CN101287501A - Nod1 as an anti-tumor agent - Google Patents

Abstract

The present invention provides compositions and methods for treating tumors that involve increasing the expression of Nod1 and/or the activity of NOD1.

Description

As antitumor agent NOD1

The application requires the interim series number NO.60/656 of the U.S. of submission on February 25th, 2005, the interim series number No.60/752 of the U.S. of 175,2005 on Decembers submission in 22,, and the interests of 794 submission day, its content is incorporated in this by reference.

Government fund

The appropriation that invention described here is authorized in the National Institutes of Health is numbered under the AI15136 and is made by the support of U.S. government.U.S. government has some right in the present invention.

Invention field

The present invention relates to Nod1 and its function in apoptosis that transform, malignant cell.

Background of invention

Cancer is the disease that torments many people, and is main causes of death among human and the non-human animal.Cancer generally comprises the uncontrolled separation of a few cell, and it produces many new cells then.Thereby many cancer therapy drugs are the reagent that suppresses or stop the cell growth.Though this chemotherapeutant has improved the patient's who suffers from neoplastic disease survival rate, the serious side effects relevant with many chemotherapeutants limited their use, and destroyed the patient's who is weakened by cancer health status.Thereby needing new reagent, it represents the enhanced selectivity to cancerous cell, and can control the propagation of oncocyte.

A main difficult problem of many anticarcinogen is their specificitys.Cancer therapy drug need be distinguished carcinous cell and non-carcinous cell.Yet a large amount of in this cancer therapy drugs is indiscriminate.Usually, antitumor and anticancer agent (for example has negative hematology's effect, stop the mitosis and the disassociation of the element that is shaped in bone marrow and the lymphoid tissue) and immunosuppressive action is (for example, reduce cell counting), also can be (for example to epithelial tissue, intestinal mucosa), germinal tissue (for example, spermatogenetic damage) and nervous system seriously influence.Referring to, for example, P.Calabresi and B.A.Chabner, In:Goodman and Gilman, ThePharmacological Basis of Therapeutics (Pergamon Press, 8th Edition) is (pp.1209-1216).

What need is antitumor and anticancer agent, and it can treat selected tumor type valuably, or preferred various tumor type, and is particularly suitable for the invasive tumor.In addition, though this antitumor and anticancer agent should be that effectively they also should seldom represent or not represent toxicity.

Summary of the invention

The invention provides be used for promoting apoptotic compositions and method, it relates to and improves Nod1 and express or the NOD1 activity.

Thereby one aspect of the present invention is the method that promotes tumour regression in the mammal, and it relates to described administration raising Nod1 expresses or the active reagent of NOD1.Can comprise brain, bladder, cervix uteri, colon, gallbladder, kidney, liver, lung, pancreas, ovary, prostate, skin, stomach or thyroid tumor with the example of the tumor of method of the present invention treatment.In some embodiments, described tumor is estrogen-sensitive tumor or breast tumor.

The example that improves the active reagent of NOD1 comprises the peptide with following sequence: D-Ala-L-Glu-meso diaminopimelic acid (γ TriDAP), γ-D-glutamyl-meso-meso diaminopimelic acid (iE-DAP), γ-D-Gln-DAP (iQ-DAP), D-Ala-L-Glu-meso diaminopimelic acid (γ TriDAP) and its combination.These peptides can activate NOD1 albumen, and the Nod1 dependent pathway causes apoptosis thus.Another example that can improve the active reagent of NOD1 is the NOD1 polypeptide.In some embodiments, described NOD1 polypeptide can be human NOD1 polypeptide, for example, has the human NOD1 polypeptide of SEQ ID NO:1 or SEQ ID NO:3.

An example that can improve the reagent of Nod1 expression is the segmental nucleic acid that comprises coding NOD1 polypeptide.The example of the sequence of NOD1 polypeptide comprises SEQ ID NO:1 or SEQ IDNO:3.An example of the nucleic acid fragment of coding NOD1 polypeptide comprises SEQ ID NO:2.Described nucleic acid may further include regulating element, for example, and promoter, enhancer, transcription stop signals, or its combination.Described nucleic acid can be the part of expression cassette or expression vector or gene delivery vector.

Can express or co-administered other active component of the active reagent of NOD1 with raising Nod1.For example, can with the tumor necrosis factor of the co-administered effective dose of these reagent.In some embodiments, tumor necrosis factor can strengthen Nod dependent cell apoptosis pathway.In addition, can use the cycloheximide of effective dose, express or the NOD1 activity to improve Nod1 with described reagent.In addition, can with co-administered one or more chemotherapy compounds of described reagent.

The example that can be used for the chemotherapy compound of the compositions and methods of the invention comprises hexamethyl melamine, bleomycin, Busulfan, calcium folinate, capecitabine, carboplatin, carmustine, benzenebutanoic acid helium mustard, cisplatin, cladribine, Crisantaspase, cyclophosphamide, cytosine arabinoside, dacarbazine, D actinomycin D, daunorubicin, docetaxel, amycin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, liposomal doxorubicin, lomustine, melphalan, purinethol, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pentostatin, procarbazine, Raltitrexed, streptozotocin, UFT, Temozoromide, thiotepa, thioguanine/thioguanine, topotecan, treosulfan, vincaleucoblastine, vincristine, desacetyl vinblastine amide, vinorelbine and its combination.

Described reagent can be administered to the site of tumor partly, and/or is used for continuing to discharge by preparation.

Another aspect of the present invention is a kind of compositions; it comprises carrier, comprises the segmental nucleic acid of coding NOD1 polypeptide; and the D-Ala-L-Glu-meso diaminopimelic acid of effective dose (γ TriDAP), γ-D-glutamyl-meso-meso diaminopimelic acid (iE-DAP), γ-D-Gln-DAP (iQ-DAP) or D-Ala-L-Glu-meso diaminopimelic acid (γ TriDAP), wherein said compositions is used for local application to tumor by preparation.Described NOD1 polypeptide is passable, for example, comprises SEQ ID NO:1 or SEQ ID NO:3.The example of the nucleic acid fragment of coding NOD1 polypeptide is SEQ ID NO:2.The nucleic acid that adopts in described compositions may further include regulating element, for example, and promoter, enhancer, transcription stop signals, or its combination.Described nucleic acid can be expression cassette or expression vector.Described nucleic acid comprises gene delivery vector.Compositions of the present invention also can comprise other active component, for example, and the tumor necrosis factor of effective dose or chemotherapy compound.Described compositions can be used for being administered to partly the site of tumor by preparation, and/or is used for continuing to discharge by preparation.

Another aspect of the present invention is to promote apoptotic method in the breast tumor cell, comprises D-Ala-L-Glu-meso diaminopimelic acid (γ TriDAP) the contact breast tumor cell with effective dose.

Another aspect of the present invention is to promote apoptotic method in the estrogen-sensitive tumor cell, comprises D-Ala-L-Glu-meso diaminopimelic acid (γ TriDAP) the contact breast tumor cell with effective dose.

Description of drawings

Accompanying drawing 1A-C has illustrated the Nod1 that relates in the Apoptosis that TNF induces. Accompanying drawing 1A provides the schematic diagram of mutator, and it has produced TNF α resistant phenotype, is accredited as afterwards the Nod1 sudden change of inserting with blasticidine (blast) gene. Clone with this Nod1 sudden change is MCF7-C20 clone. Insertion from pDisrup retroviral construct body is drawn on the Nod1 gene. And the contact of the blasticidine of Nod1 gene fusion appears at 3 ' end of Nod1 gene between rich leucine zone 8 (LRR8) and the rich leucine zone 9 (LRR9). Therefore LRR9 and LRR10 zone be blasticidine insert 3 '. Accompanying drawing 1B has shown that NOD1 albumen does not exist with detectable amount in the MCF-7C20 cell. Preparation is from the cell extract of MCF-7 parent (being called " wt ") and MCF-7C20 cell, precipitate (upper picture) or directly be loaded into (lower picture) on the SDS-PAGE gel with the anti-NOD1 antibody mediated immunity of monoclonal, then transfer on the pvdf membrane. Analyze trace with the anti-membrane antibody of identical monoclonal by Western blotting. Accompanying drawing 1C has shown with the MCF-7C20 cell and has compared that the MCF-7 cell more has resistance for the Apoptosis that TNF induces. TNF (0-40ng/ml) with rising concentration processes MCF-7 and MCF-7C20 cell 20h. Measure cell survival by iodate the third ingot (PI) discharge analysis and flow cytometry. Chart shows that the MCF-7C20 cell significantly more may experience Apoptosis.

Accompanying drawing 2A-D has shown MCF-7 cell experience Apoptosis when γ TriDAP processes. Accompanying drawing 2A has illustrated that NOD1 is that the γ TriDAP cell death of inducing is required. There is (shade post) or lacking in the situation of (open tubular column) cycloheximide (CHX) (3 μ g/ml), processing MCF-7 Blasto cell, the expression of the NOD1 that expresses normal level with γ TriDAP or α TriDAP (each 50 μ g/ml) and seldom or not express the MCF-7 C20 cell of NOD1 or the MCF-7 Nod1 cell 48h of overexpression NOD1. Check analysis is accepted culture medium (Med) and is replaced γ TriDAP or α TriDAP. After 48h is hatched, harvesting, and hatch (4 μ g/ml) with iodate the third ingot (PI). Measure cell survival by Flow Cytometry. What data showed is the representativeness experiment of at least four independent experiments. Accompanying drawing 2B shown in the MCF-7 Blasto cell with independent carrier transfection, have in the MCF-7 C20 cell of endogenous Nod1 gene disruption or by through engineering approaches with the NOD1 expression in the MCF-7 Nod1 cells of overexpression NOD1. The expression of NOD1 is by analyzing with the western trace of the anti-Nod1 antibody of monoclonal in MCF-7 Blasto, MCF-7 C20 and MCF-7 Nod1 cell. Accompanying drawing 2C has illustrated the metamorphosis in the MCF-7 Nod1 cell that γ TriDAP processes. Cell is seeded on the 4 pore chamber slides, and processes with γ TriDAP/ cycloheximide (CHX) (picture b, d, f) or independent CHX (picture a, c, e). Cell is with DAPI (picture c, d) or TUNEL (picture e, f) dyeing, and is fixing and observe differing (picture a, b) or fluorescence (picture c-f) microscopically. Accompanying drawing 2D shown, by the caspase inhibitor of two kinds of wide spectrums, and z-VAD-FMK and Boc-D-FMK, the Apoptosis that the γ TriDAP in MCF-7 Nod1 cell induces reduces or eliminates. With z-VAD or Boc-D-FMK caspase inhibitor (each 50 μ M) preliminary treatment MCF-7 Nod1 cell 30 minutes, add afterwards γ TriDAP/CHX 48h. With iodate the third ingot (PI) incubated cell, measure apoptotic cell death by flow cytometry.

Accompanying drawing 3 has passed through the western analytic explanation, add γ TriDAP to the MCF-7 cell, rather than invalid tester tripeptides α TriDAP, caused the proteolytic cleavage of poly-(ADP-ribose) polymerase (PARP) and capases 6,7,8 and 9 proteolytic cleavage. After existing or lacking in the situation of CHX (0.5 μ g/ml) with γ TriDAP, α TriDAP or culture medium (contrast) stimulation, the PARP that the Western engram analysis by MCF-7 Blasto, MCF-7 C20 and MCF-7 Nod1 cell detects and the cracking of various caspases. Harvesting, experience western trace uses antibody test PARP, caspases, p20, p41/43 and p35 with its reaction.

Accompanying drawing 4A-C has shown that NOD1 mutant V41Q reacts to γ TriDAP, and has kept the function in the apoptotic pathways, and NOD1 mutant K208R is insensitive for γ TriDAP, and in apoptotic pathways be do not have activated. Accompanying drawing 4A illustrates the percentage of Apoptosis cell in the clone different after processing with culture medium (Med., contrast) or γ TriDAP. NOD1 V41Q and K208R mutant make up by direct mutagenesis. The V41Q sudden change is in the CARD domain of NOD1, and the K208R sudden change is considered to block the required conformation change of oligomerization by the mediation of Nod/NBD domain. The construct of these NOD1 V41Q and K208R mutant polypeptide of encoding is transfected in the MCF-7 C20 cell, carries out the Apoptosis analysis. As shown, the V41Q mutant has kept NOD1 active, but the K208R mutant does not have. Accompanying drawing 4B has shown that the expression of NOD1 mutant and wild type peptide is identical basically. Accompanying drawing 4C is by the western analytic explanation, and it is that the proteolytic cleavage of PARP and capases 6,7,8 and 9 proteolytic cleavage are required that the NOD1 in the MCF-7 cell expresses. The cracking of PARP and various caspases detects by the MCF-7 C20 cell of not expressing NOD1 and the Western engram analysis that restructuring of Nod1 construct is imported the MCF-7C20 Nod1 cell in the MCF-7 C20 cell. Exist or lacking in the situation of cycloheximide (CHX) (0.5 μ g/ml) with γ TriDAP or culture medium (contrast) irritation cell 24h. Then harvesting carries out the western trace. Use antibody test PARP, caspases, p20, p41/43 and the p35 of with it reaction.

Accompanying drawing 5A-C has illustrated in the MCF-7 cell not cell death inducing of Nod2. In accompanying drawing 5A, process MCF-7 Blasto, MCF-7 Nod1 and MCF-7 Nod2 48h with NOD1 part γ TriDAP or Nod2 part muramyl dipeptide (MDP) (each 20 μ g/ml) existing or lack in the situation of CHX. Then use the PI incubated cell, measure the Apoptosis cell death by flow cytometry. As shown, γ TriDAP irritation cell apoptosis, but the Nod2 part is not. In accompanying drawing 5B, the expression of NOD1 and NOD2 is confirmed by the Western engram analysis, uses the anti-Myc antibody of the detection that is used for recombinant protein. Accompanying drawing 5C has shown MCF-7 Nod2 cellular response MDP, as detecting by interleukin 8 (IL-8) secretion. Stimulate MCF-7 Blasto, MCF-7 Nod1 and MCF-7 Nod2 cell 24h with γ TriDAP or MDP existing or lack in the situation of CHX (0.5 μ g/ml). Then the harvesting supernatant is analyzed the IL-8 secretion.

Accompanying drawing 6A-D illustrated, caspase 8 and caspase 9 are that the γ TriDAP Apoptosis of inducing is required. Accompanying drawing 6A has shown the apoptotic impact that different caspase inhibitor are induced γ TriDAP. Before stimulating 48h with γ TriDAP/CHX, use the inhibitor preliminary treatment MCF-7 Nod1 cell 30 minutes of the caspase that lists along the x axle of accompanying drawing 6A. Then use iodate the third ingot incubated cell, measure cell survival by flow cytometry. All inhibitor use in the concentration of 100 μ M. Accompanying drawing 6B has shown, when Nod1 and caspase 9 coexpression, detects the HMW form of NOD1, shows that NOD1 and caspase 9 interact. In this experiment, in the situation that has empty carrier, Myc-Nod1 or Myc-NOD2, the 293 cells carrier cotransfection of coding FLAG-caspase 9. With anti--FLAG antibody mediated immunity precipitation (IP) cell extract, use Anti-TNF-α-Myc antibody to disclose the protein of co-precipitation by Western blotting (WB). Accompanying drawing 6C has shown, the Apoptosis that CLARP fully stops γ TriDAP to induce, and Bcl2 only partly suppresses the Apoptosis that γ TriDAP induces. Do not process or process MCF-7CLARP, MCF-7CLARP/Nod1, MCF-7Bcl2 and MCF-7Bcl2/Nod1 cell with γ TriDAP existing or lack in the situation of CHX (3 μ g/ml). Then use iodate the third ingot incubated cell, measure the Apoptosis cell death by flow cytometry. Accompanying drawing 6D has shown the Western trace, illustrated at MCF-7 cells CLARP, Bcl2 and NOD1, show that the result who observes is owing to the function difference between CLARP and the Bcl2 in accompanying drawing 6C, rather than the difference in these two kinds of protein expression levels.

Accompanying drawing 7A-C has illustrated that (KD) sudden change of the kinase deficiency of wild type RIP2 and RIP2 is functional in NOD 1 apoptotic pathways. Yet the RIP2 that lacks the CARD domain works as the dominant negative inhibitor of NOD1 signal transduction. The percentage of apoptotic cell in wild type RIP2, RIP2KD and RIP2 Δ CARD cell has been described accompanying drawing 7A figure. With wild type Myc-RIP2, Myc-RIP2 KD and Myc-RIP2 Δ CARD transfection MCF-7 cell stably, existing or lacking in the situation of CHX (3 μ g/ml), do not process or process 48h with γ TriDAP. Use the PI incubated cell, measure the Apoptosis cell death by flow cytometry. Accompanying drawing 7B has shown that the RIP2 polypeptide is expressed, as by with Anti-TNF-α Myc antibody to the immunoprecipitation of cell extract and use the Western blotting of the anti-Myc 9E10 of monoclonal antibody to confirm. Accompanying drawing 7C has illustrated that the γ TriDAP of the phosphorylation of JNK in RIP2 wild type, MCF-7 RIP2 KD and RIP2 Δ CARD cell induces. As shown, there is in the situation of cycloheximide the MCF-7 cell of expression wild type RIP2 or RIP2 KD is induced JNK to the exposure 2h of γ TriDAP phosphorylation. Yet, in the RIP2 Δ CARD cell that the same manner is processed, do not observe this phosphorylation of JNK.

Accompanying drawing 8A-D has illustrated, has collaborative correlation between NOD1 and TNF α. Illustrated that when the concentration of NOD1 was brought up to 1ng/ml from 0.0 concentration of bringing up to 100 μ g/ml and TNF α from 0.5ng/ml, the percentage of Apoptosis cell improved in dosage specificity mode accompanying drawing 8A figure. Illustrated that when the concentration of cycloheximide was brought up to 1ng/ml from 0.0 concentration of bringing up to 3 μ g/ml and TNF α from 0.5ng/ml, the percentage of Apoptosis cell improved in dosage specificity mode accompanying drawing 8B figure. Illustrated that although γ TriDAP improves Apoptosis in the situation that has TNF α, invalid contrast tripeptides α TriDAP in fact can inhibited apoptosis accompanying drawing 8C figure, even under higher TNFi dosage. Accompanying drawing 8D has illustrated that the NOD1 at each time point expresses after the MCF-7 cell is to the exposure of TNF α.

Accompanying drawing 9A-C has illustrated that the NOD1 in another human breast cancer cell line, SKBR3 cancerous cell line expresses. Illustrated as the percentage function of TNF α concentration, apoptotic SKBR3 wild type and SKBR3 Nod1 cell accompanying drawing 9A (top) figure. Accompanying drawing 9A (bottom) bottom illustration as the apoptotic SKBR3 wild type of the function of γ TriDAP concentration and the percentage of SKBR3 Nod1 cell. The percentage of the apoptotic SKBR3 cell of observing under different condition by iodate the third ingot (PI) or Dioc6 has been described accompanying drawing 9B (top) figure. The condition that adopts is at following Western trace indicating. The abbreviation of using is as follows: CHX (cycloheximide), TNF (TNF α), γ Tri (γ TriDAP), α Tri (α TriDAP). Accompanying drawing 9B2 provides the western analysis, and the proteolytic cleavage of poly-(ADP-ribose) polymerase (PARP) and caspase 3,7 and 8 proteolytic cleavage have been described. After under the condition of following Western trace appointment, stimulating, analyze to detect the cracking of PARP and various caspases by the Western impression cytology of MCF-7 cell. Harvesting, experience western trace uses its reactive antibody to detect PARP, caspases, p20, p41/43 and p35. The wild type observed under the condition of following column diagram appointment has been described accompanying drawing 9C figure, has expressed NOD1's and has expressed the percentage of cerebral apoptosis of the SKBR3 cell of CLARP. The abbreviation of using: CHX (cycloheximide), TNF (TNF α), gTri (γ TriDAP), gTC (γ TriDAP+CHX), aTC (α TriDAP+CHX).

Accompanying drawing 10A, B and C provide the image of the mouse that the MCF-7 cell of the MCF-7 cell that knocks out with wild type MCF-7 breast cancer cell, NOD1 and Nod1-transfection inoculates respectively. With 3 * 10⁶The mankind mastopathy cell is Mice Inoculated hypodermically. The tumour that the arrow indication is subcutaneous, it is shown in the enlarged image of accompanying drawing 10B.

Accompanying drawing 11A-D has illustrated that the existence of Nod1 in the SCID mouse stops tumor growth. Illustrated accompanying drawing 11A figure, with MCF-7 C20 (left side) and MCF-7 C20/Nod1 (right side) injection cell hour in, as the gross tumor volume of the function of time. With cell (3 * 10⁶Individual cell/mouse) is expelled to the flank of female SCID mouse. Measure weekly tumor size, according to formula (W2 * L)/2 determine volume. Every tumour (n=4) that the line representative is observed in a mouse. As described, gross tumor volume reduces in the mouse of the tumour cell (C20/Nod1) of accepting expression Nod1. By contrast, gross tumor volume increases in the mouse of the tumour cell (C20 cell) of accepting not express Nod1. Illustrated at injection MCF-7 Blasto, MCF-7 C20 and MCF-7 C20/Nod1 cell (3 * 10 accompanying drawing 11B figure⁶Individual cell/mouse) implant before the estrogen bullet in mouse, (C20/Nod1 cell) all improves tumor growth during except tumor cells expression Nod1. The result of the mouse acquisition of estrogen bullet is accepted in the representative of shade post, and the result that the mouse of estrogen bullet obtains is never accepted in the open tubular column representative. Measure as mentioned above gross tumor volume. Gross tumor volume along with the time has been described in the mouse of MCF-7 Blasto, MCF-7 C20 and MCF-7 RIP2 Δ CARD injection cell accompanying drawing 11C figure. As shown, accept MCF-7RIP2 Δ CARD cell (^*) mouse in gross tumor volume approximately express (Blasto, solid diamond) in the mouse of certain Nod1, but less than (C20, filled squares) in the mouse of not expressing Nod1. Growth of tumour cell as the function of estrogen concentrations has been described accompanying drawing 11D figure. As shown, do not express the tumour cell 9MCF-7 C20 cell of Nod1) and to express the tumour cell (MCF-7 RIP2 Δ CARD cell) of RIP2 of sudden change more responsive for estrogen, and represented the tumor growth that improves. MCF-7 Blasto, MCF-7 C20, MCF-7 C20/Nod1 and MCF-7 RIP2 Δ CARD cell are exposed to 17 beta estradiols that improve concentration, with the pulse of 3H-thymidine, measure Growth of Cells by liquid scintillation. Data are representatives of at least 3 independent experiments.

Accompanying drawing 12A-C has illustrated that estrogen improves the inductive apoptosis of γ TriDAP in various MCF-7 tumor cell lines, and zitazonium suppresses the inductive apoptosis of γ TriDAP.Illustrated at the estrogen of elevated amounts and cycloheximide (CHX) or CHX accompanying drawing 12A figure and added the MCF-7Blasto, the MCF-7C20 that cultivate among the γ TriDAP (γ Tri) and the percentage of cerebral apoptosis in the MCF-7C20/Nod1 cell.Illustrated at the zitazonium of elevated amounts and cycloheximide (CHX) or CHX accompanying drawing 12B figure and added the MCF-7 Blasto, the MCF-7 C20 that cultivate among the γ TriDAP (γ Tri) and the percentage of cerebral apoptosis in the MCF-7 C20/Nod1 cell.For estrogen research, in the culture medium of charcoal treatment, cultivate the MCF-7 cell, and under the situation of the estrogen that has rising concentration (E2), stimulate with γ TriDAP/CHX, eject the measurement cell survival by iodate third ingot (PI).For zitazonium research, before adding γ TriDAP/CHX, handle cell with zitazonium, eject the measurement cell survival by PI.Accompanying drawing 12C has shown the immunoblotting of the cellular lysate of cell (left side) from In vitro culture and interior tumor cell (right side), has illustrated that being expressed in the cell of expressing Nod1 of estrogen receptor reduce.From MCF-7 Blasto, MCF-7 C20 and MCF-7C20/Nod1 cell, and next total protein extract since the tumor isolated cells, use is analyzed at the immunoblotting of the antibody of estrogen receptor (ER α), and ERK2 is as loading contrast.

Accompanying drawing 13A-D has illustrated that RIP2 is the important component of Nod1 apoptotic pathways.Accompanying drawing 13A shown, the stably express sensitization of kinase deficiency RIP2 in tumor cell line (RIP2 KD) these cells to Nod1 or the inductive apoptosis cell death of Nod2 activator.With Myc-Nod1, Myc-RIP2 (wild type), Myc-RIP2 KD and Myc-RIP2 Δ CARD stable transfection MCF-7 cell.Handle test cell with γ TriDAP, α TriDAP and MDP (each 20 μ g/ml) existing or lack under the situation of CHX (3 μ g/ml) then, and control cells is without these agent treated.Use iodate third ingot (PI) incubated cell then, measure the apoptosis cell death by flow cytometry.The influence of TNF when accompanying drawing 13B has illustrated apoptosis in the identical MCF-7 cell line of describing in accompanying drawing 13A.Handled these cell lines 18 hours with TNF (10ng/ml), as the evaluation apoptosis cell death of accompanying drawing 13A description.TNF improves apoptosis in the cell line of all tests.Accompanying drawing 13C has shown, compares with MCF-7 Nod1 cell, and MCF-7 RIP2 KD cell has represented the inductive apoptosis of γ TriDAP that improves.There is CHX, having incubated cell 48hr under the situation of the γ TriDAP of rising concentration or MDP, the evaluation apoptosis cell death of describing as accompanying drawing 13A.Accompanying drawing 13D has illustrated the IL-8 secretion that stimulates back MCF-7 Blasto, MCF-7 Nod1 and MCF-7 RIP2 wild-type cell under the situation that has CHX (0.5mg/ml) or TNF at γ TriDAP.After hatching, the harvesting supernatant is analyzed the IL-8 secretion.

Detailed Description Of The Invention

According to the present invention, the Nod1 of raising expresses or the NOD1 activity causes tumour regression.Thereby the present invention relates to the experimenter use improve that Nod1 expresses or the active NOD1 polypeptide of NOD1, Nod1 nucleic acid and reagent as antineoplaston.In some embodiments; improve the peptide activator that Nod1 expression or active reagent comprise NOD1; for example γ-D-glutamyl-meso-meso diaminopimelic acid (iE-DAP), γ-D-Gln-DAP (iQ-DAP), D-Ala-L-Glu-meso diaminopimelic acid (γ TriDAP) and its combination.Though the present invention expects the tumor of treatment any kind, the compositions and methods of the invention also have special practicality for the tumor and/or the breast cancer tumour of treatment estrogen sensitivity.

NOD1

Inborn immune system is made up of the receptor family of the host cell of discerning microorganism, virus, unusual/damage or the like composition, starts host response and eliminates and kill the organism of intrusion or remove abnormal host cell.Recently, be called in the cell of Nod/Caterpillar family, the cell cytosol protein family interrelates with innate immune response.All members of this family have two conservative domains; Nucleotide repeats (LRR) zone in conjunction with oligomerization domain (NBD/NOD) and the rich leucine of carboxyl terminal.Family member's amino terminal area is called the effector domain, contains variable structure, and it comprises that caspase raises domain (CARD), pyrin, BIR and other domains.

Two members of Nod/Caterpillar family interrelate especially with at the born immunoreation of infecting.These members are called as NOD1 (CARD4) and NOD2 (CARD15).The effector domain of NOD1 and NOD2 is made up of one and two CARD domains respectively.It is intracellular protein as bacteria lipopolysaccharide receptor (LPS) that NOD1 and NOD2 are prompted at first.Subsequently, discovery is that the NOD part is from bacterial peptide polysaccharide (PGN) rather than from LPS.Thereby NOD1 and NOD2 may work as the cell interoceptor of antibacterial between infection period or bacterial product.Yet that carries out up to now studies show that, the mice of NOD1 or NOD2 defective is not the tangible dominant phenotype relevant with the infection sensibility of immunodeficiency or raising.

Other members' of NOD1 and NOD/Caterpillar family nucleic acid and aminoacid sequence can for example find in ncbi database in the art.Referring to website ncbi.nlm.nih.gov.For example, the aminoacid sequence of human NOD1 provides so that reference is SEQ ID NO:1 (NCBI registration number Q9Y239 following; Gi:20137579).

1 MEEQGHSEME?IIPSESHPHI?QLLKSNRELL?VTHIRNTQCL

41 VDNLLKNDYF?SAEDAEIVCA?CPTQPDKVRK?ILDLVQSKGE

81 EVSEFFLYLL?QQLADAYVDL?RPWLLEIGFS?PSLLTQSKVV

121 VNTDPVSRYT?QQLRHHLGRD?SKFVLCYAQK?EELLLEEIYM

161 DTIMELVGFS?NESLGSLNSL?ACLLDHTTGI?LNEQGETIFI

201 LGDAGVGKSM?LLQRLQSLWA?TGRLDAGVKF?FFHFRCRMFS

241 CFKESDRLCL?QDLLFKHYCY?PERDPEEVFA?FLLRFPHVAL

281 FTFDGLDELH?SDLDLSRVPD?SSCPWEPAHP?LVLLANLLSG

321 KLLKGASKLL?TARTGIEVPR?QFLRKKVLLR?GFSPSHLRAY

361 ARRMFPERAL?QDRLLSQLEA?NPNLCSLCSV?PLFCWIIFRC

401 FQHFRAAFEG?SPQLPDCTMT?LTDVFLLVTE?VHLNRMQPSS

441 LVQRNTRSPV?ETLHAGRDTL?CSLGQVAHRG?MEKSLFVFTQ

481 EEVQASGLQE?RDMQLGFLRA?LPELGPGGDQ?QSYEFFHLTL

521 QAFFTAFFLV?LDDRVGTQEL?LRFFQEWMPP?AGAATTSCYP

561 PFLPFQCLQG?SGPAREDLFK?NKDHFQFTNL?FLCGLLSKAK

601 QKLLRHLVPA?AALRRKRKAL?WAHLFSSLRG?YLKSLPRVQV

641 ESFNQVQAMP?TFIWMLRCIY?ETQSQKVGQL?AARGICANYL

681 KLTYCNACSA?DCSALSFVLH?HFPKRLALDL?DNNNLNDYGV

721 RELQPCFSRL?TVLRLSVNQI?TDGGVKVLSE?ELTKYKIVTY

761 LGLYNNQITD?VGARYVTKIL?DECKGLTHLK?LGKNKITSEG

801 GKYLALAVKN?SKSISEVGMW?GNQVGDEGAK?AFAEALRNHP

841 SLTTLSLASN?GISTEGGKSL?ARALQQNTSL?EILWLTQNEL

881 NDEVAESLAE?MLKVNQTLKH?LWLIQNQITA?KGTAQLADAL

921 QSNTGITEIC?LNGNLIKPEE?AKVYEDEKRI?ICF

The nucleotide sequence of human Nod1 also is obtainable, and NCBI registration number BC040339 (gi:25955660) below is listed as SEQ ID NO:2 so that reference.

1 CCCGGCCCCG?GCGTCCCCGG?ACCATGGCGC?TCTCCGGGCT

41 CTTCTCTAGC?TCTCAGCGGC?TGCGAAGTCT?GTAAACCTGG

81 TGGCCAAGTG?ATTGTAAGTC?AGGAGACTTT?CCTTCGGTTT

121 CTGCCTTTGA?TGGCAAGAGG?TGGAGATTGT?GGCGGCGATT

161 ACAGAAAACG?TCTGGGAAGA?CAAGTTGCTG?TTTTTATGGG

201 AATCGCAGGC?TTGGAAGAGA?CAGAAGCAAT?TCCAGAAATA

241 AATTGGAAAT?TGAAGATTTA?AACAATGTTG?TTTTAAAACA

281 TTCTAACTTC?AAAGAATGAT?GCCAGAAACT?TAAAAAGGGG

321 CTGCGCAGAG?TAGCAGGGGC?CCTGGAGGGC?GCGGCCTGAA

361 TCCTGATTGC?CCTTCTGCTG?AGAGGACACA?CGCAGCTGAA

401 GATGAATTTG?GGAAAAGTAG?CCGCTTGCTA?CTTTAACTAT

441 GGAAGAGCAG?GGCCACAGTG?AGATGGAAAT?AATCCCATCA

481 GAGTCTCACC?CCCACATTCA?ATTACTGAAA?AGCAATCGGG

521 AACTTCTGGT?CACTCACATC?CGCAATACTC?AGTGTCTGGT

561 GGACAACTTG?CTGAAGAATG?ACTACTTCTC?GGCCGAAGAT

601 GCGGAGATTG?TGTGTGCCTG?CCCCACCCAG?CCTGACAAGG

641 TCCGCAAAAT?TCTGGACCTG?GTACAGAGCA?AGGGCGAGGA

681 GGTGTCCGAG?TTCTTCCTCT?ACTTGCTCCA?GCAACTCGCA

721 GATGCCTACG?TGGACCTCAG?GCCTTGGCTG?CTGGAGATCG

761 GCTTCTCCCC?TTCCCTGCTC?ACTCAGAGCA?AAGTCGTGGT

801 CAACACTGAC?CCAGTGAGCA?GGTATACCCA?GCAGCTGCGA

841 CACCATCTGG?GCCGTGACTC?CAAGTTCGTG?CTGTGCTATG

881 CCCAGAAGGA?GGAGCTGCTG?CTGGAGGAGA?TCTACATGGA

921 CACCATCATG?GAGCTGGTTG?GCTTCAGCAA?TGAGAGCCTG

961 GGCAGCCTGA?ACAGCCTGGC?CTGCCTCCTG?GACCACACCA

1001?CCGGCATCCT?CAATGAGCAG?GGTGAGACCA?TCTTCATCCT

1041?GGGTGATGCT?GGGGTGGGCA?AGTCCATGCT?GCTACAGCGG

1081?CTGCAGAGCC?TCTGGGCCAC?GGGCCGGCTA?GACGCAGGGG

1121?TCAAATTCTT?CTTCCACTTT?CGCTGCCGCA?TGTTCAGCTG

1161?CTTCAAGGAA?AGTGACAGGC?TGTGTCTGCA?GGACCTGCTC

1201?TTCAAGCACT?ACTGCTACCC?AGAGCGGGAC?CCCGAGGAGG

1241?TGTTTGCCTT?CCTGCTGCGC?TTCCCCCACG?TGGCCCTCTT

1281?CACCTTCGAT?GGCCTGGACG?AGCTGCACTC?GGACTTGGAC

1321?CTGAGCCGTG?TGCCTGACAG?CTCCTGCCCC?TGGGAGCCTG

1361?CCCACCCCCT?GGTCTTGCTG?GCCAACCTGC?TCAGTGGGAA

1401?GCTGCTCAAG?GGGGCTAGCA?AGCTGCTCAC?AGCCCGCACA

1441?GGCATCGAGG?TCCCGCGCCA?GTTCCTGCGG?AAGAAGGTGC

1481?TTCTCCGGGG?CTTCTCCCCC?AGCCACCTGC?GCGCCTATGC

1521?CAGGAGGATG?TTCCCCGAGC?GGGCCCTGCA?GGACCGCCTG

1561?CTGAGCCAGC?TGGAGGCCAA?CCCCAACCTC?TGCAGCCTGT

1601?GCTCTGTGCC?CCTCTTCTGC?TGGATCATCT?TCCGGTGCTT

1641?CCAGCACTTC?CGTGCTGCCT?TTGAAGGCTC?ACCACAGCTG

1681?CCCGACTGCA?CGATGACCCT?GACAGATGTC?TTCCTCCTGG

1721?TCACTGAGGT?CCATCTGAAC?AGGATGCAGC?CCAGCAGCCT

1761?GGTGCAGCGG?AACACACACA?GCCCAGTGGA?GACCCTCCAC

1801?GCCGGCCGGG?ACACTCTGTG?CTCGCTGGGG?CAGGTGGCCC

1841?ACCGGGGCAT?GGAGAAGAGC?CTCTTTGTCT?TCACCCAGGA

1881?GGAGGTGCAG?GCCTCCGGGC?TGCAGGAGAG?AGACATGCAG

1921?CTGGGCTTCC?TGCGGGCTTT?GCCGGAGCTG?GGCCCCGGGG

1961?GTGACCAGCA?GTCCTATGAG?TTTTTCCACC?TCACCCTCCA

2001?GGCCTTCTTT?ACAGCCTTCT?TCCTCGTGCT?GGACGACAGG

2041?GTGGGCACTC?AGGAGCTGCT?CAGGTTCTTC?CAGGAGTGGA

2081?TGCCCCCTGC?GGGGGCAGCG?ACCACGTCCT?GCTATCCTCC

2121?CTTCCTCCCG?TTCCAGTGCC?TGCAGGGCAG?TGGTCCGGCG

2161?CGGGAAGACC?TCTTCAAGAA?CAAGGATCAC?TTCCAGTTCA

2201?CCAACCTCTT?CCTGTGCGGG?CTGTTGTCCA?AAGCCAAACA

2241?GAAACTCCTG?CGGCATCTGG?TGCCCGCGGC?AGCCCTGAGG

2281?AGAAAGCGCA?AGGCCCTGTG?GGCACACCTG?TTTTCCAGCC

2321?TGCGGGGCTA?CCTGAAGAGC?CTGCCCCGCG?TTCAGGTCGA

2361?AAGCTTCAAC?CAGGTGCAGG?CCATGCCCAC?GTTCATCTGG

2401?ATGCTGCGCT?GCATCTACGA?GACACAGAGC?CAGAAGGTGG

2441?GGCAGCTGGC?GGCCAGGGGC?ATCTGCGCCA?ACTACCTCAA

2481?GCTGACCTAC?TGCAACGCCT?GCTCGGCCGA?CTGCAGCGCC

2521?CTCTCCTTCG?TCCTGCATCA?CTTCCCCAAG?CGGCTGGCCC

2561?TAGACCTAGA?CAACAACAAT?CTCAACGACT?ACGGCGTGCG

2601?GGAGCTGCAG?CCCTGCTTCA?GCCGCCTCAC?TGTTCTCAGA

2641?CTCAGCGTAA?ACCAGATCAC?TGACGGTGGG?GTAAAGGTGC

2681?TAAGCGAAGA?GCTGACCAAA?TACAAAATTG?TGACCTATTT

2721?GGGTTTATAC?AACAACCAGA?TCACCGATGT?CGGAGCCAGG

2761?TACGTCACCA?AAATCCTGGA?TGAATGCAAA?GGCCTCACGC

2801?ATCTTAAACT?GGGAAAAAAC?AAAATAACAA?GTGAAGGAGG

2841?GAAGTATCTC?GCCCTGGCTG?TGAAGAACAG?CAAATCAATC

2881?TCTGAGGTTG?GGATGTGGGG?CAATCAAGTT?GGGGATGAAG

2921?GAGCAAAAGC?CTTCGCAGAG?GCTCTGCGGA?ACCACCCCAG

2961?CTTGACCACC?CTGAGTCTTG?CGTCCAACGG?CATCTCCACA

3001?GAAGGAGGAA?AGAGCCTTGC?GAGGGCCCTG?CAGCAGAACA

3041?CGTCTCTAGA?AATACTGTGG?CTGACCCAAA?ATGAACTCAA

3081?CGATGAAGTG?GCAGAGAGTT?TGGCAGAAAT?GTTGAAAGTC

3121?AACCAGACGT?TAAAGCATTT?ATGGCTTATC?CAGAATCAGA

3161?TCACAGCTAA?GGGGACTGCC?CAGCTGGCAG?ATGCGTTACA

3201?GAGCAACACT?GGCATAACAG?AGATTTGCCT?AAATGGAAAC

3241?CTGATAAAAC?CAGAGGAGGC?CAAAGTCTAT?GAAGATGAGA

3281?AGCGGATTAT?CTGTTTCTGA?GAGGATGCTT?TCCTGTTCAT

3321?GGGGTTTTTG?CCCTGGAGCC?TCAGCAGCAA?ATGCCACTCT

3361?GGGCAGTCTT?TTGTGTCAGT?GTCTTAAAGG?GGCCTGCGCA

3401?GGCGGGACTA?TCAGGAGTCC?ACTGCCTCCA?TGATGCAAGC

3441?CAGCTTCCTG?TGCAGAAGGT?CTGGTCGGCA?AACTCCCTAA

3481?GTACCCGCTA?CAATTCTGCA?GAAAAAGAAT?GTGTCTTGCG

3521?AGCTGTTGTA?GTTACAGTAA?ATACACTGTG?AAGAGACTTT

3561?ATTGCCTATT?ATAATTATTT?TTATCTGAAG?CTAGAGGAAT

3601?AAAGCTGTGA?GCAAACAGAG?GAGGCCAGCC?TCACCTCATT

3641?CCAACACCTG?CCATAGGGAC?CAACGGGAGC?GAGTTGGTCA

3681?CCGCTCTTTT?CATTGAAGAG?TTGAGGATGT?GGCACAAAGT

3721?TGGTGCCAAG?CTTCTTGAAT?AAAACGTGTT?TGATGGATTA

3761?GTATTATACC?TGAAATATTT?TCTTCCTTCT?CAGCACTTTC

3801?CCATGTATTG?ATACTGGTCC?CACTTCACAG?CTGGAGACAC

3841?CGGAGTATGT?GCAGTGTGGG?ATTTGACTCC?TCCAAGGTTT

3881?TGTGGAAAGT?TAATGTCAAG?GAAAGGATGC?ACCACGGGCT

3921?TTTAATTTTA?ATCCTGGAGT?CTCACTGTCT?GCTGGCAAAG

3961?ATAGAGAATG?CCCTCAGCTC?TTAGCTGGTC?TAAGAATGAC

4001?GATGCCTTCA?AAATGCTGCT?TCCACTCAGG?GCTTCTCCTC

4041?TGCTAGGCTA?CCCTCCTCTA?GAAGGCTGAG?TACCATGGGC

4081?TACAGTGTCT?GGCCTTGGGA?AGAAGTGATT?CTGTCCCTCC

4121?AAAGAAATAG?GGCATGGCTT?GCCCCTGTGG?CCCTGGCATC

4161?CAAATGGCTG?CTTTTGTCTC?CCTTACCTCG?TGAAGAGGGG

4201?AAGTCTCTTC?CTGCCTCCCA?AGCAGCTGAA?GGGTGACTAA

4241?ACGGGCGCCA?AGACTCAGGG?GATCGGCTGG?GAACTGGGCC

4281?AGCAGAGCAT?GTTGGACACC?CCCCACCATG?GTGGGCTTGT

4321?GGTGGCTGCT?CCATGAGGGT?GGGGGTGATA?CTACTAGATC

4361?ACTTGTCCTC?TTGCCAGCTC?ATTTGTTAAT?AAAATACTGA

4401?AAACACTAAA?AAAAAAAAAA?AA

NOD1 albumen seems to have important function in antibacterial identification, and the specificity host pattern recognition receptor that can be used as in the cellular compartment works.Recent studies show that, NOD1 be necessary in host's identification of the bacterial peptide polysaccharide that contains meso diaminopimelic acid (people such as Chamaillard., Nature Immunology, DOI:10.1038/ni945, June 8,2003).The machine of NOD1 identification is a dipeptides, γ-D-glutamyl-meso-meso diaminopimelic acid (being also referred to as iE-DAP).This dipeptides is known to exist only in the antibacterial of limited quantity (Escherichia coli and several gram-positive bacterium, for example Bacillus subtilis and Listeria monocytogenes).

The inventor has found that NOD1 makes cell to the inductive apoptosis sensitization of TNF α, and under the situation that lacks any other known apoptosis triggering, the apoptosis in the NOD1 ligands specific inducing tumor cell.Use the interior research explanation of body of animal model and emphasized the effect that NOD1 has in tumour regression.Especially, as shown here, the xenograft of inserting the MCF-7 breast tumor cell of SCID mice generally forms tumor.Yet after a while, these tumors generally can be degenerated, even without antineoplaston.But, work as NOD1 ^-/-The MCF-7 tumor cell transplantation is in mice the time, and tumor can not degenerated, continued growth on the contrary (referring to accompanying drawing 10).When the NOD1 function is added back Nod1 ^-/-During the MCF-7 tumor cell (by the transfection of suitable genetic constructs), will degenerate now by the tumor of transplanting these NOD1 express cells generations.Therefore, NOD1 expresses can help to control growth of tumour cell, and can cause the apoptosis of tumor cell.

The present invention also provides and has kept the active NOD1 sudden change of apoptosis (V41Q) polypeptide.The sequence of this V41Q sudden change NOD1 polypeptide is following to be provided as SEQ ID NO:3, and the V41Q sudden change is runic and underscore.

1 MEEQGHSEME?IIPSESHPHI?QLLKSNRELL?VTHIRNTQCL

41 QDNLLKNDYF?SAEDAEIVCA?CPTQPDKVRK?ILDLVQSKGE

81 EVSEFFLYLL?QQLADAYVDL?RPWLLEIGFS?PSLLTQSKVV

121 VNTDPVSRYT?QQLRHHLGRD?SKFVLCYAQK?EELLLEEIYM

161 DTIMELVGFS?NESLGSLNSL?ACLLDHTTGI?LNEQGETIFI

201 LGDAGVGKSM?LLQRLQSLWA?TGRLDAGVKF?FFHFRCRMFS

241 CFKESDRLCL?QDLLFKHYCY?PERDPEEVFA?FLLRFPHVAL

281 FTFDGLDELH?SDLDLSRVPD?SSCPWEPAHP?LVLLANLLSG

321 KLLKGASKLL?TARTGIEVPR?QFLRKKVLLR?GFSPSHLRAY

361 ARRMFPERAL?QDRLLSQLEA?NPNLCSLCSV?PLFCWIIFRC

401 FQHFRAAFEG?SPQLPDCTMT?LTDVFLLVTE?VHLNRMQPSS

441 LVQRNTRSPV?ETLHAGRDTL?CSLGQVAHRG?MEKSLFVFTQ

481 EEVQASGLQE?RDMQLGFLRA?LPELGPGGDQ?QSYEFFHLTL

521 QAFFTAFFLV?LDDRVGTQEL?LRFFQEWMPP?AGAATTSCYP

561 PFLPFQCLQG?SGPAREDLFK?NKDHFQFTNL?FLCGLLSKAK

601 QKLLRHLVPA?AALRRKRKAL?WAHLFSSLRG?YLKSLPRVQV

641 ESFNQVQAMP?TFIWMLRCIY?ETQSQKVGQL?AARGICANYL

681 KLTYCNACSA?DCSALSFVLH?HFPKRLALDL?DNNNLNDYGV

721 RELQPCFSRL?TVLRLSVNQI?TDGGVKVLSE?ELTKYKIVTY

761 LGLYNNQITD?VGARYVTKIL?DECKGLTHLK?LGKNKITSEG

801 GKYLALAVKN?SKSISEVGMW?GNQVGDEGAK?AFAEALRNHP

841 SLTTLSLASN?GISTEGGKSL?ARALQQNTSL?EILWLTQNEL

881 NDEVAESLAE?MLKVNQTLKH?LWLIQNQITA?KGTAQLADAL

921 QSNTGITEIC?LNGNLIKPEE?AKVYEDEKRI?ICF

Sudden change V41Q is present in the CARD domain of Nod1, is in the news previously to destroy combining of Caspase 9 and Nod1.Yet opposite with the previous result who shows V41Q sudden change inhibition Nod1 dependent cell apoptosis, the experiment that the inventor carries out shows that the V41Q mutant polypeptide is active (accompanying drawing 4) in apoptotic pathways.

Tumor

The compositions and methods of the invention are useful in various cancers and tumor treatment, include but not limited to the estrogen-sensitive tumor, and the tumor of mammary gland, bladder, cervix uteri, colon, gallbladder, kidney, liver, lung, pancreas, ovary, prostate, skin, stomach, thyroid or the like.In some embodiments, compositions of the present invention can be used for the treatment of or prophylaxis of cancer, for example bladder, mammary gland, colon, kidney, liver, lung, comprise small cell lung cancer, esophagus, gallbladder, ovary, pancreas, stomach, cervix uteri, thyroid, prostate and skin, comprise the cancer of squamous cell carcinoma; Lymphoid hemopoietic tumor comprises leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B cell lymphoma, t cell lymphoma, Hodgkin ' s lymphoma, non-Hodgkin ' s lymphoma, hair cell lymphoma and Burkett ' s lymphoma; The hemopoietic tumor of bone marrow system comprises acute and chronic graininess leukemia, myelodysplastic syndrome and promyelocytic leukemia; Between the tumor of matter origin, comprise fibrosarcoma and rhabdomyosarcoma; The tumor of central authorities and peripheral nervous system comprises astrocytoma, neuroblastoma, glioma and Schwann-cell tumor; Other tumors comprise melanoma, spermocytoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoxanthoma, thyroid follicle cancer and Kaposi ' s sarcoma.In some embodiments, the present invention can be used for the treatment of or Breast Cancer Prevention disease and tumor.

For example, NOD1 polypeptide, Nod1 nucleic acid, can improve that NOD1 expresses or active agent, or its combination, can be individually, with other factors for example TNF cause tumor cell experience apoptosis in combination at least in the tumor or contiguous tumor.Thereby the compositions and methods of the invention can be used for the treatment of cancer.

Regulate NOD1 expression and active

According to of the present invention, the NOD1 of raising expression and/or NOD1 activity have promoted the degeneration of tumor.Therefore, the invention provides the method for the treatment of and preventing tumor growth in the mammal, by expressing and/or active reagent or its combination to described administration NOD1 polypeptide, Nod1 nucleic acid, raising NOD1.Improve that NOD1 expresses or active reagent comprises and can improve the transcribing of NOD1, translation or active any reagent.

Thereby by using the nucleic acid (for example, comprising the nucleic acid of SEQ ID NO:2) of NOD1 polypeptide (for example, SEQ ID NO:1), coding NOD1 polypeptide, the incidence rate of tumour regression can be enhanced or promote.The nucleic acid of coding NOD1 can place expression cassette, and/or keeps in carrier, so that operate, express and duplicate.Below understand in more detail and produce coding NOD1 and can express the method for the nucleic acid of NOD1 polypeptide.

Improve NOD1 expression or active reagent and comprise the active little peptide ylidene ligands of enhancing NOD1.For example, γ-D-glutamyl-meso-meso diaminopimelic acid (iE-DAP), γ-D-Gln-DAP (iQ-DAP), D-Ala-L-Glu-meso diaminopimelic acid (γ TriDAP) and its combination can improve NOD1 expression or active.In some embodiments, only γ-D-glutamyl-meso-meso diaminopimelic acid (iE-DAP) or only γ-D-Gln-DAP (iQ-DAP), or only D-Ala-L-Glu-meso diaminopimelic acid (γ TriDAP) is used or uses.In other embodiments, the combination of γ-D-glutamyl-meso-meso diaminopimelic acid (iE-DAP), γ-D-Gln-DAP (iQ-DAP) and/or D-Ala-L-Glu-meso diaminopimelic acid (γ TriDAP) is used or uses.

The present invention further provides and in the experimenter, regulated the active method of NOD1, comprised the NOD1 that can change the experimenter is provided active reagent; Use described reagent to the experimenter under certain condition, thereby change experimenter's NOD1 activity.In some embodiments, use the degeneration that described reagent causes tumor in the experimenter to the experimenter.The present invention is not limited to specific chemical compound.In fact, all cpds is expected, include but not limited to, comprise peptide, the glutamine-meso diaminopimelic acid dipeptides of D-Ala-L-Glu-meso diaminopimelic acid (γ TriDAP) and comprise the peptide of glutamic acid-meso diaminopimelic acid dipeptides (for example, the small molecule mimetics of the analog of iE-DAP, iQ-DAP, iE-DAP or iQ-DAP, iE-DAP or iQ-DAP).

Therapeutic alliance

Compositions and method that the present invention expects the combination of adopting NOD1 promoter and other available anti-tumor therapeutic agents.It is low as far as possible that the dosage of conventional antitumor agent keeps usually, because may observe side effect at higher dosage.According to the present invention, NOD1 and/or the combination that improves NOD1 expression or active reagent and available antitumor agent can improve the scope (spectrum) of the effective cancer of described antitumor agent, and reduce the dosage of required described antitumor agent.Thereby the present invention expects current NOD1 related reagent and one or more antitumor or system cancer combination of agents.Obtainable any antitumor of those skilled in the art and system cancer reagent can use with current NOD1 related reagent.Yet in some embodiments, the antitumor of selection or antitumor and anticancer agent have different mechanism of action, or at how many dissimilar cancers or tumor work.For example, NOD1 related reagent of the present invention can make up with making cancer reagent or immune activation agent, makes up the proapoptosis effect of NOD1 and the anti-superfluous fruit of coming into force of described system cancer reagent, and/or by the inductive preceding immunoreation of described immune activation agent.Further, in some cases, except these methods, carry out the effect that radiotherapy or surgical intervention improve treatment.

The example of other chemotherapeutants that can together use with NOD1 related reagent of the present invention comprises hexamethyl melamine, bleomycin, Busulfan, calcium folinate, capecitabine, carboplatin, carmustine, benzenebutanoic acid helium mustard, cisplatin, cladribine, Crisantaspase, cyclophosphamide, cytosine arabinoside, dacarbazine, D actinomycin D, daunorubicin, docetaxel, amycin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, liposomal doxorubicin, lomustine, melphalan, purinethol, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pentostatin, procarbazine, Raltitrexed, streptozotocin, UFT, Temozoromide, thiotepa, thioguanine/thioguanine, topotecan, treosulfan, vincaleucoblastine, vincristine, desacetyl vinblastine amide, vinorelbine and its combination.

In some embodiments, use the NOD1 related reagent with one or more hormons.For example, can with the co-administered NOD1 related reagent of one or more androgens, Progesterone, estrogen or estrogen antagonist.By to cell or tissue that estrogen is reacted performance antagonistic effect, or by being positioned at the acceptor site of cell surface with the estrogen competition, estrogen antagonist works.For example, the medicine zitazonium (trade name: Nolvadex) or Arimidex (Anastrozole) be operable estrogen antagonist.Zitazonium has been used for the treatment of breast carcinoma, and in order to reduce the breast cancer incidence among the excessive risk women.As shown here, add zitazonium and partly blocked the inductive cell death of γ TriDAP-Nod1.Therefore, in some cases, zitazonium can be not used in the NOD1 compositions of the present invention.Yet in other examples, in the time of in being included in the therapeutic scheme that has comprised the NOD1 agent administration, zitazonium may be useful.

In another embodiment, NOD1 related reagent of the present invention and tumor necrosis factor (TNF α) are co-administered.TNF α is commercially available, for example, and from Pro-SpecTany TechnoGene Ltd. (Israel).The sequence of tumor necrosis factor can find in the ncbi database of ncbi.nlm.nih.gov.An example of the sequence of human TNF alpha is following to be provided in SEQ ID NO:4.

1 MSTESMIRDV?ELAEEALPKK?TGGPQGSRRC?LFLSLFSFLI

41 VAGATTLFCL?LHFGVIGPQR?EESPRDLSLI?SPLAQAVRSS

81 SRTPSDKPVA?HVVANPQAEG?QLQWLNRRAN?ALLANGVELR

121 DNQLVVPSEG?LYLIYSQVLF?KGQGCPSTHV?LLTHTISRIA

161 VSYQTKVNLL?SAIKSPCQRE?TPEGAEAKPW?YEPIYLGGVF

201 QLEKGDRLSA?EINRPDYLDF?AESGQVYFGI?IAL

Apoptosis

As described here, NOD1 can promote the apoptosis of tumor cell.In order to treat or prevent tumor growth, those skilled in the art can select to adopt the combination of antitumor agent and NOD1 related reagent described here.The compositions that contains various antitumor agents and NOD1 related reagent of the present invention can be tested in the available the whole bag of tricks of those skilled in the art, determines whether these compositionss promote the apoptosis of tumour regression and/or tumor cell best.

For example, by detect 3 of the dna fragmentation that produces during the apoptosis '-TUNEL (the TdT mediated dUTP nick end labeling fracture-end labelling) labelling of OH free-end, can the analysis of cells apoptosis (people such as Gavrieli. (1992) J.Cell Biol.119:493).TUNEL analyzes to generally comprise to add to the oligo DNA fragment of 180-bp (base pair) and catalytically adds nucleotide detecting described fragment, described nucleotide and combine with the chromophore system or combine with fluorescence labels.The stagewise existence of the DNA of 180-bp oligomer is apoptotic indication.The step that detects cell death according to the TUNEL method is commercially available, for example, and from Boehringer Mannheim (Cell Death test kit) and Oncor (apoptag Plus).

Current obtainable another apoptosis mark is annexin, with trade mark APOPTEST ^TMSell.The annexin mark is used in " apoptosis test regent box ", and it also is commercially available, for example, and from R ﹠amp; D Systems.During apoptosis, the phospholipid unsymmetry of cell membrane changes, thereby phospholipid is exposed on the adventitia.Annexins is in the proteinic congener group that exists under the situation of calcium in conjunction with phospholipid.Second reagent can be used for being connected with reagent iodate third ingot (PI) that detects annexin, and it is the DNA binding fluorescent dyes.When cell colony was exposed to two kinds of reagent, apoptotic cell dyeed for annexin positively, dyeed negatively for PI, and downright bad cell is a stained positive for the both, and living cells is that dyeing is negative for both.The additive method of test cell apoptosis is known in the art, can be used for method of the present invention.

Tumour regression can be evaluated by using animal model, for example, and the obtainable any animal model of those skilled in the art, or at the heteroplastic transplantation model of this description and explanation.

Heteroplastic transplantation model

The present invention also provides heteroplastic transplantation model, and it comprises the cell line that can form tumor in mice.When with these xenotransplantation cell inoculation mices, tumor occurs.Xenotransplantation cell line of the present invention lacks the Nod1 function, is sometimes referred to as Nod1 at this ^-/-Cell.Surprisingly, have identical genetic background, the cell except having the wild type relative, form the tumor of very fast degeneration with zero Nod1 allele.The Nod1 that only lacks the Nod1 function ^-/-Cell forms the tumor of continued growth.According to of the present invention, these isolating Nod1 ^-/-Cell is useful for research tumor and tumour regression.Nod1 of the present invention ^-/-Cell thereby can be used to develop chemotherapeutant and be used to study tumorigenic mechanism.

Isolating Nod1 of the present invention ^-/-An example of cell line is a MCF-7C20 cell line.When the C20 clone was transplanted in the male mice, the inventor had observed more healthy and stronger tumor growth.Polymerase chain reaction (PCR) amplification has been determined, and has imported in the allelic MCF-7C20 cell of functional NOD1 (that is, the C20Nod1 cell) in the MCF-7C20 cell and in reorganization, does not have progesterone receptor.Further PCR research has disclosed, and estrogen receptor alpha is present in each of all three kinds of MCF7 cell types (wild type MCF-7 cell, MCF-7C20 cell and MCF-7C20Nod1 cell).

Nod1 expression cassette and carrier

According to of the present invention, the NOD1 polypeptide can be recombinantly produced, and purification is used for using to the experimenter as antitumor agent then.In another embodiment, the nucleic acid of coding NOD1 can place expression cassette and/or expression vector.These NOD1 expression cassettes and expression vector also can be used as antitumor agent and use to the experimenter.Therefore, the invention provides Nod1 expression cassette and Nod1 expression vector.

The mammal of NOD1 polypeptide is expressed can be as people such as Dijkema., EMBO J. (1985) 4:761, people such as Gorman., Proc.Natl.Acad.Sci.USA (1982b) 79:6777, people such as Boshart., the realization of describing in Cell (1985) 41:521 and the U.S. Patent No. 4,399,216.Other features that mammal is expressed can be as Ham and Wallace, Meth.Enz. (1979) 58:44, Barnes and Sato, Anal.Biochem. (1980) 102:255, United States Patent(USP) Nos. 4,767,704,4,657,866,4,927,762,4,560,655, that describes among WO 90/103430, WO 87/00195 and the U.S. Patent No. RE 30,985 comes judicial convenience.Use this Nod1 nucleic acid can increase or replace endogenous Nod1 expression of gene.

Nod1 nucleic acid can place in linearity or the ring molecule.They can place the intramolecularly of self-replicating, or do not have the intramolecularly of replication sequence.They can be by they self, or regulates sequences by other and regulate, and this is known in the art.The nucleic acid construct of coding NOD1 can comprise transcription regulatory element, and for example, promoter element, enhancer or UAS element and tanscription termination subsignal are used for controlling Nod1 sequence transcribing at cell.

Nod1 nucleic acid can be used for expression cassette or gene delivery vector, in cell, preferred eukaryotic cell, sending Nod1 mRNA, total length NOD1 albumen, NOD1 fusion rotein, NOD1 polypeptide, or the fragment of NOD1 polypeptide.According to the present invention, gene delivery vector can be, for example, naked plasmid dna, virus expression carrier, or together with the Nod1 nucleic acid of the present invention of liposome or flocculating agent.

Nod1 nucleic acid can use the obtainable various technology in this area to import in the host cell that is fit to, for example the DNA of transferrins polycation mediation shifts, with nucleic acid transfection naked or sealing, liposome-mediated DNA transfer, the intracellular transport of emulsion pearl, protoplast fusion, viral infection, electroporation, the use of nucleic acid microinjection step and the transfection of calcium phosphate mediation of DNA bag quilt.

In an embodiment of the invention, described gene delivery vector comprises promoter and NOD 1 code nucleic acid.The example of operable promoter comprises tissue-specific promoter and passes through the activated promoter of cell proliferation, for example thymidine kinase and thymidylate synthetase promoter.Other preferred promoteres comprise by the activated promoter of the infection of virus, for example α-and beta-interferon promoter and can be by the hormone activated promoter of estrogen for example.Operable other promoteres comprise Moloney virus LTR, CMV promoter and mice albumin promoter.

Gene delivery vector can comprise virus sequence, for example virus replication starting point or packaging signal.These virus sequences can be selected from for example virus of Astrovirus, coronavirus, influenza virus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, retrovirus, togavirus or adenovirus.In some embodiments, described gene delivery vector is a recombinant retroviral vector.Recombinant retrovirus and its various uses and described in many lists of references comprise, for example people such as Mann., Cell 33:153,1983, Cane and Mulligan, Proc.Natl.Acad.Sci.USA 81:6349,1984, people such as Miller., Human Gene Therapy 1:5-14,1990, United States Patent(USP) Nos. 4,405,712,4,861,719 and 4,980,289 and PCT application Nos.WO 89/02,468, WO 89/05,349 and WO 90/02,806.Can use many reverse transcription virus gene delivery vectors in the present invention, comprise for example in EP 0,415,731; WO 90/07936; WO 94/03622; WO93/25698; WO 93/25234; U.S. Patent No. 5,219,740; WO 9311230; WO9310218; Vile and Hart, Cancer Res.53:3860-3864,1993; Vile and Hart, Cancer Res.53:962-967,1993; People such as Ram, Cancer Res.53:83-88,1993; People such as Takamiya., J.Neurosci.Res.33:493-503,1992; People such as Baba., J.Neurosurg.79:729-735,1993 (U.S. Patent No. 4,777,127, GB2,200,651, describe among EP 0,345,242 and the WO91102805 those.

Utilizable retroviral example comprises avian leukosis virus (ATCC Nos.VR-535 and VR-247), bovine leucosis poison (VR-1315), murine leukemia poison (MLV), ermine cell focus-induction virus (Koch el al., J.Vir.49:828,1984; With people such as Oliff., J.Vir.48:542,1983), murine sarcoma virus (ATCC Nos.VR-844,45010 and 45016), reticuloendotheliosis virus (ATCC Nos.VR-994, VR-770 and 45011), rous sarcoma virus, Mason-Pfizer monkey disease poison, baboon endogenous virus, endogenous cat family retrovirus are (for example, RD114) with as the mice or the rat gL30 sequence of retroviral vector.The MLV strain that therefrom can produce recombinant retrovirus comprises 4070A and 1504A (Hartley and Rowe, J.Vir.19:19,1976), Abelson (ATCC No.VR-999), Friend (ATCC No.VR-245), Graffi (people such as Ru., J.Vir.67:4722,1993; With Yantchev Neopksma 26:397,1979), Gross (ATCC No.VR-590), Kirsten (people such as Albino., J.Exp.Med.164:1710,1986), Harvey sarcoma virus (people such as Manly, J.Vir 62:3540,1988; With people such as Albino, J.Exp.Med.164:1710,1986) and Rauscher (ATCC No.VR-998) and MoloneyMLV (ATCC No.VR-190).Operable non-Murine retroviral is a rous sarcoma virus, for example Bratislava (people such as Manly., J.Vir.62:3540,1988; With people such as Albino., J.Exp.Med.164:1710,1986), the high titre of Bryan (for example, ATCC Nos.VR-334, VR-657, VR-726, VR-659 and VR-728), Bryan standard (ATCC No.VR-140), Carr-Zilber (people such as Adgighitov., Neoplasma 27:159,1980), Engelbreth-Holm (people such as Laurent., Biochem Biophys Acta 908:241,1987), Harris, Prague (for example, ATCC Nos.VR-772 and 45033) or Schmidt-Ruppin (ATCC Nos.VR-724 for example, VR-725, VR-354) virus.

According to the recombinant technique of the open and standard that provides at this (for example, people such as Sambrook., Molecular Cloning:A Laboratory Manual, 2 ^NdEdition (1989), people such as Sambrook., Molecular Cloning:A Laboratory Manual, 3 ^RdEdition (2001) and Kunkle, Proc.Natl.Acad.Sci.U.S.A.82:488,1985), can easily use above-mentioned retroviral any to make up or make up contrary expressing gene delivery vector.The contrary part of expressing expression vector can be from different retrovirus.For example, retrovirus LTR can be derived from murine sarcoma virus, from the tRNA binding site of rous sarcoma virus, and from the packaging signal of murine leukemia poison, and from the synthetic starting point of second chain of avian leukosis virus.By importing suitable package cell line, these recombinant retroviral vectors can be used for producing transduction competence retroviral vector particle (referring to, on November 29th, 1991 volume serial No.071800,921).

Can produce recombinant retrovirus, it instructs the site-specific integration of recombinant retrovirus genome to the specific region of host cell DNA.This site-specific integration is useful for sudden change or replacement endogenous NO D1 gene.By being incorporated into the chimeric intergrase in the retroviral particle, can mediate site-specific integration (referring to the serial No.08/445 that submits on, May 22 nineteen ninety-five, 466).Preferably, the recombinant viral genome delivery vector is the recombinant virus of replication defective.

The package cell line that is fit to use with above-mentioned reverse transcription virus gene delivery vector can easily prepare (referring to WO 92/05266), and is used to produce the production that Producer cell line (being also referred to as carrier cell system or " VCL ") is used for recombinant virus particle.In some embodiments of the present invention, package cell line is made by human (for example HT1080 cell) or ermine blast cell system, thereby allows the recombinant retrovirus gene delivery vector that generation can be survived in the passivation of human serum.Being structured among the WO 91/02805 of this recombinant retrovirus gene delivery vector described in detail.These recombinant retrovirus gene delivery vectors can be used for producing the competent retroviral particle of transduction by they being imported suitable package cell line.Similarly, according to provide at this open (also referring to, Berkner, Biotechniques 6:616-627,1988 and people such as Rosenfeld, Science 252:431-434,1991, WO 93/07283, WO 93/06223 and WO 93/07282), can easily prepare and use the adenoviral gene delivery vector.

Gene delivery vector also can be the recombinant adenovirus gene delivery vector.According to the open and obtainable information in this area that provides at this (referring to, Berkner for example, Biotechniques 6:616,1988 and people such as Rosenfeld., Science 252:431,1991, WO 93/07283, WO 93/06223 and WO 93/07282), can easily prepare and use this instrument.Also can make up and use the adeno-associated virus gene delivery vector to come body to send protein of the present invention or nucleic acid in cell interior or externally.People such as Chatteijee., Science 258:1485-1488 (1992), people such as Walsh., Proc.Natl.Acad.Sci.89:7257-7261 (1992), people such as Walsh., J.Clin.Invest.94:1440-1448 (1994), people such as Flotte., J.Biol.Chem.268:3781-3790 (1993), people such as Ponnazhagan., J.Exp.Med.179:733-738 (1994), people such as Miller, Proc.Natl Acad.Sci.91:10183-10187 (1994), people such as Einerhand, Gene Ther.2:336-343 (1995), people such as Luo, people such as Exp.Hematol.23:1261-1267 (1995) and Zhou have described the external use of adeno-associated virus gene delivery vector among the Gene Therapy 3:223-229 (1996).Use people such as Flotte in the body of these instruments., people such as Proc.Natl Acad.Sci.90:10613-10617 (1993) and Kaplitt, Nature Genet.8:148-153 has described in (1994).

In yet another embodiment of the present invention, gene delivery vector is from tunicle virus.This tunicle virus comprises Alphavirus, the U.S. serial No.08/405 that adds in March 15 nineteen ninety-five for example, and 627, describe among the WO 95/07994.Alphavirus comprises Sindbis and ELVS virus, can be the gene delivery vector that is used for nucleic acid of the present invention.In WO 94/21792, WO 92/10578 and WO 95/07994, Alphavirus has been described.Can make up and use several different alphavirus gene delivery vectors to come according to the present invention delivery of nucleic acids in cell.The representative example of this system is included in United States Patent (USP) NO.5, those that describe in 091,309 and 5,217,879.In some embodiments, be used for alphavirus gene delivery vector of the present invention and be included in those that WO 95/07994 describes.

Recombinant viral vector also can be based on the reorganization alphavirus viral vector of Sindbis virus.Can easily prepare the Sindbis construct, and many similar constructs.Sindbis viral gene delivery carrier generally comprise the 5 ' sequence that can start the Sindbis virus transcription, coding Sindbis non-structural protein nucleotide sequence, be passivated viral bonding pad, and Sindbis rna polymerase recognition sequence to prevent that fragment from transcribing.Choose wantonly, viral bonding pad can be modified, thereby transcribed nucleic acid is lowered, improves or keeps.Those of ordinary skill in the art will be appreciated that from the respective regions of other Alphaviruses and can use in those above-mentioned.

The viral bonding pad of the deutero-gene delivery vector of Alphavirus can comprise the first viral bonding pad, and it has been passivated stoping transcribing and the second viral bonding pad of nucleic acid, thereby it is modified transcribed nucleic acid and is lowered.The deutero-carrier of Alphavirus also can comprise the synthetic 5 ' promoter that can start viral RNA from cDNA, and 3 ' sequence of control tanscription termination.

Other reorganization tunicle viral gene delivery carriers that can use in the present invention comprise from Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), ross river virus (ATCC VR-373; ATCC VR-1246), equine encephalitis virus (ATCC VR923 is drawn in committee the inner; ATCC VR-1250; ATCC VR-1249; ATCC VR-532) those, and at United States Patent (USP) 5,091, in 309 and 5,217,879 and in WO 92/10578, describe those.

Be applicable to that other viral gene delivery carriers of the present invention comprise, for example, from poliovirus (people such as Evans., Nature 339:385,1989 and people such as Sabin, J.Biol.Standardization 1:115,1973) (ATCC VR-58); Rhinovirus (people such as Arnold., J.Cell.Biochem.L401,1990) (ATCC VR-1110); Poxvirus, for example canary pox virus or vaccinia virus (people such as Fisher-Hoch, PROC.NATL.ACAD.SCI.U.S.A.86:317,1989; People such as Flexner., Ann.N.Y.Acad.Sci.569:86,1989; People such as Flexner, Vaccine 8:17,1990; United States Patent(USP) Nos. 4,603,112 and 4,769,330; WO 89/01973) (ATCC VR-111; ATCC VR-2010); SV40 (people such as Mulligan., Nature 277:108,1979) (ATCC VR-305), (people such as Madzak, J.Gen.Vir 73:1533,1992); Influenza virus (people such as Luytjes., Cell 59:1107,1989; People such as McMicheal., The New England Journal of Medicine309:13,1983; With people such as Yap., Nature 273:238,1978) (ATCC VR-797); Parvovirus, for example adeno-associated virus (AAV) (people such as Samulski., J.Vir.63:3822,1989 and people such as Mendelson, Virology 166:154,1988) (ATCC VR-645); Herpes simplex virus (people such as Kit., Adv.Exp.Med.Biol.215:219,1989) (ATCCVR-977; ATCC VR-260); Nature 277:108,1979); The Human Immunodeficiency Viruses (EPO 386,882, people such as Buchschacher., J.Vir.66:2731,1992); Measles virus (EPO 440,219) (ATCC VR-24); A (ATCC VR-67; ATCCVR-1247), Aura (ATCC VR-368), Bebaru virus (ATCC VR-600; ATCCVR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCCVR-64; ATCC VR-1241), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369; ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR-66), Mucambo virus (ATCC VR-580; ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR-372; ATCC VR-1245), Tonate (ATCC VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374), Whataroa (ATCC VR-926), Y-62-33 (ATCC VR-375), O ' Nyong virus, east encephalitis disease (ATCC VR-65; ATCC VR-1242), west encephalitis (ATCCVR-70; ATCC VR-1251; ATCC VR-622; ATCC VR-1252), and coronavirus (people such as Hamre., Proc.Soc.Exp.Biol.Med.121:190,1966) (ATCCVR-740).

Nucleic acid of the present invention also can make up with flocculating agent and form gene delivery vector.In some embodiments, described flocculating agent is a polycation, for example polylysine, poly arginine, poly ornithine, protamine, spermine, spermidine and slough amine.The many suitable method that between flocculating agent and nucleic acid, produce to connect be known in the art (referring to, for example, the series number No.08/366 that December in 1994 was submitted on the 30th, 787).

In embodiment optionally, Nod1 nucleic acid or Nod1 polypeptide and liposome associate and form gene delivery vector.Liposome is little, lipid vesicles, is made up of the moisture compartment that is sealed by lipid bilayer, generally is spherical or microscler a little structure, the hundreds of dusts of diameter.Under the condition that is fit to, liposome can merge with the plasma membrane of cell, or with internalization the film of intracellular endocytosis vesicle of liposome merge, thereby the content that discharges it is in Cytoplasm.Yet, with cell show before the interaction that liposome membrane works as impermeable relatively layer, its isolation has also been protected its inclusions, for example, away from the degradability enzyme.In addition, because liposome is synthetic structure, can produce the specially designed liposome of the feature that comprises expectation.Referring to Stryer, Biochemistry, pp.236-240,1975 (W.H.Freeman, San Francisco, Calif); People such as Szoka., Biochim.Biophys.Acta 600:1,1980; People such as Bayer., Biochim.Biophys.Acta.550:464,1979; People such as Rivnay., Meth.Enzymol.149:119,1987; People such as Wang., PROC.NATL.ACAD.SCI.U.S.A.84:7851,1987, people such as Plant., Anal.Biochem.176:420,1989, and U.S. Patent No. 4,762,915.Liposome can seal various nucleic acid and peptide molecule, comprises DNA, RNA, plasmid, comprises those disclosed expression of nucleic acids construct and Nod1 polypeptide in the present invention.

Be used for liposome product of the present invention and comprise cationic (positively charged), anionic (electronegative) and neutral goods.Cationic-liposome has shown mediation plasmid DNA (people such as Feigner, Proc.Natl.Acad.Sci.USA 84:7413-7416,1987), mRNA (people such as Malone., Proc.Natl.Acad.Sci.USA 86:6077-6081,1989) and the transcription factor of purification (people such as Debs, J.Biol.Chem.265:10189-10192,1990) send in the cell with functional form, cationic-liposome is to obtain easily.For example, N[1-2,3-dioleyloxy) propyl group]-N, N, N-triethylamine (DOTMA) liposome is with trade mark Lipofectin ^TMCan be from GIBCO BRL, Grand Island, N.Y obtains.Also referring to people such as Feigner, Proc.Natl.Acad.Sci.US491:5148-5152.87,1994.Other commercially available liposomees comprise Transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger).Other cationic-liposomes can use the available technology in this area to prepare from the material that is easy to obtain.Synthetic for DOTAP (1,2-two (oleoyl oxygen base)-3-(Trimethylamine) propane) liposome, referring to, people such as Szoka for example., Proc.Natl.Acad.Sci.USA 75:4194-4198,1978; With WO 90/11092.

Similarly, anion and neutral fat plastid are to obtain easily, and for example (Birmingham Ala.), maybe can use the material that is easy to obtain easily to prepare from Avanti PolarLipids.These materials comprise phosphatidylcholine, cholesterol, PHOSPHATIDYL ETHANOLAMINE, dioleyl phosphatidyl choline (DOPC), dioleoyl phosphatidyl glycerol (DOPG), dioleoyl PHOSPHATIDYL ETHANOLAMINE (DOPE) etc.These materials also can with DOTMA and DOTAP parent material mixed to be fit to.The method of using these material manufacture liposomees is well known in the art.

Liposome can comprise multilamelar vesicles (MLV), little monolayer vesicle (SUV) or big monolayer vesicle (LUV).Use methods known in the art to prepare various liposome-nucleic acid complexes.Referring to, people such as Straubinger for example, METHODS OF IMMUNOLOGY (1983), Vol.101, pp.512-527; People such as Szoka., Proc.Natl.Acad.Sci.USA 87:3410-3414,1990; People such as Papahadjopoulos, Biochim.Biophys.Acta 394:483,1975; People such as Wilson., Cell 17:77,1979; Deamer and Bangham, Biochim.Biophys.Acta 443:629,1976; People such as Ostro., Biochem.Biophys.Res.Commun.76:836,1977; People such as Fraley., Proc.Natl.Acad Sci.USA76:3348,1979; Enoch and Strittmatter, Proc.Natl.Acad Sci.USA 76:145,1979; People such as Fraley., J.Biol.Chem.255:10431,1980; Szoka andPapahadjopoulos, Proc.Natl.Acad.Sci.USA 75:145,1979; And people such as Schaefer-Ridder., Science 215:166,1982.

In addition, can comprise that lipoprotein and nucleic acid of the present invention together are used for sending to cell.The example of this lipoprotein comprises Chylomicron, HDL, IDL, LDL and VLDL.Also can use these proteic mutants, fragment or fusions.Also can use the modification body of the lipoprotein of natural generation, for example acetylizad LDL.These lipoproteins can be with the cell of sending the targeted expression lipoprotein receptor of nucleic acid.In some embodiments, if lipoprotein and nucleic acid together comprise, do not comprise other targeting parts in compositions.

Also can use the receptor-mediated targeted delivery of Nod1 nucleic acid to particular organization.People such as for example Findeis. (1993), Trends in Biotechnol.11,202-05; People such as Chiou. (1994), Gene Therapeutics:Methods and Applications of Direct GeneTransfer (J.A.Wolff, ed.); Wu ﹠amp; Wu (1988), J.Biol.Chem.263,621-24; People such as Wu. (1994), J.Biol.Chem.269,542-46; People such as Zenke. (1990), Proc.Natl.Acad.Sci.U.S.A.87,3655-59; People such as Wu. (1991), J.Biol.Chem.266 has described receptor-mediated DNA delivery technique among the 338-42.

In another embodiment, exposed nucleic acid molecules is used as gene delivery vector, for example, and as what describe in WO 90/11092 and the U.S. Patent No. 5,580,859.This gene delivery vector can be DNA or RNA, in some embodiments, is connected with the adenovirus of deactivation.People such as Curiel., Hum.Gene.Ther.3:147-154,1992.Other carriers that are fit to comprise the DNA-part (people such as Wu., J.Biol.Chem.264:16985-16987,1989), lipid-DNA compositions (people such as Feigner., Proc.Natl.Acad.Sci.USA 84:74137417,1989), liposome (people such as Wang, Proc.Natl.Acad Sci.84:7851-7855,1987) and microinjection (people such as Williams., Proc.Natl.Acad.Sci.88:2726-2730,1991).

By nucleic acid being coated on the biodegradable emulsion pearl, can improving exposed nucleic acid and absorb effectiveness into cell.This method has been utilized observed result, and when hatching with cell in cultivation, the nuclear week zone of cell is transported and be concentrated in to the emulsion pearl effectively.In the time of in being expelled to muscle, pearl will be transported in the cell then.After the endocytosis that is started by the emulsion pearl, the emulsion pearl of nucleic acid bag quilt will be by in the transporte to cells effectively, thereby improves gene transfer and express and render a service.This method can improve their hydrophobicity by handling pearl, thereby promotes the destruction and the release of nucleic acid in Cytoplasm of endosome further to improve.

The nucleic acid of coding NOD1 is in a similar manner in the transfered cell.Mechanical means, for example microinjection, liposome-mediated transfection, electroporation or calcium phosphate precipitation can be used for NOD1 nucleic acid construct transfered cell, promote apoptosis and/or death of neoplastic cells.Alternatively, keep DNA construct if wish cytotostatic ground, DNA construct can provide on plasmid, and keeps as element independently, or is incorporated in the genome of cell, and this is known in the art.

The expression of endogenous NO D1 gene in cell also can import DNA construct by reading frame ground by homologous recombination and endogenous NO D1 gene and change, described DNA construct comprises the NOD1 target sequence, regulates sequence, exon and unpaired donor splicing site position, thereby forms the homologous recombination cell that comprises described DNA construct.New transcript unit can be used for opening or closing as desired the NOD1 gene.Influence this method that endogenous gene expresses in U.S. Patent No. 5,641, instructed in 670.

The integration of NOD1 nucleic acid in the genome of cell line or tissue that discharges can monitor by methods known in the art.The Southern trace of the NOD1 nucleic acid that for example, can send.The change indication of the segmental big or small aspect of the nucleic acid of sending is integrated.The nucleic acid of sending duplicate can be especially by certification mark the mixing of nucleotide, in conjunction with detecting with the NOD1 probe hybridization.The NOD1 expression of nucleic acids can be by detecting the generation with the NOD1mRNA of the nucleic acid hybridization of sending, or monitor by detecting NOD1 albumen.NOD1 albumen can immune detection.

The RIP2 regulator

According to of the present invention, when " kinase deficiency " RIP2 expresses in cell, cell for apoptosis by sensitization.As used herein, " kinase deficiency " RIP2 is meant does not have the RIP2 of kinase function polypeptide basically.Therefore, the invention provides does not have the RIP2 of kinase function polypeptide, and RIP2 inhibitors of kinases and being used to is expressed expression cassette and the expression vector of the RIP2 of kinase deficiency.

RIP2 is a serine/threonine kinase, and its carboxyl terminal at it contains the CARD domain, and has shown induce the NF-kB activation in the overexpression system.RIP2 also shown regulate congenital and the adaptive immunity reaction in play effect.The mice of Rip2 defective only has at Toll sample receptor stimulating agent, for example the immunoreation that weakens of lipopolysaccharide (LPS).

The sequence of RIP2 can for example obtain in the ncbi database.Referring to website ncbi.nlm.nih.gov.For example, the aminoacid sequence of human RIP2 provides so that reference is SEQ ID NO:5 (NCBI registration number AAC27722 following; Gi:3342910).

1 MNGEAICSAL?PTIPYHKLAD?LRYLSRGASG?TVSSARHADW

41 RVQVAVKHLH?IHTPLLDSER?KDVLREAEIL?HKARFSYILP

81 ILGICNEPEF?LGIVTEYMPN?GSLNELLHRK?TEYPDVAWPL

121 RFRILHEIAL?GVNYLHNMTP?PLLHHDLKTQ?NILLDNEFHV

161 KIADFGLSKW?RMMSLSQSRS?SKSAPEGGTI?IYMPPENYEP

201 GQKSRASIKH?DIYSYAVITW?EVLSRKQPFE?DVTNPLQIMY

241 SVSQGHRPVI?NEESLPYDIP?HRARMISLIE?SGWAQNPDER

281 PSFLKCLIEL?EPVLRTFEEI?TFLEAVIQLK?KTKLQSVSSA

321 IHLCDKKKME?LSLNIPVNHG?PQEESCGSSQ?LHENSGSPET

361 SRSLPAPQDN?DFLSRKAQDC?YFMKLHHCPG?NHSWDSTISG

401 SQRAAFCDHK?TTPCSSAIIN?PLSTAGNSER?LQPGIAQQWI

441 QSKREDIVNQ?MTEACLNQSL?DALLSRDLIM?KEDYELVSTK

481 PTRTSKVRQL?LDTTDIQGEE?FAKVIVQKLK?DNKQMGLQPY

521 PEILVVSRSP?SLNLLQNKSM

As noted above, and illustrate in greater detail in an embodiment, when another domain of RIP2 and function (for example, the CARD domain) when mainly being functional, the cell of RIP2 of expressing kinase deficiency for apoptosis by sensitization.Therefore, the present invention relates to suppress the method that the kinase whose reagent exposing cell of RIP2 makes the sensitization of cell pair cell apoptosis by using.In another embodiment, the present invention relates to by in animal, treating method for cancer to the RIP2 inhibitors of kinases of administration treatment effective dose.The RIP2 inhibitors of kinases also can with NOD1 polypeptide or Nod1 nucleic acid, or it is co-administered to regulate the active reagent of NOD1.

Any available RIP2 inhibitor can use in the compositions and methods of the invention, in some embodiments, can use the p38 inhibitor to suppress the RIP2 kinase function.For example, the p38 inhibitor that can be used to suppress RIP2 comprises 2-(4-chlorphenyl)-4-(4-fluorophenyl)-5-pyrimidine-4-base-1,2-dihydro-pyrazolone-3, SC68376, SB203580 (Iodo), SB202190, SB203580, SB203580 (sulfone), PD169316, SB220025, SKF-86002, SB239063 or ML 3163.The structure of SB220025, SB203580 and PD169316 below is provided.

These inhibitor can with the comparable concentration of concentration that is used to suppress p38 under use.

In another embodiment, the invention provides by observation test reagent whether can suppress the method that the RIP2 kinase activity is identified the RIP2 inhibitor.This method can be carried out in external or body.When under the situation that lacks test agent, carrying out the RIP2 analysis, can comprise tester.The active reduction of RIP2 shows that described test agent is the RIP2 inhibitor under the situation of test agent existing.

Compositions

Use NOD1 polypeptide and NOD1 part, the salt and NOD1 nucleic acid and/or the RIP2 inhibitors of kinases that comprise them promote apoptosis and tumour regression, adjusting NOD1 activity, or realize at least a symptom that and cancer condition or the other diseases relevant with inappropriate cell growth is correlated with.As described here, can comprise other reagent, for example, the nucleic acid of antitumor agent, chemotherapeutant, TNF, RIP2 inhibitors of kinases, RIP2 kinase deficiency polypeptide, coding RIP2 kinase deficiency polypeptide, or the like.

In order to realize desired effects, can be used as dosage independent or that separate use NOD1 polypeptide, part, nucleic acid and with other combination of agents.For example, NOD1 polypeptide, part and nucleic acid can with at least about 0.01mg/kg to about 500 to 750mg/kg, at least about 0.01mg/kg to about 300 to 500mg/kg, at least about 0.1mg/kg to about 100 to 300mg/kg or use to the dosage of about 50 to 100mg/kg body weight, although other dosage can provide useful result at least about 1mg/kg.The quantity of using will depend on that various factors changes, include but not limited to, polypeptide, part or nucleic acid, mammiferous disease, body weight, health, health, the age selected, realize that prevention is still treated and whether described polypeptide, part or nucleic acid are chemically modified.These factors can adopt available other test macros of animal model or this area easily to determine by the clinicist.

Use according to treatment reagent of the present invention can be with single dose, with multiple dose, with continuous or intermittent mode, depends on that for example, receiver's physical qualification, the purpose of using are therapeutic or preventative, and known other factors of skilled practitioners.Using of polypeptide of the present invention, part, nucleic acid and other reagent can be successive basically in predetermined periods, maybe can be the dosage at a series of intervals.Local and general is used all and is expected.

In order to prepare compositions, synthetic or obtain polypeptide, part, nucleic acid and reagent, as required or expectation come purification, lyophilizing and stabilisation then.Can regulate described polypeptide, part or nucleic acid then to suitable concentration, and optional and other agent combination.The absolute weight that is included in given polypeptide, part or nucleic acid in the unit dose can change widely.For example, can use about 0.01 to about 2g or about 0.1 to about 500mg polypeptide at least a of the present invention, nucleic acid or antibody, or multiple polypeptides, part and nucleic acid.Alternatively, unit dose can be from about 0.01g to about 50g, from about 0.01g to about 35g, from about 0.1g to about 25g, from about 0.5g to about 12g, from about 0.5g to about 8g, about 0.5g is to about 4g, or changes to about 2g from about 0.5g.

The daily dose of polypeptide of the present invention, part or nucleic acid also can change.This daily dose can for example about 0.1g/ days to about 50g/ days, from about 0.1g/ days to about 25g/ days, from about 0.1g/ days to about 12g/ days, from about 0.5g/ days to about 8g/ days, from about 0.5g/ days to about 4g/ days with in about 0.5g/ days to about 2g/ days scope.

Thereby, one or more unit dosage forms that are fit to that comprise therapeutical peptide of the present invention, part or nucleic acid can be used by all means, comprise oral, parenteral (comprise subcutaneous, intravenous, intramuscular and endoperitoneal), rectum, skin, wear (respiratory) approach of epidermis, intrathoracic, non-nutrient film and intranasal.In some embodiments, NOD1 polypeptide, part or nucleic acid are used to tumor or cancer site partly.

Treatment reagent also can be prepared and be used for continuing to discharge (for example, using microencapsulation, referring to WO 94/07529 and U.S. Patent No. 4,962,091).When suitable, described preparation can present with dispersive unit dosage forms easily, can prepare by the known any method of drug world.These methods can comprise treatment reagent and liquid-carrier, solid matrix, semi-solid carrier, finely divided solid carrier or its combined hybrid, then, if desired, with the product importing or be configured as the delivery system of expectation.

When treatment reagent of the present invention is prepared to be used for when Orally administered, their general and pharmaceutically acceptable carrier, diluent or the incompatible formation pharmaceutical preparation of vehicle group or unit dosage forms.For Orally administered, described treatment reagent can be rendered as powder, granular preparation, solution, suspension, Emulsion, or be in natural or synthetic polymer or resin in be used for from chewing gum picked-up active component.Treatment reagent also can be used as bullet, eclegm or paste and exists.Orally administered treatment reagent of the present invention also can be used for continue discharging by preparation, and for example, treatment reagent can coated, micro encapsulation seal, or places and continue delivery apparatus.Gross activity composition in this preparation comprises 0.001 to 99.9% of weight of formulation.

" pharmaceutically acceptable " is meant that other compositions of carrier, diluent, excipient and/or salt and preparation are compatible, and harmless for its receiver.

The pharmaceutical preparation that contains treatment reagent of the present invention can be used known and composition that be easy to obtain prepares by step known in the art.For example, can treat reagent with common excipient, diluent or carrier preparation, and form tablet, capsule, solution, suspension, powder, aerosol or the like.The example that is suitable for excipient, diluent and the carrier of these preparations comprises buffer, and filler and extender, for example starch, cellulose, saccharide, mannitol and silicon derivative.Also can comprise binding reagents, for example carboxymethyl cellulose, hydroxy methocel, hydroxypropyl emthylcellulose and other cellulose derivatives, alginate, gelatin and polyethylene-ketopyrrolidine.Can comprise humidification reagent, glycerol for example, disintegrating agent is calcium carbonate and sodium bicarbonate for example.Also can comprise and be used to delay dissolved reagent, for example paraffin.Can also comprise and heavily absorb accelerator, for example quaternary ammonium compound.Can comprise surfactant, for example hexadecanol and glyceryl monostearate.Can add adsorptive support, for example Kaolin and bentonite.Can also comprise lubricant, for example Pulvis Talci, calcium and magnesium stearate, and solid polyethylene glycol.Can also add antiseptic.Compositions of the present invention can also contain thickening agent, for example cellulose and/or cellulose derivative.They also can contain natural gum, for example xanthan gum, guar gum or carbon natural gum or Radix Acaciae senegalis, or as selecting, Polyethylene Glycol, bentonite and montorillonite clay, or the like.

For example, the tablet or the lozenge that contain treatment reagent of the present invention can comprise buffer reagent, for example calcium carbonate, magnesium oxide and magnesium carbonate.Lozenge and tablet also can comprise the non-activity composition, for example cellulose, pregelatinized Starch, silicon dioxide, HYDROXY PROPYL METHYLCELLULOSE, magnesium stearate, microcrystalline Cellulose, starch, Pulvis Talci, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol, sodium phosphate, zinc stearate, or the like.Hard or the soft gelatin capsule that contains at least a treatment reagent of the present invention can contain the non-activity composition, for example gelatin, microcrystalline Cellulose, sodium lauryl sulphate, starch, Pulvis Talci and titanium dioxide or the like, and liquid-carrier for example Polyethylene Glycol (PEG) and vegetable oil.In addition, the lozenge or the tablet that contain the intestinal bag quilt of one or more treatment reagent of the present invention are designed to resist under one's belt disassociation, and are dissolved in duodenal more neutral and alkaline environment.

Treatment reagent of the present invention also can be formulated as elixir or solution, is used for Orally administeredly easily, or is formulated as and is suitable for parenteral administration, for example by solution intramuscular, subcutaneous, endoperitoneal or that intravenous route is used.The pharmaceutical preparation of treatment reagent of the present invention also can be adopted form moisture or anhydrous solution or dispersion, or as selecting, adopts the form of Emulsion or suspension or ointment.

Thereby, treatment reagent can (for example be used for parenteral administration by preparation, by injection, for example, bolus injection or continuous infusion) and can present with syringe, the infusion container of small size or the unit dosage form in multi-dose container of filling in the ampoule bottle, in advance.As mentioned above, can add antiseptic and help keep shelving the phase of dosage form.Active polypeptide, nucleic acid or antibody and other compositions can be formed on suspension, solution or the Emulsion in oil or the water carrier, can contain prescription reagent, for example suspend, stablize and/or dispersion reagent.Alternatively, movable polypeptide, nucleic acid or antibody and other compositions can be with the formation of powder, by the aseptic solid of aseptic separation, or by from the solution lyophilizing, are used for before use and suitable carriers, for example aseptic pyrogen-free water structure.

These preparations can contain pharmaceutically acceptable carrier well known in the art, excipient and adjuvant.For example, possible be to use that acceptable organic solvent prepares solution on one or more physiology's viewpoints, except water, described organic solvent is selected from solvent, for example acetone, ethanol, isopropyl alcohol, glycol ether are for example with title " Dowanol " polyglycols and Polyethylene Glycol product sold, the C of short chain acids ₁-C ₄Arrcostab, ethyl or isopropyl lactic acid, fatty acid triglycercide are for example with title " Miglyol " product sold, isopropyl myristate, animal, mineral and vegetable oil and polysiloxanes.

If wish, might add and select antioxidant, surfactant, other antiseptic, film former, keratolytic agent or antiblocking (comedolytic) reagent, aromatic, flavoring agent and coloring agent.Can add antioxidant, for example tert-butyl hydroquinone, butylated hydroxyanisole (BHA), Yoshinox BHT and alpha-tocopherol and its derivant.

In addition, polypeptide, part and nucleic acid are well suited for preparation for the dosage form that continue to discharge etc.Described preparation can be formed like this, thereby they may be over a period to come, and for example the specific part at intestinal or respiratory tract discharges treatment reagent.Can be from polymer, for example polyactide-oxyacetate, liposome, microemulsion, microgranule, receive grain or wax and make bag by, peplos and protectiveness substrate.By inherent equipment, for example stent, conduit, peritoneal dialysis bucket, drainage device or the like are useful for bag for these bag quilts, peplos and protectiveness substrate.

For surface applied, treatment reagent can be used for directly using to the target area by preparation known in the art.The form that is mainly local application and regulates takes for example ointment, milk, gel, dispersion or microemulsion, thicken to bigger or than the washing liquid of low degree, the form of soaking into plate, ointment or rod, aerosol formulations (for example, spraying or foam), soap, detergent, washing liquid or soap-cake.Other conventionally forms that are used for this purpose comprise binder or other polymeric covers, ointment, ointment, washing liquid, paste, jelly, spraying and the aerosol of wound dressing, bag quilt.Thereby treatment reagent of the present invention can be sent via paster that is used for dermal administration or binder.Alternatively, polypeptide, part and/or nucleic acid can be mixed with the adhesiveness polymer, for example the part of polyacrylate or acrylate/vinylacetic acid ester copolymer.For prolonged application, that may expect is to use liner lamination micropore and/or that can suck, thereby the aquation of skin or dipping can be minimized.Laying can be to provide any suitable thickness of desired protection and support function.The thickness that is fit to generally is from about 10 to about 200 microns.

For example, ointment and ointment can be prepared with aqueous or oleaginous base, add the thickening agent and/or the gellant that are fit to.Washing liquid can be prepared with aqueous or oleaginous base, generally also will contain one or more emulsifying agents, stabilizing agent, dispersant, suspending agent, thickening agent or coloring agent.Treatment reagent also can be sent via iontophoresis, for example, and as United States Patent (USP) NO.4,140,122; 4,383,529; Or it is disclosed in 4,051,842.The percetage by weight of the treatment reagent of the present invention that exists in surface preparation will depend on various factors, generally be 0.001% to 95% of total formulation weight amount, generally be 0.01-85% by weight.

Drop, for example eye drop or nasal drop can be prepared with one or more treatment reagent in moisture or anhydrous substrate, also contain one or more dispersants, solubilizing agent or suspending agent.Spray from the packages sealed delivering liquid easily.Drop can be via the simple eye drop bottle that lid is arranged or via the plastic bottle that is suitable for dripping shape delivering liquid content, send via the closure of given shape.

Treatment reagent can further be prepared and be used in the oral cavity or the throat surface applied.For example, active component can be formulated as the substrate that further comprises seasoning, the lozenge that is generally sucrose and Radix Acaciae senegalis or tragacanth; For example comprise the lozenge of compositions in gelatin and glycerol or sucrose and the Radix Acaciae senegalis at inert base; And the mouthwash that in the liquid-carrier that is fit to, comprises compositions of the present invention.

Pharmaceutical preparation of the present invention can comprise, as optional ingredients, and pharmaceutically acceptable carrier, diluent, solubilizing agent or emulsifying agent and available in the art salt type.The example of these materials comprises normal saline solution, for example physiology's buffered saline solution and water.The specific limiting examples of useful carrier and/or diluent comprises water and the acceptable buffered saline solution of physiology, for example phosphate buffered salt solution of pH 7.0-8.0 in pharmaceutical preparation of the present invention.

Treatment reagent of the present invention also can be used to respiratory tract.Thereby the present invention also provides aerosol drug preparation and the dosage form that is used for method of the present invention.Usually, these dosage forms comprise quantity, at least a reagent of the present invention of the clinical symptoms of effective treatment or prevention particular cancers, tumor, disease or relevant disease.Weaken this treatment for cancer that is considered in the scope of the present invention significantly on any statistics according to one or more symptoms of the cancer of method of the present invention treatment.

Alternatively, for by using of sucking or be blown into, compositions can be taked the form of dry powder, for example treats for example mixture of powders of lactose or starch of reagent and the powder substrate that is fit to.The powder compositions can be present in unit dosage forms, for example capsule or box, or for example in gelatin or the blister package, therefrom powder can use by the inhaler of inhaler, insufflator or metering (referring to, for example, at Newman, S.P.in Aerosols and the Lung, Clarke, S.W.and Davia, D.eds., pp.197-224, Butterworths, London, England, the metered dose inhaler (MDI) and the Diskus of disclosed sealing in 1984).

When using with the form of aerosol or suction, treatment reagent of the present invention can also be used in aqueous solution.Thereby other aerosol drug preparations can comprise, for example, the acceptable buffered saline solution of physiology, contain have an appointment between 0.1mg/ml and the about 100mg/ml, be specific to the disease that will treat or one or more treatment reagent of the present invention of disease.The dry aerosol of insoluble or the finely divided solid polypeptide, part or the nucleic acid particle form that float on a liquid also is useful in practice of the present invention.Polypeptide of the present invention, part or nucleic acid can be formulated as face powder, and comprise and have between the about 1 and 5 μ m, as the finely divided particle of selecting the average particle size between the 2 and 3 μ m.Finely divided particle can be by using this area the pulverizing and the sieving of known technology prepare.Described granule can be used by the finely divided material that sucks scheduled volume, and described material can be a form of powder.It being understood that the unit content of the active component that contains in the single aerosol dosage of every kind of dosage form, itself needn't be configured for treating the effective dose of specific infection, disease or disease, because essential dosage can reach by using a plurality of dosage units.In addition, use the dosage that is lower than in the dosage form individually or in a series of using, can reach effective dose.

For using to upper respiratory tract (nose) or lower respiratory tract by sucking, treatment reagent of the present invention easily from aerosol apparatus or packages sealed or send aerosol injection other easily device discharge.Packages sealed can comprise suitable propellant, for example dichlorodifluoromethane, Arcton 11, Dichlorotetrafluoromethane, carbon dioxide or other gas that is fit to.For the aerosol of pressurization, dosage unit can be determined by the quantity that provides valve to send metering.Aerosol apparatus includes but not limited in United States Patent (USP) NO.4,624,251; 3,703,173; Those that describe in 3,561,444 and 4,635,627.Aerosol delivery systems type disclosed herein can obtain from the source of many commerce, comprise Fisons Corporation (Bedford, Mass.), Schering Corp. (Kenilworth, NJ) and American Pharmoseal Co. (Valencia, CA).For intranasal administration, treatment reagent also can be used via nasal drop, liquid spray, for example via plastic bottle aerosol apparatus or metered dose inhaler.Typical aerosol apparatus is Mistometer (Wintrop) and Medihaler (Riker).

In addition, active component also can use with the other treatment agent combination, for example, analgesic, anti-inflammatory agent, hydryllin, antimicrobial, bronchodilator or the like, the condition that no matter is used to describe still is some other condition.

The invention further relates to the pharmaceutical composition that is used to regulate the packing that Nod1 expresses, for example test kit or other containers.Described test kit or container have held the pharmaceutical composition of the treatment effective dose that is used to regulate Nod1 gene expression and have been used to use described pharmaceutical composition to regulate the description of Nod1 gene expression.Described pharmaceutical composition comprises at least a Nod1 nucleic acid of the present invention, thereby is conditioned for treating effective dose Nod1 gene expression.Described compositions also can contain antitumor agent or chemotherapeutant.

In another embodiment, the invention provides the pharmaceutical composition that is used to regulate the active packing of NOD1.Described test kit or container have held and have been used to regulate the pharmaceutical composition of the active treatment effective dose of NOD1 and are used to use described pharmaceutical composition to regulate the active description of NOD1.Described pharmaceutical composition comprises at least a NOD1 polypeptide of the present invention or NOD1 part, thereby is conditioned for treating effective dose NOD1 activity.

To further specify the present invention with reference to following detailed example, described embodiment provides for illustration the present invention, and is not intended to be subject to this.

Embodiment 1:NOD1 induces and causes tumour regression

This embodiment has illustrated that Nod1 makes cell to the inductive apoptosis sensitization of TNF α, and under the situation that lacks any other known apoptosis triggering, the NOD1 ligands specific is induced the apoptosis in the MCF-7 breast cancer cell.Adopt that animal model proves the effect of NOD1 in tumour regression in the body, it relates to the SCID mice and carries out xenotransplantation.These data show that Nod1 plays a key effect in the control growth of tumour cell.

Material and method

Cell culture. human breast cancer cell line MCF-7 and SKBR3 maintain in Eagle ' the s culture medium that is supplemented with 10% fetal bovine serum, 2mM glutamine, 100U/ml penicillin and 10 μ g/ml modification streptomycin, Dulbecco.

Mammal expression construct and direct mutagenesis. human FLAG-Nod1, FLAG-Nod2cDNAs be available from Dr Gabriel Nuez, and people such as Y.Ogura, and J.Biol.Chem.276 has described in 4812 (2001).Human Myc-RIP2 wt and Myc-RIP2 (K47A) are being so kind as to give from Dr.C.Vincenz (University of Michigan Medical School).Make up Nod1 mutant (V41Q and K208R) by direct mutagenesis.By deletion carboxyl terminal CARD domain and be cloned into the pcDNA4/Myc/His plasmid (Invitrogen, Carlsbad produce Myc-RIP2 Δ CARD in CA).Nucleotide sequence is all confirmed by dna sequencing.

The retrovirus transfection. the range gene that uses in this research is cloned in pMSCV-Blasto, pBabe-Puro or the pBabe-Neo retroviral vector.Use obtainable step stable transfection MCF-7 cell.Briefly, with coding selected Nod1, Nod1 and other proteinic retroviral vector transfection amphophilic 293 cells.After the transfection 24 hours, the cell overnight incubation is produced virion at 32 ℃.Second day with containing recombinant retrovirus is particulate to be contained viral 293 cell conditioned medium liquid inductances and dyes target cell.With 10 μ g/ml powder for curing rice blast-S (Calbiochem EMD Biosciences Inc., San Diego, CA), 500 μ g/ml gentamycins (Invitrogen, Carlsbad, CA) or 5 μ g/ml puromycins (Calbiochem) select cell.Confirm the expression of all constructs by the Western engram analysis.In order to determine the effect of 17 beta estradiols (Calbiochem), cultured cell in the no phenol red DMEM that is supplemented with the FCS that 5% active carbon sloughs.Cell is seeded into 24h in the 96 hole flat boards of 17 beta estradiols (E2), and uses with various concentration ³H-thymus pyrimidine (1 μ Ci/ hole, MPBiomedicals, Irvine, CA) pulse.Harvesting on glass fiber filter is measured radioactivity by liquid scintillation.

Western engram analysis and immunoprecipitation. washed cell widely, and use the lysis buffer cracking that contains 50mM Hepes, 100mM NaCl, 2mM EDTA, 10% glycerol, 1%Nonidet P-40,14 μ M Gastric inhibitory polypeptide A, 100 μ M leupeptins, 3mM benzenecarboximidamide, 1mM PMSF, 1mM tetrasodium pyrophosphate, 10mM sodium orthovanadate, 100U/ml aprotinin and 100mM sodium fluoride.After hatching 30 minutes on ice, cellular lysate is carried out centrifugal (14000rpm, 10 minutes, 4 ℃), reclaims supernatant.For immunoprecipitation, cellular lysate was mixed 3 hours at 4 ℃ under constant stirring with 5 μ g antibody.Allow that immune complex spends the night in conjunction with 20 μ l protein A-Sepharose pearls, wash pearl three times with lysis buffer.The separating immune precipitate is transferred on the pvdf membrane on SDS-PAGE.

Cell survival is analyzed. and iodate third ingot is discharged and analyzed: irritation cell is 2 days as noted.Subsequently, harvesting (contains 1%FCS and 0.1%NaN at the FACS buffer ₃PBS) in washed twice, be resuspended in the FACS buffer (4 μ g/ml) that contains iodate third ingot (PI).Use FACSCalibur flow cytometry (Becton Dickinson, Mountain View, CA) degree of analysis of cells death.DAPI dyeing: the MCF-7 cell places the chamber microscope slide, stimulates 2 days.Use the PBS washed cell, (SigmaChemical Co., St.Louis's apoptotic nuclear MO) dye with 1 μ g/ml DAPI.Fixed cell in 4% paraformaldehyde is by the fluorescence microscopy inspection.

TUNEL dyeing: handle cell with γ TriDAP, (ROCHE, Indianapolis IN) monitor apoptotic nuclear by TUNEL (the UTP fracture-end labelling of TdT mediation) analysis according to the description of producer.

The concentration of IL-8 is used 96 holes immunity dull and stereotyped (Dynatech Laboratories, Chantilly VA) is measured by ELISA in the culture supernatant of the HeLa cell of IL-8 ELISA. transfection.Anti-people IL-8Ab (the R ﹠amp of mAb MAB208 that use is used to catch and biotinylated multi-clone rabbit; D Systems, Minneapolis MN), is the Succ-PEG-DSPE HRP that is used to detect subsequently, carries out ELISA.

Human xenotransplantation in the nude mice. with MCF-7 Blasto, MCF-7 C20 and MCF-7C20/Nod1 cell trypsinized, with the PBS washing once, be resuspended to 1.5 * 10 ⁷The concentration of/ml.Every kind of suspension of 200 microlitres is inoculated into hypodermically the flank of athymism SCID/SCID or SCID/Nod (non-adiposis characteristic of disease) female mice.Evaluate the tumor size weekly.Calculate gross tumor volume.

The result

Nod1 has effect in TNF α and the inductive apoptosis of Nod1 part.Breast carcinoma epithelial cell line MCF-7 has been widely used for research by biological signals TNF α or by the inductive apoptosis of cytotoxic drug for example.MCF-7 cell line also is used as model and studies the positive breast carcinoma of estrogen people such as (, Exp.Biol.Med.228:995-1003 (2003)) Simstein.

Originally the MCF-7 cell uses in the genetic screening of the mutation of adopting retrovirus-mediated method, identifies the required gene of the inductive cell death of TNF α.After the retroviral construct body, select the clone of TNF α resistance in the MCF-7 cellular exposure, in TNF α resistance clone, identify mutant gene then.One of resistance clone contains destructive Nod1 gene; The cell line of this TNF α resistance clone is called as MCF7-C20.PDisrup from the retroviral construct body inserts the 3 ' part that is positioned to the Nod1 gene, (LRR9-10 in the zone in rich leucine zone 9 and 10; Referring to accompanying drawing 1A).This insertion is arranged in the blasticidine open reading frame of Nod1 coding region.The sketch map of blasticidine-Nod1 fusion mRNA schematically shows in accompanying drawing 1A.Whether use the Western engram analysis of anti-people Nod1 monoclonal antibody to test NOD1 albumen expresses in the MCF7-C20 cell.In parent MCF-7 cellular lysate (being labeled as " wt "), detect endogenous NOD1 albumen, but the detected expression that the Western trace of MCF-7 C20 cellular lysate fails to disclose Nod1 shows functional Nod1 allele destroyed (accompanying drawing 1B) in MCF-7 C20.

MCF-7 C20 cell line is deposited in U.S. typical case culture collection (the 10801 University Blvd. of institute according to the clause of budapest treaty on February 23rd, 2006, Manassas, Va., 20110-2209 USA (ATCC)), ATCC registration number No.ATCC Number.

Being lost in of Nod1 function has remarkable influence in the apoptosis.Especially, compare with the parent MCF-7 cell of expressing Nod1, MCF-7C20 cell (not having Nod1 to express) significantly more has resistance (accompanying drawing 1C) for the inductive apoptosis of TNF α.These data show that Nod1 is the sensitizer in the TNF α approach in the MCF-7 cell, and Nod1 has promoted the apoptosis in the MCF-7 breast cancer cell.

For the further effect of research Nod1 in apoptosis, the Nod1 part D-Ala-L-Glu-meso diaminopimelic acid (γ TriDAP) of various cell types and specific, activated Nod1 is hatched.The cell type of test comprises that thereby the MCF-7C20 clone of parent MCF-7 cell line, Nod1 defective and through engineering approaches are to contain other human Nod1 allele Nod1 by the cell line of overexpression (MCF-7Nod1).In order to determine whether the C20 cell causes by lacking Nod1 the inductive apoptotic resistance of γ TriDAP, the human Nod1 of stably express produces the competent C20 cell of Nod1 (C20/Nod1) in the MCF-7C20 cell.As another contrast, produce the MCF-7Blasto cell with empty retroviral vector transfection parent MCF-7 cell line.Handle cell with γ TriDAP existing or lack under the situation of cycloheximide (CHX), measure cell death by dyeing of iodate third ingot and flow cytometry then.

Exist under the situation of cycloheximide, wild type MCF-7 cell has been induced about 25% cell death (accompanying drawing 2A, upper left picture, shade post) to the exposure of γ TriDAP.In the wild-type cell of handling with independent cycloheximide (open tubular column) or independent γ TriDAP, do not observe cell death.By contrast, MCF-7C20 (not having Nod1 to express) cell has resistance completely to the influence that γ TriDAP adds cycloheximide, does not observe apoptosis basically and improve (accompanying drawing 2A, upper right picture) in these cells.The again importing of Nod1 in the MCF-7C20 cell repaired these cells to the inductive apoptotic complete sensitivity of γ TriDAP (accompanying drawing 2A, bottom right picture).The cell death that enlarges has been induced in being combined in of cycloheximide and γ TriDAP in the MCF-7 cell of stablizing overexpression Nod1, cell death almost reaches 60% (accompanying drawing 2A, bottom right picture) in these MCF-7Nod1 cells of 48h processing back.

Nod1 part γ TriDAP is that inducing of apoptotic the best is required.When hatching with the MCF-7 cell under the situation that has cycloheximide, the non-activity that is called α TriDAP contrasts not inducing cell death (the α Tri among the accompanying drawing 2A) of tripeptides, among the α TriDAP mesoDAP in conjunction with alpha position in Glu, rather than as the γ position among the γ TriDAP.

In contrast, use culture medium (" Med ") to replace γ TriDAP or α TriDAP.Add culture medium and do not have effect basically for apoptosis.The overexpression of the expression of endogenous Nod1, Nod1 is confirmed (accompanying drawing 2B) by immunoprecipitation and Western engram analysis in MCF-7Blasto and MCF-7Nod1 cell.In MCF-7 C20 cell, do not see this expression (accompanying drawing 2B).These data show, Nod1 makes MCF-7 breast cancer cell pair cell apoptotic sensitivityization under the situation that has γ TriDAP.

Yet, in wild-type cell, use not cell death inducing of γ TriDAP individual processing cell.On the contrary, add γ TriDAP and cycloheximide observe before the apoptosis essential.Proapoptosis reagent for example cycloheximide is observed the before frequent needs of apoptosis.Yet, the change of using that the MCF-7 cell notices to the sensitivity of γ TriDAP or TNF α, use other apoptosis to stimulate, comprise the not discovery of doxirubicin and camptothecine, wherein parent and C20 cell are equally responsive (data not shown) for cell death.

The bright microscopic examination of the MCF-7 cell of handling with γ TriDAP has disclosed the morphological change characteristics (accompanying drawing 2C, picture a-b) of apoptosis and non-necrosis.Yet, be actually apoptosis in order to confirm the inductive cell death of γ TriDAP, carry out DAPI and TUNEL dyeing.Shown in accompanying drawing 2C, the MCF-7 nucleus contains spissated chromatin after handling with γ TriDAP, shown in DAPI dyeing (picture c-d), observes karyorrhexis (picture e-f) by TUNEL dyeing.In untreated cell, do not detect nuclear staining.

Confirmed that further use two kinds of broad-spectrum caspase inhibitor, z-VAD-FMK and Boc-D-FMK have obtained the inductive apoptosis of γ TriDAP.Z-VAD-FMK and Boc-D-FMK cancel the inductive cell death of γ TriDAP (accompanying drawing 2D).At last, add γ TriDAP, rather than invalid contrast tripeptides α TriDAP to the MCF-7 cell, caused poly-(ADP-ribose) polymerase (PARP) and capases 6,7,8 and 9 proteolytic cleavage (accompanying drawing 3,9B).The known shortage of MCF-7 cell caspase 3, the expression of caspase 3 does not have to change the reaction pattern (data not shown) to γ TriDAP in parent MCF-7 cell or MCF-7C20 cell.

Thereby the increase of a plurality of cell lines shows, has the inductive specific cell apoptosis pathway of cognate ligand γ TriDAP by Nod1 in the MCF-7 cell, and this approach needs the proteic existence of Nod1.

In order further to determine the MCF-7C20 cell to the shortage of the inductive apoptotic resistance of γ TriDAP from Nod1, human Nod1 is stably express in the MCF-7C20 cell, produces the MCF-7C20 cell (MCF-7C20/Nod1) of expressing Nod1.Nod1 heavily importing in these Nod1 deficient cells repaired the complete sensitivity (accompanying drawing 2A) of these cells to γ TriDAP, and causes caspase and PARP cracking (accompanying drawing 3).

In order to determine whether there is Nod1 dependent cell apoptosis in other human cell line, checked a series of epithelial cell lines, wherein Nod1 is stabilized expression to strengthen the sensitivity for the Nod1 pathway activation.Handle cell with TNF α (T) and γ TriDAP (γ) existing or lack under the situation of CHX (C), dyeing by PI monitors viability (table 1) as apoptotic tolerance.

Table 1: the cell survival after TNF α and γ TriDAP processing

In the cell line of test, SK-BR3 and A431 cell have all disclosed Nod1 dependent cell apoptosis pathway.Yet, to compare with MCF-7 system, these cells only experience apoptosis when γ TriDAP and TNF α and CHX add simultaneously.CaCo2 and HT29 experience apoptosis in response to TNF α and CHX, but do not observe cooperative effect when simultaneously by γ TriDAP activation Nod1 approach.Other are that for example 293 cells have resistance to the inductive cell death of γ TriDAP, even when adding TNF α and CHX.The shortage of Nod1 dependent cell apoptosis is not the result that γ TriDAP can not activate NOD1, does not discharge IL-8 in response to the Nod1 part because express 293 cells of Nod1.Although there is No? albumen, HT29 and CaCo2 cell seldom react to γ TriDAP in apoptosis and IL-8 release analysis, show to lack one or more positive modulators, or have strong negative adjusting approach.Although relate to complex interactions,, comprise that in SK-BR3 and the A431 cell line, Nod1 dependent cell apoptosis is evincible at some epithelial cell lines.

NOD1 expresses in the SKBR3 human breast cancer cell line, and is as noted before, also regulates the apoptosis in those cancerous cell.Compare with the SKBR3 of overexpression Nod1, the SKBR3 wild-type cell has represented the less apoptosis (accompanying drawing 9A) as the function of TNF α or γ TriDAP concentration.In addition, the percentage ratio of apoptotic SKBR3 cell depends on condition of culture and changes (accompanying drawing 9B).Shown in accompanying drawing 9B, when cell this area γ TriDAP (γ Tri) added TNF α (TNF) and cycloheximide (CHX), the apoptosis of SKBR3 cell was the highest.The alternative apoptosis that causes reduction of non-activity γ TriDAP analog α TriDAP (α Tri).The western of lysate that has been exposed to the SKBR3 cell of these reagent analyzes and to have shown that poly-(ADP-ribose) polymerase (PARP) and capases 3,7 and 8 have experienced proteolytic cleavage.The percentage of cerebral apoptosis of cultivating observed wild type after the SKBR3 cell, expressing NOD1's and expression CLARP's SKBR3 cell has been described under having the situation of different reagent accompanying drawing 9C figure.As indicated, and when having cycloheximide (CHX or C), TNF α (TNF or T) and γ TriDAP (γ Tri) (, " gTC " combination), the apoptosis of SKBR3 cell is the highest.Yet under various condition of culture, CLARP expresses and does not improve apoptosis.

Previous structure-functional study of human Nod1 shows, it is required that P-ring residue (K208) is that the Nod1 albumen of overexpression momently activates NF-κ B in 293 cells.Other studies show that the required conformation change of oligomerization by the mediation of nucleotide combination/oligomerization (NBD/NOD) domain has been blocked in the sudden change of K208.Sudden change V41Q in the CARD of Nod1 domain has also shown the combination that destroys 9 couples of Nod1 of caspase, the inhibition that has produced Nod1 dependent cell apoptosis during the of short duration transfection research of 293 cells.

Test further to determine that NOD1 domain is that apoptosis is required.Shown in accompanying drawing 4A, the K208R mutant has been cancelled the effect that γ TriDAP adds cycloheximide, and the V41Q mutant have seldom or do not have a remarkable result.The Nod1 polypeptide of these sudden changes is expressed effectively, as by as shown in the Western trace of describing among the accompanying drawing 4B.Thereby the different-effect of these polypeptide is not because the difference in the expression.On the contrary, these tables of data understand that nucleotide-combination/oligomerization (NBD/NOD) domain of the conformation change that the mediation oligomerization is required is that the Nod1 apoptosis is required.

Further experiment shows, though the inductive IL-8 of γ TriDAP produces and is suppressed (data not shown) in the MCF-7C20 cell, when wild type Nod1 expresses in the MCF7-C20Nod1 cell (accompanying drawing 5C), or work as V41Q Nod1 mutant (promptly at MCF-7C20, when MCF-7C20V41Q) expressing in the cell (data not shown), this IL-8 produces and is resumed.Yet (that is, MCF-7C20K208R) the inductive apoptosis of γ TriDAP is not repaired in the expression in the cell to K208R Nod1 mutant polypeptide at MCF-7C20.

Nod2 activation cell death inducing not in the MCF-7 cell

Also carried out experiment and determined whether Nod2 will start apoptosis by the activation of its specific, activated dose of muramyldipeptide (MDP) in parent MCF-7 cell or in the MCF-7 of overexpression Nod2 cell (MCF-7 Nod2).Thereby each of adding γ TriDAP or MDP to these cell lines is measured apoptosis (accompanying drawing 5A) and IL-8 generation (accompanying drawing 5C).Add the apoptosis that MCF-7Nod2 cell that CHX handles does not experience raising with MDP.By contrast, as expected, γ TriDAP interpolation has produced cell death.MDP handles and cause IL-8 secretion (accompanying drawing 5C) really in MCF-7 Nod2 cell.Nod1 and the Nod2 expression in MCF-7 stable transfection is confirmed (accompanying drawing 2B) by immunoblotting.The complementation of MCF-7 C20 cell and Nod2 does not cause apoptosis (data not shown) after adding MDP or γ TriDAP.

The caspase that relates to Nod1 dependent cell apoptosis

The activation that it being understood that apoptosis approach in the cell of the uniqueness that is started by specificity caspase now takes place afterwards.In order to obtain information about the initial caspase in the Nod1 dependent pathway, use have at the variable specific pharmacology of caspase and biology inhibitor.Broad-spectrum inhibitor z-VAD almost entirely blocks the inductive apoptosis of γ TriDAP (accompanying drawing 6A).By contrast, caspase 1,2,6 and 7 specific inhibitor only the pair cell apoptosis have seldom influence (accompanying drawing 6A).Yet caspase 9 inhibitor LEHD and caspase 8 inhibitor IETD have significant inhibition effect, and the inhibitory action level is similar to z-VAD being seen (accompanying drawing 6A).These data show, caspase 8 and 9 possible effects aspect the inductive apoptosis startup of γ TriDAP.

Two kinds of main apoptotic pathways have been described, inherent (mitochondrion, pressure inducement) and external (receptor-mediated) approach.As if participate in apoptotic caspase and be grouped into fractionated cascade, caspase 9 and caspase 8 are respectively the upstream starting materials in inherent and the external approach.In order to distinguish these approach, use several protein inhibitors, it is by the transfection transfered cell.

At first, tested CLARP (Flip) transfectant.CLARP is considered to the specific inhibitor of caspase 8, has two death effector thing domains (DED) and has the caspase domain of non-activity.CLARP is known to interact with caspase 8 and FADD, thereby suppresses right by various ligand-receptors specifically, comprises Fas, TNF α and the inductive apoptosis of TRAIL.In order to determine the influence of CLARP to the inductive cell death of γ TriDAP, set up MCF-7 cell line, it is individually or have stably express CLARP under the situation of Nod1 (MCF-7 CLARP).When hatching the MCF-7CLARP cell with γ TriDAP/CHX, cell death is suppressed fully, shows inductive apoptotic pathways of Nod1 and the approach overlapping (accompanying drawing 6C, top picture) that is started by caspase 8.

Another kind of anti-apoptotic PROTEIN B cl-2 has shown that histiocyte pigment C discharges from mitochondrion, thus the activation of blocking-up Apaf1/caspase 9 complex.Produce the stable MCF-7 cell line of overexpression Bcl-2, the cell death when assessment Nod1 activates in the MCF-7 cell of this expression Bcl-2.Shown in accompanying drawing 6C (bottom field), the overexpression of Bcl-2 only partly stops the inductive cell death of γ TriDAP.

Known MCF-7 cell lacks Caspase III.The inventor has further determined, and these cells of the Caspase III expression in parent MCF-7 or MCF-7C20 cell the change are to the reaction pattern (data not shown) of Nod1 part γ TriDAP.These experiments show, Caspase 8 starts in the inductive apoptosis of γ TriDAP in the MCF-7 cell and plays an important role.Support in addition though be indirect, from such discovery, fails to influence the V41Q sudden change of the No d1 of caspase9, is supporting inductive apoptosis of γ TriDAP (accompanying drawing 4A) and IL-8 to produce aspect (data not shown) and wild type Nod1 equivalence.

CARD domain but not kinase activity is Nod1 that inductive apoptosis is required.

RIP2 is the protein kinase that contains the CARD domain.Verified RIP2 is important in the Nod1 signal transduction, and it causes NF-kB activation (people .Nature416:194-99 (2002) such as Kobayashi; People .Nature 416:190-94 (2002) such as Chin).RIP2 interacts via CARD-CARD and is considered to for the NF-kB activation with combining of Nod1 to be essential, to work as the negative inhibitor of the dominance of Nod1 signal transduction because lack the RIP2 of CARD domain.

In the MCF-7 cell, express several RIP2 mutants and assess the effect of RIP2 kinase activity in the inductive apoptosis of γ TriDAP.The expression that lacks the RIP2 of CARD domain has fully phased out the inductive cell death of Nod1 (accompanying drawing 7A).By contrast, with respect to the apoptosis level seen in the parent MCF-7Blasto cell of expressing normal level Nod1, the expression of the RIP2 of wild type RIP2 or catalytically inactive (RIP2 KD) improved apoptotic degree (accompanying drawing 7A, 13A).Compare the cell (accompanying drawing 13C) or the parent MCF-7Blasto cell (data not shown) of only expressing Nod1, the cell of expression wild type RIP2 and RIP2KD construct is responsive for the γ TriDAP of lower concentration, and faster death.In addition, MDP induces high-caliber apoptosis in the cell of expressing RIP2 KD be effectively, but be not (accompanying drawing 13A) in the cell of expressing wild type RIP2 or RIP2 Δ CARD.Compare with the cell of expressing Nod1, MDP has also induced higher levels of apoptosis (accompanying drawing 13C) in RIP2 KD cell.Yet in the cell of expressing wild type RIP2, RIP2 KD and RIP2 Δ CARD, TNF α has induced the apoptosis (accompanying drawing 13B) of similar level.The inductive IL-8 secretion of γ TriDAP is the highest (accompanying drawing 13D) in the cell of expressing wild type RIP2.That approximate equal number is expressed by every kind of cells transfected system of being studied or sudden change RIP2 (accompanying drawing 7B) shows that expression is not the reason of the difference of cell death aspect.Thereby Nod1 dependent cell apoptosis pathway needs the RIP2CARD domain, but surprisingly, does not need the RIP2 kinase activity.

Checked that also RIP2 expresses the influence (accompanying drawing 7C) to the inductive JNK phosphorylation of γ TriDAP.There is under the situation of cycloheximide the MCF-7 cell of expression wild type RIP2 or RIP2 KD is induced JNK to the exposure 2h of γ TriDAP phosphorylation (accompanying drawing 7C).Yet γ TriDAP and cycloheximide do not have this influence to RIP2 Δ CARD cell.

Thereby Nod1 needs RIP2 (scaffolding protein kinases) to be used to regulate the estrogen-sensitive tumor growth.Yet, though regulating, the Nod1-RIP2 of estrogen-sensitive tumor growth needs the RIP2CARD domain, it does not need the RIP2 kinase activity to come to provide suitable downstream signal for the inhibition of Nod1 dependent cell apoptosis and tumor growth.Obtained similar result (data not shown) with p38.The activatory shortage of MAPK is not because the mapk kinase signal transduction that changes in RIP2 Δ CARD cell because in all transfectants IL-1 induced strong JNK phosphorylation.Thereby the activity of γ TriDAP in the MCF-7 cell needs RIP2 rather than its kinase activity.

These tables of data understand that the protein kinase RIP2/RICK in Nod1 downstream may be the key component of Nod1 proapoptosis approach, because the inductive cell death of γ TriDAP has been eliminated in the expression of the negative form of the dominance of RIP2.

Nod1 control tumor forms.Nod1 dependent cell apoptosis pathway may be important in many biological processess, comprises that growth of tumour cell is regulated and the decay of malignant cell experience cell death, causes that tumor takes place.Tumor growth heteroplastic transplantation model in the SCID mice is used to the effect of checking that Nod1 plays in tumor growth and tumor rejection.The colony of MCF-7Blasto, MCF-7C20 and MCF-7C20Nod1 cell (amounts to about 3 * 10 separately ⁶Individual cell), the flank that is subcutaneously injected into female mice individually comes induced tumor growth in SCID/SCID or SCID/Nod1 mice.A week is write down the tumor formation of animal weekly up to 8 weeks after injection.

Several weeks before after injection, all three kinds of cell colonys tumor of equally growing, thereby to back 15 days of injection, the gross tumor volume 10mm that in all mices, exists approximate calculation to go out ³Thereby after importing mice about 15 days, MCF-7Blasto, MCF-7C20 and MCF-7C20Nod1 cell can produce tumor.

Yet, remaining 8 weeks experiment, almost disappear from MCF-7Blasto and the plastidogenetic tumor of MCF-7C20Nod1.By the initial tumor size reduction soon of MCF-7Blasto and the generation of MCF-7C20Nod1 cell, and degenerate (accompanying drawing 10).Only the injection site that is injected at of MCF-7C20 cell has produced big circular tumor, and it is not degenerated and continues growth.These results show that the shortage of Nod1 is allowed tumor growth, and the existence of Nod1 causes tumour regression.

Because these researchs are carried out in the SCID mice, immune effect can be ignored in heteroplastic tumor growth.In addition, the shortage of tumor is not because the multiplication potentiality that reduces in the animal of accepting MCF-7Blasto and MCF-7C20Nod1 cell, because every kind of MCF-7 system in this research, comprise the cell line of expressing MCF-7Blasto, MCF-7Nod1, MCF-7C20 and MCF-7C20Nod1, under conditions of tissue culture, have identical growth characteristics (data not shown) with forming in the analysis at the soft agar bacterium colony.

The result's of three independent experiments general introduction is displayed in Table 2, and wherein uses SCID/SCID hour in experiment 1 and 2, uses the SCID/Nod mice in experiment 3.

The inductive tumor incidence rate of table 2:MCF-7 cell line

Cell line	Experiment	1	Experiment 2	Experiment 3	Altogether
						Wild type MCF-7	?2/8	?0/8	?0/6	2/22
MCF-7C20	?7/8	?4/8	?5/6	16/22
					MCF-7C20Nod1	?1/8	?1/8	?0/6	2/22

These results show that the forfeiture of Nod1 function (when using MCF-7 C20 cell) has improved the probability that tumor is not degenerated.The replacement of Nod1 function (when using MCF-7 C20Nod1 cell), or inducing of expressing of Nod1 have improved the probability of tumour regression.

Also carried out an experiment, wherein from having the SCID/SCID mice results tumor of tumor, described mice is used MCF-7C20 and MCF-7C20Nod1 injection cell before 3.5 weeks.Shred tumor tissues then, put into tissue culture flasks.After an about week, remove any remaining entity tumor tissue, add 10 μ g/ml blasticidin Ss.There are 6 generations of cell of under standard conditions, keeping sowing under the situation of blasticidin S then.Use the natural SCID mice of these injection cells then, in 60 days time, measure the time course that gross tumor volume changes.

The mice that contains deutero-MCF-7C20 of tumor or MCF-7C20Nod1 cell all begins the tumor of growing.To back 15 days of injection, two kinds of cell types produced the proximate gross tumor volume 50mm that calculates ³In the residue of this experiment during 40 days, detectable the size (＜10mm from the plastidogenetic tumour regression of MCF7-C20Nod1 to bottom line ³), and the tumor growth that is produced by the MCF-7C20 cell is to 200-270mm ³Maximum volume (accompanying drawing 11A-C).

These results have confirmed that further the Nod1 in the tumor cell expresses can cause tumor cell apoptosis and tumour regression.

Because MCF-7 is in response to TNF α experience apoptosis, in the use and the hamster monoclonal antibody of Mus TNF α people .J.Immunol.143:127-30 (1989) such as () Bancroft further check the effect of TNF α in the inductive apoptosis of Nod1.The existence of this antibody does not suppress apoptosis, because do not form tumor when inoculation MCF-7 Blasto or MCF-7 Nod1 cell.In addition, as observed in previous experiment, when with MCF-7 C20 injection cell in mice the time, tumor growth.

Again, the shortage of tumor is not that formation has identical growth characteristics in analyzing with the soft agar bacterium colony because every kind of MCF-7 of research ties up to cultivation owing to compare the MCF-7 Blasto and the MCF-7 C20/Nod1 cell proliferation rate of reduction with MCF-7 C20 cell after the tumor cell of expressing Nod1 at least.In addition, simple cell apoptosis blocking-up by other factors may not be the mechanism that produces tumor growth, because CLARP (c-FLIP) is the specific inhibitor of caspase 8, MCF-7 c-FLIP/CLARP cell can not form tumor (data not shown) in nude mice.

Estrogenic effect

In preliminary experiment, when being transplanted to the C20 clone in the male mice, observe more healthy and stronger tumor growth, hinted the female relevant effect of hormone in tumour regression.The further research of the polymerase chain reaction (PCR) amplification by various hereditism's target spots has produced observed result, lacks progesterone receptor in C20 cell line, and it is present in the wild-type cell and more importantly be present in the C20Nod1 cell.In addition, pcr analysis has also disclosed estrogen receptor alpha and has been present in each of three kinds of MCF7 cell types.

In great majority research, the tumor of MCF-7 cell forms in nude mice needs estrogen to replenish tumorigenic, even with the high concentration inoculating cell time.In order further to check the effect of estrogen in tumor takes place, with all three kinds of cell lines together with estrogen bolus injection (accompanying drawing 11B) in mice.As expected, when having estrogen, tumor growth in the mice of injection MCF-7 Blasto cell.Mice with MCF-7 C20 injection cell produces tumor, and it grows greatlyyer when having the estrogen bullet.Enjoyably, the mice of the accepting MCF-7 C20/Nod1 tumor of under the situation that has the estrogen bullet, not growing.These data show that Nod1 suppresses the estrogen-dependent tumor growth.

For the further effect of proof Nod1 approach in tumor growth, studied the expression of the negative allele of dominance (RIP2 Δ CARD) of RIP2, determine whether this allele also can disturb the ability of MCF-7 cell growth tumor.Accompanying drawing 11C has shown, these RIP2 Δs CARD cell growth tumor, however tumor is less than observed in MCF-7 C20 cell.Combine, these tables of data understand that Nod1 has pivotal role in tumor growth, and the effect of the blockage of estrogen-dependent tumor growth has been played in the existence of Nod1.

In order to obtain other support, under the condition that cell is grown, observed the sensitivity of MCF-7 cell line in the estrogenic culture medium of shortage to the propagation of estrogen-induced to this hypothesis.Under these conditions, in response to the estrogen that adds, MCF-C20 cell and MCF-7 RIP2 Δ CARD cell have experienced strong propagation, and parent and MCF-7 C20/Nod1 cell line is not all by thorn explosive value (accompanying drawing 11D).Yet, to MCF-7 C20 cell observation to the propagation of estrogen-induced be blocked (data not shown) by in culture medium, adding zitazonium.

Whether regulate the inductive apoptotic pathways of Nod1, cultured cell in the culture medium of the serum that contains charcoal treatment in order to determine estrogenic existence.In the apoptosis of C20 cell, estrogen have seldom or not influence, described C20 cell is not expressed Nod1 basically, it does not represent apoptosis (accompanying drawing 12A, midway film) basically.When cultivating under the situation that is lacking steroid, C20/Nod1 and Blasto cell more have resistance (not have steroid C20/Nod1 cell to represent 10% apoptosis for the inductive apoptosis of γ TriDAP, 80% of steroid is arranged relatively, accompanying drawing 12A, bottom picture).Thereby, be reversed by adding estrogen at apoptotic resistance, thereby apoptosis improves (accompanying drawing 12A) in the dose dependent mode of estrogen concentrations.On the contrary, add zitazonium and partly blocked the inductive cell death of γ TriDAP-Nod1 (accompanying drawing 12B).At last, the overexpression of Nod1 has reduced the expression of endogenous estrogen receptor alpha (ER α) significantly and has not influenced as the expression (accompanying drawing 12C) that loads the ERK2 that contrasts.Similarly, at the reduction of from pre-incubated tumor isolated cells, observing the ER alpha expression (accompanying drawing 12C, right side picture).These data show that the Nod1 approach influences ER alpha expression level, and thereby influence the MCF-7 breast cancer cell for the sensitivity that develops tumor.

Quote and all patents and the publication mentioned are the performances of the present invention's those skilled in the art's level of skill of living at this, the patent of every kind of effect or publication are incorporated in this by reference, as separately intactly or in this degree of listing that intactly is incorporated in this by reference.The applicant keeps patent that any of these is quoted or all material in the publication and information and physically merges to right in this description.

Ad hoc approach described here and compositions are representatives preferred embodiment, are exemplary, and are not intended to limit the scope of the invention.When considering this description, those skilled in the art will expect other purposes, aspect and embodiment, be included within the spirit of the present invention of being delimited by the scope of claim.It will be apparent to one skilled in the art that and to carry out various substitutions and modifications and not deviate from scope and spirit of the present invention aspect disclosed herein.This suitably the invention described of illustrative can under the situation that lacks any element or element or restriction or limitation, carry out, it is concrete not open on this necessity ground.This suitably the method described of illustrative can carry out with different sequence of steps with step, they need not be confined in this or named order in the claims.As used herein and in subsidiary claim, use, singulative " a kind of ", " one " and " being somebody's turn to do " comprise plural content, unless indicated in addition significantly in the context.Thereby for example, citation " antibody " comprises multiple (for example the solution of antibody, a series of antibody preparations) such antibody, or the like.Under any circumstance this patent is not construed as limited at this specific disclosed specific embodiment or embodiment yet.Under any circumstance this patent is not interpreted as being limited by any statement that other officials of any auditor or patent and trademark office or employee carry out yet, unless by particularly and unconditionally, or clear and definite reservation adopts in the answer that the applicant writes in this statement.

Term that has adopted and statement are used as the term of description, and not restrictive, when using these terms and statement, be not intended to get rid of any equivalent or its part shown and feature of describing, but being recognized that, is possible in the various scope of the invention that are modified in prescription.Thereby, should be understood that, though the present invention is by preferred embodiment disclosing particularly with optional feature, the modifications and variations of notion disclosed herein can be adopted by those skilled in the art, and this modifications and variations considered to be in by in the subsidiary defined scope of the invention of claim.

At this present invention has been described widely and usually.Fall into the classification of disclosed various narrower classifications of generality and subgenus and also constitute part of the present invention.This comprises of the present invention general description that has the conditionality of removing any theme from described genus or negativity restriction, and whether the material of no matter being removed is narrated particularly at this.

Other embodiments are within the following claim.In addition, when describing feature of the present invention or aspect, person of skill in the art will appreciate that, for any independent member of Ma Kushi cohort or member's subgroup, also described the present invention thus by the mode of Ma Kushi cohort.

Sequence table

<110>The?Scripps?Research?Institute

Ulevitch，Richard?J.

da?Silva，Jean

Han，Jiahuai

＜120〉as antitumor agent NOD1

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Met?Glu?Glu?Gln?Gly?His?Ser?Glu?Met?Glu?Ile?Ile?Pro?Ser?Glu?Ser

1 5 10 15

His?Pro?His?Ile?Gln?Leu?Leu?Lys?Ser?Asn?Arg?Glu?Leu?Leu?Val?Thr

20 25 30

His?Ile?Arg?Asn?Thr?Gln?Cys?Leu?Val?Asp?Asn?Leu?Leu?Lys?Asn?Asp

35 40 45

Tyr?Phe?Ser?Ala?Glu?Asp?Ala?Glu?Ile?Val?Cys?Ala?Cys?Pro?Thr?Gln

50 55 60

Pro?Asp?Lys?Val?Arg?Lys?Ile?Leu?Asp?Leu?Val?Gln?Ser?Lys?Gly?Glu

65 70 75 80

Glu?Val?Ser?Glu?Phe?Phe?Leu?Tyr?Leu?Leu?Gln?Gln?Leu?Ala?Asp?Ala

85 90 95

Tyr?Val?Asp?Leu?Arg?Pro?Trp?Leu?Leu?Glu?Ile?Gly?Phe?Ser?Pro?Ser

100 105 110

Leu?Leu?Thr?Gln?Ser?Lys?Val?Val?Val?Asn?Thr?Asp?Pro?Val?Ser?Arg

115 120 125

Tyr?Thr?Gln?Gln?Leu?Arg?His?His?Leu?Gly?Arg?Asp?Ser?Lys?Phe?Val

130 135 140

Leu?Cys?Tyr?Ala?Gln?Lys?Glu?Glu?Leu?Leu?Leu?Glu?Glu?Ile?Tyr?Met

145 150 155 160

Asp?Thr?Ile?Met?Glu?Leu?Val?Gly?Phe?Ser?Asn?Glu?Ser?Leu?Gly?Ser

165 170 175

Leu?Asn?Ser?Leu?Ala?Cys?Leu?Leu?Asp?His?Thr?Thr?Gly?Ile?Leu?Asn

180 185 190

Glu?Gln?Gly?Glu?Thr?Ile?Phe?Ile?Leu?Gly?Asp?Ala?Gly?Val?Gly?Lys

195 200 205

Ser?Met?Leu?Leu?Gln?Arg?Leu?Gln?Ser?Leu?Trp?Ala?Thr?Gly?Arg?Leu

210 215 220

Asp?Ala?Gly?Val?Lys?Phe?Phe?Phe?His?Phe?Arg?Cys?Arg?Met?Phe?Ser

225 230 235 240

Cys?Phe?Lys?Glu?Ser?Asp?Arg?Leu?Cys?Leu?Gln?Asp?Leu?Leu?Phe?Lys

245 250 255

His?Tyr?Cys?Tyr?Pro?Glu?Arg?Asp?Pro?Glu?Glu?Val?Phe?Ala?Phe?Leu

260 265 270

Leu?Arg?Phe?Pro?His?Val?Ala?Leu?Phe?Thr?Phe?Asp?Gly?Leu?Asp?Glu

275 280 285

Leu?His?Ser?Asp?Leu?Asp?Leu?Ser?Arg?Val?Pro?Asp?Ser?Ser?Cys?Pro

290 295 300

Trp?Glu?Pro?Ala?His?Pro?Leu?Val?Leu?Leu?Ala?Asn?Leu?Leu?Ser?Gly

305 310 315 320

Lys?Leu?Leu?Lys?Gly?Ala?Ser?Lys?Leu?Leu?Thr?Ala?Arg?Thr?Gly?Ile

325 330 335

Glu?Val?Pro?Arg?Gln?Phe?Leu?Arg?Lys?Lys?Val?Leu?Leu?Arg?Gly?Phe

340 345 350

Ser?Pro?Ser?His?Leu?Arg?Ala?Tyr?Ala?Arg?Arg?Met?Phe?Pro?Glu?Arg

355 360 365

Ala?Leu?Gln?Asp?Arg?Leu?Leu?Ser?Gln?Leu?Glu?Ala?Asn?Pro?Asn?Leu

370 375 380

Cys?Ser?Leu?Cys?Ser?Val?Pro?Leu?Phe?Cys?Trp?Ile?Ile?Phe?Arg?Cys

385 390 395 400

Phe?Gln?His?Phe?Arg?Ala?Ala?Phe?Glu?Gly?Ser?Pro?Gln?Leu?Pro?Asp

405 410 415

Cys?Thr?Met?Thr?Leu?Thr?Asp?Val?Phe?Leu?Leu?Val?Thr?Glu?Val?His

420 425 430

Leu?Asn?Arg?Met?Gln?Pro?Ser?Ser?Leu?Val?Gln?Arg?Asn?Thr?Arg?Ser

435 440 445

Pro?Val?Glu?Thr?Leu?His?Ala?Gly?Arg?Asp?Thr?Leu?Cys?Ser?Leu?Gly

450 455 460

Gln?Val?Ala?His?Arg?Gly?Met?Glu?Lys?Ser?Leu?Phe?Val?Phe?Thr?Gln

465 470 475 480

Glu?Glu?Val?Gln?Ala?Ser?Gly?Leu?Gln?Glu?Arg?Asp?Met?Gln?Leu?Gly

485 490 495

Phe?Leu?Arg?Ala?Leu?Pro?Glu?Leu?Gly?Pro?Gly?Gly?Asp?Gln?Gln?Ser

500 505 510

Tyr?Glu?Phe?Phe?His?Leu?Thr?Leu?Gln?Ala?Phe?Phe?Thr?Ala?Phe?Phe

515 520 525

Leu?Val?Leu?Asp?Asp?Arg?Val?Gly?Thr?Gln?Glu?Leu?Leu?Arg?Phe?Phe

530 535 540

Gln?Glu?Trp?Met?Pro?Pro?Ala?Gly?Ala?Ala?Thr?Thr?Ser?Cys?Tyr?Pro

545 550 555 560

Pro?Phe?Leu?Pro?Phe?Gln?Cys?Leu?Gln?Gly?Ser?Gly?Pro?Ala?Arg?Glu

565 570 575

Asp?Leu?Phe?Lys?Asn?Lys?Asp?His?Phe?Gln?Phe?Thr?Asn?Leu?Phe?Leu

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Cys?Gly?Leu?Leu?Ser?Lys?Ala?Lys?Gln?Lys?Leu?Leu?Arg?His?Leu?Val

595 600 605

Pro?Ala?Ala?Ala?Leu?Arg?Arg?Lys?Arg?Lys?Ala?Leu?Trp?Ala?His?Leu

610 615 620

Phe?Ser?Ser?Leu?Arg?Gly?Tyr?Leu?Lys?Ser?Leu?Pro?Arg?Val?Gln?Val

625 630 635 640

Glu?Ser?Phe?Asn?Gln?Val?Gln?Ala?Met?Pro?Thr?Phe?Ile?Trp?Met?Leu

645 650 655

Arg?Cys?Ile?Tyr?Glu?Thr?Gln?Ser?Gln?Lys?Val?Gly?Gln?Leu?Ala?Ala

660 665 670

Arg?Gly?Ile?Cys?Ala?Asn?Tyr?Leu?Lys?Leu?Thr?Tyr?Cys?Asn?Ala?Cys

675 680 685

Ser?Ala?Asp?Cys?Ser?Ala?Leu?Ser?Phe?Val?Leu?His?His?Phe?Pro?Lys

690 695 700

Arg?Leu?Ala?Leu?Asp?Leu?Asp?Asn?Asn?Asn?Leu?Asn?Asp?Tyr?Gly?Val

705 710 715 720

Arg?Glu?Leu?Gln?Pro?Cys?Phe?Ser?Arg?Leu?Thr?Val?Leu?Arg?Leu?Ser

725 730 735

Val?Asn?Gln?Ile?Thr?Asp?Gly?Gly?Val?Lys?Val?Leu?Ser?Glu?Glu?Leu

740 745 750

Thr?Lys?Tyr?Lys?Ile?Val?Thr?Tyr?Leu?Gly?Leu?Tyr?Asn?Asn?Gln?Ile

755 760 765

Thr?Asp?Val?Gly?Ala?Arg?Tyr?Val?Thr?Lys?Ile?Leu?Asp?Glu?Cys?Lys

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Gly?Leu?Thr?His?Leu?Lys?Leu?Gly?Lys?Asn?Lys?Ile?Thr?Ser?Glu?Gly

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Gly?Lys?Tyr?Leu?Ala?Leu?Ala?Val?Lys?Asn?Ser?Lys?Ser?Ile?Ser?Glu

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Val?Gly?Met?Trp?Gly?Asn?Gln?Val?Gly?Asp?Glu?Gly?Ala?Lys?Ala?Phe

820 825 830

Ala?Glu?Ala?Leu?Arg?Asn?His?Pro?Ser?Leu?Thr?Thr?Leu?Ser?Leu?Ala

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Ser?Asn?Gly?Ile?Ser?Thr?Glu?Gly?Gly?Lys?Ser?Leu?Ala?Arg?Ala?Leu

850 855 860

Gln?Gln?Asn?Thr?Ser?Leu?Glu?Ile?Leu?Trp?Leu?Thr?Gln?Asn?Glu?Leu

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Asn?Asp?Glu?Val?Ala?Glu?Ser?Leu?Ala?Glu?Met?Leu?Lys?Val?Asn?Gln

885 890 895

Thr?Leu?Lys?His?Leu?Trp?Leu?Ile?Gln?Asn?Gln?Ile?Thr?Ala?Lys?Gly

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Thr?Ala?Gln?Leu?Ala?Asp?Ala?Leu?Gln?Ser?Asn?Thr?Gly?Ile?Thr?Glu

915 920 925

Ile?Cys?Leu?Asn?Gly?Asn?Leu?Ile?Lys?Pro?Glu?Glu?Ala?Lys?Val?Tyr

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Glu?Asp?Glu?Lys?Arg?Ile?Ile?Cys?Phe

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cgcaatactc?agtgtctggt?ggacaacttg?ctgaagaatg?actacttctc?ggccgaagat?600

gcggagattg?tgtgtgcctg?ccccacccag?cctgacaagg?tccgcaaaat?tctggacctg?660

gtacagagca?agggcgagga?ggtgtccgag?ttcttcctct?acttgctcca?gcaactcgca?720

gatgcctacg?tggacctcag?gccttggctg?ctggagatcg?gcttctcccc?ttccctgctc?780

actcagagca?aagtcgtggt?caacactgac?ccagtgagca?ggtataccca?gcagctgcga?840

caccatctgg?gccgtgactc?caagttcgtg?ctgtgctatg?cccagaagga?ggagctgctg?900

ctggaggaga?tctacatgga?caccatcatg?gagctggttg?gcttcagcaa?tgagagcctg?960

ggcagcctga?acagcctggc?ctgcctcctg?gaccacacca?ccggcatcct?caatgagcag?1020

ggtgagacca?tcttcatcct?gggtgatgct?ggggtgggca?agtccatgct?gctacagcgg?1080

ctgcagagcc?tctgggccac?gggccggcta?gacgcagggg?tcaaattctt?cttccacttt?1140

cgctgccgca?tgttcagctg?cttcaaggaa?agtgacaggc?tgtgtctgca?ggacctgctc?1200

ttcaagcact?actgctaccc?agagcgggac?cccgaggagg?tgtttgcctt?cctgctgcgc?1260

ttcccccacg?tggccctctt?caccttcgat?ggcctggacg?agctgcactc?ggacttggac?1320

ctgagccgtg?tgcctgacag?ctcctgcccc?tgggagcctg?cccaccccct?ggtcttgctg?1380

gccaacctgc?tcagtgggaa?gctgctcaag?ggggctagca?agctgctcac?agcccgcaca?1440

ggcatcgagg?tcccgcgcca?gttcctgcgg?aagaaggtgc?ttctccgggg?cttctccccc?1500

agccacctgc?gcgcctatgc?caggaggatg?ttccccgagc?gggccctgca?ggaccgcctg?1560

ctgagccagc?tggaggccaa?ccccaacctc?tgcagcctgt?gctctgtgcc?cctcttctgc?1620

tggatcatct?tccggtgctt?ccagcacttc?cgtgctgcct?ttgaaggctc?accacagctg?1680

cccgactgca?cgatgaccct?gacagatgtc?ttcctcctgg?tcactgaggt?ccatctgaac?1740

aggatgcagc?ccagcagcct?ggtgcagcgg?aacacacaca?gcccagtgga?gaccctccac?1800

gccggccggg?acactctgtg?ctcgctgggg?caggtggccc?accggggcat?ggagaagagc?1860

ctctttgtct?tcacccagga?ggaggtgcag?gcctccgggc?tgcaggagag?agacatgcag?1920

ctgggcttcc?tgcgggcttt?gccggagctg?ggccccgggg?gtgaccagca?gtcctatgag?1980

tttttccacc?tcaccctcca?ggccttcttt?acagccttct?tcctcgtgct?ggacgacagg?2040

gtgggcactc?aggagctgct?caggttcttc?caggagtgga?tgccccctgc?gggggcagcg?2100

accacgtcct?gctatcctcc?cttcctcccg?ttccagtgcc?tgcagggcag?tggtccggcg?2160

cgggaagacc?tcttcaagaa?caaggatcac?ttccagttca?ccaacctctt?cctgtgcggg?2220

ctgttgtcca?aagccaaaca?gaaactcctg?cggcatctgg?tgcccgcggc?agccctgagg?2280

agaaagcgca?aggccctgtg?ggcacacctg?ttttccagcc?tgcggggcta?cctgaagagc?2340

ctgccccgcg?ttcaggtcga?aagcttcaac?caggtgcagg?ccatgcccac?gttcatctgg?2400

atgctgcgct?gcatctacga?gacacagagc?cagaaggtgg?ggcagctggc?ggccaggggc?2460

atctgcgcca?actacctcaa?gctgacctac?tgcaacgcct?gctcggccga?ctgcagcgcc?2520

ctctccttcg?tcctgcatca?cttccccaag?cggctggccc?tagacctaga?caacaacaat?2580

ctcaacgact?acggcgtgcg?ggagctgcag?ccctgcttca?gccgcctcac?tgttctcaga?2640

ctcagcgtaa?accagatcac?tgacggtggg?gtaaaggtgc?taagcgaaga?gctgaccaaa?2700

tacaaaattg?tgacctattt?gggtttatac?aacaaccaga?tcaccgatgt?cggagccagg?2760

tacgtcacca?aaatcctgga?tgaatgcaaa?ggcctcacgc?atcttaaact?gggaaaaaac?2820

aaaataacaa?gtgaaggagg?gaagtatctc?gccctggctg?tgaagaacag?caaatcaatc?2880

tctgaggttg?ggatgtgggg?caatcaagtt?ggggatgaag?gagcaaaagc?cttcgcagag?2940

gctctgcgga?accaccccag?cttgaccacc?ctgagtcttg?cgtccaacgg?catctccaca?3000

gaaggaggaa?agagccttgc?gagggccctg?cagcagaaca?cgtctctaga?aatactgtgg?3060

ctgacccaaa?atgaactcaa?cgatgaagtg?gcagagagtt?tggcagaaat?gttgaaagtc?3120

aaccagacgt?taaagcattt?atggcttatc?cagaatcaga?tcacagctaa?ggggactgcc?3180

cagctggcag?atgcgttaca?gagcaacact?ggcataacag?agatttgcct?aaatggaaac?3240

ctgataaaac?cagaggaggc?caaagtctat?gaagatgaga?agcggattat?ctgtttctga?3300

gaggatgctt?tcctgttcat?ggggtttttg?ccctggagcc?tcagcagcaa?atgccactct?3360

gggcagtctt?ttgtgtcagt?gtcttaaagg?ggcctgcgca?ggcgggacta?tcaggagtcc?3420

actgcctcca?tgatgcaagc?cagcttcctg?tgcagaaggt?ctggtcggca?aactccctaa?3480

gtacccgcta?caattctgca?gaaaaagaat?gtgtcttgcg?agctgttgta?gttacagtaa?3540

atacactgtg?aagagacttt?attgcctatt?ataattattt?ttatctgaag?ctagaggaat?3600

aaagctgtga?gcaaacagag?gaggccagcc?tcacctcatt?ccaacacctg?ccatagggac?3660

caacgggagc?gagttggtca?ccgctctttt?cattgaagag?ttgaggatgt?ggcacaaagt?3720

tggtgccaag?cttcttgaat?aaaacgtgtt?tgatggatta?gtattatacc?tgaaatattt?3780

tcttccttct?cagcactttc?ccatgtattg?atactggtcc?cacttcacag?ctggagacac?3840

cggagtatgt?gcagtgtggg?atttgactcc?tccaaggttt?tgtggaaagt?taatgtcaag?3900

gaaaggatgc?accacgggct?tttaatttta?atcctggagt?ctcactgtct?gctggcaaag?3960

atagagaatg?ccctcagctc?ttagctggtc?taagaatgac?gatgccttca?aaatgctgct?4020

tccactcagg?gcttctcctc?tgctaggcta?ccctcctcta?gaaggctgag?taccatgggc?4080

tacagtgtct?ggccttggga?agaagtgatt?ctgtccctcc?aaagaaatag?ggcatggctt?4140

gcccctgtgg?ccctggcatc?caaatggctg?cttttgtctc?ccttacctcg?tgaagagggg?4200

aagtctcttc?ctgcctccca?agcagctgaa?gggtgactaa?acgggcgcca?agactcaggg?4260

gatcggctgg?gaactgggcc?agcagagcat?gttggacacc?ccccaccatg?gtgggcttgt?4320

ggtggctgct?ccatgagggt?gggggtgata?ctactagatc?acttgtcctc?ttgccagctc?4380

atttgttaat?aaaatactga?aaacactaaa?aaaaaaaaaa?aa 4422

<210>3

<211>953

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic NOD1 mutant

<400>3

Met?Glu?Glu?Gln?Gly?His?Ser?Glu?Met?Glu?Ile?Ile?Pro?Ser?Glu?Ser

1 5 10 15

His?Pro?His?Ile?Gln?Leu?Leu?Lys?Ser?Asn?Arg?Glu?Leu?Leu?Val?Thr

20 25 30

His?Ile?Arg?Asn?Thr?Gln?Cys?Leu?Gln?Asp?Asn?Leu?Leu?Lys?Asn?Asp

35 40 45

Tyr?Phe?Ser?Ala?Glu?Asp?Ala?Glu?Ile?Val?Cys?Ala?Cys?Pro?Thr?Gln

50 55 60

Pro?Asp?Lys?Val?Arg?Lys?Ile?Leu?Asp?Leu?Va1?Gln?Ser?Lys?Gly?Glu

65 70 75 80

Glu?Val?Ser?Glu?Phe?Phe?Leu?Tyr?Leu?Leu?Gln?Gln?Leu?Ala?Asp?Ala

85 90 95

Tyr?Val?Asp?Leu?Arg?Pro?Trp?Leu?Leu?Glu?Ile?Gly?Phe?Ser?Pro?Ser

100 105 110

Leu?Leu?Thr?Gln?Ser?Lys?Val?Val?Val?Asn?Thr?Asp?Pro?Val?Ser?Arg

115 120 125

Tyr?Thr?Gln?G1n?Leu?Arg?His?His?Leu?Gly?Arg?Asp?Ser?Lys?Phe?Val

130 135 140

Leu?Cys?Tyr?Ala?Gln?Lys?Glu?Glu?Leu?Leu?Leu?Glu?Glu?Ile?Tyr?Met

145 150 155 160

Asp?Thr?Ile?Met?Glu?Leu?Val?Gly?Phe?Ser?Asn?Glu?Ser?Leu?Gly?Ser

165 170 175

Leu?Asn?Ser?Leu?Ala?Cys?Leu?Leu?Asp?His?Thr?Thr?Gly?Ile?Leu?Asn

180 185 190

Glu?Gln?Gly?Glu?Thr?Ile?Phe?Ile?Leu?Gly?Asp?Ala?Gly?Val?Gly?Lys

195 200 205

Ser?Met?Leu?Leu?Gln?Arg?Leu?Gln?Ser?Leu?Trp?Ala?Thr?Gly?Arg?Leu

210 215 220

Asp?Ala?Gly?Val?Lys?Phe?Phe?Phe?His?Phe?Arg?Cys?Arg?Met?Phe?Ser

225 230 235 240

Cys?Phe?Lys?Glu?Ser?Asp?Arg?Leu?Cys?Leu?Gln?Asp?Leu?Leu?Phe?Lys

245 250 255

His?Tyr?Cys?Tyr?Pro?Glu?Arg?Asp?Pro?Glu?Glu?Val?Phe?Ala?Phe?Leu

260 265 270

Leu?Arg?Phe?Pro?His?Val?Ala?Leu?Phe?Thr?Phe?Asp?Gly?Leu?Asp?Glu

275 280 285

Leu?His?Ser?Asp?Leu?Asp?Leu?Ser?Arg?Val?Pro?Asp?Ser?Ser?Cys?Pro

290 295 300

Trp?Glu?Pro?Ala?His?Pro?Leu?Val?Leu?Leu?Ala?Asn?Leu?Leu?Ser?Gly

305 310 315 320

Lys?Leu?Leu?Lys?Gly?Ala?Ser?Lys?Leu?Leu?Thr?Ala?Arg?Thr?Gly?Ile

325 330 335

Glu?Val?Pro?Arg?Gln?Phe?Leu?Arg?Lys?Lys?Val?Leu?Leu?Arg?Gly?Phe

340 345 350

Ser?Pro?Ser?His?Leu?Arg?Ala?Tyr?Ala?Arg?Arg?Met?Phe?Pro?Glu?Arg

355 360 365

Ala?Leu?Gln?Asp?Arg?Leu?Leu?Ser?Gln?Leu?Glu?Ala?Asn?Pro?Asn?Leu

370 375 380

Cys?Ser?Leu?Cys?Ser?Val?Pro?Leu?Phe?Cys?Trp?Ile?Ile?Phe?Arg?Cys

385 390 395 400

Phe?Gln?His?Phe?Arg?Ala?Ala?Phe?Glu?Gly?Ser?Pro?Gln?Leu?Pro?Asp

405 410 415

Cys?Thr?Met?Thr?Leu?Thr?Asp?Val?Phe?Leu?Leu?Val?Thr?Glu?Val?His

420 425 430

Leu?Asn?Arg?Met?Gln?Pro?Ser?Ser?Leu?Val?Gln?Arg?Asn?Thr?Arg?Ser

435 440 445

Pro?Val?Glu?Thr?Leu?His?Ala?Gly?Arg?Asp?Thr?Leu?Cys?Ser?Leu?Gly

450 455 460

Gln?Val?Ala?His?Arg?Gly?Met?Glu?Lys?Ser?Leu?Phe?Val?Phe?Thr?Gln

465 470 475 480

Glu?Glu?Val?Gln?Ala?Ser?Gly?Leu?Gln?Glu?Arg?Asp?Met?Gln?Leu?Gly

485 490 495

Phe?Leu?Arg?Ala?Leu?Pro?Glu?Leu?Gly?Pro?Gly?Gly?Asp?Gln?Gln?Ser

500 505 510

Tyr?Glu?Phe?Phe?His?Leu?Thr?Leu?Gln?Ala?Phe?Phe?Thr?Ala?Phe?Phe

515 520 525

Leu?Val?Leu?Asp?Asp?Arg?Val?Gly?Thr?Gln?Glu?Leu?Leu?Arg?Phe?Phe

530 535 540

Gln?Glu?Trp?Met?Pro?Pro?Ala?Gly?Ala?Ala?Thr?Thr?Ser?Cys?Tyr?Pro

545 550 555 560

Pro?Phe?Leu?Pro?Phe?Gln?Cys?Leu?Gln?Gly?Ser?Gly?Pro?Ala?Arg?Glu

565 570 575

Asp?Leu?Phe?Lys?Asn?Lys?Asp?His?Phe?Gln?Phe?Thr?Asn?Leu?Phe?Leu

580 585 590

Cys?Gly?Leu?Leu?Ser?Lys?Ala?Lys?Gln?Lys?Leu?Leu?Arg?His?Leu?Val

595 600 605

Pro?Ala?Ala?Ala?Leu?Arg?Arg?Lys?Arg?Lys?Ala?Leu?Trp?Ala?His?Leu

610 615 620

Phe?Ser?Ser?Leu?Arg?Gly?Tyr?Leu?Lys?Ser?Leu?Pro?Arg?Val?Gln?Val

625 630 635 640

Glu?Ser?Phe?Asn?Gln?Val?Gln?Ala?Met?Pro?Thr?Phe?Ile?Trp?Met?Leu

645 650 655

Arg?Cys?Ile?Tyr?Glu?Thr?Gln?Ser?Gln?Lys?Val?Gly?Gln?Leu?Ala?Ala

660 665 670

Arg?Gly?Ile?Cys?Ala?Asn?Tyr?Leu?Lys?Leu?Thr?Tyr?Cys?Asn?Ala?Cys

675 680 685

Ser?Ala?Asp?Cys?Ser?Ala?Leu?Ser?Phe?Val?Leu?His?His?Phe?Pro?Lys

690 695 700

Arg?Leu?Ala?Leu?Asp?Leu?Asp?Asn?Asn?Asn?Leu?Asn?Asp?Tyr?Gly?Val

705 710 715 720

Arg?Glu?Leu?Gln?Pro?Cys?Phe?Ser?Arg?Leu?Thr?Val?Leu?Arg?Leu?Ser

725 730 735

Val?Asn?Gln?Ile?Thr?Asp?Gly?Gly?Val?Lys?Val?Leu?Ser?Glu?Glu?Leu

740 745 750

Thr?Lys?Tyr?Lys?Ile?Val?Thr?Tyr?Leu?Gly?Leu?Tyr?Asn?Asn?Gln?Ile

755 760 765

Thr?Asp?Val?Gly?Ala?Arg?Tyr?Val?Thr?Lys?Ile?Leu?Asp?Glu?Cys?Lys

770 775 780

Gly?Leu?Thr?His?Leu?Lys?Leu?Gly?Lys?Asn?Lys?Ile?Thr?Ser?Glu?Gly

785 790 795 800

Gly?Lys?Tyr?Leu?Ala?Leu?Ala?Val?Lys?Asn?Ser?Lys?Ser?Ile?Ser?Glu

805 810 815

Val?Gly?Met?Trp?Gly?Asn?Gln?Val?Gly?Asp?Glu?Gly?Ala?Lys?Ala?Phe

820 825 830

Ala?Glu?Ala?Leu?Arg?Asn?His?Pro?Ser?Leu?Thr?Thr?Leu?Ser?Leu?Ala

835 840 845

Ser?Asn?Gly?Ile?Ser?Thr?Glu?Gly?Gly?Lys?Ser?Leu?Ala?Arg?Ala?Leu

850 855 860

Gln?Gln?Asn?Thr?Ser?Leu?Glu?Ile?Leu?Trp?Leu?Thr?Gln?Asn?Glu?Leu

865 870 875 880

Asn?Asp?Glu?Val?Ala?Glu?Ser?Leu?Ala?Glu?Met?Leu?Lys?Val?Asn?Gln

885 890 895

Thr?Leu?Lys?His?Leu?Trp?Leu?Ile?Gln?Asn?Gln?Ile?Thr?Ala?Lys?Gly

900 905 910

Thr?Ala?Gln?Leu?Ala?Asp?Ala?Leu?Gln?Ser?Asn?Thr?Gly?Ile?Thr?Glu

915 920 925

Ile?Cys?Leu?Asn?Gly?Asn?Leu?Ile?Lys?Pro?Glu?Glu?Ala?Lys?Val?Tyr

930 935 940

Glu?Asp?Glu?Lys?Arg?Ile?Ile?Cys?Phe

945 950

<210>4

<211>233

<212>PRT

＜213〉mankind

<400>4

Met?Ser?Thr?Glu?Ser?Met?Ile?Arg?Asp?Val?Glu?Leu?Ala?Glu?Glu?Ala

1 5 10 15

Leu?Pro?Lys?Lys?Thr?Gly?Gly?Pro?Gln?Gly?Ser?Arg?Arg?Cys?Leu?Phe

20 25 30

Leu?Ser?Leu?Phe?Ser?Phe?Leu?Ile?Val?Ala?Gly?Ala?Thr?Thr?Leu?Phe

35 40 45

Cys?Leu?Leu?His?Phe?Gly?Val?Ile?Gly?Pro?Gln?Arg?Glu?Glu?Ser?Pro

50 55 60

Arg?Asp?Leu?Ser?Leu?Ile?Ser?Pro?Leu?Ala?Gln?Ala?Val?Arg?Ser?Ser

65 70 75 80

Ser?Arg?Thr?Pro?Ser?Asp?Lys?Pro?Val?Ala?His?Val?Val?Ala?Asn?Pro

85 90 95

Gln?Ala?Glu?Gly?Gln?Leu?Gln?Trp?Leu?Asn?Arg?Arg?Ala?Asn?Ala?Leu

100 105 110

Leu?Ala?Asn?Gly?Val?Glu?Leu?Arg?Asp?Asn?Gln?Leu?Val?Val?Pro?Ser

115 120 125

Glu?Gly?Leu?Tyr?Leu?Ile?Tyr?Ser?Gln?Val?Leu?Phe?Lys?Gly?Gln?Gly

130 135 140

Cys?Pro?Ser?Thr?His?Val?Leu?Leu?Thr?His?Thr?Ile?Ser?Arg?Ile?Ala

145 150 155 160

Val?Ser?Tyr?Gln?Thr?Lys?Val?Asn?Leu?Leu?Ser?Ala?Ile?Lys?Ser?Pro

165 170 175

Cys?Gln?Arg?Glu?Thr?Pro?Glu?Gly?Ala?Glu?Ala?Lys?Pro?Trp?Tyr?Glu

180 185 190

Pro?Ile?Tyr?Leu?Gly?Gly?Val?Phe?Gln?Leu?Glu?Lys?Gly?Asp?Arg?Leu

195 200 205

Ser?Ala?Glu?Ile?Asn?Arg?Pro?Asp?Tyr?Leu?Asp?Phe?Ala?Glu?Ser?Gly

210 215 220

Gln?Val?Tyr?Phe?Gly?Ile?Ile?Ala?Leu

225 230

<210>5

<211>540

<212>PRT

＜213〉mankind

<400>5

Met?Asn?Gly?Glu?Ala?Ile?Cys?Ser?Ala?Leu?Pro?Thr?Ile?Pro?Tyr?His

1 5 10 15

Lys?Leu?Ala?Asp?Leu?Arg?Tyr?Leu?Ser?Arg?Gly?Ala?Ser?Gly?Thr?Val

20 25 30

Ser?Ser?Ala?Arg?His?Ala?Asp?Trp?Arg?Val?Gln?Val?Ala?Val?Lys?His

35 40 45

Leu?His?Ile?His?Thr?Pro?Leu?Leu?Asp?Ser?Glu?Arg?Lys?Asp?Val?Leu

50 55 60

Arg?Glu?Ala?Glu?Ile?Leu?His?Lys?Ala?Arg?Phe?Ser?Tyr?Ile?Leu?Pro

65 70 75 80

Ile?Leu?Gly?Ile?Cys?Asn?Glu?Pro?Glu?Phe?Leu?Gly?Ile?Val?Thr?Glu

85 90 95

Tyr?Met?Pro?Asn?Gly?Ser?Leu?Asn?Glu?Leu?Leu?His?Arg?Lys?Thr?Glu

100 105 110

Tyr?Pro?Asp?Val?Ala?Trp?Pro?Leu?Arg?Phe?Arg?Ile?Leu?His?Glu?Ile

115 120 125

Ala?Leu?Gly?Val?Asn?Tyr?Leu?His?Asn?Met?Thr?Pro?Pro?Leu?Leu?His

130 135 140

His?Asp?Leu?Lys?Thr?Gln?Asn?Ile?Leu?Leu?Asp?Asn?Glu?Phe?His?Val

145 150 155 160

Lys?Ile?Ala?Asp?Phe?Gly?Leu?Ser?Lys?Trp?Arg?Met?Met?Ser?Leu?Ser

165 170 175

Gln?Ser?Arg?Ser?Ser?Lys?Ser?Ala?Pro?Glu?Gly?Gly?Thr?Ile?Ile?Tyr

180 185 190

Met?Pro?Pro?Glu?Asn?Tyr?Glu?Pro?Gly?Gln?Lys?Ser?Arg?Ala?Ser?Ile

195 200 205

Lys?His?Asp?Ile?Tyr?Ser?Tyr?Ala?Val?Ile?Thr?Trp?Glu?Val?Leu?Ser

210 215 220

Arg?Lys?Gln?Pro?Phe?Glu?Asp?Val?Thr?Asn?Pro?Leu?Gln?Ile?Met?Tyr

225 230 235 240

Ser?Val?Ser?Gln?Gly?His?Arg?Pro?Val?Ile?Asn?Glu?Glu?Ser?Leu?Pro

245 250 255

Tyr?Asp?Ile?Pro?His?Arg?Ala?Arg?Met?Ile?Ser?Leu?Ile?Glu?Ser?Gly

260 265 270

Trp?Ala?Gln?Asn?Pro?Asp?Glu?Arg?Pro?Ser?Phe?Leu?Lys?Cys?Leu?Ile

275 280 285

Glu?Leu?Glu?Pro?Val?Leu?Arg?Thr?Phe?Glu?Glu?Ile?Thr?Phe?Leu?Glu

290 295 300

Ala?Val?Ile?Gln?Leu?Lys?Lys?Thr?Lys?Leu?Gln?Ser?Val?Ser?Ser?Ala

305 310 315 320

Ile?His?Leu?Cys?Asp?Lys?Lys?Lys?Met?Glu?Leu?Ser?Leu?Asn?Ile?Pro

325 330 335

Val?Asn?His?Gly?Pro?Gln?Glu?Glu?Ser?Cys?Gly?Ser?Ser?Gln?Leu?His

340 345 350

Glu?Asn?Ser?Gly?Ser?Pro?Glu?Thr?Ser?Arg?Ser?Leu?Pro?Ala?Pro?Gln

355 360 365

Asp?Asn?Asp?Phe?Leu?Ser?Arg?Lys?Ala?Gln?Asp?Cys?Tyr?Phe?Met?Lys

370 375 380

Leu?His?His?Cys?Pro?Gly?Asn?His?Ser?Trp?Asp?Ser?Thr?Ile?Ser?Gly

385 390 395 400

Ser?Gln?Arg?Ala?Ala?Phe?Cys?Asp?His?Lys?Thr?Thr?Pro?Cys?Ser?Ser

405 410 415

Ala?Ile?Ile?Asn?Pro?Leu?Ser?Thr?Ala?Gly?Asn?Ser?Glu?Arg?Leu?Gln

420 425 430

Pro?Gly?Ile?Ala?Gln?Gln?Trp?Ile?Gln?Ser?Lys?Arg?Glu?Asp?Ile?Val

435 440 445

Asn?Gln?Met?Thr?Glu?Ala?Cys?Leu?Asn?Gln?Ser?Leu?Asp?Ala?Leu?Leu

450 455 460

Ser?Arg?Asp?Leu?Ile?Met?Lys?Glu?Asp?Tyr?Glu?Leu?Val?Ser?Thr?Lys

465 470 475 480

Pro?Thr?Arg?Thr?Ser?Lys?Val?Arg?Gln?Leu?Leu?Asp?Thr?Thr?Asp?Ile

485 490 495

Gln?Gly?Glu?Glu?Phe?Ala?Lys?Val?Ile?Val?Gln?Lys?Leu?Lys?Asp?Asn

500 505 510

Lys?Gln?Met?Gly?Leu?Gln?Pro?Tyr?Pro?Glu?Ile?Leu?Val?Val?Ser?Arg

515 520 525

Ser?Pro?Ser?Leu?Asn?Leu?Leu?Gln?Asn?Lys?Ser?Met

530 535 540

Claims (48)

1. compositions; described compositions comprises D-Ala-L-Glu-meso diaminopimelic acid (γ TriDAP), γ-D-glutamyl-meso-meso diaminopimelic acid (iE-DAP), γ-D-Gln-DAP (iQ-DAP), D-Ala-L-Glu-meso diaminopimelic acid (γ TriDAP) or its combination of carrier and treatment effective dose, and wherein said treatment effective dose is effective for the degeneration of tumor.

2. the compositions of claim 1, described compositions also comprises nucleic acid, and this nucleic acid comprises the nucleic acid fragment of coding NOD1 polypeptide.

3. the compositions of claim 2, wherein said NOD1 polypeptide comprises SEQ ID NO:1 or SEQ ID NO:3.

4. the compositions of claim 2, the fragment of wherein said coding NOD1 polypeptide comprises SEQ ID NO:2.

5. the compositions of claim 2, wherein said nucleic acid further comprises regulating element.

6. the compositions of claim 2, wherein said regulating element is promoter, enhancer, transcription stop signals or its combination.

7. the compositions of claim 2, wherein said nucleic acid is expression cassette or expression vector.

8. the compositions of claim 2, wherein said nucleic acid comprises gene delivery vector.

9. the compositions of claim 1, wherein said compositions also comprises the NOD1 polypeptide of effective dose.

10. the compositions of claim 1, wherein said compositions also comprises the tumor necrosis factor of effective dose.

11. the compositions of claim 1, wherein said compositions also comprises the cycloheximide of effective dose.

12. the compositions of claim 1, wherein said compositions also comprise the hormone or the hormone antagonist of effective dose.

13. the compositions of claim 1, wherein said compositions also comprise the RIP2 inhibitors of kinases of effective dose.

14. the compositions of claim 1, wherein said compositions is used for to tumor or cancerous tissue local application by preparation.

15. a method that promotes tumour regression in mammal, described method comprise that improving Nod1 to described administration expresses or the active reagent of NOD1.

16. the method for claim 15, wherein said tumor are brain, bladder, cervix uteri, colon, gallbladder, kidney, liver, lung, pancreas, ovary, prostate, skin, stomach or thyroid tumor.

17. the method for claim 15, wherein said tumor are the estrogen-sensitive tumors.

18. the method for claim 15, wherein said tumor is a breast tumor.

19. the method for claim 15, wherein said reagent are D-Ala-L-Glu-meso diaminopimelic acid (γ TriDAP), γ-D-glutamyl-meso-meso diaminopimelic acid (iE-DAP), γ-D-Gln-DAP (iQ-DAP), D-Ala-L-Glu-meso diaminopimelic acid (γ TriDAP) or its combination.

20. the method for claim 15, wherein said reagent is nucleic acid, and it comprises the nucleic acid fragment of coding NOD1 polypeptide.

21. the method for claim 20, wherein said NOD1 polypeptide comprise SEQ ID NO:1 or SEQ ID NO:3.

22. the method for claim 20, the nucleic acid fragment of wherein said coding NOD1 polypeptide comprises SEQ ID NO:2.

23. the method for claim 20, wherein said nucleic acid further comprises regulating element.

24. the method for claim 23, wherein said regulating element are promoter, enhancer, transcription stop signals or its combination.

25. the method for claim 20, wherein said nucleic acid are expression cassette or expression vector.

26. the method for claim 20, wherein said nucleic acid comprises gene delivery vector.

27. the method for claim 15, wherein said reagent are the NOD1 polypeptide.

28. the method for claim 27, wherein said NOD1 polypeptide comprise SEQ ID NO:1 or SEQ ID NO:3.

29. the method for claim 15, wherein said reagent are administered to the site of tumor or cancerous tissue partly.

30. the method for claim 15, wherein said reagent is used for continuing to discharge by preparation.

31. the method for claim 15, the tumor necrosis factor of wherein said reagent and effective dose is co-administered.

32. the method for claim 15, the cycloheximide of wherein said reagent and effective dose is co-administered.

33. the method for claim 15, wherein said reagent and RIP2 inhibitors of kinases are co-administered.

34. the method for claim 15, the hormone or the hormone antagonist of wherein said reagent and effective dose are co-administered.

35. the method for claim 34, wherein said hormone is an estrogen.

36. the method for claim 34, wherein said hormone antagonist is an estrogen antagonist.

37. the method for claim 15, the chemotherapy compound of wherein said reagent and effective dose is co-administered.

38. the method for claim 37, wherein said chemotherapy compound is selected from hexamethyl melamine, bleomycin, Busulfan, calcium folinate, capecitabine, carboplatin, carmustine, benzenebutanoic acid helium mustard, cisplatin, cladribine, Crisantaspase, cyclophosphamide, cytosine arabinoside, dacarbazine, D actinomycin D, daunorubicin, docetaxel, amycin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, liposomal doxorubicin, lomustine, melphalan, purinethol, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pentostatin, procarbazine, Raltitrexed, streptozotocin, UFT, Temozoromide, thiotepa, thioguanine/thioguanine, topotecan, treosulfan, vincaleucoblastine, vincristine, desacetyl vinblastine amide, vinorelbine and its combination.

39. one kind lacks the active isolating Nod1 of NOD1 ^-/-Cell line.

40. the isolating Nod1 of claim 39 ^-/-Cell line, it also lacks progesterone receptor.

41. the isolating Nod1 of claim 39 ^-/-Cell line, wherein said cell line comprises the MCF-7C20 cell.

42. the isolating Nod1 of claim 39 ^-/-Cell line, it also comprises reorganization Nod1 allele.

43. method of identifying chemotherapeutant, described method comprises the enzyme with test agent contact RIP2, and determine whether described test agent suppresses the RIP2 kinase activity, wherein indicating described test agent by the active inhibition of the RIP2 of described test agent in cell is chemotherapeutant.

44. the method for claim 43, wherein said method is carried out external.

45. the method for claim 43, wherein said method is carried out in vivo.

46. one kind promotes the apoptotic method in the breast tumor cell, described method comprises D-Ala-L-Glu-meso diaminopimelic acid (γ TriDAP) the contact breast tumor cell with effective dose.

47. identify the apoptotic compositions and methods that improves in the cell for one kind, described method comprises with test agent and contacts Nod1 ^-/-Cell, and determine whether described test agent improves the apoptosis of described cell.

48. the method for claim 47 wherein improves apoptotic test agent and can be used as chemotherapeutant.

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