Analyses and methods with application of biomarkers

FIELD: medicine.

SUBSTANCE: what is presented is a method for Apo2L/TRAIL sensitivity prediction of a malignant tissue or cell sampled from a mammal, involving the stages as follows: sampling a malignant tissue or cell from a mammal; analysing the sample malignant tissue or cell for detecting expression of one or more biomarkers selected from a group of fucosyl transferase 3, fucosyl transferase 6, sialyl-Lewis A and/or X antigen (antigens) where expression of one or more specified biomarkers is an indicator of the fact that the specified sampled tissue or cell is sensitive to apoptosis-inducing activity Apo2L/TRAIL. Also, what is described is a method of apoptosis induction in the sampled malignant tissue or cell of a mammal. What is offered is a method of treating a malignant tumour in a mammal. The inventions enables using the detection of expression of one or more biomarkers as the indicator of the fact that a sample is sensitive to apoptosis-inducing agents, such as Apo2L/TRAIL and DR5 agonist antibodies. Specific biomarkers to be examined include fucosyl transferases, particularly fucosyl transferase 3 (FUT3) and/or fucosyl transferase 6 (FUT6), as well as sialyl-Lewis A and/or X antigens.

EFFECT: method improvement.

35 cl, 22 dwg, 1 ex

 

Related applications

Under this proposal p(e) the priority of the provisional application U.S. number 60/599425, filed August 6, 2004, the full contents of which are hereby incorporated by reference.

The scope of the invention

This invention relates to methods and assays for the detection of biomarkers that predict sensitivity of mammalian cells to Apo2L/TRAIL and/or antibody - receptor agonists death.

Background of the invention

In this area identified a variety of ligands and receptors belonging to the superfamily of factors tumor necrosis (TNF). Such ligands include tumor necrosis factor-alpha ("TNF-alpha), tumor necrosis factor-β ("TNF-β" or "lymphotoxin-alpha"), lymphotoxin-β ("LT-β"), CD30 ligand, a CD27 ligand, a CD40 ligand, a ligand, OX-40 ligand, 4-1BB, LIGHT, ligand, Apo-1 (denoted as Fas ligand or CD95 ligand), the ligand Apo-2 (denoted as Apo2L or TRAIL), the ligand, Apo-3 (marked as TWEAK), APRIL, OPG ligand (denoted as RANK ligand, ODF or TRANCE), and TALL-1 (also marked as BlyS, BAFF or THANK) (see, for example, Ashkenazi, Nature Review, 2:420-430 (2002); Ashkenazi and Dixit, Science 281:1305-1308 (1998); Ashkenazi and Dixit, Curr. Opin. Cell Biol., 11:255-260 (2000); Golstein, Curr. Biol., 7:750-753 (1997) Wallach, Cytokine Reference, Academic Press, 2000, pages 377-411; Locksley et al., Cell, 104:487-501 (2001); Gruss and Dower, Blood, 85:3378-3404 (1995); Schmid et al., Proc. Natl. Acad. Sci., 83:1881 (1986); Dealtry et al, Eur. J. Immunol., 17:689 (1987); Pitti et al., J. Biol. Chem., 271:12687-12690 (1996); Wiley et al., Immunity, 3:673-682 (1995); Browning et al., Cell 72:847-856 (1993); Armitage et al. Nature, 357:80-82 (1992); WO 97/01633, published January 16, 1997; WO 97/25428, published July 17, 1997; Marsters et al., Curr. Biol., 8:525-528 (1998); Chicheportiche et al., Biol. Chem., 272:32401-32410 (1997); " Hahne " et al., J. Exp. Med., 188:1185-1190 (1998); WO 98/28426, published 2 July 1998; WO 98/46751, published October 22, 1998; WO 98/18921, published may 7, 1998; Moore et al., Science, 285:260-263 (1999); Shu et al., J. Leukocyte Biol., 65:680 (1999); Schneider et al., J. Exp. Med., 189:1747-1756 (1999); Mukhopadhyay et al., J. Biol. Chem., 274:15978-15981 (1999)).

Induction of various cellular responses mediated by such ligands of the TNF family, usually initiated by their binding to specific cell receptors. Some, but not all, of the ligands of the TNF family are associated with the "death receptors" on the surface of the cells and induce through different biological activity for the activation of caspases or enzymes that carry out the cell death or apoptotic path (Salvesen et al., Cell 91:443-446 (1997)). Identified to date, members of the superfamily of TNF receptor include TNFR1, TNFR2, TACI, GITR, CD27, OX-40, CD30, CD40, HVEM, Fas (labelled as Apo-1 or CD95), DR4 (marked as TRAIL-R1), DR5 (labelled as Apo-2 or TRAIL-R2), DcR1, DcR2, osteoprotegerin (OPG), RANK and Apo-3 (denoted as DR3 or TRAMP) (see, for example, Ashkenazi, Nature Reviews, 2:420-430 (2002); Ashkenazi and Dixit, Science 281:1305-1308 (1998); Ashkenazi and Dixi, Curr. Opin. Cell Biol., 11:255-260 (2000); Golstein, Curr. Biol., 7:750-753 (1997) Wallach, Cytokine Reference, Academic Press, 2000, pp. 377-411; Locksley et al. Cell, 104:487-501 (2001); Gruss and Dower, Blood, 85:3378-3404 (1995); Hohman et al., J. Biol. Chem., 264:14927-14934 (1989); Brockhaus et al., Proc. Natl. Acad. Sci., 87:3127-3131 (1990); EP 417563 published March 20, 1991; Loetscher et al., Cell, 61:351 (1990); Schall et al., Cell 61:361 (1990); Smith et al., Science, 248:1019-1023 (1990); Lewis et al., Proc. Natl. Acad. Sci., 88:2830-2834 (1991); Goodwin et al., Mol. Cell. Biol., 11:3020-3026 (1991); Stamenkovic et al., EMBO J. 8:1403-1410 (1989); Mallett et al., EMBO J. 9:1063-1068 (1990); Anderson et al., Nature, 390:175-179 (1997); Chicheportiche et al., J. Biol. Chem., 272:32401-32410 (1997); Pan et al., Science, 276:111-113 (1997); Pan et al., Science, 277:815-818 (1997); Sheridan et al., Science, 277:818-821 (1997); Degli-Esposti et al., J. Exp. Med., 186:1165-1170 (1997); Marsters et al., Curr. Biol., 7:1003-1006 (1997); Tsuda et al., BBRC, 234:137-142 (1997); Nocentini et al., Proc. Natl. Acad. Sci., 94:6216-6221 (1997); vonBulow et al., Science 278:138-141 (1997)).

Most of these members of the family of TNF receptors share a common structure of cell surface receptors, including extracellular, transmembrane and intracellular region, while others are found in nature as a soluble protein with no transmembrane and intracellular domain. The extracellular part of the typical TNFR contains a repeating pattern of amino acid sequences of multiple cysteine-rich domains (CRD), starting from N-end.

The ligand is labelled as Apo-2L) or TRAIL, identified several years ago as a member of a family of cytokines TNF (see, e.g., Wiley et al., Immunity, 3:673-682 (1995); Pitti et al., . Biol. Chem., 271:12697-12690 (1996); WO 97/01633; WO 97/25428; U.S. patent 5763223, published June 9, 1998; U.S. patent 6284236, published September 4, 2001). Full size natural sequence of the polypeptide of human Apo2L/TRAIL is a transmembrane protein type II length of 281 amino acids. Some cells can produce a natural soluble form of the polypeptide due to enzymatic cleavage of the extracellular region of the polypeptide (Mariani et al., J. Cell. Biol., 137:221-229 (1997)). Crystallographic studies of soluble Apo2L/TRAIL revealed homotrimer structure similar to the structures of TNF and other related proteins (Hymowitz et al., Molec. Cell, 4:563-571 (1999); Cha et al., Immunity, 11:253-261 (1999); Mongkolsapaya et al., Nature Structural Biology, 6:1048 (1999); Hymowitz et al., Biochemistry, 39:633-644 (2000)). However, found that, unlike other members of the TNF family, Apo2L/TRAIL has the unique structural property that three cysteine residue (at position 230 of each subunit in homotrimer) together coordinate the zinc atom and the binding of zinc is important for the stability of the trimer and biological activity (Hymowitz et al., above, Bodmer et al., J. Biol. Chem., 275:20632-20637 (2000)).

In the literature it was reported that Apo2L/TRAIL may play a role in modulation of the immune system, including autoimmune diseases such as rheumatoid arthritis (see, for example, Thomas et al., J. Immunol., 161:2195-2200 (1998); Johnsen et al., Cytokine, 11:664-672 (1999); Griffith et al., J. xp. Med., 189:1343-1353 (1999); Song et al., J. Exp. Med., 191:1095-1103 (2000)).

Also published that soluble forms of Apo2L/TRAIL induce apoptosis in several cancer cells, including tumors of the colon, lung, breast, prostate, bladder, kidney, ovary and brain, as well as melanoma, leukemia, and multiple myeloma (see, e.g., Wiley et al., above; Pitti et al., above; U.S. patent 6030945, published on February 29, 2000; U.S. patent 6746668, published June 8, 2004; Rieger et al., FEBS Letters 427:124 to 128 (1998); Ashkenazi et al., J. Clin. Invest., 104:155-162 (1999); Walczak et al., Nature Med., 5:157-163 (1999); Keane et al., Cancer Research, 59:734-741 (1999); Mizutani et al., Clin. Cancer Res., 5:2605-2612 (1999); Gazitt, Leukemia, 13:1817-1824 (1999); Yu et al., Cancer Res., 60:2384-2389 (2000); Chinnaiyan et al., Proc. Natl. Acad. Sci., 97:1754-1759 (2000)). Researchin vivoon models of tumors in mice allow further assumed that Apo2L/TRAIL, alone or in combination with chemotherapy or radiotherapy, can have significant antitumor effects (see, e.g., Ashkenazi et al., above; Walzcak et al., above; Gliniak et al., Cancer Res., 59:6153-6158 (1999); Chinnaiyan et al., above; Roth et al., Biochem. Biophys. Res. Comm., 265:1999 (1999); patent application PCT US/00/15512; patent application PCT US/0l/23691). Unlike many types of cancer cells, most normal types of human cells, apparently, are resistant to induction of apoptosis specific recombinant forms of Apo2L/TRAIL (Ashkenazi et al., above; Walzcak et al., above). Jo et al. published that labeled polyhistidine the m a soluble form of Apo2L/TRAIL induces apoptosis in vitroin normal dedicated human hepatocytes, in contrast to non-human (Jo et al., Nature Med., 6:564-567 (2000); see also Nagata, Nature Med., 6:502-503 (2000)). Consider that the specific preparations of recombinant Apo2L/TRAIL may vary in relation to the biochemical properties and biological activities of cells affected by the disease, in contrast to normal cells, depending on, for example, from the presence or absence of the label molecules, zinc content and the % content of the trimer (see Lawrence et al., Nature Med., Letter to the Editor, 7:383-385 (2001); Qin et al., Nature Med., Letter to the Editor, 7:385-386 (2001)).

Discovered that Apo2L/TRAIL associated with at least five different receptors. At least two of the receptors that bind Apo2L/TRAIL, contain a functional cytoplasmic domain of death. One of these receptors is indicated by "DR4" (and alternative, as TR4 or TRAIL-R1) (Pan et al., Science, 276:111-113 (1997); see also WO 98/32856, published July 30, 1998; WO 99/37684, published on July 29, 1999; WO 00/73349, published 7 December 2000; US 6433147, published August 13, 2002; US 6461823, published October 8, 2002, and US 6342383, published on 29 January 2002).

Another receptor for Apo2L/TRAIL marked DR5 (alternative denote as Apo-2; TRAIL-R or TRAIL-R2, TR6, Tango-63, hAPO8, TRICK2 or KILLER) (see, e.g., Sheridan et al., Science, 277:818-821 (1997), Pan et al., Science, 277:815-818 (1997), WO 98/51793, published on November 19, 1998; WO 98/41629, SDA is likovnog 24 September 1998; Screaton et al., Curr. Biol., 7:693-696 (1997); Walczak et al., EMBO J. 16:5386-5387 (1997); Wu et al., Nature Genetics, 17:141-143 (1997); WO 98/35986, published August 20, 1998; EP 870827, published on October 14, 1998; WO 98/46643, published October 22, 1998; WO 99/02653 published January 21, 1999; WO 99/09165, published on 25 February 1999; WO 99/11791, published 11 March 1999; US 2002/0072091, published August 13, 2002; US 2002/0098550, published 7 December 2001; US 6313269 published on 6 December 2001; US 2001/0010924 published 2 August 2001; US 2003/01255540, published 3 July 2003; US 2002/0160446, published October 31, 2002, US 2002/0048785, published April 25, 2002; US 6342369, published in February 2002; US 6569642, published 27 may 2003, US 6072047, published June 6, 2000, US 6642358 published 4 November 2003; US 6743625, published June 1, 2004). Published that, like DR4, DR5 contains a cytoplasmic death domain and is able to signal apoptosis upon binding of the ligand (or binding molecules, such as antibody-agonist, which mimicries activity of the ligand). Crystal structure of the complex formed between the Apo-2L/TRAIL and DR5, are described in Hymowitz et al., Molecular Cell, 4:563-571 (1999).

When the binding of the ligand as DR4 and DR5 can independently run apoptosis through the involvement and activation of the initiator of apoptosis, caspase-8, through containing the domain of death adapting molecule, denoted FADD/Mrt1 (Kischkel et al., Immunity, 12:611-620 (2000); and Sprick et al., Immunity, 12:599-609 (2000); Bodmer et al., Nature Cell Biol., 2:241 to 243 (2000)).

Published that Apo2L/TRAIL also binds to such receptors, designated DcR1, DcR2 and OPG, which are believed to function as inhibitors, but not transmitters of the signal (see, for example, DCR1 (denoted as TRID, LIT or TRAIL-R3) (Pan et al., Science, 276: 111-113 (1997); Sheridan et al., Science, 277:818-821 (1997); McFarlane et al., J. Biol. Chem., 272:25417-25420 (1997); Schneider et al., FEBS Letters 416:329-334 (1997); Degli-Esposti et al., J. Exp. Med., 186:1165-1170 (1997); Mongkolsapaya et al., J. Immunol., 160:3-6 (1998)); DCR2 (also called TRUNDD or TRAIL-R4) (Marsters et al., Curr. Biol., 7:1003-1006 (1997); Pan et al., FEBS Letters 424:41-45 (1998); Degli-Esposti et al., Immunity, 7:813-820 (1997)), OPG (Simonet et al., above). In contrast to DR4 and DR5, receptors DcR1 and DcR2 not transmit a signal apoptosis.

Specific antibodies that bind receptors DR4 and/or DR5, published in the literature. For example, anti-DR4 antibodies directed against the receptor DR4 and having agonistic or apoptotic activity in a particular mammalian cells, are described, for example, in WO 99/37684, published on July 29, 1999; WO 00/73349, published July 12, 2000; WO 03/066661, published August 14, 2003 Cm. also, for example, Griffith et al., J. Immunol., 162:2597-2605 (1999); Chuntharapai et al., J. Immunol., 166:4891-4898 (2001); WO 02/097033, published December 2, 2002; WO 03/042367, published may 22, 2003; WO 03/038043, published may 8, 2003; WO 03/037913, published may 8, 2003, in a Similar way have been described specific anti-DR5 antibodies, see, for example the EP, WO 98/51793, published November 8, 1998; Griffith et al., J. Immunol., 162:2597-2605 (1999); Ichikawa et al., Nature Med., 7:954-960 (2001); Hylander et al., "An Antibody to DR5 (TRAIL-Receptor 2) Suppresses the Growth of Patient Derived Gastrointestinal Tumors Grown in SCID mice", Abstract, 2d International Congress on Monoclonal Antibodies in Cancers, Aug. 29-Sept. 1, 2002, Banff, Alberta, Canada; WO 03/038043, published may 8, 2003; WO 03/037913, published may 8, 2003, were also described some of the antibodies with cross-reactivity as the receptor DR4 and receptor DR5 (see, for example, U.S. patent 6252050, published on 26 June 2001).

Neoplastic transformation of some mammalian cells, in certain cases, is associated with characteristic changes in the expression of antigens sialyl-Lewis A and sialyl-Lewis X. a Relatively high number sialyl-Lewis A/X are, for example, in some adenocarcinomas of the colon, pancreas and stomach of a man, and analyses using antibodies directed against carbohydrate structures of these antigens, applied as a means for detection of malignant tumors of the pancreas and the gastrointestinal tract (see, for example, Ugorski et al., Acta Biochimica Polonica, 49:2:303-311 (2002)). The expression level data of carbohydrate tumor marker correlates with clinical outcome, survival time of the patient and the signs of metastasis.

It is shown that as sialyl-Lewis A and sialyl-Lewis X contacted the family of carbohydrate-binding proteins, involved in the radiolabeled cells from the bloodstream, called selectins. Some reports suggest that sialyl-Lewis A and X are ligands for E-selectin and may be responsible for the adhesion of tumor cells of a person on the endothelium. Valerevna the Lewis structure present on the surface of cancer cells, are carbohydrate chains of glycoproteins and glycolipids and bind E-selectin present on endothelial cells. The selectins and their carbohydrate ligands, respectively, can play an important role in the electoral homing of tumor cells during metastasis.

Believe that the biosynthesis sialyl-Lewis A and X depends on the final addition of fucose from guanozintrifosfat-fucose (GDP - Fuc) in the alpha (1,3) and α (1,4) linkage cialisovernight precursors specific for the cell type and the stage of development of enzymes, phase, catalyzed alpha-1,3/1,4-fucosyltransferase (alpha 1,3/1,4 Fuc-T, FUT).

To date, cloned and characterized several genes fucosyltransferase person. Expression data of genes (FUT 3-7) and their enzymatic products (Fuc-TIII-VII), apparently, are tissue-specific. The enzymes encoded by five genes identified FUTIII, FUTIV, FUTV, FUTVI and FUTVII. Three genes encoding FUTIII, FUTV and FUTVI, physically localized in close positions on the chromosome 19pl3.3. Biochemically the research and molecular cloning suggest what linespecific the expression of molecules sialyl-Lewis A/X defines linespecific gene expression of alpha-1,3-fucosyltransferase, enzymatic products which operate on the constitutive expressed oligosaccharide precursor for obtaining localized on the surface of the determinants sialyl-Lewis A/x Fucosyltransferase person responsible for the activity in the epithelial tissues are FUT3 and FUT6. Transcripts FUT3 (also called gene Lewis alpha(1,3/1,4)fucosyltransferase) and FUT6 (gene alpha(1,3)fucosyltransferase plasma) are present in both normal and transformed tissues. Transcripts fucosyltransferase are also common in numerous adenocarcinoma cell lines with significantly increased expression of FUT3 and 6 in carcinoma of the colon (see, for example, Ugorski et al.,Acta Biochimica Polonica,49:303-311 (2002); Nakamori et al.,Dis. ColonThe Rectum., 40:420-431 (1997); Takada et al.,Cancer Res.,53:354-361 (1993); Ichikawa et al.,J. Surg. Oncol.,75:98-102 (2000); Nakagoe et al., J Exp Clin Cancer Res., 2002 Mar; 21(1):107-13; Matsumoto et al., Br J Cancer. 2002 Jan 21; 86(2):161-7; Ito et al., J Gastroenterol. 2001 Dec; 36(12):823-9; Nakagoe et al., Cancer Detect Prev. 2001; 25(3):299-308; Kumamoto et al., Cancer Res. 2001 Jun 1; 61 (11:4620-7; Murata et al., Dis Colon Rectum. 2001 Apr; 44 (4):A2-A4; Nakagoe etal.,J Exp Clin Cancer Res. 2001 Mar; 20(1):85-90; Nakagoe et al., J Gastroenterol. 2001 Mar; 36(3):166-72; Nakagoe et al., Tumour Biol. 2001 Mar-Apr; 22(2):115-22; Nakagoe et al., Can J Gastroenterol. 2000 Oct; 14(9):753-60; Izawa et al., Cancer Res. 2000 Mar 1; 60(5):1410-6; Tanaka et al., Hepatogastroenterology. 1999 Ma-Apr; 46(26):875-82; Matsushita et al., Cancer Lett. 1998 Mar 27; 133 (2):151-60; Sato et al., Anticancer Res. 1997 Sep-Oct; 17 (5A):3505-11; Yamada et al., Br J Cancer. 1997; 76(5):582-7; Nakamori et al., Dis Colon Rectum. 1997 Apr; 40(4):420-31; Srinivas et al., Scand J Immunol. 1996 Sep; 44(3):197-203; Matsushita et al., Lab Invest. 1990 Dec; 63(6):780-91; Ashizawa et al., J Exp Clin Cancer Res. 2003 Mar; 22 (1):91-8; Nakagoe et al., J Exp Clin Cancer Res. 2002 Sep; 21(3):363-9; Nakagoe et al., Anticancer Res. 2002 Jan-Feb; 22(1A):451-8; Nakagoe et al., J Clin Gastroenterol. 2002 Apr; 34(4):408-15; Nakagoe et al., Cancer Lett. 2002 Jan 25; 175(2):213-21; Tatsumi et al., Clin Exp Metastasis. 1998 Nov; 16(8):743-50; Ikeda et al., J Surg Oncol. 1996 Jul; 62(3):171-6; Ikeda et al., Eur J Surg Oncol. 1995 Apr; 21 (2):168-75; Togayachi et al., Int J Cancer. 1999 Sep 24; 83 (1):70-9; Satoh et al., Clin Cancer Res. 1997 Apr; 3(4):495-9; Satoh et al., Respiration. 1998; 65(4):295-8; Satoh et al., Anticancer Res. 1998 Jul-Aug; 18(4B):2865-8; Fukuoka et al., Lung Cancer. 1998 May; 20(2):109-16; Fujiwara et al., Anticanefer Res. 1998 Mar-Apr; 18(2A):1043-6; Ogawa et al., Int J Cancer. 1997 Apr 22; 74 (2):189-92; Ogawa et al., J Thorac Cardiovasc Surg. 1994 Aug; 108(2):329-36; Asao et al., Cancer. 1989 Dec 15; 64 (12):2541-5; Narita et al., Breast Cancer. 1996 Mar 29; 3(1):19-23; Yamaguchi et al., Oncology. 1998 Jul-Aug; 55(4):357-62; Sikut et al., Int J Cancer. 1996 May 29; 66 (5):617-23; Saito et al., Anticancer Res. 2003 Jul-Aug; 23(4):3441-6; Fujii et al., Urol Int. 2000; 64 (3):129-33; Idikio et al., Glycoconj J. 1997 Nov; 14 (7):875-7; Inoue et al., Obstet Gynecol. 1992 Mar; 79(3):434-40; Yamashita et al., Eur J Cancer. 2000 Jan; 36(1):113-20; Hamanaka et al., Pancreas. 1996 Aug; 13(2):160-5; Ho et al., Cancer Res. 1995 Aug 15; 55(16):3659-63).

The invention

This invention relates to methods and analyses to study the expression of one or more biomarkers in the sample of tissue or cells of a mammal, where the expression of one or more of these biomarkers is indicative of the fact that Bud is t if a sample of tissue or cells sensitive to apoptosis-inducing agents, such as Apo2L/TRAIL and antibody-agonists anti-DR5. In various embodiments of the invention these methods and analyses investigate the expression of such biomarkers as specific fucosyltransferase, in particular fucosyltransferase 3 (FUT3) and/or fucosyltransferase 6 (FUT6), as well as antigens sialyl-Lewis A and/or X.

As discussed above, most normal types of human cells, apparently, are resistant to induction of apoptosis specific recombinant forms of Apo2L/TRAIL (Ashkenazi et al., above, Walzcak et al., above). Also found that some populations affected types of human cells (such as specific populations of cancer cells) are resistant to induction of apoptosis specific recombinant forms of Apo2L/TRAIL (Ashkenazi et al., J. Clin. Invest., 1999, above; Walczak et al., Nature Med., 1999, above). Therefore, in the study by analyzing the expression of specific biomarkers in the tissue sample or cells of a mammal can be conveniently and effectively to obtain information applicable to determining the appropriate or effective therapy for the treatment of patients. For example, information obtained from the analysis for detecting expression of FUT3 or FUT6 in a sample of tissue or cells of a mammal, can provide physicians with useful data that can be used to determine the optimal therapeutic regime (with the use of Apo2L/TRIL or antibody-receptor agonists death) for patients with a violation, such as a malignant tumor.

The invention relates to a method of predicting the sensitivity of a tissue sample or cells (such as cancer cells) of the mammal to Apo2L/TRAIL or the antibody-receptor agonist death. In specific embodiments, the implementation of the methods include obtaining a sample of tissue or cells of a mammal and validation in tissue or cell expression fucosyltransferase 3 or fucosyltransferase 6. The methods may also include validation in tissue or cell expression of other biomarkers, such as the antigen(s) sialyl-Lewis A and/or x-Ways can be done in a variety of formats analysis, including analyses, detecting expression of mRNA, enzyme assays, detecting the presence of enzymatic activity, immunohistochemical analyses and others discussed here. Determination of the expression of these biomarkers in the above tissues or cells is an indication that such tissues or cells will be sensitive to the apoptosis-inducing activity of Apo2L/TRAIL or antibody-receptor agonist death. In optional embodiments, the implementation in the tissues or cells can be investigated also the expression of the receptors DR4, DR5, DcR1 or DcR2.

Additional methods of the invention include methods of inducing apoptosis in a tissue sample or cells of a mammal, including what Tadei a sample of tissue or cells of a mammal, research in tissue or cell expression of one or more biomarkers, such as fucosyltransferase 3, fucosyltransferase 6, the antigen(s) sialyl-Lewis A and/or X, and when determining that the given sample of tissue or cell expresses one or more of these biomarkers, exposure to a specified sample of tissue or cells an effective amount of Apo2L/TRAIL or antibody-receptor agonist death. Steps in how to verify the expression of one or more biomarkers can be performed in a variety of formats analysis, including analyses, detecting expression of mRNA, enzyme assays, detecting the presence of enzymatic activity, and immunohistochemical analyses. In optional embodiments, the implementation of the methods also include checking in a tissue sample or cells the expression of receptors DR4, DR5, DcR1 or DcR2. Optionally, a tissue sample or cells contains cancerous tissue or cells.

Additional methods of the invention include methods of treatment of mammalian disorders, such as associated with immune disorders or malignant tumor, including the stage of obtaining a sample of tissue or cells from a mammal, check in tissues or cells of the expression of one or more biomarkers, such as fucosyltransferase 3, fucosyltransferase 6, the antigen(s) sialyl-Lewis A is/or X, and when determining that the given sample of tissue or cell expresses one or more of these biomarkers, the introduction of the specified mammal an effective amount of Apo2L/TRAIL or antibody-receptor agonist death. Steps in how to verify the expression of one or more biomarkers can be performed in a variety of formats analysis, including analyses, detecting expression of mRNA, enzyme assays, detecting the presence of enzymatic activity, and immunohistochemical analyses. In optional embodiments, the implementation of the methods also include checking in a tissue sample or cells the expression of receptors DR4, DR5, DcR1 or DcR2.

Additionally, the methods provide for treatment of malignant tumors in a mammal. Additionally, the methods include, in addition to the introduction of the specified mammal an effective amount of Apo2L/TRAIL and/or antibody-receptor agonist death, the introduction of chemotherapeutic drugs (money) or radiotherapy.

Additional options for implementation are described in more detail in the following claims.

1. A method of predicting the sensitivity of a sample of tissue or mammalian cells to Apo2L/TRAIL, which includes stages:

a sample of tissue or cells of a mammal;

the research sample of tissue or cells for detection of expression of the underwater or more biomarkers, selected from the group of fucosyltransferase 3, fucosyltransferase 6, antigen (antigens) sialyl-Lewis A and/or X, where the expression of one or more of these biomarkers is an indication that the sample of tissue or cells is sensitive to the apoptosis-inducing activity of Apo2L/TRAIL.

2. The method according to claim 1, where the specified expression of one or more biomarkers examined by detection of mRNA expression fucosyltransferase 3 or fucosyltransferase 6.

3. The method according to claim 1, where the specified expression of one or more biomarkers examined by immunohistochemistry for the detection of expression of the antigen (antigens) sialyl-Lewis A and/or X.

4. The method according to claim 1, further comprising a stage of study the expression of receptors DR4, DR5, DcR1 or DcR2 in a specified sample of tissue or cells.

5. The method according to claim 1, where a sample of tissue or cells includes tissue or cells of a malignant tumor.

6. The method according to claim 5, where these malignant tumors are malignant cells or tissue of the colon, colorectal, gastric, intestinal, or pancreas.

7. Method of inducing apoptosis in a tissue sample or cells of a mammal, comprising the stage of:

a sample of tissue or cells of a mammal;

the research sample of tissue or cells for detection of the expression of the nogo or more biomarkers, selected from the group of fucosyltransferase 3, fucosyltransferase 6, antigen (antigens) sialyl-Lewis A and/or X, and

after detection of expression of one or more of these biomarkers of exposure at a specified sample of tissue or cells an effective amount of Apo2L/TRAIL.

8. The method according to claim 7, where the specified expression of one or more biomarkers examined by testing the expression of mRNA fucosyltransferase 3 or fucosyltransferase 6.

9. The method according to claim 7, where the specified expression of one or more biomarkers examined by immunohistochemistry for the detection of expression of the antigen(antigens) sialyl-Lewis A and/or X.

10. The method according to claim 7, further comprising a stage of study the expression of receptors DR4, DR5, DcR1 or DcR2 in a specified sample of tissue or cells.

11. The method according to claim 7, where the specified sample of tissue or cells includes tissue or cells of a malignant tumor.

12. The method according to claim 11, where these malignant tumors are malignant cells or tissue of the colon, colorectal, gastric, intestinal, or pancreas.

13. The method according to claim 7, where these cells influence the effective amount of the polypeptide Apo2L/TRAIL containing amino acids 114-281 1.

14. The method of treatment of mammalian disorders, such as associated with disturbance and immunity and malignancy, incorporating the following stages:

a sample of tissue or cells from the mammal;

the research sample of tissue or cells for detection of expression of one or more biomarkers selected from the group of fucosyltransferase 3, fucosyltransferase 6, antigen (antigens) sialyl-Lewis A and/or X, and

after detection of expression of one or more of these biomarkers introduction to the specified mammal an effective amount of Apo2L/TRAIL.

15. The method according to 14, where the specified expression of one or more biomarkers examined by detection of mRNA expression fucosyltransferase 3 or fucosyltransferase 6.

16. The method according to 14, where the specified expression of one or more biomarkers examined by immunohistochemistry for the detection of expression of the antigen (antigens) sialyl-Lewis A and/or X.

17. The method according to 14, further comprising a stage of study the expression of receptors DR4, DR5, DcR1 or DcR2 in a specified tissue or cell.

18. The method according to 14, where a sample of tissue or cells includes tissue or cells of a malignant tumor.

19. The method according to p, where these cells or tissue malignant tumors contain cancer cells or tissue of the colon, colorectal, gastric, intestinal, or pancreas.

20. The method according to 14, where the specified mammal is administered an effective quantity is of polypeptide Apo2L/TRAIL, containing amino acids 114-281 1.

21. The method according to 14, where the specified mammal also gets a chemotherapeutic agent (assets) or radiotherapy.

22. The method according to 14, where the specified mammal also introduce a cytokine, a cytotoxic agent or a growth inhibitory agent.

23. The method according to claim 7, where the specified polypeptide Apo2L/TRAIL attached to a molecule of polyethylene glycol.

24. The method according to 14, where the specified polypeptide Apo2L/TRAIL attached to a molecule of polyethylene glycol.

25. The method according to claim 6, where these malignant tumors are malignant cells in the colon or colorectal.

26. The method according to claim 1, where the specified Apo2L/TRAIL is a polypeptide containing amino acids 41-281 figure 1 (SEQ ID NO:1), or its biologically active fragment.

27. The method according to p where specified Apo2L/TRAIL is a polypeptide containing amino acids 114-281 figure 1 (SEQ ID NO:1).

28. The method according to item 12, where these malignant tumors are malignant cells in the colon or colorectal.

29. The method according to claim 20, where the specified polypeptide Apo2L/TRAIL consists of amino acids 114-281 figure 1 (SEQ ID NO:1).

30. A method of predicting the sensitivity of malignant tumor cells of a mammal, of the colon, or colorectal, to Apo2L/TRAIL, which includes stages:

obtain CL is current malignant tumor of a mammal, from the colon or colorectal;

studies of malignant tumor cells for detection of expression of one or more biomarkers selected from the group of fucosyltransferase 3, fucosyltransferase 6, antigen (antigens) sialyl-Lewis A and/or X, where the expression of one or more of these biomarkers is an indication that these cells are malignant tumors are sensitive to apoptosis-inducing activity of Apo2L/TRAIL.

31. Method of induction of apoptosis in malignant tumor cells of a mammal, of the colon, or colorectal, incorporating the following stages:

receipt of malignant tumor cells of the mammal, from the colon or colorectal;

studies of malignant tumor cells for detection of expression of one or more biomarkers selected from the group of fucosyltransferase 3, fucosyltransferase 6, antigen (antigens) sialyl-Lewis A and/or X, and

after detection of expression of one or more of these biomarkers of effect on these cells of a malignant tumor effective amount of the polypeptide Apo2L/TRAIL.

32. The method according to p, where the specified polypeptide Apo2L/TRAIL contains amino acids 41-281 figure 1 (SEQ ID NO:1) or a fragment having apoptotic activity.

33. The method according to p, where the specified polypeptide Apo2L/TRAIL tool is to a molecule of polyethylene glycol.

34. The method according to p, where the specified polypeptide Apo2L/TRAIL contains amino acids 114-281 figure 1 (SEQ ID NO:1).

35. The method of treatment of mammalian malignant tumors of the colon, or colorectal, incorporating the following stages:

a sample of malignant tumor cells specified mammal, from the colon or colorectal;

studies of malignant tumor cells for detection of expression of one or more biomarkers selected from the group of fucosyltransferase 3, fucosyltransferase 6, antigen (antigens) sialyl-Lewis A and/or X, and

after detection of expression of one or more of these biomarkers introduction to the specified mammal an effective amount of Apo2L/TRAIL.

36. The method according to p, where the specified polypeptide Apo2L/TRAIL contains amino acids 41-281 figure 1 (SEQ ID NO:1) or a fragment having apoptotic activity.

37. The method according to p, where the specified polypeptide Apo2L/TRAIL attached to a molecule of polyethylene glycol.

38. The method according to p, where the specified polypeptide Apo2L/TRAIL contains amino acids 114-281 figure 1 (SEQ ID NO:1).

Brief description of figures

Figure 1 shows the nucleotide sequence of cDNA ligand Apo-2 (SEQ ID NO:2) and its derived amino acid sequence (SEQ ID NO:1). "N" in nucleotide position 447 is used to indicate that the nucleotide base could do is add a "T" or "G".

On figa and 2B shows the nucleotide sequence of the cDNA (SEQ ID NO:4) for full-length DR4 person and its derived amino acid sequence (SEQ ID NO:3). The corresponding nucleotide and amino acid sequence of human DR4 published in Pan et al., Science, 276:111 (1997).

On figa shows the sequence of human DR5 of 411 amino acids (SEQ ID NO:5), as published in WO 98/51793 on November 19, 1998 In this area known variant transcription of splicing human DR5. This alternative splicing encodes the sequence of human DR5 of 440 amino acids (SEQ ID NO:6)shown in figv and 3C, as published in WO 98/35986 20 August 1998

On fig.3D shows the nucleotide sequence of the cDNA (SEQ ID NO:7) for full-DcRl person and its derived amino acid sequence (SEQ ID NO:8). The corresponding nucleotide and amino acid sequences for DcRl man (and his specific domains) shown and described in WO 98/58062.

On file shows the nucleotide sequence of the cDNA (SEQ ID NO:9) for full-length human DcR2 and its derived amino acid sequence (SEQ ID NO:10). The corresponding nucleotide and amino acid sequences for human DcR2 (and its specific domain) is shown and described in WO 99/10484.

Figure 4 shows the nucleotide sequence of the cDNA (SEQ ID N:11) for a full-sized (1,3/1,4) fucosyltransferase (FUT3) of man and its derived amino acid sequence (SEQ ID NO:12). These sequences correspond to the inventory number in GenBank HSU27328 and described, for example, Kukowska-Latallo et al., Genes Dev. 1990 Aug; 4 (8):1288-303.

Figure 5 shows the nucleotide sequence of the cDNA (SEQ ID NO:13) full-size alpha (1,3) fucosyltransferase (FUT6) of man and its derived amino acid sequence (SEQ ID NO:14). These sequences correspond to the inventory number in GenBank HSU27333 and described, for example, Koszdin and Bowen, Biochem Biophys Res Commun. 1992 Aug 31; 187 (1):152-7.

Figure 6 shows the summary table of data obtained during the analysis of 28 lines of cancer cells, colon, or colorectal, sensitivity or resistance to apoptotic activity of Apo2L (a+0.5% fetal bovine serum "FBS" or 10% FBS) or monoclonal antibody to DR5 mab", cross-stitched "XL" or not cross-linked (+0,5% fetal bovine serum "FBS" or 10% FBS) and by expression of FUT 3, FUT 6, sialyl-Lewis A and sialyl-Lewis X.

7 shows the comparison of sensitivity of different lines of cancer cells, colon, or colorectal, for the antibody to DR5 and expression FUT 3, as measured by quantitative PCR.

On Fig shows the comparison of sensitivity of different lines of cancer cells, colon, or colorectal, sensitivity or resistance to the antibody to DR5 (plus cross-linking agent) and the expression of SIA is Il-Lewis X or A, as determined by FACS.

On figa shows the rank correlation coefficient of Spermine in the analysis of the sensitivity or resistance of different lines of cancer cells, colon, or colorectal, and correlation with expression of FUT3.

On FIGU shows the results of the exact test of Fisher for the sensitivity analysis ("feelings.") or stability ("mouth.") various lines of cancer cells, colon, or colorectal, and statistical significance between the expression of FUT 3 and sialyl-Lewis A/X and the sensitivity of the respective lines of the cells to the apoptotic activity of the antibody to DR5.

Figure 10 shows the comparison of different lines of cancer cells, colon, or colorectal, on the expression of receptors DcRl or DcR2 (as determined by quantitative PCR) and status (sensitive or resistant) of specific cell lines in relation to Apo2L or the antibody to DR5.

Figure 11 shows the comparison of different lines of cancer cells, colon, or colorectal, on the expression of receptors DcRl or DcR2 (as determined by FACS) and status (sensitive or resistant) of specific cell lines in relation to Apo2L or the antibody to DR5.

On Fig shows immunohistochemical staining sialyl-Lewis A and X in four lines of colorectal cancer cells, CaCo2, 1417 SW, DLD-1 and Colo 205, and its correlation with expression sialyl-Lewis A and X, as change is prohibited FACS, and its correlation with sensitivity to Apo2L.

On Fig shows the aggregated results of IHC experiments showing the expression of sialyl-Lewis A and X in the tissue samples of normal mucosa of the colon, normal liver tissue, primary colon cancer and metastatic cancer of the colon.

Detailed description of the invention

The methods and procedures described or shown in the links here, as a rule, are well understood specialists in this field and usually doable by them, using the common methodology, such as, for example, a widely used methodology, molecular cloning, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kits and reagents, usually carried out in accordance with specific manufacturer's protocols and/or parameters, unless otherwise specified.

Before describing these methods and analyses should be understood that the present invention is not limited to a particular described methodology, protocols, cell lines, species or genera of animals constructs and reagents, as they certainly can be changed. It should be understood that the terminology used here is intended solely for the purpose of describing particular is of embodiments and is not intended to limit the scope of the present invention, which will be limited only by the attached claims.

It should be noted that, as used herein and in the appended claims, the singular number include references in the plural, unless the context clearly requires otherwise. So, for example, reference to "genetic modification" includes many such changes, and the reference to "a probe" includes reference to one or more probes and cash equivalents, known to specialists in this field, and so on.

All publications mentioned here are listed here as a reference to disclose and describe the methods and/or materials in connection with which the cited publication. Quoted here the publication is included here to describe them before the filing date of this application. Nothing here should not be interpreted as recognition that the authors of the present invention do not have the right to name the date of publication retroactively on the basis of an earlier priority date or before the date of invention. In the future, the actual date of publication may vary and require independent verification.

I. DEFINITIONS

The terms "Apo2L/TRAIL", "Apo-2L" and "TRAIL" is used here to denote the sequence of the polypeptide containing amino acid residues 114-281, inclusive, 95-281, inclusive, residues 92-281, inclusive, shall mistakes 91-281, inclusive, residues 41-281, inclusive, residues 15-281, inclusive, or residues 1-281, inclusive, of the amino acid sequence shown in figure 1, as well as biologically active fragments, deletion, insertion or substituted variants of the above sequences. In one embodiment, the implementation sequence of the polypeptide includes residues 114-281 1 and, optionally, consists of residues 114-281 figure 1. Optional, sequence of the polypeptide includes residues 92-281 or residues 91-281 figure 1. Polypeptides Apo-2L can encode natural nucleotide sequence shown in figure 1. Optionally, the codon encoding residue Proll9 (figure 1), can represent "CCT" or "CCG". In other embodiments, implementation of the fragments or variants are biologically active and have at least about 80% amino acid sequence identity, more preferably at least about 90% sequence identity and even more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity with any of the above sequences Apo2L/TRAIL. Optionally, the polypeptide Apo2L/TRAIL encodes a nucleotide sequence that hybridizes in stringent conditions with a polynucleotide coding consistently is thew, presented in figure 1. The definition covers substituted variants of Apo2L/TRAIL in which at least one of its natural amino acids replaced by an alanine residue. Specific substituted variants of Apo2L/TRAIL include those in which at least one substituted amino acid residue is alanine. Data substituted variants include, for example, is labeled "D203A"; "D218A" and "D269A". This nomenclature is used to denote options Apo2L/TRAIL, where the aspartic acid residues at positions 203, 218, and/or 269 (using the numbering shown in figure 1) is substituted by alanine residues. Optional, options Apo2L can contain one or more alanine substitutions listed in table I or published PCT application WO 01/00832. Substituted variants include one or more substitutions of residues listed in table I or WO 01/00832, published on 4 January 2001, the Definition also includes natural sequence of Apo2L/TRAIL, isolated from the source of Apo2L/TRAIL or received recombinant or synthetic methods. Apo2L/TRAIL according to the invention includes polypeptides, designated as Apo2L/TRAIL or TRAIL, described in PCT publications WO 97/01633 and WO 97/25428. The terms "Apo2L/TRAIL or Apo2L" is used to denote in a General sense forms of Apo2L/TRAIL, including Monomeric, dimeric or trimeric forms of the polypeptide. For the whole numbering of amino acid OST the Cove, marked on the sequence Apo2L, using the numbering according to figure 1, unless specifically stated otherwise. For example, "D203" or "Asp203" refers to the residue of aspartic acid at position 203 in the sequence shown in figure 1.

The term "extracellular domain Apo2L/TRAIL or Apo2L/TRAIL ECD" refers to a form of Apo2L/TRAIL, which is essentially free of the transmembrane and cytoplasmic domains. Usually ECD will have less than 1% of such transmembrane and cytoplasmic domains and preferably, will have less than 0.5% of such domains. It will be understood that any transmembrane domain(s)identified for the polypeptides of the present invention, identified according to the criteria usually used in this field to identify the hydrophobic domains of this type. The exact boundaries of the transmembrane domain can vary, but most likely not more than about 5 amino acids at either end of the domain, as identified initially. In preferred embodiments, the implementation of the ECD will comprise a sequence of the soluble extracellular domain of the polypeptide free of the transmembrane and cytoplasmic or intracellular domains (and not associated with the membrane). The specific sequence of the extracellular domain of Apo-2L/TRAIL described in publ is the brand PCT WO 97/01633 and WO 97/25428.

The term "monomer Apo2L/TRAIL" or "monomer Apo2L refers to a sequence covalent chain extracellular domain Apo2L.

The term "dimer Apo2L/TRAIL" or "dimer Apo2L" refers to two molecules of Apo-2L connected by covalent bond via disulfide bonds. The term, as used here, includes freely located dimers Apo2L and dimers Apo2L inside the trimeric forms of Apo2L (i.e., associated with another, the third monomer Apo2L).

The term "trimer Apo2L/TRAIL" or "trimer Apo2L" refers to the three monomers Apo2L related ecovalence.

The term "unit Apo2L/TRAIL" is used to denote samoassotsiiruyutsya higher oligomeric forms of Apo2L/TRAIL, such as trimers Apo2L/TRAIL, which form, for example, review and nanoscale forms of Apo2L/TRAIL. The presence and quantity of monomer, dimer or trimer (or other units) Apo2L/TRAIL can be performed using methods and assays known in this area (and with the use of commercially available materials, such as native exclusion HPLC (SEC), denaturing exclusion with the use of sodium dodecyl sulfate ("SDS " SEC"), obremenitve HPLC and capillary electrophoresis.

"Receptor ligand Apo-2 includes the receptors identified in this area as "DR4 and DR5", polynucleotide and polypeptide sequences are shown in figure 2 and 3 meet the but. Pan et al. described member of the family of TNF receptors, designated as "DR4" (Pan et al., Science, 276:111-113 (1997); see also WO 98/32856, published July 30, 1998; WO 99/37684, published on July 29, 1999; WO 00/73349, published 7 December 2000; US 6433147, published August 13, 2002; US 6461823, published October 8, 2002, and US 6342383, published on January 29, 2002). Sheridan et al., Science, 277:818-821 (1997) and Pan et al., Science, 277:815-818 (1997) described another receptor for Apo2L/TRAIL (see also WO 98/51793, published on November 19, 1998; WO 98/41629 published on September 24, 1998). This receptor was identified as DR5 (alternative receptor identified as Apo-2; TRAIL-R, TR6, Tango-63, hAPO8, TRICK2 or KILLER; Screaton et al., Curr. Biol., 7:693-696 (1997); Walczak et al., EMBO J. 16:5386-5387 (1997); Wu et al., Nature Genetics, 17:141-143 (1997); WO 98/35986, published August 20, 1998; EP 870827, published on October 14, 1998; WO 98/46643, published October 22, 1998; WO 99/02653 published January 21, 1999; WO 99/09165, published on 25 February 1999; WO 99/11791, published 11 March 1999; US 2002/0072091, published August 13, 2002; US 2002/0098550, published 7 December 2001; US 6313269 published on 6 December 2001; US 2001/0010924 published 2 August 2001; US 2003/01255540, published 3 July 2003; US 2002/0160446, published October 31, 2002, US 2002/0048785, published April 25, 2002; US 6569642, published 27 may 2003, US 6072047, published June 6, 2000, US 6642358 published 4 November 2003). As described above, other receptor is for Apo-2L include DcRl, DcR2 and OPG (see Sheridan et al., above; Marsters et al., above; and Simonet et al., above). The term "receptor Apo-2L", when used here, covers the natural sequence of the receptor and receptor variants. These terms cover the receptor for Apo-2L, expressed in many mammals, including humans. The receptor for Apo-2L may be endogenous downregulation, as naturally occurs in many lines of human tissue, or may be expressed by recombinant or synthetic methods. "The natural sequence of the receptor Apo-2L" includes a polypeptide having the same amino acid sequence as the receptor for Apo-2L, can be found in nature. Thus, the natural sequence of the receptor Apo-2L can have the amino acid sequence of naturally occurring receptor Apo-2L of any mammal. This natural sequence of the receptor Apo-2L can be distinguished from a natural source or can be obtained recombinant or synthetic methods. The term "natural sequence of the receptor Apo-2L" specifically encompasses naturally occurring truncated or secreted forms of the receptor (for example, soluble form containing, for example, the sequence of the extracellular domain), a naturally occurring form of options (for example, alternative splicing, and naturally occurring allelic variants. Variants of the receptor may include fragments or deletion mutants of the natural sequence of the receptor Apo-2L. On figa shows the sequence of human DR5 of 411 amino acids, as published in WO 98/51793 on November 19, 1998 In this area known variant transcription of splicing human DR5. This alternative splicing encodes the DR5 DR5 sequence of a person of 440 amino acids shown in figv and 3C, as published in WO 98/35986 20 August 1998

"The antibody to the receptor of death" is used here to denote in a General sense antibody or antibodies directed against the receptor of the superfamily of receptors of the tumor necrosis factor containing a death domain that is able to transmit the signal apoptosis, and such antibodies include antibody to DR5 and antibody to DR4.

"The antibody to the receptor DR5", "antibody to DR5 or anti-DR5 antibody" is used in a broad sense to refer to antibodies that bind at least one form of the receptor DR5 or its extracellular domain. Optionally, the antibody to DR5 fused or linked to a heterologous sequence or molecule. Heterologous sequence preferably allows or helps the antibody to form oligomeric complexes or complexes of the highest order. Antibody to DR5, optional, binds to the receptor DR5, but not bound or is harekrishna does not react with any additional receptor Apo-2L (for example, DR4, DcRl or DcR2). The antibody optionally is agonist signaling activity of DR5.

Antibody to DR5 according to the invention, optionally, binds to a receptor DR5 in the concentration range from about 0.1 nm to about 20 mm, as measured by BIAcore analysis of binding. For antibody to DR5 according to the invention, optionally, show the value of the IC50from approximately 0.6 nm to approximately 18 mm, as measured by BIAcore analysis of binding.

"The antibody to the receptor DR4", "antibody to DR4 or anti-DR4 antibody" is used in a broad sense to refer to antibodies that bind at least one form of the receptor DR4 or its extracellular domain. Optionally, the antibody to DR4 fused or linked to a heterologous sequence or molecule. Heterologous sequence preferably allows or helps the antibody to form oligomeric complexes or complexes of the highest order. Antibody to DR4, optional, binds to the receptor DR4, but not linked or not cross-react with any additional receptor Apo-2L (for example, DR5, DcR1 or DcR2). The antibody optionally is agonist signaling activity of DR4.

Antibody to DR4 according to the invention, optionally, binds to a receptor DR4 in the concentration range from about 0.1 nm to about 20 mm, as measured by the analysis attributed the project BIAcore. For antibodies to DR4 according to the invention, optionally, show the value of the IC50from approximately 0.6 nm to approximately 18 mm, as measured by BIAcore analysis of binding.

The term "agonist" is used here in its broadest sense and includes any molecule that partially or fully enhances, stimulates or activates one or more of the biological activities of Apo2L/TRAIL, DR4 or DR5in vitro,in situorin vivo. Examples of such biological action binding Apo2L/TRAIL to DR4 or DR5 include apoptosis, as well as activity, additionally described in the literature. The agonist may function directly or indirectly. For example, the agonist may act to partially or fully enhance, stimulate or activate one or more of the biological activities of DR4 or DR5,in vitro,in situorin vivoin the result of direct binding to DR4 or DR5, which leads to activation of the receptor or signal transmission. The agonist may also operate indirectly to partially or fully enhance, stimulate or activate one or more of the biological activities of DR4 or DR5,in vitro,in situorin vivoas a result, for example, stimulation of other effector molecules, which then leads to the activation signal DR4 or DR5. Assume that the agonist can act as alcula-enhancer, which acts indirectly to enhance or increase the activation or activity of DR4 or DR5. For example, the agonist may enhance the activity of endogenous Apo-2L in a mammal. This can be accomplished, for example, the preliminary formation of complexes DR4 or DR5 or stabilization of the complexes of the corresponding ligand with the receptor DR4 or DR5 (such as the stabilization of the native complex formed between the Apo-2L and DR4 or DR5).

The term "biomarker"as used in this application generally refers to a molecule, including gene, protein, carbohydrate structure, or glycolipid, the expression of which in or on a tissue or cell of a mammal can be detected by standard methods (or methods described herein), and it is an indicator for determining the sensitivity of cells or tissue of a mammal to Apo2L/TRAIL or the antibody to the receptor of death. Such biomarkers considered in this invention include as non-limiting examples "(1,3/1,4)fucosyltransferase" or "FUT3", "alpha(1,3)fucosyltransferase or FUT6", "sialyl-Lewis A and sialyl-Lewis X". Not necessarily determine that the expression of this biomarker is higher than observed in the control sample of tissue or cells. Optional, for example, in the analysis of PCR or FACS will determine that the expression of this biomarker one is camping at least 50 times, or preferably at least 100 times higher, than observed for the control sample of tissue or cells. Optionally, the expression of this biomarker will be determined in the analysis IHC score of staining intensity of at least 2 or above.

"(1,3/1,4)fucosyltransferase" or "FUT3" is used here to refer to molecules having structural properties described herein, and, optionally, catalyzing the transfer of a fucose residue from a donor substrate, GDP-fucose, to the acceptor substrate on α3 or α4 - link GlcNAc (FUT III-VII and IX). The DNA sequence and amino acid sequence of FUT3 person presented on figure 4. These sequences correspond to the inventory number in GenBank HSU27328 and described, for example, Kukowska-Latallo et al., Genes Dev. 1990 Aug; 4(8):1288-303. FUT, as a rule, are transmembrane glycoproteins in the Golgi vesicles, and usually consist of N-terminal cytoplasmic tail, a transmembrane region and a catalytic domain facing the lumen of the Golgi apparatus. Between the transmembrane region and the catalytic domain, there is a region called the stem (Paulson and Colley, J. Biol. Chem., 264:17615-17618 (1989)).

Alpha (1,3) fucosyltransferase or FUT6" is used here to refer to molecules, structurally related, for example, the DNA sequence and amino acid sequence of FUT6 person, shown in figure 5 sequence Data correspond to the inventory number in GenBank HSU27333 and described, for example, in Koszdin and Bowen, Biochem Biophys Res Commun. 1992 Aug 31;187 (1):152-7. FUT 6 is normally expressed in epithelial cells, in the liver, kidney and gastrointestinal tissues, specifically, stomach, small intestine and colon (and usually minimally expressed in spleen, lung and cervix). FUT 6 is not normally found in the brain, the adrenal cortex or peripheral blood leukocytes.

"Sialyl-Lewis A" is used here to denote tetrasaccharide carbohydrate structures or antigen having the following sequence or structure that may be present in membrane-bound or soluble form, circulating, for example, in serum:

NeuAcα2-->3Galβl-->3[Fucαl-->4]GlcNAcβl-->R (NeuAcα2-->3Galβl-->3 (Fucαl-->4)GlcNAcβl-->R)

"Sialyl-Lewis X" is used here to denote tetrasaccharide carbohydrate structures or antigen having the following sequence or structure that may be present in membrane-bound or soluble form, circulating, for example, in serum:

NeuAcα2-->3Galβl-->4[Fucαl-->3]GlcNAcβl-->R (NeuAcα2-->3Galβl-->4 (Fucαl-->3)GlcNAcβl-->R)

Under "subject" or "patient" understand any separate entity for which the desired therapy, including humans, cattle, dogs, Guinea pigs, rabbits, s is let, insects, etc. For inclusion as subjects are any subjects involved in clinical research that does not have any clinical signs of disease, or the actors involved in epidemiological studies or subjects used as controls.

The term "mammal"as used here, refers to any animal classified as a mammal, including humans, cows, horses, dogs and cats. In a preferred embodiment of the invention the mammal is a human.

Under "sample of tissue or cells is a set of identical cells derived from a tissue of the subject or patient. The source of the tissue sample or cells may be dense tissue, such as fresh, frozen and/or fixed organ or tissue sample or biopsy or aspirate; blood or any blood components; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells of any period of gestation or development of the subject. The tissue sample may also be a primary or cultured cells or cell lines. A sample of tissue or cells, not necessarily, derived from primary or metastatic tumors. The tissue sample can in order to contain the components of course not mixed with cloth in nature, such as preservatives, anticoagulants, buffers, latches, nutrients, antibiotics or the like.

For purposes here, a "slice" of tissue sample indicates a single part or piece of a tissue sample, for example a thin slice of tissue or cells cut from the tissue sample. It is clear that we can take many sections of tissue samples and to analyze the present invention, provided that it is clear that the present invention relates to a method in which the same section of tissue sample analyzed both at morphological and molecular levels, or analyze with respect to both protein and nucleic acids.

Under the "correlate" or "correlation" understand the comparison, in any way, output and/or results of the first analysis or Protocol with output and/or results of the second analysis or Protocol. For example, you can use the results of the first analysis or Protocol in performing the secondary protocols and/or you can use the results of the first analysis or Protocol to determine whether to perform a second analysis or Protocol. With regard to the implementation immunohistochemical analysis or Protocol, you can use the results of IHC to determine whether to perform a specific treatment regimen.

The term "nucleic acid is intended, to include any DNA or RNA, such as chromosomal, mitochondrial, viral and/or bacterial nucleic acid present in the tissue sample. The term "nucleic acid" encompasses any of the two or both strands of double-stranded molecules of nucleic acid, and includes any fragment or part of intact molecules of nucleic acids.

By "gene" is understood any sequence of nucleic acid or part thereof, the functional role of which is coding or transcription or protein regulation of expression of another gene. The gene can be completely consist of nucleic acids, responsible for encoding a functional protein, or only partially of the nucleic acids responsible for coding or expression of the protein. The sequence of the nucleic acid can contain a genetic abnormality in exons, introns, regions of initiation or termination, promoter sequences and other regulatory sequences or unique related with genome regions.

The word "label" when used there refers to a compound or composition that is conjugated or fused directly or indirectly with a reagent, such as a nucleic acid probe or antibody, and facilitate the detection reagent with which they are conjugated or fused. The label can be dete is together as such (for example, radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of the composition of the substrate or of a composition which is detektivami.

The term "antibody" is used here in its broadest sense and specifically applied to intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bespecifically antibodies)formed from at least two intact antibodies, and fragments of antibodies, while they possess the desired biological activity.

"Fragments of the antibodies contain a portion of an intact antibody, preferably containing antigennegative or variable region. Examples of fragments of antibodies include Fab fragments, Fab', F(ab')2and Fv; dimeric antibodies; linear antibodies; single-chain molecule antibodies; and multispecific antibodies formed from fragments of antibodies.

"Native antibodies" are usually heterotetrameric the glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different isotypes of immunoglobulins is different. Each heavy and light shall EPL also has regularly spaced disulfide bridges within the chain. Each heavy chain has at one end contains a variable domain (VH), followed by a number of constant domains. Each light chain contains a variable domain (VL) on one end and a constant domain at the other end; the constant domain of the light chain is connected with the first constant domain of the heavy chain and the variable domain of the light chain is connected with the variable domain of the heavy chain. Consider that the boundary between the variable domains of the light chain and heavy chains form a specific amino acid residues.

The term "variable" refers to the fact that a particular part of the variable domains differ significantly in sequence among antibodies and are used for binding and specificity of each particular antibody with its specific antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three areas, called hypervariable or complementarity determining regions in the variable domains, both light chain and heavy chain. More highly conservative part of the variable domains are called the frame regions (FR). Each of the variable domains of the natural heavy and light chain contains four FR, largely taking a configuration of a β-sheets connected by three g is pervariabilis areas forming a connecting loop, and in some cases forming part of the structure of β-sheet. Hypervariable region of each chain are held together in close proximity by the FR and, with the hypervariable regions from the other chain, involved in the formation antigennegative site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). The constant domains are not involved directly in binding the antibody to the antigen, but have different effector functions, such as participation of the antibody in antibody-dependent cell-mediated cytotoxicity (ADCC).

The cleavage of antibodies with papain get two identical antigenspecific fragment, called "Fab"fragments, each with one antigennegative plot, and the remaining "Fc"fragment, whose name reflects its ability to easily crystallize. Treatment with pepsin get F(ab')2a fragment with two antihistamine sites and is still capable of cross-link the antigen.

"Fv" is the minimum antibody fragment that contains a complete recognize antigen and antigennegative plot. This region consists of a dimer variable domain of one heavy chain and one light chain in tight, non-covalent linkages. That is, in this configuration, Tr is the hypervariable regions of each variable domain interact, determining antigennegative area on the surface of the dimer VH-VL. Together, the six hypervariable regions reported the antibody antigennegative specificity. However, even a single variable domain (or half of an Fv, containing only three hypervariable region that is specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

Fab-fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab'fragments differ from Fab fragments by the addition of a few residues at the C-end of the heavy chain CH1 domain including one or more cysteines from the hinge region of the antibody. Fab'-SH refers Fab'in which the residue (remainder) of cysteine constant domains have at least one free thiol group. F(ab')2-fragments of the antibodies were initially obtained as pairs of Fab'fragments with swivel cysteine between them. There are also other chemical joining fragments of antibodies.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be attributed to one of two clearly distinct types, called Kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

Depending on the amino acid th the sequences of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (isotypes), e.g. IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The constant domains of the heavy chains, corresponding to different classes of antibodies, designated α, δ, ε, γ and μ, respectively. The structure of the subunits and three-dimensional configurations of different classes of immunoglobulins are well known.

"Single-chain Fv" or "scFv fragments of antibodies contain VHand VLdomains of antibodies, where these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between domains VHand VLthat enables the scFv to form the desired structure for antigen binding. A review of scFv see Plückthun in The Pharmacology of Monoclonal antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994).

The term "dimeric antibody" refers to small antibody fragments with two antihistamine areas where the fragments contain the variable domain of the heavy chain (VH)connected to the variable domain light chain (VL) in the same polypeptide chain (VH- VL). The use of a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to mate with sets the elemental domains of another chain and create two antigenspecific plot. Dimeric antibodies are more fully described in, for example, in EP 404097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

The term "monoclonal antibody", as used here, refers to an antibody obtained from a population of mostly homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single antigen determinants. In addition to their specificity, monoclonal antibodies are preferred because they are synthesized in the culture of hybridoma, not contaminated by other immunoglobulins. The definition of "monoclonal" indicates the characteristic of the antibody as being obtained from an essentially homogeneous population of antibodies, and it should not be seen as a requirement for antibodies to any particular method. For example, monoclonal antibodies for use in the present invention can be obtained by way of hybridoma originally described by Kohler et al., Nature, 256:495 (1975), or can be obtained by means of recombinant DNA (see, for example, U.S. patent No. 4816567). "Monoclonal antibodies" can be distinguished from phage libraries of antibodies using methods described in, for example, Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

Monoclonal antibodies here specifically include "chimeric" antibodies (immunoglobulins)in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibody, while the remainder of the chain (chain) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass antibodies as well as fragments of such antibodies, as they possess the desired biological activity (U.S. patent No. 4816567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies described here, include "primaryservername" antibodies containing antigennegative sequence of the variable domain obtained from a non-human Primate (for example, monkeys of the Old world, such as the baboon, rhesus or monkeys-having), and sequences of the constant regions of human (U.S. patent No. 5693780).

"Humanizer the data" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (antibody-recipient), in which residues from a hypervariable region of the recipient are replaced residues from a hypervariable region of non-human species (the antibody-donor), such as a mouse, rat, rabbit or non-human Primate having the desired specificity, affinity and capacity. In some cases, remnants of the frame region (FR) of a human immunoglobulin replace the corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the antibody-receptor or antibody-donor. Data modifications are made to further improve the functioning of antibodies. In General, humanitariannet antibody will contain basically all or at least one, and typically two, variable domain, in which all or substantially all of the hypervariable loops correspond to loops non-human immunoglobulin and all or substantially all of the FR is FR sequence of the human immunoglobulin. Humanitariannet antibody optionally also will contain at least the least part of the constant region of immunoglobulin (Fc), usually from a human immunoglobulin. For more details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

The term "hypervariable region" in the application refers to amino acid residues of the antibody responsible for binding to the antigen. Hypervariable region contains amino acid residues from a "complementarity determining region" or "CDR" (e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the variable domain light chain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the variable domain of the heavy chain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the variable domain light chain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the variable domain of the heavy chain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Framework" or "FR" residues are variable domain residues other than residues from a hypervariable region, as defined here.

An antibody that "binds" the desired antigen is an antibody capable of binding that antigen with sufficient affinity and/or avidity, so that the antibody is useful as a therapeutic or diagnostic tools for targeting a cell expressing the antigen.

For the purposes of section is camping, "immunotherapy" is to indicate a method of treating a mammal (preferably, the patient is human) antibody, where the antibody may be unconjugated or naked antibody or antibody may be conjugated or fused to a heterologous molecule (molecules) or a tool (equipment)such as one or more cytotoxic agents, thereby obtaining "immunoconjugate".

"Isolated" antibody is a fact that is identified and separated or extracted from its natural environment. Contaminant components of its natural environment are substances which would interfere with diagnostic or therapeutic applications of the antagonist or antibody, and may include enzymes, hormones and other protein and non-protein solutions. In preferred embodiments, the implementation of the antibody will be clear (1) to more than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain the amino acid sequence of at least 15 residues from the N-terminal, or internal, using sequencing machine with rotating bowl, or (3) to homogeneity in SDS-PAGE in reducing and non conditions using colour, Kumasi blue or, preferably, took silver at the om. The selected antibody includes antibodyin situwithin recombinant cells, since ceases to attend at least one component of the natural environment antibodies. Typically, however, the selected antibody will get through at least one stage of cleaning.

The expression "effective amount" denotes an amount of a substance (for example, Apo2L/TRAIL, anti-DR4 or anti-DR5 and so on), which is effective to prevent, alleviate or treat a specific disease or condition.

The terms "treatment", "treatment" and "therapy", as used here, denotes a healing therapy, prophylactic therapy, and preventative therapy. Sequential treatment or introduction means processing at least on daily basis without interruption of processing on one or more days. Periodic treatment or the introduction of, or treatment or the introduction of a periodic manner, means treatment that is not consistent, but rather cyclical in nature.

The term "cytokine" is a generic term for proteins released by one population of cells, which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, Monokini and conventional polypeptide hormones. Among the cytokines include Gorm the us growth such as human growth hormone, N-methionyl the human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prolactin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyrostimulin hormone (TSH), and luteinizing hormone (LH); a growth factor for hepatocytes; fibroblast growth factor; prolactin; placental lactogenic; tumor necrosis factor-α and-β; müller inhibiting substance; gonadotropin-associated peptide mouse; inhibin; activin, growth factor vascular endothelial; integrin; thrombopoietin (TPO); nerve growth factors; platelet growth factor; transforming growth factors (TGF)such as TGF-α and TGF-β; insulin-like growth factor-I and-II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β and-γ; colony stimulating factors (CSFs)such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF); interleukins (IL)such as IL-1, IL-2, IL-3, IL - 4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17; and other polypeptide factors including LIF and kit ligand (KL). As used here, the term cytokine includes proteins from natural sources or from a culture of recombinant cells and biologically active equivalents of the cytokines with the natural sequence.

The term "cytotoxic agent"as used here, refers to prophetic the creation, inhibitory or warning functioning of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., I131I125, Y90and Re186), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal, or fragments thereof.

"Chemotherapeutic agent" is a chemical compound used in the treatment of malignant tumors. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulphonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzodepa, carboquone, matureup and uredepa; ethylenimines and methylmelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and triethylenemelamine; acetogenins (especially bullatacin, bullatacin); camptothecin (including the synthetic analogue topotecan); bryostatin; callistemon; CC-1065 (including its synthetic analogues of adozelesin, carzelesin and bizelesin); cryptophycin (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard gas analogues, such as chlorambucil of chlornaphazine, chlorophosphate, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novemberin, finestein, prednimustine, trofosfamide, uramustine; nitrosoanatabine, such as carmustine, chlorozotocin, fotemustine, lomustin, nimustine, ranimustine; antibiotics such as enediyne antibiotics (for example, calicheamicin, especially calicheamicin gamma and calicheamicin Fi, see, e.g., Agnew, Chem Intl. Ed. Engl., 33:183-186 (1994); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; spiramycin; and the chromophore neocarzinostatin and related chromophores chromoproteins enediynes antibiotics), aclacinomycin, actinomycin, autralian, azaserine, bleomycin, actinomycin, carubicin, karminomitsin, calcination, chromomycin, dactinomycin, daunorubicin, demoralizing, 6-diazo-5-oxo-L-norleucine, doxorubicin (Adriamycin™) (including morpholino doxorubicin, cyanomethane doxorubicin, 2-pyrroline doxorubicin and desoxidation), epirubicin, zorubicin, idarubitsin marsellaise, mitomycin, such as mitomycin C, mycofenolate acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, colomycin, radiobeacon, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); analogs of folic acid, such as deeperin, the methods of rexach, peripherin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, timipre, tioguanin; pyrimidine analogues such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, propionate dromostanolone, epitiostanol, mepitiostane, testolactone; means, depressing the function of the adrenal cortex, such as aminoglutethimide, mitotane, trilostane; compensator folic acid, such as prolinnova acid; Eagleton; glycoside aldophosphamide; aminolevulinic acid; eniluracil; amsacrine; astroball; bisantrene; edatrexate; defaming; demecolcine; diazinon; alternity; the acetate slipline; epothilone; etoposide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoid, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitrogen; pentostatin; penomet; pirarubicin; losoxantrone; podofillina acid; 2-acylhydrazides; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogermanium; tinoisamoa acid; triaziquone; 2,2',2"-trihlortrietilamin; trichothecenes (especially toxin T-2, verrucarin a, roridin A and unguided); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; Galitsin; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoid, such as paclitaxel (AXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (Gemzar®); 6-tioguanin; mercaptopurine; methotrexate; platinum analogues, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (Navelbine™); Novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; deformational (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition antihormone means that act to regulate or inhibit hormone action on tumors such as antiestrogens and selective modulators of estrogen receptor (SERM), including, for example, tamoxifen (including Nolvadex™), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (Fareston™); aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, acetate megestrol (Megace™), exemestane, formestane, fadrozole, vorozole (Rivisor™), letrozole (Femara™) and anastrozole (Arimidex™); and antiandrogens, such as flutamide, nilutamide, bikalutamid, leuprolide and goserelin; and the pharmaceutically suitable salts, acids or derivatives of any of the above.

"Growth inhibitory agent" when applied here means a compound or composition, inhibiting growth of cells, particularly cancer cells, sverkhekspressiya any of the genes mentioned here, orin vitroorin vivo. Thus, inhibiting the growth of the tool is a fact, which significantly reduces the percentage of cells sverkhekspressiya these genes in S-phase. Examples of inhibiting growth means include means blocking the passage of the cell cycle (point other than S phase), such as a means of inducing Gl-arrest and arrest in M-phase. Classic blockers M-phases include the Vinca alkaloids (vincristine and vinblastine), Taxol and inhibitors of topo II, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those tools that block G1, shall also apply to stop S-phase, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil and Aga-C. further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogens, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p.13.

The terms "apoptosis" and "apoptotic activity" used in a broad sense and refer to an ordered or controlled fo the th cell death in mammals, usually accompanied by one or more characteristic cell changes, including condensation of cytoplasm, loss of plasma membrane microvilli, fragmentation of the nucleus, degradation of chromosomal DNA or loss of mitochondrial function. This activity can be defined and measured, for example, analysis of the viability of the cells (such as analysis of Alamar blue or MTT), FACS analysis, activation of caspase, DNA fragmentation (see, for example, Nicoletti et al., J. Immunol. Methods, 139:271-279 (1991)), and analyses of the cleavage of poly(ADP-ribose)polymerase "PARP", known in this field.

As used here, the term "violation" in General refers to any condition that will improve from treatment with the compositions described herein, including any disease or condition that can be treated with effective amounts of Apo2L/TRAIL or anti-DR4 antibody and/or anti-DR5 antibody. It includes chronic and acute disorders, as well as such pathological conditions that lead to a mammal of interest violation. Violations that are subject to treatment here include as non-limiting examples of benign and malignant tumors; inflammatory, angiogenic and immunologic disorders, autoimmune disorders, arthritis (including rheumatoid arthritis), multiple sclerosis and HIV/AIDS.

The terms "malignant op is hol", "cancer" or "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Malignant tumors include as non-limiting examples of carcinoma, lymphoma, leukemia, blastoma and sarcoma. More specific examples of such malignant tumors include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer (gastrointestinal tract), renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colon cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, the multiform glioma, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, carcinoma of the colon and head and neck cancer.

The term "associated with immune disease" means a disease in which component of the immune system causes, mediates or otherwise contributes to the incidence of a mammal. Also included are diseases in which stimulation of the immune response or intervention it has a relieving effect on the progression of the disease. In this term included autoimmune diseases, immune is espeletiinae disease, nimmanoradee inflammatory diseases, infectious diseases and immunodeficiency diseases. Examples of immune-related and inflammatory diseases, some of which are immune or mediated by T-cells that can be treated according to the present invention include systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjogren syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal night hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purple, immunopositive thrombocytopenia), thyroiditis (diffuse toxic goiter, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immunopositive renal disease (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the Central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelinizing polyneuropathy or Guillain-Barre syndrome, chronic inflammatory demyelinizing polyneuropathy, hepatobiliary diseases such as infecti the config hepatitis (hepatitis A, B, C, D, E and others, nagapattanam, viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory and fibrotic lung disease, inflammatory bowel disease (ulcerative colitis: Crohn's disease), glutensensitive enteropathy, and Whipple's disease, autoimmune or immune skin diseases including bullous skin disease, polymorphic erythema, and contact dermatitis, psoriasis, allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, hypersensitivity to food and urticaria, immunologic diseases of the lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitive pneumonitis, diseases associated with transplantation, including graft rejection and graft-versus-host. Infectious diseases include AIDS (HIV infection), hepatitis A, B, C, D and E, bacterial infection, fungal infection, protozoan infection and parasitic infection.

"Autoimmune disease" is used here in the broad, generic sense to refer to diseases or conditions in mammals, in which occurs the destruction of normal or healthy tissue due to humoral or cellular immune responses of the individual mlekovita is it on his or her own tissue components. They include as non-limiting examples lupus erythematosus, thyroiditis, rheumatoid arthritis, psoriasis, multiple sclerosis, autoimmune diabetes, and inflammatory bowel disease (IBD).

The term "labeled" in the application refers to a chimeric molecule containing the antibody or polypeptide fused with a polypeptide-tagged". Polypeptide-label has enough residues to provide an epitope against which to obtain the antibody, or to provide some other function, such as the ability to oligomerizate (for example, as in the case of peptides, possessing the domains latinovich lightning), but is short enough so that, as a rule, does not interfere with activity of the antibody or polypeptide. Polypeptide-label preferably is sufficiently unique so that the antibody specific to the tag, basically does not cross react with other epitopes. Suitable polypeptides tags, generally have at least six amino acid residues and usually between about 8 and about 50 amino acid residues (preferably, between approximately 10 and approximately 20 residues).

The term "divalent metal ion" means a metal ion with two positive charges. Divalent metal ions include as ogranichivaya examples of zinc, cobalt, Nickel, cadmium, magnesium and manganese. Specific forms of such metals that can be used include salt form (e.g., pharmaceutically suitable salt form such as the form of the chloride, acetate, carbonate, citrate and sulfate above-mentioned divalent metal ions. Divalent metal ion for use in the present invention, optional, is a zinc and preferably in the form of a salt, zinc sulfate or zinc chloride.

"Isolated" when used to describe the various peptides or proteins described herein, means a peptide or protein that is identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that normally would interfere with diagnostic or therapeutic applications of peptide or protein, and may include enzymes, hormones and other protein and non-protein solutions. In preferred embodiments, the implementation of the peptide or protein will be clear (1) to a degree sufficient to obtain the amino acid sequence of at least 15 residues from the N-terminal, or internal, using sequencing machine with rotating bowl, or (2) to homogeneity in SDS-PAGE in reducing and non conditions using colour, Kumasi blue and the and preferably, silver, or (3) to homogeneity by means of mass spectroscopy or peptide mapping. The selected substance includes a peptide or proteinin situwithin recombinant cells, since ceases to attend at least one component of its natural environment. However, usually, the selected peptide or protein will get through at least one stage of cleaning.

"Percent (%) amino acid sequence identity" with respect to the sequences herein, is defined as the percentage of amino acid residues in the sequence of the candidate, which is identical amino acid residues in the control sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the identity sequence. Alignment to determine percent identity of amino acid sequences can be accomplished in various ways available to professionals in this field who can determine appropriate parameters for measuring alignment, including setting algorithms needed to achieve maximal alignment over the full-sized comparing the subject sequences. For the purposes here, the percentage identity of the amino acids can be obtained using a computer program to compare sequences, ALIGN-2, which is developed by Genentech, Inc. and the source code of which has been filed with user documentation in the office for the protection of U.S. copyright, Washington, DC, 20559, registered under No. TXU510087 registration of copyright in the USA. The program ALIGN-2 publicly available at Genentech, Inc., South San Francisco, CA. All parameters comparison of the sequences set by the program ALIGN-2 was not changed.

"Stringency" of hybridization reactions is readily determinable ordinary person skilled in the art, typically, empirical calculation, depending on the length of the probe, the temperature of washing and concentration of salt. In General, longer probes require for proper annealing to higher temperatures, while shorter probes require lower temperatures. Hybridization, in General, depends on the ability of denatured DNA to re-hybridisierung, when the complementary chain present in the medium at a temperature below their melting point. The higher the desired degree of identity between the probe and hybridization sequence, the higher the relative temperature that can be used. Resulting from this it follows that the higher the relative temperature is URS tend to make the reaction conditions more stringent, while lower temperatures are less strict. Additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

"Conditions of high stringency, as defined here, represent those in which: (1) employ low ionic strength and high temperature for washing; of 0.015 M sodium chloride/0,0015 M sodium citrate/0.1% sodium dodecyl sulphate at 50°C; (2) using denaturing tool during hybridization; 50% (vol./about.) formamide with 0.1% bovine serum albumin/0.1% picollo/0.1% polyvinylpyrrolidone/50 mm buffer phosphate at pH 6.5 with 750 mm sodium chloride, 75 mm sodium citrate at 42°C; or (3) employ 50% of formamide, 5 × SSC (0.75 M NaCl, Of 0.075 M sodium citrate), 50 mm sodium phosphate (pH of 6.8), 0.1% sodium pyrophosphate, 5 × denhardt's solution, processed by the ultrasound DNA salmon sperm (50 µg/ml), 0.1% of SDS and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 × SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by rinsing high severity, consisting of 0.1 × SSC containing EDTA at 55°C.

"Moderately stringent conditions" may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the incubation over night at 37°C in a solution comprising: 20% formamide, 5 × SSC (150 mm NaCl, 15 mm trinitrate sodium), 50 mm sodium phosphate (pH of 7.6), 5 × rastv the R. Denhardt, 10% dextran sulfate, and 20 mg/ml denatured fragmented sperm DNA, salmon, followed by washing the filters in 1 × SSC at about 37-50°C. the skilled in the art will understand how to adjust the temperature, ionic strength, etc. as necessary to adapt to factors such as probe length and the like

The term "primer" or "primer" means an oligonucleotide sequences that hybridize with complementary polynucleotide RNA or DNA target and provide a starting point for stepwise synthesis of polynucleotide of mononucleotides by act nucleotidyltransferase, as occurs, for example, with polymerase chain reaction.

The term "control sequences" refers to DNA sequences necessary for the expression of the operatively linked coding sequence in a particular organism, the host. Control sequences that are suitable, for example, prokaryotes, include a promoter, optionally, the sequence operator and the binding site of the ribosome. It is known that eukaryotic cells utilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is operatively linked", when its lead in the functional interaction with another sequence of nucleic KIS is the notes. For example, DNA for proposedvalue or secretory leader is functionally linked to DNA for a polypeptide if it is expressed as preblock that participates in the secretion of the polypeptide; a promoter or enhancer is functionally linked with a coding sequence if it affects the transcription of the sequence; or the binding site of the ribosome is functionally linked with a coding sequence if it is to contribute to the broadcast. In General, "functionally linked" means that linked DNA sequences are contiguous and, in the case of a secretory leader, contiguous and in the phase of reading. However, the enhancers should not be continuous. Linking is carried legirovaniem appropriate restriction sites. If such sites do not exist, use synthetic oligonucleotide adapters or linkers in accordance with common practice.

"Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells that Express Fc receptors (FcR) (e.g., cell - natural killer cells (NK), neutrophils and macrophages), learn of bound peroxidase antibody on the target cell and then cause lysis of the target cells. Primary cells, op is sleduushie ADCC, NK cells, Express FcγRIII only, whereas monocytes Express FcγRI, FcγRII and FcγRIII. The FcR expression on hematopoietic cells is summarized in table 3 str in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the activity of molecules of interest in ADCC analysis can be performed ADCCin vitrosuch as described in U.S. patent No. 5500362 or 5821337. Applicable effector cells for such assays include mononuclear cells of peripheral blood (PBMC) and cells - natural killer cells (NK). Alternative or additionally, the active molecules of interest in ADCC can be evaluatedin vivofor example, in animal models, such as described in Clynes et al. PNAS (USA) 95:652-656 (1998).

"Effector cells" are leukocytes which Express one or more FcR and perform effector functions. Preferably, the cells Express at least FcγRIII and realize the function of ADCC effector. Examples of human leukocytes mediating ADCC include mononuclear cells of peripheral blood (PBMC), cells - natural killer cells (NK), monocytes, cytotoxic T cells and neutrophils; where PBMC and NK-cells are preferred.

The terms "Fc receptor" or "FcR" is used to describe a receptor, to bind to the Fc region of antibodies. Preferred FcR FcR is the man with the natural sequence. In addition to t the th, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the subclasses of the FcγRI, FcγRII and FcyRIII, including allelic variants and forms of alternative splicing of these receptors. The FcγRII receptors include FcγRIIA (an"activating receptor") and FcγRIIB (an"inhibiting receptor"), which has a similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains a cytoplasmic domain activating motif immunoreceptor (ITAM) on the basis of tyrosine. Inhibiting receptor FcγRIIB contains a cytoplasmic domain inhibiting motif immunoreceptor (ITIM) on the basis of tyrosine (see Daëron, Annu. Rev. Immunol. 15:203-234 (1997)). Review of FcR is given in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term "FcR" here covers other FcR, including those that will be identified in the future. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). FcR here include polymorphisms, such as genetic dimorphism in gene, encoding a FcγRIIIa, leading to phenylalanine (F) or valine (V) at amino acid position 158, localized in the region of the receptor, binding to IgG1. It is shown that homozygous for valine FcγRIIIa (FcγRIIa-158V) has a higher affinity for IgGl and promotes increased ADCC in vitrocompared to homozygous for phenylalanine FcγRIIIa (FcγRIIIa-158F) or heterozygous (FcγRIIIa-158F/V) receptors.

"Complementability cytotoxicity" or "CDC" means the ability of a molecule to lyse target in the presence of complement. Path activation of complement is initiated by binding of the first component of the complement system (C1q) with a molecule (e.g. antibody)that forms a complex with cognate antigen. To assess activation of complement can be analyzed by the CDC, for example, as described in Gazzano-three-bet et al., J. Immunol. Methods 202:163 (1996).

II. TYPICAL MATERIALS AND METHODS ACCORDING to the INVENTION

The methods and analyses described here aims to investigate the expression of one or more biomarkers in the sample of tissue or cells of a mammal, where the determining the expression of one or more such biomarkers is indicative of whether a tissue sample or cells sensitive to apoptosis-inducing tools such as Apo2L/TRAIL and antibody agonists anti-DR5. Methods and analyses include examining the expression of biomarkers, such as specific fucosyltransferase, in particular fucosyltransferase 3 (FUT3) and/or fucosyltransferase 6 (FUT6), as well as antigens sialyl-Lewis A and/or X.

As discussed above, there are some populations affected types of human cells (such as oncrete populations of cancer cells), which is resistant to induction of apoptosis. Therefore, I believe that the described methods and analyses can provide appropriate, effective and potentially cost-effective means of obtaining data and information that is applicable to determine the appropriate or effective therapy for the treatment of patients. For example, patients whose cancer was diagnosed or is associated with immune status, you can perform a biopsy to obtain a tissue sample or cells and the sample can be investigated by various testsin vitroto determine whether cells of a patient sensitive to a drug, such as Apo2L/TRAIL or the antibody to the receptor of death.

The invention relates to methods for predicting the sensitivity of a sample of tissue or mammalian cells (such as cancer cells to Apo2L/TRAIL or the antibody-receptor agonist death. In these methods, get a sample of tissue or cells of a mammal and examined for expression of one or more biomarkers. The methods can be implemented in a variety of formats analysis, including analyses, detecting expression of mRNA, enzyme assays, detecting the presence of enzymatic activity, and immunohistochemical analyses. Determination of the expression of these biomarkers in the above tissues or cells would predict that such TKA and or cells are sensitive to apoptosis-inducing activity of Apo2L/TRAIL and/or antibodies to receptor of death. The authors of the present invention unexpectedly found that the expression of these specific biomarkers correlates with the sensitivity of these tissues and cells to apoptosis-inducing tools such as Apo2L/TRAIL and antibody-receptor agonists death.

As discussed below, the expression of various biomarkers in a sample can be analyzed through a number of methodologies, many of which are known in this field and understandable to the expert, including as non-limiting examples of immunohistochemical and/or Western analysis, quantitative blood tests (eg, ELISA serum) (for research, for example, levels of protein expression), biochemical assays of enzyme activity, the hybridization of thein situ, Northern analysis and/or anilis PCR mRNA and genomic analysis by Southern (for research, for example, deletions or gene amplification), as well as any of a wide variety of analyses that can be performed through the analysis of gene and/or tissue on the biochip. Typical protocols to determine the status of genes and gene products are found, for example, in Ausubel et al. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis).

The protocols below, related to the detection in a sample of specific biomarkers, such as fucosyltransferase 3 (FUT3), fucosyltransferase 6 (FUT6), sialyl-Lewis A and sialyl-Lewis X, which are shown below for illustrative purposes.

Optional methods of the invention include protocols for research or testing the presence of proteins sialyl-Lewis A and/or sialyl-Lewis X in a sample of tissue or cells of a mammal. Can be used to include multiple methods of detection of proteins related sialyl-Lewis A and/or sialyl-Lewis X, for example, immunohistochemical analysis, immunoprecipitation, analysis, Western blotting, analysis of molecular binding, ELISA, ELIFA, fluorescently-activated sorting of cells (FACS), etc. for Example, an optional method of detecting expression of a protein related sialyl-Lewis A and/or sialyl-Lewis X in the tissue or sample which includes bringing the sample into contact with the antibody to sialyl-Lewis A and/or sialyl-Lewis X, with its fragment, reactive sialyl-Lewis A and/or sialyl-Lewis X, or with a recombinant protein containing antigennegative region antibodies to sialyl-Lewis A and/or sialyl-Lewis X; and then the detection in the sample binding protein related sialyl-Lewis A and/or sialyl-Lewis X.

In specific embodiments, the implementation according to the invention the expression of the proteins in the sample sialyl-Lewis A and/or sialyl-Lewis X were studied by means of immunohistochemistry protocols and staining. It is shown that immunohistochemical staining of tissue section is a reliable method of estimating or detecting the presence of proteins in the image is E. In the methods of immunohistochemistry ("IHC") use of the antibody as a probe for the visualization of cellular antigensin situas a rule, chromogenic or fluorescent methods.

For a sample you can use a sample of tissue or cells of a mammal (usually a patient person). Samples include as non-limiting examples of cells of a malignant tumor, such as cancer cells of the colon, breast, prostate, ovary, lung, stomach, pancreas, lymphoma and leukemia. The sample can be obtained in a variety of ways known in this area, including as non-limiting examples of surgical excision, aspiration or biopsy. The tissue may be fresh or frozen. In one of the embodiments the sample is fixed and enclosed in paraffin or similar.

The tissue sample may be fixed (i.e. save) by conventional methodology (see, for example, "Manual of Histological Staining Method of the Armed Forces Institute of Pathology," 3rdedition (1960) Lee G. Luna, HT (ASCP) Editor, The Blakston Division McGraw-Hill Book Company, New York; The Armed Forces Institute of Pathology Advanced Laboratory Methods in Histology and Pathology (1994) Ulreka V. Mikel, Editor, Armed Forces Institute of Pathology, American Registry of Pathology, Washington, DC). The person skilled in the art will recognize that the choice of a fixative is determined by the purpose for which the sample should histology the Cesky be painted or otherwise analyzed. The person skilled in the art will also take into account that the duration of fixation depends on the size of the tissue sample and used the lock. As an example, for fixing the sample, you can use neutral buffered formalin, Bouin or paraformaldehyde.

In General, the sample is first fixed and then dehydration series of alcohols of increasing concentration, permeate and flood paraffin or other medium for slices, so you can get slices of the tissue sample. Alternatively, you can cut the fabric and to record the received slices. As an example, a tissue sample can be embedded in paraffin and processed it through the conventional methodology (see, for example, "Manual of Histological Staining Method of the Armed Forces Institute of Pathology", above). Paraffin, which can be used includes, as non-limiting examples paraplast, bioloid and Tissuemay. After the conclusion of the tissue sample, the sample can be cut on a microtome or the like (see, for example, "Manual of Histological Staining Method of the Armed Forces Institute of Pathology", above). As an example, for this method, the thickness of the slices may be within the range of from about three microns to about five microns. After cutting the slices can be attached to glass in several standard ways. Adhesive substances include as non-limiting examples of the silane, the same is Athyn, poly-L-lysine, etc. as an example, enclosed in paraffin sections can be attached to the positively charged glass and/or glass coated with poly-L-lysine.

If the enclosing material use paraffin sections of tissue, usually deparaffinizing and rehydrating in the water. The tissue section can be deparaffinizing through several standard methodologies. For example, you can use xylene and a series of alcohols with a gradually decreasing concentration (see, for example, "Manual of Histological Staining Method of the Armed Forces Institute of Pathology",' above). Alternatively, you can use commercially available deparaffinizing inorganic means, such as Hemo-De7 (CMS, Houston, Texas).

Not necessarily, after receiving a sample, a tissue section, you can analyze the IHC. IHC can be performed in combination with additional means, such as morphological staining and/or fluorescent in situ hybridisationin situ. There are two main ways IHC; direct or indirect analyses. In the first analysis of the binding of an antibody to a target antigen (for example, sialyl-Lewis A and/or sialyl-Lewis X) is determined directly. In the direct analysis using labeled reagent, such as a primary antibody, labeled with a fluorescent label or an enzyme, which can be visualized without interaction with an additional antibody. Typically the indirect analysis of unconjugated primary antibody binds to the antigen, and then labeled secondary antibody binds to the first antibody. Where the secondary antibody is conjugated with an enzyme label, to provide visualization of antigen add chromogenic or fluorogenic substrate. Amplification of the signal, as several secondary antibodies can react with different epitopes of the primary antibodies.

Primary and/or secondary antibody used for immunohistochemistry, typically will be labeled with detectable group. There are many labels that can basically be grouped into the following categories.

(a) Radioactive isotopes, such as35S14C,125I3H and131I. Antibody can be marked with radioactive isotope using the methods described, for example, in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, New York, Pubs. (1991), and radioactivity can be measured using scintillation counting.

(b) Particles of colloidal gold.

(c) Fluorescent labels, including as non-limiting examples of chelates of rare earth elements (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, lissamine, umbelliferone, picogreen, phycocyanin, or commercially available fluorophores such as SPECTRUM ORANGE7 and SPECTRUM GREEN7 and/or derivatives of one or more of the above. Fluorescent labels can in order to anywherebut with the antibody using methods described, for example, in Current Protocols in Immunology, above. Fluorescence can be quantified using fluorometry.

(d) Different tags - substrates for the enzymes are available, and an overview of some of them is given in U.S. patent No. 4275149. The enzyme generally catalyzes a chemical alteration of the chromogenic substrate, which can be measured using different methods. For example, the enzyme may catalyze a color change of the substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence substrate. Methods to quantify the measurement of fluorescence described above. Chemiluminescent substrate acquires the electronic excitation due to a chemical reaction and may then emit light which can be measured (for example, using chemiluminometer), or is the donor of energy to a fluorescent acceptor. Examples of enzymatic labels include luciferase (e.g., Firefly luciferase and bacterial luciferase; U.S. patent No. 4737456), luciferin, 2,3-dihydropteridine, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase (e.g., glucose oxidase, galactosidase and glucose-6-phosphatedehydrogenase), heterocyclics oxidase (such as uricase and xanthine oxidase), lactoperoxidase, microbiocides and other Methods of conjugation of enzymes to antibodies are described in O'sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (ed. J. Langone &H. Van Vunakis), Academic press, New York, 73:147-166 (1981).

Examples of combinations of enzyme-substrate include, for example:

(i) horseradish peroxidase (HRPO) with hydrogen peroxide as a substrate, where the hydrogen peroxide oxidizes a dye precursor (e.g., orthophenylene (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB));

(ii) alkaline phosphatase (AP) with paranitrophenylphosphate as chromogenic substrate; and

(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g. p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate (for example, 4-methylumbelliferyl-β-D-galactosidase).

Professionals in this field are more combinations of enzyme-substrate. Their overview, see U.S. patent No. 4275149 and 4318980. Sometimes the label is not directly kongugiruut with the antibody. Various ways of achieving this are known to the experienced specialist in this field. For example, the antibody can be konjugierte with Biotin and any of the above four broad categories of labels can be konjugierte with Avidya and Vice versa. Biotin selectively binds to avidin, and so the label can be konjugierte with the antibody data indirectly Alternative, to achieve indirect conjugation of the label with the antibody the antibody kongugiruut with a small hapten, and one of the above-mentioned various types of labels kongugiruut with antiJapanese antibody. Thus it is possible to achieve indirect conjugation of the label with the antibody.

In addition to discussing the above methods of obtaining the sample may be desirable additional processing of the tissue section prior to, during or after IHC. For example, you can implement the methods damascenone epitopes, such as heating the tissue sample in citrate buffer (see, e.g., Leong et al. Appl. Immunohistochem. 4(3):201 (1996)).

After an optional stage of blocking the cut tissue is exposed to primary antibody for a sufficient period of time and under suitable conditions, so that the primary antibody binds to the protein antigen target in the tissue sample. Suitable conditions to achieve this, you can define common experiments. The degree of binding of the antibody with the sample determined using any of the detectable labels discussed above. Preferably, the label is an enzymatic label (such as HRPO)that catalyzes a chemical alteration of the chromogenic substrate such as 3,3'-diaminobenzidine Chromogen. Preferably, the enzymatic label kongugiruut with antibodies that specifically bind to the primary antibody is m (for example, the primary antibody is a polyclonal antibody rabbit, and the secondary antibody is an antibody goat antibodies against rabbit).

Optionally, the antibodies used in IHC analysis for detecting expression sialyl-Lewis A or anti-sialyl-Lewis X, are anti-sialyl-Lewis A and anti-sialyl-Lewis X antibodies, respectively. Optional anti-sialyl-Lewis A and anti-sialyl-Lewis X antibody is a monoclonal antibody. Anti-sialyl-Lewis A and anti-sialyl-Lewis X antibodies are readily available in this area, including from various commercial sources.

The preparations thus obtained can be secure and seal the cover glass. Then the glass estimate, for example, using a microscope, and you can apply criteria of staining intensity generally accepted in this field. Where the antigen is a protein sialyl-Lewis A and/or sialyl-Lewis X, the criteria of staining intensity can be defined as follows:

Table 1
Character coloringScore
Not detected staining in cells0
Weak the e/barely perceptible staining is detected in more than 10% of cells 1+
Painting, from weak to moderate, detected in more than 10% of cells2+
Staining, moderate to strong, detected in more than 10% of cells3+

Usually consider that point the nature of the staining approximately 2+ or higher in this analysis IHC is prognostic or indicating the sensitivity of mammalian cells (such as cancer cells of a mammal) to Apo2L/TRAIL or the antibody-receptor agonist death.

In alternative methods, the sample can be brought into contact with an antibody specific for a given biomarker in conditions suitable for formation of a complex of antibody-biomarker, and then to detect this complex. The presence of the biomarker can be assessed in a number of ways, such as how the Western blot and ELISA for the analysis of a wide variety of tissues and samples, including plasma or serum. Available in a wide range of immunoassay methods using such analyses, see, for example, U.S. patent No. 4016043, 4424279 and 4018653. They include both monovalent and divalent, or "sandwich"assays non-competitive types, as well as traditional competitive analysis link. These analyses also include direct swazilan the e labeled antibody to the biomarker target.

Sandwich assays are among the most useful and widely used tests. There are many variations of the way the sandwich-analysis, and all of them are intended to be covered by the present invention. Briefly, in a typical direct analysis of unlabeled antibody immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of a complex of antibody-antigen, then add the second antibody specific to the antigen, the labeled reporter molecule capable of giving the detected signal, and incubated, allowing sufficient time for the formation of another complex of antibody-antigen-labeled antibody. Any unreacted substances are washed and the presence of the antigen is determined by detecting the signal produced by the reporter molecule. The results can be qualitative, by simple observation of the visible signal, or they can be quantified in comparison with a control sample containing a known quantity of the biomarker.

Options direct analysis include simultaneous analysis in which the sample and the labeled antibody are added simultaneously to the bound antibody. These methods are well known to specialists in the given field including any minor changes, as will be immediately obvious. In a typical direct sandwich the analysis of the first antibody having specificity for biomarker is covalently or passively bound to a solid surface. Solid surface is typically a glass or a polymer, where the most commonly used polymers are cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid substrate can be in the form of tubes, beads, discs or microplates, or any other surface suitable for conducting immunoassay. The linking techniques well known in the field and generally consist of cross-linking covalent binding or physical adsorption, complex polymer-antibody is washed in preparation for the test sample. Then to solid phase complex add an aliquot of the test sample and incubated for a sufficient period of time (e.g., 2-40 minutes or overnight if more convenient) and under the right conditions (for example, from room temperature to 40°C, for example, between 25°C and 32°C, inclusive)to allow binding of any subunit present in the antibody. After an incubation period, the solid phase with subunit antibody is washed, dried and incubated with a second antibody specific to the part of the of biomarker. The second antibody is associated with a reporter molecule which is used to show the binding of the second antibody with a molecular marker.

An alternative method involves the immobilization of biomarkers of target in the sample and then the impact on immobilized target-specific antibody, which may or may not be labeled reporter molecule. Depending on the number of targets and signal strength of reporter molecules may be possible to detect an associated target direct labelling of the antibody. Alternatively, the complex target-the first antibody is subjected to the action of the second labeled antibody specific for the first antibody to form a ternary complex target-first antibody-second antibody. Complex detects the signal emitted by the reporter molecule. By "reporter molecule", as used in the present description, understand the molecule, which, by their chemical nature, provides a signal, which can analytically be identified, allowing the detection associated with antigen antibodies. The most commonly used reporter molecules for this type of analysis are enzymes, fluorophores or molecules containing radionuclides (i.e. radioactive isotopes), and chemiluminescent molecules.

In the case of enzyme immunoassay fernandohierro with the second antibody, usually using glutaraldehyde or periodate. How easy it will be clear, however, there is a wide variety of different ways conjugation, easily accessible to the person skilled in the art. Commonly used enzymes include, among others, horseradish peroxidase, glucose oxidase, β-galactosidase and alkaline phosphatase. For use with the specific enzymes are generally chosen substrates to obtain, after hydrolysis by the corresponding enzyme detected color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. Instead of the above chromogenic substrates can also be used fluorogenic substrates, giving a fluorescent product. In all cases, the enzyme labeled antibody is added to the complex of the first antibody-molecular marker to allow contact and wash off the excess reagent. Then the complex of antibody-antigen-antibody add a solution containing a suitable substrate. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visible signal, which can then be quantified, usually spectrophotometrically, to get an idea of the amount of biomarker present in the sample. Alternatively, fluorescent compounds such as fluorescein and rhodamine, can chemically attach the to antibodies without altering their binding capacity. When activated by the irradiation of light with a specific wavelength labeled fluorochrome antibody absorbs the light energy to the induction of a state of excitation in the molecule, followed by emission of light of a characteristic color, which can be visually detected by a light microscope. As in the EIA, a fluorescently labeled antibody is allowed to communicate with the complex of antibody-molecular marker. After washing the unbound reagent, the remaining ternary complex is then exposed to light of a suitable wavelength, the observed fluorescence indicates the presence of interest molecular marker. Methods immunofluorescence assay and EIA both are common in this area. However, you can also use other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecule.

Assume that the above methods can also be used for detecting the expression of polypeptides FUT3 or FUT 6.

The methods of the present invention further include protocols for investigation of the presence and/or mRNA expression, such as mRNA FUT3 and/or FUT6, in a sample of tissue or cells. Methods for the study of mRNAs in cells are well known and include, for example, analyses of hybridization using probes complementary DNA (such as hybridizationin situwith the use of what Finance labeled robopro FUT3 and/or FUT6, Northern blot and related methods) and various analyses of nucleic acid amplification (such as RT-PCR using complementary primers specific for FUT3 and/or FUT6, and other ways of detecting amplification type, such as, for example, branching DNA, SISBA, TMA and the like).

A sample of tissue or cells from mammals can be conveniently analyzed, for example, mRNA FUT3 and/or FUT6 using Northern, dot blot or PCR analysis. For example, analyses of RT-PCR, such as quantitative PCR analysis, well known in this field. In the illustrative embodiment according to the invention a method of detecting mRNA FUT3 and/or FUT6 in a biological sample involves obtaining cDNA from the sample by reverse transcription using at least one primer; amplifying the thus obtained cDNA using polynucleotide FUT3 and/or FUT6 as sense and antisense primers for amplification there cDNA FUT3 and/or FUT6; and detecting the presence of amplified cDNA FUT3 and/or FUT6. In addition, such methods can include one or several stages, allowing to determine the mRNA level of FUT3 and/or FUT6 in a biological sample (for example, through the simultaneous study of levels comparative control mRNA sequence of a gene "household", such as a member of a family of actin. Optionally, you can define the sequence of the amplified cDNA FUT3 and/or FUT6.

Materials for embodiments of the present invention include primers and a pair of primers FUT3 and/or FUT6, allowing specific amplification of polynucleotides of the present invention or any part thereof, and probes that selectively or specifically for hybridization with the nucleic acid molecules according to the invention or any part of them. The probes can be marked detectable label such as a radioactive isotope, a fluorescent compound, bioluminescent compound, chemiluminescent compound, metal chelator or enzyme. Such probes and primers can be used for detection of the presence of polynucleotides FUT3 and/or FUT6 in the sample and as a means for detecting cells expressing proteins FUT3 and/or FUT6. As will be clear to the person skilled in the art, based on these sequences, we can obtain a large number of different primers and probes and applied effectively to amplify, clone and/or determine the presence and/or levels of mRNA FUT3 and/or FUT6.

Optional methods of the invention include protocols for investigation or detection of mRNA, such as mRNA FUT3 and FUT6 or other mRNA fucosyltransferase, in a sample of tissue or cells by means of microarrays. When used in the research Institute of microchips with nucleic acid samples of test and control mRNA from the test and control tissue samples are subjected to reverse transcription and tagging to obtain cDNA probes. Then the probes hybridized with the array of nucleic acids immobilized on a solid substrate. The array is configured so that a known sequence and position of each member of the array. For example, on a solid substrate it is possible to get an array of assignments of genes with the ability to be expressed in specific disease States. Hybridization of the labeled probe with a specific member of the array indicates that the sample from which the probe expresses this gene. Analysis of differential gene expression in disease-affected tissues can provide valuable information. In the way of microchips used methods of hybridization of nucleic acid and computational methods to assess the profile of mRNA expression of thousands of genes in one experiment (see, for example, WO 01/75166, published on October 11, 2001; (see, for example, the discussion of making chips in U.S. patent 5700637, U.S. patent 5445934 and U.S. patent 5807522, Lockart, Nature Biotechnology, 14:1675-1680 (1996); Cheung, V.G. et al., Nature Genetics 21 (Suppl):15-19 (1999)). The DNA microarray are miniature arrays containing gene fragments, or synthesized directly, or rastapana on glass or other substrates. In a single array typically contains thousands of genes. A typical experiment with a microchip includes the following stages: 1. the production of fluorescently labeled target RNA, vydeleny the th sample, 2. hybridization of the labeled target with a microchip, 3. washing, staining and scanning of the array 4. analysis of the scanned image and 5. obtaining gene expression profiles. Currently use two main types of DNA microarrays: arrays of oligonucleotides (typically from 25 to 70 Mer) and arrays to study gene expression, containing the PCR products derived from cDNA. When forming the array oligonucleotides can either be made in advance and dispense on the surface, or synthesized directly on the surface (in situ).

System Affymetrix GeneChip® is a commercially available system microchips containing arrays obtained by direct synthesis of oligonucleotides on a glass surface. Chips with probes/genes: oligonucleotides, typically 25-chinnici, are synthesized directly on the glass plate by a combination of methods based semiconductor photolithography and solid-phase chemical synthesis. Each array contains up to 400,000 different oligonucleotides, each oligonucleotide is present in millions of copies. Because oligonucleotide probes synthesized in known positions of the chip, the pattern of hybridization and signal intensity can be interpreted in relation to identity genes and the relative levels of expression through software Affymetrix icroarray Suite. Each gene represented on the chip by a series of different oligonucleotide probes. Each pair of probes consists of an exact matching of the oligonucleotide and the oligonucleotide with inconsistencies. Exact matching probe has a sequence exactly complementary to a specific gene, and thus it is used for measuring gene expression. Probe with inconsistencies differs from the exact match probe replacement of one base in a Central position, preventing the binding of transcription of the target genes. This helps to determine the background and non-specific hybridization, contribute to the signal measured for an exact matching of the oligonucleotide. Software Microarray Suite subtracts the intensity of hybridization of the probes with mismatches of the intensities of hybridization exact match probes to determine the absolute or specific intensity for each set of probes. The probes selected on the basis of modern information from Genbank and other databases nucleotides. Consider that sequence will learn a unique region on the 3'end of the gene. For carrying out hybridization up to 64 chips simultaneously using hybridization oven GeneChip (oven "spit"). Washing and staining of the chips with probes is carried out in the system for automatic operation with liquids. It is completely automated and contains four modules, where each module holds one chip with probes. Each module is controlled independently via software Microarray Suite using pre-programmed protocols of the flow. The scanner is a confocal fluorescent scanner, measuring the fluorescence intensity emitted by the labeled crnc, contacting the array of probes. The computer workstation control system for automatic operation with liquids and scanner. Software Microarray Suite can control up to eight systems for automatic operation with liquids with the use of pre-programmed protocols for hybridization, washing and staining of the chip with probes. The software also collects data on the intensity of hybridization and converts into a signal presence/absence for each gene using suitable algorithms. Finally, the software detects changes in gene expression between experiments through comparative analysis and formats the output in files with .txt you can use other software for further analysis of the data.

The expression of selected biomarkers can also be assessed by the study deletion of the gene or gene amplification. A deletion or amp is ificatio gene can be measured by any of a wide variety of protocols, known in this field, such as conventional blotting for the Southern, Northern-blotting to quantify the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)), dot-blotting (DNA analysis), or by hybridization of thein situ(for example, FISH) using a suitable labeled probe, cytogenetic methods or methods comparative genomic hybridization (CGH) using the appropriate labeled probe. As an example, these methods can be applied for detection of deletion or amplification of genes FUT3 and/or FUT6.

In addition, you can examine the methylation status of a biomarker, such as a gene FUT3 and/or FUT6, in a sample of tissue or cells. Aberrant demethylation and/or hypermethylation of the CpG Islands in the 5'-regulatory regions of genes occurs frequently in immortalized and transformed cells and may lead to altered expression of different genes. Many analyses to study the methylation status is well known in this field. For example, in the methods of hybridization on Southern to assess the status of methylation of CpG-Islands can be used is sensitive to methylation of restriction enzymes, which cannot cleave sequences that contain methylated CpG sites. In addition, MSP (methyl-specific PCR), you can quickly get a profile of the status of methylated what I all CpG sites, present in the CpG island of the given gene. The method includes the initial modification of DNA by sodium bisulfite (which translates all neetilirovannye cytosine in uracil), followed by amplification using primers specific for the methylated, unlike demetilirovanny, DNA. Protocols involving the influence of methylation can also be found, for example, in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et al. eds., 1995; De Marzo et al., Am. J. Pathol. 155(6):1985-1992 (1999); Brooks et al, Cancer Epidemiol. Biomarkers Prev., 1998, 7:531-536; and Lethe et al., Int. J. Cancer 76(6):903-908 (1998).

The expression of selected biomarkers in the tissue sample or cells can be investigated by functional assays or analysis activity. For example, if the biomarker is an enzyme that can be known in this field analyses for determination of this enzyme activity in a tissue sample or cells.

Assume that in the methods of the present invention in a tissue sample or cells can be investigated in the sample the expression of Apo2L/TRAIL or receptors that bind Apo2L/TRAIL, or antibodies to receptor of death. As described above, and in this area, currently believe that Apo2L/TRAIL associated with at least five different receptors: DR4, DR5, DcR1, DcR2 and OPG. Using methods known in this area, including those described here, the expression of Apo2L/TRAIL, D4, DR5, DcR1, DcR2 and/or OPG can be detected at the level of mRNA and at the protein level. As shown in figure 10 and 11, the data suggest that the study of the expression of receptors DcR1 and/or DcR2 in a tissue sample or cells you can get more information about whether a tissue sample or cells sensitive to Apo2L/TRAIL or the antibody to the receptor of death. As an example, the IHC methods described above can be used for detection of the presence of one or more of these molecules in the sample. Assume that the ways in which the tissue or sample, exploring not only the presence of the marker FUT or antigen Lewis, but also the presence of, for example, DR4, DR5 or DcR1, for the same tissue or sample can be obtained from a separate glass and test each glass with a reagent specific to each biomarker or receptor. Alternatively, for a sample of tissue or cells you can get one glass, and antibodies directed against each biomarker or receptor, can be applied according to the Protocol multicolor staining, which allows the visualization and detection of relevant biomarkers or receptors.

After determining that the sample of tissue or cells downregulation of one or more of the biomarkers, showing that a sample of tissue or cells to be sensitive to Apo2L/TRAIL or the antibody to Rotz the Torah of death, assume that the effective amount of Apo2L/TRAIL or antibodies to receptor of death, you can enter the mammal for the treatment of disorders, such as cancer or associated with immune disorders, striking a mammal. An experienced practitioner can put in mammals diagnosis of various pathological conditions herein described. In this area the available diagnostic methods, allowing, for example, diagnosis or detection of cancer or svjazanno immune disease in a mammal. For example, cancers can be identified by methods that include as non-limiting examples palpation, blood test, x-ray, NMR, etc. Associated with immune system disorders are also easy to identify.

Apo2L/TRAIL or the antibody to the receptor of death can be entered in accordance with known methods, such as intravenous administration in the form of a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, vnutrizonovoy, subcutaneous, intra-articular, intra-articular, intrathecal, oral, local or inhalation methods. Optionally, the introduction can be performed by infusion through minimalis using various commercially available devices.

Effective dosages and schedules for the introduction of Apo2L/TRAIL or EN is the body to the receptor of death can be determined empirically, and such definitions within the competence of specialists in this field. You can apply single or multiple doses. Now consider that the effective dose or amount of Apo2L/TRAIL, used alone, may lie in the range from about 1 μg/kg to about 100 mg/kg of body weight or more per day. Interspecies scaling of dosages, you can spend way known in this field, for example, as described in Mordenti et al., Pharmaceut. Res., 8:1351 (1991).

When using the introduction of Apo2L/TRAILin vivonormal values dosing can vary from about 10 ng/kg to up to 100 mg/kg of body weight of the mammal or more per day, preferably about 1 μg/kg/day to 10 mg/kg/day, depending on the method of introduction. Guidance on specific doses and routes of administration granted in the literature; see, for example, U.S. patent No. 4657760; 5206344; 5225212. Expect that different formulations will be effective for a variety of medicinal compounds and various diseases, introduction sent, for example, by one organ or tissue, may require the introduction of a manner different from the way another organ or tissue.

Assume that in ways you can apply additional therapy. One or more other therapies may include as non-limiting is reamers introduction radiotherapy, the cytokines (cytokines)that inhibits the growth funds (the funds), chemotherapeutic agents (tools), cytotoxic funds (funds), tyrosine kinase inhibitors, inhibitors of ras-farnesyltransferase, angiogenesis inhibitors and inhibitors of cyclin-dependent kinases, which are known in this field and are further defined and especially above. Assume such other therapeutic agent can be applied in the form of funds, separate from the Apo2L/TRAIL or antibodies to receptor of death. In addition, the methods of treatment based on therapeutic antibodies directed against tumor antigens, such as Rituxan™ or Herceptin™, as well as anti-angiogenic antibodies, such as anti-VEGF.

You can use drugs and dosing regimens for chemotherapy drugs according to the manufacturer's instructions or as empirically determined practising professional. Drugs and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992). Chemotherapeutic agent can be entered before or after the introduction of Apo2L/TRAIL or antibodies to receptor of death or you can enter at the same time.

It may also be desirable to introduce antibodies against other antigens, such as antibodies which bind to CD20, CD11a, CD18, CD40, ErbB2, EGFR, ErbB3, ErbB4, a growth factor, vascular endothelial (VEGF) other members of the TNFR family (such as OPG, TNFR1, TNFR2, GITR, Apo-3, TACI, BCMA, BR3). Alternative or additionally, two or more antibodies binding the same or two or more of the antigens described herein, it is possible to enter the patient. Sometimes it may be favorable to enter the patient one or more cytokines. After the introduction of processedin vitrocells can be analyzed. Attended treatmentin vivofor the treated mammal can be observed in a variety of ways, well known to practitioners. For example, tumor cells can be investigated pathologically for analysis of necrosis or can be analyzed in serum responses of the immune system.

For use in the applications described or suggested above, the invention also relates to kits products. Such kits may contain a means for transportation, divided into compartments to hold in close confinement one or more containers such as vials, tubes and the like, where each of the containers contains one of the separate elements for use in the method. For example, one container may contain a probe labeled for detection or that you can mark. Such probe may be an antibody or polynucleotide-specific protein FUT3 and/or FUT6, or gene, or mRNA FUT3 and/or FUT6, respectively. Where in the set use hibri is Italy nucleic acid for detection of nucleic acid target, the kit may also contain containers containing nucleotide(s) for amplification of nucleic acid sequence target, and/or the container containing the reporter means, such as linking Biotin protein, such as avidin or streptavidin, bound with a reporter molecule such as an enzymatic, fluorescent or radioisotope label.

Set according to the invention will usually contain the container described above and one or more other containers containing substances with desirable commercial or user standpoint, including buffers, solvents, filters, needles, syringes and investments in packaging with instructions for use. The container can include a label, to indicate that the composition is used for a specific therapeutic or non-therapeutic application, and can be specified instructions for usein vivoorin vitrosuch as described above.

Kits according to the invention have a number of options for implementation. A typical implementation is a set containing a container, a label on the specified container and a composition contained within the specified container; where the composition comprises a primary antibody binding sequence polypeptide FUT3 and/or FUT6, the label on the specified container indicates that the com is ositio can be used to assess the presence of proteins FUT3 and/or FUT6 at least one type of mammalian cells, and contains instructions for using the antibodies FUT3 and/or FUT6 to assess the presence of proteins FUT3 and/or FUT6 at least one type of mammalian cells. The kit may optionally contain a set of instructions and materials for obtaining a tissue sample and application of antibodies and probes to the same slice of the tissue sample. The set can contain both primary and secondary antibody, the secondary antibody is conjugated with a label, for example an enzymatic label.

In another embodiment, the kit includes a container, a label on the specified container and a composition contained within the specified container; where the composition comprises polynucleotide that hybridizes with complementary him from polynucleotides FUT3 and/or FUT6 in stringent conditions, the label on the specified container indicates that the composition can be applied to assess the presence of FUT3 and/or FUT6 at least one type of mammalian cells, and contains instructions for use of polynucleotide FUT3 and/or FUT6 to assess the presence of RNA or DNA FUT3 and/or FUT6 at least one type of mammalian cells.

Other optional components of the kit can include one or more buffers (e.g., blocking buffer, washing buffer, the buffer for substrate and so on), other reagents, such as the substrate (for example, a Chromogen), which Henichesk the alter enzymatic label, the solution to damascenone epitopes, control samples (positive and/or negative controls), the control glass (glass), etc.

EXAMPLES

Various aspects of the invention hereinafter described and illustrated by the following examples, none of which is intended to limit the scope of the invention.

MATERIALS AND METHODS

Cell culture and cell line

The following cell lines of colorectal adenocarcinoma person: HCT-8, COLO 205, HCT 116, SW403, LoVo, SW948, Caco-2, COLO 201, SW1417, DLD-1, CX-1, HCT-15, LS 180, RKO, RKO-AS45-1, SK-CO-1, SW480, SW620, SW837, CL-40, COLO-206F, COLO 320DM, COLO 320HSR, COLO-678, HT-29, KMl2, LS1034, SW1116 received from the Bank ATCC (Manassas, Virginia), DSMZ (German collection of microorganisms and cell cultures), JCRB (Japanese Bank for cellular resources) or ECACC (European collection of cell cultures) and were cultured in medium RPMI-1640, supplemented with 10% V / V heat inactivated fetal bovine serum, 2 mm L-glutamine and 10 mm HEPES.

Analysis of cytotoxicity

Analysis of MTT (non-radioactive analysis of cell proliferation CellTiter 96®from Promega), which is a colorimetric analysis, based on the ability of viable cells to recover soluble yellow salt tetrazole (MTT) to a blue crystals formazan used to determine the number of viable cells after treatment Apo2L/TRAIL or antibody to D5. The MTT analysis was performed by adding pre-mixed solution optimized dye in the culture wells of 96-hole of tablet containing different concentrations (0-1000 ng/ml) Apo2L/TRAIL or antibody to DR5. During a 4-hour incubation, the cells were transferred tetrazolium component of the dye solution in formosanus product. Then in wells with cell culture was added solubilizers/stop solution for dissolving formisano product and record the optical density at 570 nm using a spectrophotometer for reading 96-well plates (SpectraMax). The rate of optical density at 570 nm is directly proportional to the number of cells, normally used in assays of cell proliferation. Although the maximum optical density for formisano product is 570 nm and clean solutions look blue, the color at the end of the analysis may not be blue and depends on the number of the present formazan relative to other components of culture medium (including serum oxidized phenol red and unrestored MTT).

The number of cells optimize the titration cell to obtain in the analysis of the signal close to the upper value of the linear range of the assay. Because different types of cells have different levels of metabolic activity that is done for each cell line separately. For most of the investigated tumor cells used from 5000 cells per well up to 20,000 cells per well. Following is a step-by-step description of the applied tests.

1. Cells used for biological analysis, from the mother cultures.

2. Determining the number of cells and viability using Trypanosoma blue and the suspension of cells to a finite number of from 5,000 to 20,000 cells per well.

3. Pipetting 50 ál of cell suspension in a 96-well plate.

4. Incubation tablets at 37°C in a humidified atmosphere of 5% CO2throughout the night.

5. Add 50 ál of culture medium containing different concentrations of Apo2L/TRAIL or antibody to DR5 in the range from 0 to 1000 ng/ml for samples in 96-well pad. Controls were 50 µl of culture medium (without Apo2L/TRAIL or antibody to DR5) and 100 µl of culture medium (without cells). Each experiment was performed in the triple set of holes and for three independent days. The total volume in each well was 100 μl/well.

6. Incubation tablets at 37°C for 72 hours in a humidified atmosphere of 5% CO2.

7. Add 15 μl of dye solution to each well.

8. Incubation tablets at 37°C up to 4 hours in a humidified atmosphere of 5% CO2.

9. Add 100 ál solubilizing/stopping solution into each well.

10. Incubation tablets in t is the increase night at 37°C.

11. Check the optical density at a wavelength of 570 nm using a spectrophotometer for reading 96-well plates. The reference wavelength of 750 nm was used to reduce background, the reported fragments of cells, fingerprints, and other nonspecific absorption.

12. The average values of the optical density of the negative control was used as a zero value and subtracted from all other indicators. The mean values of optical density for each concentration of Apo2L/TRAIL or antibody to DR5 divided by the average of the values of optical density of the positive control (100% viable cells - untreated) to calculate the number of viable cells (%).

13. Build a graph of the percentage of viable cells (Y-axis) depending on the concentration of Apo2L/TRAIL or antibody to DR5 (the X-axis, logarithmic scale) and determined the value of the IC50localization values on the X-axis (ng/ml), corresponding to 50% of viable cells.

Protocol introduction label Affymetrix

For all samples received indicators OD260/280 and treated samples on the BioAnalyzer. Used 5 μg of total RNA of high quality.

A. Synthesis of the first chain cDNA:

1. Hybridization of the primer

DEPC-H2O x ál mix well by shaking. Quickly ottsentrirovat.

RNA (5 μg) y ál incubate for 10 minutes at 70°C.

Marker (5 µg dilute mother liquor 1:4) 1 ál quickly ottsentrirovat and placed in ice.

T7-(dT)24 primer 1 ál

volume of 12 ál

2. Temperature control

5X buffer for 1 chain cDNA 4 ál

Add 7 ál (from a mixture left) to each sample.

0.1 M DTT 2 ál mix well by shaking. Quickly ottsentrirovat.

10 mm dNTP mix 1 ál incubate at 42°C for 2 minutes.

volume of 7 ál

3. The synthesis of the first chain

Add 1 ál of SSII RT to each sample.

SSII RT 1 ál mix well by pipetting up and down or light shaking.

Quickly ottsentrirovat.

Total volume of 20 ál incubate at 42°C for 1 hour.

B. Synthesis of the second chain cDNA

1. Place the reaction mixture for the first circuit in the ice. Briefly ottsentrirovat to lower condensate from the walls of the tube.

2. Prepare the following General reaction mixture for the second chain.

DEPC treated H2O 91 ál

5X-reaction buffer for 2 circuit 30 ál

10 mm dNTP mix 3 ál

10 units/μl of DNA ligase 1 ál

10 units/ál DNA polymerase I 4 ál

2 units/ál Mcasa H 1 ál

Total volume of 130 μl

3. Add 130 µl of total reaction mixture for the second circuit to 20 ál of the first chain cDNA. (Final volume = 150 ál)

4. Mix by pipetting up and down or light shaking. Quickly ottsentrirovat.

5. Incubate at 16°C for 2 hours, the cooled water bath.

6. Add 2 ál [10 units] T4 DNA polymerase. Mix by pipetting up and down or light shaking. Quickly ottsentrirovat.

7. Incubate for 5 minutes at 16°C.

8. Add 10 ál of 0.5 M EDTA. Easy to shake. Quickly ottsentrirovat.

9. Go to the procedure cDNA purification or stored at -20°C for later use.

Purification of double-stranded cDNA (module for cleaning samples GeneChip)

1. Add 600 ál buffer for the binding of cDNA to 162 ál final preparation of the synthesis of double-stranded cDNA.

Mix by shaking for 3 seconds.

2. Check that the color of the mixture is yellow (similar to buffer for binding cDNA without reaction mixture for the synthesis of cDNA).

If the color of the mixture is orange or violet, add 10 ál of 3 M sodium acetate, pH 5.0, and mix.

The color of the mixture turns yellow.

3. Apply 500 ál of the sample on zentrifugenbau column for purification of cDNA inserted into a 2 ml collection tube, and centrifuged for 1 minute at ≥8000 x g (≥10,000 rpm). Throw breakthrough as a hazardous waste.

4. Re-apply to zentrifugenbau column remaining mixture (262 μl) and centrifuged as above.

Throw breakthrough as a hazardous waste and discard the collection tube.

5. Move zentrifugenbau column in a new 2 ml collection tube (provided). Pour 750 clbuffer for washing cDNA in zentrifugenbau column. Centrifuged 1 minute at > 8000 x g (≥10,000 rpm).

Throw a breakthrough.

6. Open the lid zentrifugenbau column and centrifuged for 5 minutes at maximum speed (≤25000×g). Place the tubes in a centrifuge using each of the second socket. Positioning the cover over the adjacent slots so that they were oriented in the opposite direction to the rotation, i.e. if the rotation is clockwise, to align the cover in a counterclockwise direction. This prevents the destruction of the covers. Throw slippage and the collection tube.

7. Move zentrifugenbau column in a 1.5 ml collection tube. Apply 10 µl of buffer for elution cDNA directly on the membrane zentrifugenbau column. Make sure that the buffer for elution cDNA distributed directly on the membrane.

Incubate for 1 minute at room temperature and centrifuged for 1 minute at maximum speed (≤25000×g) for the elution.

Preparing and conducting the reaction of IVT

Enzo: set for efficient labeling of RNA transcripts for biochips (Catalog No. 900182)

1. Use 10 μl of purified double-stranded cDNA.

2. Prepare the following General reaction mixture for IVT:

Distilled or deionized H2O 12 ál

10X HY reaction buffer 4 ál

10x labeled with Biotin ribonucleotides 4 ál

10X DTT 4 ál

10X a Mixture of RNase inhibitors 4 ál

20X T7 RNA polymerase 2 ál

Total volume: 30 ál

3. Add 30 ál total reaction mixture IVT to 10 μl of double-stranded cDNA. (Total = 40 ál)

4. Mix by pipetting up and down or light shaking. Quickly ottsentrirovat.

5. Immediately place the tube in a water bath at 37°C. Incubate for 5 hours.

6. Store at -20°C, if RNA is not immediately clear.

Purification of labeled Biotin crnc (module for cleaning samples GeneChip)

1. Add 60 ál of H2O to the IVT reaction mixture, and mix by shaking for 3 seconds.

2. To add to the sample of 350 ál of buffer for binding IVT crnc, mix by shaking for 3 seconds.

3. To add to the lysate 250 ál of ethanol (96-100%) and mix well by pipetting. Not centrifuged.

4. Apply the sample (700 ál) to zentrifugenbau column for purification IVT crnc inserted into a 2 ml collection tube.

Centrifuged for 15 seconds at ≥8000 x g (≥10,000 rpm).

5. Once again miss the eluate through the column.

Centrifuged for 15 seconds at ≥8000 x g (≥10,000 rpm).

Throw breakthrough as a hazardous waste and discard the collection tube.

6. Move zentrifugenbau column in a new 2 ml collection tube (provided).

7. Add 500 ál of buffer for washing IVT crnc and centrifuged for 15 seconds at ≥000×g (≥10,000 rpm) to wash.

Throw a breakthrough.

8. Pour 500 μl of 80% (vol./about.) ethanol zentrifugenbau column and centrifuged for 15 seconds at ≥8000 x g (≥10,000 rpm). Throw a breakthrough.

9. Open the lid zentrifugenbau column and centrifuged for 5 minutes at maximum speed (≤25000×g).

Throw slippage and the collection tube.

10. Move zentrifugenbau column in a new 1.5 ml collection tube.

11. Apply 11 ál-free RNase water directly to the membrane zentrifugenbau column. Let stand 1 minute.

Centrifuged for 1 minute at maximum speed (≤25000×g) for the elution.

12. Apply 10 µl-free RNase water directly to the membrane zentrifugenbau column. Let stand 1 minute.

Centrifuged for 1 minute at maximum speed (≤25000×g) for the elution.

Quantitative assessment crnc (IVT product)

To use spectrophotometric analysis to determine the yield of RNA. To apply the rule that 1 OD at 260 nm is equivalent to 40 μg/ml RNA.

Check the OD at 260 nm and 280 nm to determine the concentration and purity of the sample.

To maintain the ratio of A260/A280 close to 2.0 for pure RNA (relations between 1,9 and 2,1 are acceptable).

To quantify crnc using the total RNA as starting material it is necessary to calculate the adjusted output crnc, chemioterapico its admixture of total RNA. Using assessment 100% impurities to apply the formula below to determine the adjusted output crnc:

the adjusted output crnc = Rncm - (total RNK) (y)

Rncm = number crnc measured after IVT (µg)

total RNK = initial amount of total RNA (ág)

y = the fraction of the reaction mixture for cDNA used in IVT

Fragmentation crnc to obtain the target

For fragmentation, use the corrected concentration crnc.

1. Add 2 ál of 5x buffer for fragmentation for each 8 ál of RNA plus H2O.

20 µg crnc 1-32 ál

5X buffer for fragmentation 8 ál

free from RNase water to 40 μl

Total volume: 40 ál

2. Incubate at 94°C for 30 minutes. Immediately after incubation, place in ice.

Receiving target for hybridization

1. Heat 20X eukaryotic controls for hybridization and oligonucleotide B2 for 5 minutes at 65°C.

Set eukaryotic controls for the hybridization of Affymetrix GeneChip, catalogue #900362 (150 reactions)

2. Easy to shake precipitate by centrifugation.

3. The total reaction mixture Having a concentration of fragmented crnc equal to 0.5 μg/μl):

Standard chip (ál) Final conc.

Fragmented crnc 15 ág 30 0.05 µg/µl

Oligonucleotide B2 (3 nm) 5 50 PM

20x reference marker 15 is 1.5, 5, 25, 100 PM

(Bio B, C, D, Cre)

DNA JV is my salmon 3 0.1 mg/ml

Acetylated BSA 3 0.5 mg/ml

Hu cot-1 DNA (1 mg/ml) 30 0.1 mg/ml

2X MES buffer for hybridization 150 1X

H2O 64

The final volume 300

4. Transfer aliquots to 270 ál total reaction mixture in a test tube and add each 30 ál fragmented crnc. This is a mixture for hybridization.

5. To balance the chips with probes to room temperature prior to use.

6. Fill in the chips with probes 1x MES buffer for hybridization and incubate in the oven with the "spit" for 10 minutes at 45°C, 60 rpm

7. Heat the mixture for hybridization in a water bath at 99°C for 5 minutes.

8. Transfer the mixture to hybridization in a water bath at 45°C for 5 minutes.

9. Centrifuge the mixture for hybridization of 5 minutes at maximum speed.

10. Remove 1x MES buffer for hybridization with chips with probes.

11. To fill the chip with probes to top 200 μl of the mixture for hybridization.

12. Close the foil with the protrusions Tough Spots.

13. To hybridizat chip with probes at 45°C, 60 rpm for 19 hours.

14. Wash, stain and scan the chip with probes according to Affymetrix protocols.

Materials Affymetrix

Product Manufacturer Catalogue #

T7-(dT)24 primer Biosearch Technologies in order

Control markers of own production

Superscript II/5X buffer for the first circuit/0.1 M DTT Invitrogen 18064-014

5X buffer for the second circuit Initrogen 10812-014

10 mm dNTP Invitrogen 18427-088

10 units/μl DNA ligaseE. coliInvitrogen 18052-019

10 units/ál DNA polymerase IE. coliInvitrogen 18010-025

2 units/ál Mcasa H Invitrogen 18021-071

10 units/μl T4 DNA polymerase, Invitrogen 18005-025

of 0.5 M EDTA Sigma E-7889

ENZO kit highly efficient labeling of RNA transcripts Affymetrix or ENZO 900182 (ENZO)

Module for purification of samples Affymetrix GeneChip 900371

Acetylated bovine serum albumin Invitrogen 15561-020

Goat IgG - chemically pure Sigma I-5256

Antistreptokinase antibody (goat), biotinylated Vector Labs BA-0500

R-phycoerythrin-streptavidin Molecular Probes S-866

20X SSPE BioWhittaker 51214

Set eukaryotic controls Affymetrix 900362

Water for molecular biology Ambion 9934

DNA human Cot-1 Roche 1-581-074

5 M NaCl-free RNase free from Gnkazy Ambion 9760

Antifoam 0-30 Sigma A-8082

10% Tween-20 Pierce Chemical 28320

MES monohydrate of the free acid Sigma M5287

MES, sodium salt Sigma M3885

EDTA, disodium salt, 0.5 M solution Sigma E7889

Tough Spots, Label Dots USA Scientific 9902

Bake for GeneChip hybridization Oven 640 Affymetrix 800139 GeneChip Scanner 3000 workstation Affymetrix 00-0074

The system for automatic operation with liquids Fluidics Station Affymetrix 00-0081

Automatic loading device with an external bar code reader Affymetrix 00-0129

Quantitative PCR

Synthesis of cDNA:

ComponentVolume (μl)
10 X buffer for RT10
25 X dNTP mix4
10x random primers10
MultiScribe RT (50 units/μl)5
Free from RNase H2O21
RNA (100 ng)50
Finite volume100

Conditions of incubation

25° 10 minutes

37° 2 hours

The TaqMan reaction using securitysage detector (ABI Prism 7700 Sequencing Detector:

ComponentVolume (μl)
Universal General reaction mixture for PCR TaqMan (2 X)25
The TaqMan probe (20 X) (Assays-on-Demand™)2,5
cDNA (100 ng)2
H2O20,5
Finite volume100

thermal Cycling conditions

95° for 10 minutes

40 cycles: 95° for 15 seconds

60° 1 minute

the TaqMan probes: Assays-on-Demand™ (probes TaqMan® MGB labeled with dye FAM™)

- was used to amplify the endogenous control, GAPDH (concentration of probe 100 nm, the concentration of forward and reverse primer, 200 nm) to standardize the amount of sample RNA (cDNA), added to each reaction.

Relative quantification was performed using the calibration curve method. For quantitative evaluation, normalized to the endogenous control, calibration curves were obtained for the target and endogenous control. For each experimental sample was determined by the number of target and endogenous control for the corresponding calibration curve. Then the number of the target divided by the number of endogenous control to obtain a normalized target value. One of the experimental samples served as a calibrator, or 1x sample. Then each of the normalized target values were divided by the normalized desired value for the calibrator to obtain the relative levels of expression.

FACS/Flow cytometry Protocol (staining 2° antibody)

All incubation and centrifugation was carried out at 4°C and the tubes were kept in ice, if not in the refrigerator.

1. To determine the type of tubes by definition is of the used cell lines, interested antibodies and any special conditions or treatments.

a. controls.

i. unpainted, 2° antibody, and adjustment, if the fluorochromes have overlapping spectra of radiation.

b. Example:

TubeCell lineTime (min)1°antibody2°antibody
1for example, COLO-2050--
2for example, COLO-2050-anti-mouse-FITC
3for example, COLO-2050anti-sialyl-Lewis Aanti-mouse-FITC
4for example, COLO-2050anti-CD15s (sialyl-Lewis X)anti-mouse-FITC

2. Label tubes for FACS.

a. BD Falcon 12×75 mm polystyrene round-bottom, catalog #: 352052

3. To prepare the cell is for painting.

a. Handle attached cells accurate or trypsin.

i. Innovative Cell Technologies Inc., accutane.

ii. Gibco, Trypsin. Catalog #: 25200-106.

b. Continue with the remaining stage, when the cells are in suspension.

4. Transfer an aliquot of the cells in 15 ml or 50 ml conical tube.

5. Centrifuge cells 5 min, 1200 rpm, 4°C.

6. Take the supernatant.

7. To resuspending cells in 5 ml of buffer for FACS.

8. Centrifuge cells 5 min, 1200 rpm, 4°C.

9. Take the supernatant.

10. To resuspending cells in blocking buffer.

a. To determine the amount of blocking buffer:

i. The number of tubes on the cell line/treatment X 100 ál of blocking buffer per tube.

ii. Requires 1×106cells in 100 ál of blocking buffer.

11. Transfer an aliquot of 100 ál of cells into a suitable test tube.

a. Assume a predefined type tubes.

12. Add 1° antibody in appropriate tube.

a. Lewis A:

i. Use 10 µl of the stock solution of 0.2 µg/µl per tube.

1. The final concentration is 2 mg.

ii. Chemicon: anti-sialyl-Lewis A. Catalog #: MAB2095.

b. Lewis X:

i. Use 5 ál of the stock solution of 0.5 µg/µl per tube.

1. The final concentration is 2.5 mg.

ii. BD Pharmingen: CD15s (sialyl-Lewis X). Catalog #: 551344.

13. Incubate for 30 mi is at 4°C.

14. Add 1 ml of buffer for FACS in each tube.

15. Centrifuge cells 5 min, 1200 rpm, 4°C.

16. Select the supernatant

17. Gently shake the sediment in the tubes using a tripod.

a. Using a tripod is to Hold the tubes on the surface of the tripod for tubes 12×75 mm

18. Repeat stage 14-17.

19. Add 100 ál of blocking buffer to each tube.

20. Add 2° antibody in appropriate tube.

a. Use 10 ál per tube.

b. Jackson, antibodies goat antibodies against mouse FITC. Catalog #: 115-096-068.

21. Incubate for 30 min at 4°C.

22. Repeat stage 14-17 twice.

23. To resuspending cells in the buffer for FACS/PI.

a. To determine the necessary volume:

i. Requires 1 ml per tube.

ii. PI = 1 µl per 1 ml of the buffer.

b. Molecular Probes, iodide of propecia. Catalog #: P3566.

24. Place the tubes in a container with ice or ice tripod for tubes.

25. Cover with aluminum foil and transported to the laboratory for FACS for data collection and analysis of samples by a qualified operator.

5% blocking buffer:

1. FBS under 5% of the total.

2. The buffer for FACS.

3. Filter the solution through a 0.2 μm filter.

The buffer for FACS:

1. 980 ml of PBS.

2. 8 ml of 0.25 M EDTA.

3. 20 ml of FBS.

4. Filter the solution through a 0.2 μm filter.

Immunohistochemical procedure: sialyl-Lewis A/u>

Antibody: sialyl-Lewis A AB-1

Clone: 121SLE

Supplier of: NeoMarkers

Catalogue number MS-279-P

Species Ig: mouse

The IHC method: paraffin

Pre-treatment: no

Performing IHC: a system for automatic staining Autostainer

The isotype: mouse IgM

Types for procedure: human

The IgG concentration: 200 μg/ml

The usual procedure

To deparaffinizing and hydrate to distilled water.

To block endogenous Biotin blocking system Vector Avidin Biotin.

Rinse TBS: 2 shifts, 5 minutes each.

Block with 10% normal horse serum for 30 minutes at room temperature.

Incubate sections with mouse monoclonal antibody to sialyl-Lewis A, diluted to 5 μg/ml of 10% normal horse serum for 60 minutes at room temperature.

Use mouse antibodies of isotype IgM, diluted to 5 μg/ml of 10% normal horse serum as a negative control.

Rinse TBS: 2 shifts, 5 minutes each.

Incubate sections with biotinylated antibody horses antibodies against mouse; diluted 1:200 in 10% normal horse serum, for 30 minutes at room temperature.

Rinse TBS: 2 shifts, 5 minutes each.

Incubate sections with diluted system Vector ABC Elite 30 minutes at room temperature.

Rinse TBS: 2 shifts, 5 minutes each.

And sebirouth sections with reinforced metal DAB from Pierce 5 minutes.

Rinse in running tap water for 5 minutes.

To carry out contrast staining with Mayer hematoxylin for 1 minute.

Rinse in running tap water for 5 minutes.

Podsinee hematoxylin podsinivajut reagent Richard-Allan 1 minute.

Rinse in running tap water for 2 minutes.

To degidratiruth, clean, and put in synthetic enclosing environment.

Immunohistochemical procedure: sialyl-Lewis X

Antibody: mouse anti-sialyl-Lewis X

Clone: KM93

Supplier of: Chemicon

Catalogue No. MAB2096

Species Ig: mouse

The IHC method: paraffin

Pre-processing: demeterova epitopes DAKO Target Retrieval

Performing IHC: a system for automatic staining Autostainer

The isotype: mouse IgM

Types for procedure: human

The IgG concentration: 100 μg/ml

The usual procedure

To deparaffinizing and hydrate to distilled water.

To block the activity of endogenous peroxidase blocking solution KPL - dilute the concentrate 1:10 in dH2O, 4 minutes at room temperature;

Rinse in distilled water for 5 minutes.

Incubate the solution for damascenone epitopes from DAKO (S1700), preheated to 99 ° C for 20 minutes in a boiling water bath. Remove from the boiling bath and allow to cool 20 minutes.

Block endo is built Biotin blocking system Vector Avidin Biotin.

Block with 10% normal horse serum for 30 minutes at room temperature.

Incubate sections with mouse monoclonal antibody to sialyl-Lewis X, diluted to 5 μg/ml of 10% normal horse serum for 60 minutes at room temperature.

Use mouse antibodies of isotype IgM, diluted to 5 μg/ml in 10% normal horse serum as a negative control.

Rinse TBS: 2 shifts, 5 minutes each.

Incubate sections with biotinylated antibody horses against mouse antibodies from Vector; diluted 1:200 in 10% normal horse serum, for 30 minutes at room temperature.

Rinse TBS: 2 shifts, 5 minutes each.

Incubate sections with diluted system Vector ABC Elite 30 minutes at room temperature.

Rinse TBS: 2 shifts, 5 minutes each.

Incubate sections with reinforced metal DAB from Pierce 5 minutes.

Rinse in running tap water for 5 minutes.

To carry out contrast staining with Mayer hematoxylin for 1 minute.

Rinse in running tap water for 5 minutes.

Podsinee hematoxylin podsinivajut reagent Richard-Allan 1 minute.

Rinse in running tap water for 2 minutes.

To degidratiruth, clean, and put in synthetic enclosing environment.

Experimental results

The experiments were conducted with the use the of materials and methods described above. The results of these experiments are illustrated in Fig.6-13, as discussed below.

Figure 6 shows the summary table of data obtained during the analysis of 28 lines of cancer cells, colon, or colorectal, sensitivity or resistance to apoptotic activity of Apo2L (a+0.5% fetal bovine serum "FBS" or 10% FBS) or monoclonal antibody to DR5 mab", crosslinking "XL" or separatistskogo (a+0.5% fetal bovine serum "FBS" or 10% FBS) and the expression of FUT 3, FUT 6, sialyl-Lewis A and sialyl-Lewis X.

7 shows the comparison of sensitivity of different lines of cancer cells, colon, or colorectal, for the antibody to DR5 and expression FUT 3 (as measured by quantitative PCR).

On Fig shows the comparison of sensitivity of different lines of cancer cells, colon, or colorectal, sensitivity or resistance to the antibody to DR5 (plus cross-linking agent) and expression sialyl-Lewis X or A, as determined by FACS.

On figa shows the rank correlation coefficient of Spermine in the analysis of the sensitivity or resistance of different lines of cancer cells, colon, or colorectal, and correlation with expression of FUT3.

On FIGU shows the results of the exact test of Fisher for the sensitivity analysis ("feelings.") or sustainability ("the mustache is.") various lines of cancer cells, from the colon, or colorectal, and statistical significance between the expression of FUT 3 and sialyl-Lewis A/X and the sensitivity of the respective lines of the cells to the apoptotic activity of the antibody to DR5.

Figure 10 shows the comparison of different lines of cancer cells, colon, or colorectal, on the expression of receptors DcRl or DcR2 (as determined by quantitative PCR) and status (sensitive or resistant) of specific cell lines in relation to Apo2L or the antibody to DR5.

Figure 11 shows the comparison of different lines of cancer cells, colon, or colorectal, on the expression of receptors DcRl or DcR2 (as determined by FACS) and status (sensitive or resistant) of specific cell lines in relation to Apo2L or the antibody to DR5.

On Fig shows immunohistochemical staining sialyl-Lewis A and X in four lines of colorectal cancer cells, CaCo2 (Colo 2), 1417 SW, DLD-1 and Colo 205, and its correlation with expression sialyl-Lewis A and X, as measured by FACS, and its correlation with sensitivity to Apo2L/TRAIL. For lines of colorectal cancer cells Colo 2 and SW1417 shows no staining, weak staining, respectively, or when they were FACS respectively negative or weakly positive for antigens sialyl-Lewis and was resistant to Apo2L/TRAIL. For lines of colorectal cancer cells DLD-1 and Colo 205 shows the mind of the military and strong staining, respectively, when they were FACS, respectively, moderately and strongly positive for antigens sialyl-Lewis and was sensitive to Apo2L/TRAIL.

On Fig shows the aggregated results of IHC experiments showing the expression of sialyl-Lewis A and X in the tissue samples of normal mucosa of the colon, normal liver tissue, primary colon cancer and metastatic cancer of the colon. Tissue samples of normal colon and primary cancer of the colon, ordered on the microchip to the tissues tested in IHC experiment, while tissue samples of normal liver and metastases of colon cancer was on a separate glass. The distribution of expression sialyl-Lewis A and X and the intensity of immunohistochemical staining was increased from normal tissue of the colon to the primary colon cancer to metastasis of colon cancer. Normal liver cells were not stained for either of sialyl-Lewis A or X.

1. A method of predicting the sensitivity of a tissue sample or cells of a malignant tumor of a mammal to Apo2L/TRAIL, which includes stages:
a sample of tissue or cells of a malignant tumor of a mammal;
the research sample of tissue or cells of a malignant tumor for detecting expression of one or more biomarkers selected from the group of fu is oiltransfer 3, fucosyltransferase 6, antigen(antigens) sialyl-Lewis a and/or X, where the expression of one or more of these biomarkers is an indication that the sample of tissue or cells of a malignant tumor is
sensitive to the apoptosis-inducing activity of Apo2L/TRAIL.

2. The method according to claim 1, where the specified expression of one or more biomarkers examined by detection of mRNA expression fucosyltransferase 3 or fucosyltransferase 6.

3. The method according to claim 1, where the specified expression of one or more biomarkers examined by immunohistochemistry for the detection of expression of the antigen(antigens) sialyl-Lewis a and/or X.

4. The method according to claim 1, further comprising a stage of study the expression of receptors DR4, DR5, DcRl or DcR2 in a specified sample of tissue or cells.

5. The method according to claim 1, where the specified malignant tumors are malignant cells or tissue of the colon, colorectal, gastric, intestinal, or pancreas.

6. Method of inducing apoptosis in a tissue sample or cells of a malignant tumor of a mammal, comprising the stage of:
a sample of tissue or cells of a malignant tumor of a mammal;
the research sample of tissue or cells of a malignant tumor for detecting expression of one or more biomarkers selected the C group of fucosyltransferase 3, fucosyltransferase 6, antigen(antigens) sialyl-Lewis a and/or X, and
after detection of expression of one or more of these biomarkers of exposure at a specified sample of tissue or cells of a malignant tumor an effective amount of Apo2L/TRAIL.

7. The method according to claim 6, where the specified expression of one or more biomarkers examined by testing the expression of mRNA fucosyltransferase 3 or fucosyltransferase 6.

8. The method according to claim 6, where the specified expression of one or more biomarkers examined by immunohistochemistry for the detection of expression of the antigen(antigens) sialyl-Lewis a and/or X.

9. The method according to claim 6, further comprising a stage of study the expression of receptors DR4, DR5, DcRl or DcR2 in a specified sample of tissue or cells.

10. The method according to claim 6, where these malignant tumors are malignant cells or tissue of the colon, colorectal, gastric, intestinal, or pancreas.

11. The method according to claim 6, where these cells influence the effective amount of the polypeptide Apo2L/TRAIL containing amino acids 114-281 figure 1 (SEQ ID NO:1).

12. A method of treating a malignant tumor in a mammal, comprising the stage of:
a sample of tissue or cells from the mammal;
the research sample of tissue or cells for detection of expression of one who or several biomarkers, selected from the group of fucosyltransferase 3, fucosyltransferase 6, antigen(antigens) sialyl-Lewis a and/or X, and
after detection of expression of one or more of these biomarkers
the introduction of the specified mammal an effective amount of Apo2L/TRAIL.

13. The method according to item 12, where the specified expression of one or more biomarkers examined by detection of mRNA expression fucosyltransferase 3 or fucosyltransferase 6.

14. The method according to item 12, where the specified expression of one or more biomarkers examined by immunohistochemistry for the detection of expression of the antigen(antigens) sialyl-Lewis a and/or X.

15. The method according to item 12, further comprising a stage of study the expression of receptors DR4, DR5, DcR1 or DcR2 in a specified tissue or cell.

16. The method according to item 12, where these cells or tissue malignant tumors contain cancer cells or tissue of the colon, colorectal, gastric, intestinal, or pancreas.

17. The method according to item 12, where the specified mammal is administered an effective amount of the polypeptide Apo2L/TRAIL containing amino acids 114-281 figure 1 (SEQ ID NO:1).

18. The method according to p. 12, where the specified mammal also gets a chemotherapeutic agent(assets) or radiotherapy.

19. The method according to item 12, where the specified mammal also introduce cytokine, cytotoxic agent is whether the growth inhibitory agent.

20. The method according to claim 6, where the specified polypeptide Apo2L/TRAIL attached to a molecule of polyethylene glycol.

21. The method according to item 12, where the specified polypeptide Apo2L/TRAIL attached to a molecule of polyethylene glycol.

22. The method according to claim 5, where these malignant tumors are malignant cells in the colon or colorectal.

23. The method according to claim 1, where the specified Apo2L/TRAIL is a polypeptide containing amino acids 41-281 figure 1 (SEQ ID NO:1), or its biologically active fragment.

24. The method according to item 23, where the specified Apo2L/TRAIL is a polypeptide containing amino acids 114-281 figure 1 (SEQ ID NO:1).

25. The method according to claim 10, where these malignant tumors are malignant cells in the colon or colorectal.

26. The method according to 17, where the specified polypeptide Apo2L/TRAIL consists of amino acids 114-281 figure 1 (SEQ ID NO:1).

27. A method of predicting the sensitivity of malignant tumor cells of a mammal from the colon or colorectal to Apo2L/TRAIL, which includes stages:
receipt of malignant tumor cells of a mammal from the colon or colorectal;
studies of malignant tumor cells for detection of expression of one or more biomarkers selected from the group of fucosyltransferase 3, fucosyltransferase 6, antigen(antigens) sialyl-Lewis a and/and and X, where the expression of one or more of these biomarkers is an indication that these cells are malignant tumors are sensitive to apoptosis-inducing activity of Apo2L/TRAIL.

28. Method of induction of apoptosis in malignant tumor cells
mammal of the colon, or colorectal, including stage:
receipt of malignant tumor cells of the mammal, from the colon or colorectal;
studies of malignant tumor cells for detection of expression of one or more biomarkers selected from the group of fucosyltransferase 3, fucosyltransferase 6, antigen(antigens) sialyl-Lewis a and/or X, and
after detection of expression of one or more of these biomarkers of effect on these cells of a malignant tumor effective amount of the polypeptide Apo2L/TRAIL.

29. The method according to p, where the specified polypeptide Apo2L/TRAIL contains amino acids 41-281 figure 1 (SEQ ID NO:1) or a fragment having apoptotic activity.

30. The method according to clause 29, where the specified polypeptide Apo2L/TRAIL attached to a molecule of polyethylene glycol.

31. The method according to clause 29, where the specified polypeptide Apo2L/TRAIL contains amino acids 114-281 of Fig. 1 (SEQ ID NO:1).

32. The method of treatment of mammalian malignant tumors of the colon, or colorectal, including stage:
getting about is Azza of malignant tumor cells specified mammal from the colon or colorectal;
the research sample of malignant tumor cells for detection of expression of one or more biomarkers selected from the group of fucosyltransferase 3, fucosyltransferase 6, antigen(antigens) sialyl-Lewis a and/or X, and
after detection of expression of one or more of these biomarkers introduction to the specified mammal an effective amount of Apo2L/TRAIL.

33. The method according to p, where the specified polypeptide Apo2L/TRAIL contains amino acids 41-281 figure 1 (SEQ ID NO:1) or a fragment having apoptotic activity.

34. The method according to p, where the specified polypeptide Apo2L/TRAIL attached to a molecule of polyethylene glycol.

35. The method according to p, where the specified polypeptide Apo2L/TRAIL contains amino acids 114-281 figure 1 (SEQ ID NO:1).



 

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