. 2B). However, like the effect on oligomerization, treatment with MUC1 / CQC peptide, but not MUC1 / AQA, was associated with a decrease in MUC1-C levels in the nucleus (FIG. 2B). A similar effect was observed in MCF-7 cells with reduced levels of MUC1-C in the nucleus in response to treatment with MUC1 / CQC peptide (FIG. 2C). These results indicate that the peptide

MUC1 / CQC blocks MUC1-C oligomerization and thus nuclear targeting of MUC1-C.

[0109] MUC1 / CQC peptide blocks growth and causes necrosis. To determine if the MUC1 / CQC peptide was affecting growth, ZR-75-1 cells were treated with 5 μM MUC1 / CQC for 72 hours and monitored for cell cycle distribution. Importantly, there was significant S-phase arrest compared to cells that were untreated or treated with MUC1 / AQA peptide (FIG. 3A). After 96 h, the population of S-phase cells decreased, probably due to destruction due to cell death (FIG. 3A). No or small accumulation of cells with sub-G1 DNA content occurred to confirm the induction of apoptosis (FIG. 3A). However, treatment of ZR-75-1 cells with MUC1 / CQC peptide, but not MUC1 / AQA, was associated with necrosis induction, which was detectable after 72 h and more pronounced after 96 h (FIG. 3B). MCF-7 cells responded similarly to the MUC1 / CQC peptide with S-phase arrest (FIG. 3C) and necrosis (FIG. 3D). These results indicate that the MUC1 / CQC peptide inhibits growth and necrosis of human breast cancer cells.

[0110] MUC1 / CQC peptide specificity for MUC1-expressing tumor cells. To determine whether the MUC1 / CQC peptide has selective activity against breast cancer cells that overexpress endogenous MUC1, the inventors treated ZR-75-1 cells in which MUC1 expression is permanently silenced using MUC1siRNA (FIG. 4A). In contrast to arresting the growth and death of ZR-75-1 / vector control cells, the MUC1 / CQC peptide had significantly less effect on ZR-75-1 / MUC1siRNA cells (FIG. 4B). Furthermore, MUC1 / CQC peptide had no apparent effect on the growth of MUC1-293 negative cells (FIG. 4C). Studies have also been carried out using non-transformed MCF-10A breast epithelial cell lines (Muthuswamy, 2001; Soule, 1990), which show MUC1 expression, but at a lower level than ZR-75-1 and MCF-7 cells (Ahmad et al., 2007 ). It is worth noting that, unlike ZR-75-1 and MCF-7 cells, the MUC1 / CQC peptide had no effect on cell cycle distribution (FIG. 4D) and growth (FIG. 4E) of MCF-10A. These results indicate that the MUC1 / CQC peptide has selective activity against breast cancer cells that overexpress endogenous MUC1.

[0111] MUC1 / CQC peptide inhibits oncogenicity in vivo. To determine whether MUC1 / CQC peptide administration was associated with body weight effects, five female nude (nu / nu) mice were injected intraperitoneally (IP) with a daily dose of 50 mg / kg. Peptide administration for 11 days had no apparent effect on the body weight of individual mice. In addition, there was no further effect on body weight within the next 28 days after MUC1 / CQC suspension (data not shown). To assess the anti-tumor effect, ZR-75-1 cells (1x10⁷) implanted subcutaneously in the flank of nude mice. After 12 days, mice with tumors about 150 mm in size³ MUC1 / CQC peptide was administered at doses of 10 and 50 mg / kg / day. As controls, mice were dosed with vehicle alone or MUC1 / AQA peptide. Administration of MUC1 / CQC peptide at 10 mg / kg / day x 21 days slowed down growth compared to that obtained with MUC1 / AQA peptide administered at 50 mg / kg / day (FIG. 5A). In addition, administration of MUC1 / CQC peptide at a dose of 50 mg / kg / day blocked growth for the first 7 days of treatment (FIG. 5A). As a result, treatment was discontinued and the mice were monitored for re-growth. Importantly, there was no detectable tumor growth over the next 17 days (FIG. 5A). To partially assess the underlying cause of this effect, tumors collected from control and experimental mice were subjected to histopathological examination. Tumors from MUC1 / CQC-treated mice (10 and mg / kg) showed a significant degree of necrosis compared to those from vehicle or MUC1 / AQA peptide-treated mice (FIG. 5B and data not shown). It is worth emphasizing, however, that tumor cells were also detectable around necrosis areas (FIG. 5B). Tumor sections were also stained with MUC1 antibody. MUC1 / CQC peptide treatment was associated with a significant reduction in MUC1 expression compared to control tumors and those treated with MUC1 / AQA peptide (FIG. 5C and data not shown).

[0112] Studies were also carried out on larger tumors (~ 275 mm³) (FIG. 6A). Administration of MUC1 / CQC peptide at an average dose of 30 mg / kg / day x 21 days was associated with tumor growth arrest (FIG. 6A). In addition, no re-growth was observed for the next 31 days after treatment (FIG. 6A), which further indicates that MUC1 / CQC peptide is effective in stopping tumor growth. Tumors harvested on day 52 showed extensive necrosis (FIG. 6B) and a decrease in MUC1 expression. These results indicate that the MUC1 / CQC peptide reduces MUC1 expression and is associated with necrosis induction and prolonged tumor growth arrest.

[0113] Clipped MUC1-C-terminal CQC peptides. Intracellular protein-protein interactions that drive many biological pathways often occur through α-helix protein structures. Helical peptides can also interfere with or stabilize protein-protein interactions. Native helical peptides have significant disadvantages as therapeutic agents because of their low potency, instability and inefficient delivery into cells. Recent studies have shown that these problems can be overcome by chemical modifications of α-helical peptides called hydrocarbon clamping.

[0114] The inventors of the invention used the C-terminal endogenous MUC1 peptide sequence (AIVYLIALAVCQCRRKNYG) and produced two α-helical peptides, GO200-1B and GO-200-2B using the hydrocarbon clamp method:

GO-200-1B: Ac-AIVYL-S5-ALA-S5-CQCRRKNYG-NH2

GO-200-2B: Ac-AKKYL-S5-ALA-B5-CQC-S5-RKNY-NH2

To determine if GO-200-1B exposure affects the growth of non-small cell lung cancer cells, H-1650 cells were treated with 1 and 5 μΜ GO-200-1B for 7 days and their growth monitored. The results show that treatment with 5 μΜ GO200-1 B cells was associated with significant growth inhibition (FIG. 9A). In addition, another non-small cell lung cancer cell line, H-1975, was treated with 5 μΜ GO-2002B for 3 days and cell growth as well as cell death were monitored. The results show that treatment of H-1975 GO-200-2B cells for 3 days was associated with over 80% inhibition of cell proliferation. In addition, GO-200-2B was also associated with significant cell death induction (FIG. 9B). These results indicate that clamped MUC1-C peptides are effective in inducing growth arrest and death of human MUCl positive tumor cells.

[0115] GO-203 analogues. Recent studies of the inventors have shown that the MUC1 C-terminal peptide (CQCRRKNYGQLDIFP) acts by inhibiting the growth of many tumor cell lines. They also showed that the shorter MUC1 C-terminal peptide, CQCRRKN, also works to kill cancer cells. However, these MUC1 C-terminal peptides are composed of L-amino acids. Importantly, L-amino acid peptides are susceptible to degradation by proteolytic enzymes, while those containing D-amino acids have been shown to be more stable. Accordingly, they generated the right-handed form of the above-described shorter C-terminal MUC1 peptide, in which the L-amino acids were replaced with D-amino acids (GO-203). In addition, to determine the minimum number of amino acid residues from the MUC1 C-terminal region required to preserve cell killing activity, they also produced many different versions of GO-203, as described in FIG. 8.

[0116] Many tumor cell lines (hormone dependent breast cancer ZR75-1; triple negative breast cancer MDA-MB-231; A549 non-small cell lung cancer; H-1975 non-small cell lung cancer) were cultured in ΚΓΜΙ-1640 supplemented with 10% heat inactivated fetal bovine serum, 100 units / ml penicillin and 100 μg / ml streptomycin and 2 mmol / l L-glutamine. Cells were treated separately with 5 μΜ of different GO-203 analogues (FIG. 8) for 3 to 7 days and viability determined using Trypan Blue. The proliferation of various cell lines was compared to cells treated with vehicle alone. The results show that the treatment of many 5 μΜ tumor cell lines of various GO-203 analogues was associated with significant growth inhibition (FIGS. 10-14).

[0117] Combination therapy. The association index (CI) values ​​generated by CombiTool were plotted as a function of the fraction with the effect of the combination of drugs. CI values ​​were close to 1 for Cisplatin and GO-203, indicating an additive interaction. The values ​​of CI obtained for Doxorubicin (25, 50, 100, 200 πΜ), Taxol (25, 50, 100 πΜ) and TNF (10, 20, 40 ng / ml) from GO-203 were less than 1, which confirms the strong effect additive or synergism (FIGS. 15A-D and 16).

Example 3 - Discussion [0118] MUC1 / CQC peptide blocks MUC1 oligomerization. Overexpression of ΜυΟ is sufficient to elicit anchorage independent of growth and oncogenicity (Li et al., 2003a; Huang et al., 2003; Huang et al., 2005). Importantly, the transforming activity of Μϋί1 is abolished by mutation of the CQC motif in the cytoplasmic domain on AQA (Leng et al., 2007). ΜυΟ forms oligomers, and the CQC motif is necessary for its oligomerization (Leng et al., 2007). In addition, oligomer formation is necessary to direct the ΜυΟ-C subunit to the nucleus (Leng et al., 2007). Other activities of the ΜυΟ-C subunit, such as activation of the Wnt / e-catenin and II &lt;I &lt;e&gt; \ I pathways^;-i &lt;B, also depend on the formation of ΜϋΟ-C oligomers (unpublished data). On the basis of these discoveries, the inventors have concluded that the disruption of oligomerization of ΜϋΠ by a small molecule can potentially block the transforming action of ΜϋΠ. In this context, they synthesized a ΜυΟ-derived peptide that contains the CQC motif and the poly-Arg cell targeting domain for entry into the cells. Preliminary studies with this MUC1 / CQC peptide have shown that it inhibits ig-CD oligomerization in vitro. As shown earlier in the BIAcore analysis, ΜυΟ-CD creates dimers with a dissociation constant (Kd) of 33 πΜ (Leng et al., 2007). MUC1 / CQC peptide similarly bound to ΜυΟ-CD with a Kd of 30 πΜ. In addition, demonstrating that the MUC1 / AQA peptide has little or no effect on Μϋί1 oligomerization has provided support for CQC motif dependence. MUC1 / CQC, but not MUC1 / AQA, was also effective in blocking ΜυΟ-CD oligomerization in cells. These results therefore indicated that the MUC1 / CQC peptide could be used to disrupt igί1 oligomerization and thus potentially działaniaυΟ activity in human breast cancer cells.

[0119] Selectivity of MUC1 / CQC peptide for tumor cells overexpressing MUC1. Cell targeting domains, such as poly-DArg, and their conjugates enter the cells, at least in large part, by endocytosis and then must reach their intended destination (Fischer, 2007). The penetration of MUC1 / CQC peptide into ZR-75-1 breast cancer cells was detectable immediately and persisted for at least 24 h. Importantly, and in line with the fact that nuclear targeting of ΜυΟ is dependent on oligomerization (Leng et al., 2007), MUC1 / CQC peptide uptake was associated with a reduction in ΜυΟ-C levels in the nucleus. Similar results were obtained using MCF-7 breast cancer cells, indicating that this MUC1 / CQC peptide response is not cell specific. Furthermore, which is noteworthy, the exposure of these cells to MUC1 / CQC, but not MUC1 / AQA, was associated with growth arrest and necrosis. It is important whether MUC1 / CQC causes death in a mechanism dependent on the expression of its intended purpose, or whether it is non-specific cytotoxin. In this context, MUC1 silencing in ZR-75-1 cells abolished the cytotoxic effect of MUC1 / CQC. In contrast, the exposure of non-cancer mammalian MCF-10A breast epithelial cells to the MUC1 / CQC peptide had little effect. These results indicate that MUC1 / CQC peptide sensitivity is dependent on MUC1 overexpression and MUC1 activity associated with the malignant phenotype. It turns out, therefore, that the MUC1 / CQC peptide has a dominant negative effect that is selective for cancer cells that overexpress MUC1.

[0120] Antitumor effect of MUC1 / CQC peptide. Cell targeting domains are used to deliver therapeutic charges (Fischer, 2007). However, as for each of these factors, the overarching question is whether the MUC1 / CQC peptide can be delivered in vivo with an effective therapeutic index, i.e., anti-tumor activity, and acceptable toxicity profile. Responding to this issue, the inventors found that administration of MUC1 / CQC peptide at 10 and 30 mg / kg / day for 21 days was well tolerated with no apparent acute toxicity. They also found that treatment with these doses was effective in inhibiting tumor growth. These results were opposed to administration of MUC1 / AQA peptide at a dose of 50 mg / kg / day for 21 days, which showed no anti-tumor activity. Furthermore, which was somewhat surprising, there was no evidence of tumor re-growth after administration of a 30 mg / kg / day dose for 21 days. Administration of MUC1 / CQC peptide at 50 mg / kg / day for 7 days also showed that tumor growth remained stopped for a long time after treatment. These results can be explained, at least in part, by the finding that treatment with MUC1 / CQC peptide is associated with induction of tumor necrosis.

[0121] The in vitro effect of MUC1 / CQC 7-mer is as strong as MUC1 / CQC 15-mer. Based on these results, it can be predicted that the 7-mer MUC1 / CQC will also be active as an antitumor agent in in vivo tumor models.

[0122] All compositions and / or methods disclosed and claimed herein may be prepared and carried out without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes can be made to these compositions and / or methods, and in the steps and sequences of the steps of the method described herein.

VII. References [0123] The following references, to the extent that they provide details of exemplary procedures or other supplementary to those given in the description, are specifically incorporated herein by reference:

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List of sequences / 177109 &lt;151&gt; 2009-05-11 &lt;150&gt; 61/106380 &lt;151&gt; 2008-10-17 &lt;160&gt; 62 &lt;170&gt; PatentIn version 3.5 &lt;210&gt; 1 &lt;211&gt; 72 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 1

Cys Gin Cys Arg Arg Lys Asn Tyr Gly Gin Leu Asp Ile Phe Pro Ala 15 10 15

Arg Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His 20 25 30

Gly Arg Tyr Val Pro Pr o Ser Cheese Thr Asp Arg Ser Pro Tyr Glu Lys 35 40 45

Val Ser Ala Gly Asn Gly Gly Cheese Cheese Leu Cheese Tyr Thr Asn Pro Ala 50 55 60

Val Ala Ala Thr Cheese Ala Asn Leu

70 &lt;210&gt; 2 &lt;211&gt; 159 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 2

Gly Ser Val Val Val Gin Leu Thr Leu Ala Phe Arg Glu Gly Thr Ile 15 10 15

Asn Val His Asp Val Glu Thr Gin Phe Asn Gin Tyr Lys Thr Glu Ala 20 25 30

Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Val Ser Asp Val 35 40 45

Pro Phe Pro Phe Cheese Ala Gin Cheese Gly Ala Gly Val Pro Gly Trp Gly 50 55 60

Ile Ala Leu Leu Val Leu Val Cys Val Leu Val Ala Leu Ala Ile Val 65 70 75 80

Tyr Leu Ile Ala Leu Ala Val Cys Gin Cys Arg Arg Lys Asn Tyr Gly 85 90 95

Gin Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr His Pro Met Ser Glu 100 105 110

Tyr Pro Thr Tyr His Thr His Gly Arg Tyr Val Pro Pro Ser Ser Thr 115 120 125

Asp Arg Ser Pro Tyr Glu Lys Val Cheese Ala Gly Asn Gly Gly Cheese 130 135 140

Leu Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr Ser Ala Asn Leu 145 150 155 &lt;210&gt; 3 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 3

Cys Gin Cys Arg Arg Lys Asn Tyr Gly Gin Leu Asp Ile Phe Pro 15 10 15 &lt;210&gt; 4 &lt;211&gt; 6 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 4

Cys Gin Cys Arg Arg Lys 1 5 &lt;210&gt; 5 15 &lt;211&gt; 34 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 5

Gin Ala Ala Thr Ala Thr Arg Gly Arg Ser Ala Ala Ser Arg Pro Thr 15 10 15

Glu Arg Pro Arg Ala Pro Ala Arg Cheese Ala Ser Arg Pro Arg Arg Pro 20 25 30

Val Glu &lt;210&gt; 6 &lt;211&gt; 16 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 6

Arg Gin Ile Lys Ile Trp Phe Gin Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 &lt;210&gt; 7 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 7

Arg Arg Met Lys Trp Lys Lys 1 5 &lt;210&gt; 8 &lt;211&gt; 16 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 8

Arg Arg Trp Arg Arg Trp Trp Arg Arg Trp Trp Arg Arg Trp Arg Arg 15 10 15 &lt;210&gt; 9 &lt;211&gt; 18 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 9

Arg Gly Gly Arg Leu Ser Tyr Ser Arg Arg Arg Phe Ser Thr Ser Thr 15 10 15

Gly Arg &lt;210&gt; 10 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 10

Tyr Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg 1 5 10 &lt;210&gt; 11 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 11

Arg Lys Lys Arg Arg Gin Arg Arg Arg 1 5 &lt;210&gt; 12 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 12

Tyr Ala Arg Ala Ala Ala Arg Gin Ala Arg Ala 15 10 &lt;210&gt; 13 &lt;211&gt; 8 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 13

Arg Arg Arg Arg Arg Arg Arg Arg 1 5 &lt;210&gt; 14 &lt;211&gt; 8 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 14

Lys Lys Lys Lys Lys Lys Lys Lys 1 5 &lt;210&gt; 15 &lt;211&gt; 27 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (25) .. (25) &lt;223&gt; Xaa can be any naturally occurring amino acid &lt;400&gt; 15

Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu 15 10 15

Lys Ala Leu Ala Ala Leu Ala Lys Xaa Ile Leu 20 25 &lt;210&gt; 16 &lt;211&gt; 18 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 16

Leu Leu Ile Leu Leu Arg Arg Arg Ile Arg Lys Gin Ala Asn Ala His 15 10 15

Lys &lt;210&gt; 17 &lt;211&gt; 16 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 17

Ser Arg Arg His His Cys Arg Ser Lys Ala Lys Arg Ser Arg His His 15 10 15 &lt;210&gt; 18 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 18

Asn Arg Ala Arg Arg Asn Arg Arg Arg Val Arg 1 5 10 &lt;210&gt; 19 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 19

Arg Gin Leu Arg Ile Ala Gly Arg Arg Leu Arg Gly Arg Ser Arg

5 10 &lt;210&gt; 20 &lt;211&gt; 13 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 20

Lys Leu Ile Lys Gly Arg Thr Pro Ile Lys Phe Gly Lys 15 10 &lt;210&gt; 21 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 21

Arg Arg Ile Pro Asn Arg Arg Pro Arg Arg 1 5 10 &lt;210&gt; 22 &lt;211&gt; 18 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 22

<img file="PL2352508T3_D0001.tif" />

&lt;210&gt; 23 &lt;211&gt; 14 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 23

Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys 15 10 &lt;210&gt; 24 &lt;211&gt; 27 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 24

Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Asn Gly 15 10 15

Ala Trp Ser Gin Pro Lys Lys Lys Arg Lys Val 20 25 &lt;210&gt; 25 &lt;211&gt; 21 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 25

Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gin Pro Lys 15 10 15

Lys Lys Arg Lys Val 20 &lt;210&gt; 26 &lt;211&gt; 23 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 26

Gly Ala Leu Phe Leu Gly Trp Leu Gly Ala Ala Gly Ser Thr Met Gly 15 10 15

Ala Lys Lys Lys Arg Lys Val 20 &lt;210&gt; 27 &lt;211&gt; 23 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 27

Met Gly Leu Gly Leu His Leu Leu Val Leu Ala Ala Ala Leu Gin Gly 15 10 15

Ala Lys Ser Lys Arg Lys Val 20 &lt;210&gt; 28 &lt;211&gt; 26 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 28

Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro 15 10 15

Ala Ala Ala Asn Tyr Lys Lys Pro Lys Leu 20 25 &lt;210&gt; 29 &lt;211&gt; 28 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 29

Met Ala Asn Leu Gly Tyr Trp Leu Leu Ala Leu Phe Val Thr Met Trp 15 10 15

Thr Asp Val Gly Leu Cys Lys Lys Arg Pro Lys Pro 20 25 &lt;210&gt; 30 &lt;211&gt; 24 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 30

Leu Gly Thr Tyr Thr Gin Asp Phe Asn Lys Phe His Thr Phe Pro Gin 15 10 15

Thr Ala Ile Gly Val Gly Ala Pro &lt;210&gt; 31 &lt;211&gt; 26 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (24) .. (24) &lt;223&gt; Xaa can be any naturally occurring amino acid &lt;400&gt; 31

Asp Pro Lys Gly Asp Pro Lys Gly Val Thr Val Thr Val Thr Val Thr 15 10 15

Val Thr Gly Lys Gly Asp Pro Xaa Pro Asp 20 25 &lt;210&gt; 32 &lt;211&gt; 14 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 32

Pro Pro Pro Pro Pro Pro Pro Pro. Pro Pro Pro Pro Pro Pro 15 10 &lt;210&gt; 33 &lt;211&gt; 18 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 33

Val Arg Leu Pro Pro Pro Val Arg Leu Pro Pro Pro Val Arg Leu Pro 15 10 15

Pro Pro &lt;210&gt; 34 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 34

Pro Arg Pro Leu Pro Pro Pro Arg Pro Gly 1 5 10 &lt;210&gt; 35 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 35

Cheese Val Arg Arg Arg Pro Arg Pro Pro Tyr Leu Pro Arg Pro Arg Pro 15 10 15

Pro Pro Phe Phe Pro Pro Arg Leu Pro Pro Arg Ile Pro Pro 20 25 30 &lt;210&gt; 36 &lt;211&gt; 21 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 36

Thr Arg Ser Ser Arg Ala Gly Leu Gin Phe Pro Val Gly Arg Val His 15 10 15

Arg Leu Leu Arg Lys 20 &lt;210&gt; 37 &lt;211&gt; 23 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide 5 &lt;400&gt; 37

Gly Ile Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Phe 15 10 15

Val Gly Glu Ile Met Asn Ser 20 &lt;210&gt; 38 &lt;211&gt; 37 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 38

Lys Trp Lys Leu Phe Lys Lys Ile Glu Lys Val Gly Gin Asn Ile Arg 15 10 15

Asp Gly Ile Ile Lys Ala Gly Pro Ala Val Ala Val Val Gly Gin Ala 20 25 30

Thr Gin Ile Ala Lys 35 &lt;210&gt; 39 15 &lt;211&gt; 28 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide 20 &lt;400&gt; 39

Ala Leu Trp Met Thr Leu Leu Lys Lys Val Leu Lys Ala Ala Ala Lys 15 10 15

Ala Ala Leu Asn Ala Val Leu Val Gly Ala Asn Ala 20 25 &lt;210&gt; 40 &lt;211&gt; 26 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 40

Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu 15 10 15

Ile Ser Trp Ile Lys Arg Lys Arg Gin Gin 20 25 &lt;210&gt; 41 &lt;211&gt; 14 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 41

How much Asn Leu Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 1 5 10 &lt;210&gt; 42 &lt;211&gt; 33 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 42

Gly Phe Phe Ala Leu Ile Pro Lys Ile Ile Cheese Ser Pro Leu Pro Lys 15 10 15

Thr Leu Leu Cheese Ala Val Gly Cheese Ala Leu Gly Gly Cheese Gly Gly Gin 20 25 30

Glu &lt;210&gt; 43 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 43

Leu Ala Lys Trp Ala Leu Lys Gin Gly Phe Ala Lys Leu Lys Ser 15 10 15 &lt;210&gt; 44 &lt;211&gt; 27 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (23) .. (23) &lt;223&gt; Xaa can be any naturally occurring amino acid &lt;400&gt; 44

Cheese Met Ala Gin Asp Ile Ile Cheese Thr Ile Gly Asp Leu Val Lys Trp 15 10 15 /

How much How much Gin Thr Val Asn Xaa Phe Thr Lys Lys 20 25 &lt;210&gt; 45 &lt;211&gt; 41 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 45

<td colspan="4">Leu Leu Gly Asp 1</td><td>phe 5</td><td colspan="3">Phe Arg Lys</td><td>Cheese</td><td>lys 10</td><td>Glu</td><td>lys</td><td>How much</td><td>Gly</td><td>lys 15</td><td>Glu</td>

<td>phe</td><td>lys</td><td>Arg</td><td>How much</td><td>val</td><td>Gin</td><td>Arg</td><td>How much</td><td>lys</td><td>Gin</td><td>Arg</td><td>How much</td><td>lys</td><td>Asp</td><td>phe</td><td>Leu</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>ala</td><td>own</td><td>Leu</td><td>val</td><td>Pro</td><td>Arg</td><td>Thr</td><td>Glu</td><td>Cheese</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

40 &lt;210&gt; 46 &lt;211&gt; 20 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 46

Leu Lys Lys Leu Leu Lys Lys Leu Leu Lys Lys Leu Leu Lys Lys Leu 15 10 15

Leu Lys Lys Leu 20 &lt;210&gt; 47 &lt;211&gt; 18 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 47

Lys Leu Lys Leu Lys Leu Lys Leu Lys Leu Lys Leu Lys Leu Lys Leu 15 10 15

Lys Leu &lt;210&gt; 48 &lt;211&gt; 18 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 48

Pro Ala Trp Arg Lys Ala Phe Arg Trp Ala Trp Arg Met Leu Lys Lys 15 10 15

Ala Ala &lt;210&gt; 49 &lt;211&gt; 6 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 49

Lys Arg Arg Cys Gin Cys 1 5 &lt;210&gt; 50 &lt;211&gt; 5 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 50

Cys Gin Cys Arg Arg 1 5 &lt;210&gt; 51 &lt;211&gt; 6 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 51

Cys Gin Cys Arg Arg Arg 1 5 &lt;210&gt; 52 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 52

Cys Gin Cys Arg Arg Arg Arg 1 5 &lt;210&gt; 53 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 53

Cys Gin Cys Arg Arg Lys Asn 1 5 &lt;210&gt; 54 &lt;211&gt; 4 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 54

Cys Gin Cys Arg 1 &lt;210&gt; 55 &lt;211&gt; 19 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 55

Ala Ile Val Tyr Leu Ile Ala Leu Ala Val Cys Gin Cys Arg Arg Lys 1 5 10 15

Asn Tyr Gly &lt;210&gt; 56 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 56

Ala Ile Val Tyr Leu Ala Leu Ala Cys Gin Cys Arg Arg Lys Asn Tyr 15 10 15

Gly &lt;210&gt; 57 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 57

Ala Lys Lys Tyr Leu Ala Leu Ala Cys Gin Cys Arg Lys Asn Tyr 15 10 15 &lt;210&gt; 58 &lt;211&gt; 8 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 58

Cys Gin Cys Arg Arg Lys Asn Arg 1 5 &lt;210&gt; 59 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 59

Asn Lys Arg Arg Cys Gin Cys 1 5 &lt;210&gt; 60 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 60

Ala Gin Ala Arg Arg Lys Asn Tyr Gly Gin Leu Asp Ile Phe Pro 15 10 15 &lt;210&gt; 61 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 61

Ala Gin Ala Arg Arg Lys Asn 1 5 &lt;210&gt; 62 &lt;211&gt; 69 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 62

<td>Cys</td><td>Gin</td><td>Cys</td><td>Arg</td><td>Arg</td><td>lys</td><td>own</td><td>Tyr</td><td>Gly</td><td>Gin</td><td>Leu</td><td>Asp</td><td>How much</td><td>phe</td><td>Pro</td><td>ala</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Arg</td><td>Asp</td><td>Thr</td><td>Tyr</td><td>His</td><td>Pro</td><td>Underworld</td><td>Cheese</td><td>Glu</td><td>Tyr</td><td>Pro</td><td>Thr</td><td>Tyr</td><td>His</td><td>Thr</td><td>His</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Gly</td><td>Arg</td><td>Tyr</td><td>val</td><td>Pro</td><td>Pro</td><td>Cheese</td><td>Cheese</td><td>Thr</td><td>Asp</td><td>Arg</td><td>Cheese</td><td>Pro</td><td>Tyr</td><td>Glu</td><td>lys</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>val</td><td>Cheese</td><td>ala</td><td>Gly</td><td>own</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Tyr</td><td>Thr</td><td>own</td><td>Pro</td><td>ala</td><td>val</td>

55 60

Ala Ala Ala Cheese Leu 65