Analyses and methods of biomarker application

FIELD: medicine.

SUBSTANCE: detecting expression of GalNac-T14 molecules enables to predict sensitivity or indicates that a tissue or cell sample is sensitive to apoptosis inducing agents, such as DR4 or DR5 agonist antibodies. The information obtained by the analysis aimed at detecting GalNac-T14 expression in the mammal's tissue or cell sample can provide a hospital doctor with data which can be used for prescribing an optimal treatment schedule for patients suffering such diseases as pancreas cancer, lymphoma, non-small cell carcinoma of lung, colon cancer, rectal cancer, melanoma or chondrosarcoma.

EFFECT: expanded scope of the compounds.

16 cl, 53 dwg, 14 tbl

 

RELATED APPLICATIONS

This application claims the priority of provisional patent application U.S. number 60/708677, filed August 16, 2005, and provisional patent application U.S. number 60/808076, filed may 24, 2006.

The technical FIELD TO WHICH the INVENTION RELATES.

The described invention relates to methods and analytical techniques to identify biomarkers that predict sensitivity of mammalian cells to Apo2L/TRAIL and/or an agonistic antibody receptors (cell) death. In more detail the invention relates to methods and analytical techniques that detect molecules associated with the family of proteins GalNac-T, which allows to predict the sensitivity of cancerous mammalian cells to Apo2L/TRAIL and/or an agonistic antibody receptors (cell) death, such as agonistic antibodies to DR4 or DR5.

The prior art INVENTIONS

In this field of technology identified a variety of ligands and receptors belonging to the superfamily of factor tumor necrosis (TNF). These ligands include factor-alpha tumor necrosis ("TNF-alpha), beta-factor tumor necrosis ("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 (also referred to as Fas ligand or ligand CD9), ligand Apo-2 (also called Apo2L or TRAIL), the ligand, Apo-3 (also referred to as TWEAK), APRIL, OPG ligand (also called RANK ligand, ODF or TRANCE), and TALL-1 (also referred to 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, page 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 (publication dated January 16, 1997); WO 97/25428 (publication dated 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 (publication dated July 2, 1998); WO 98/46751 (publication dated 22 October 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 induce different types of biological activity, contacting located on the cell surface "death receptors" and activating caspase or enzymes, when adamie in the execution of the metabolic pathway of cell death or apoptosis (Salvesen et al., Cell, 91:443-446 (1997)). Among the members of the superfamily of TNF receptors identified to date, you must specify TNFR1, TNFR2, TACI, GITR, CD27, OX-40, CD30, CD40, HVEM, Fas (also called Apo-1 or CD95), DR4 (also known as TRAIL-R1), DR5 (also referred to as Apo-2 or TRAIL-R2), DcR1, DcR2, osteoprotegerin (OPG), RANK and Apo-3 (also known as DR3 or TRAMP) (see, for example, Ashkenazi,Nature Reviews, 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, page 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., 82:3127-3131 (1990); EP 417563 (publication dated 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 members of the family of TNF receptors have a common characteristic structure of the receptor cell surface the displacement, including extracellular, transmembrane and intracellular region, but some of them are found in nature in the form of soluble proteins that have lost the transmembrane and intracellular domain. The extracellular part of the characteristic of the TNF receptors contains a picture of the repeating amino acid sequence with multiple domains, rich in cysteine (CRD), starting with NH2-end.

The ligand, known as Apo-2L) or TRAIL, a few years ago was identified as a member of a family of cytokines TNF (see, e.g., Wiley et al., Immunity, 3:673-682 (1995); Pitti et al., J. Biol. Chem., 271:12697-12690 (1996); WO 97/01633; WO 97/25428; U.S. patent 5763223, issued June 9, 1998, U.S. patent 6284236, issued September 4, 2001). Full length natural sequence of the polypeptide of human Apo2L/TRAIL is a 281 amino acid residue, of which formed a transmembrane protein type II. Some cells can produce a natural soluble form of the polypeptide that exists due to enzymatic cleavage of its extracellular region (Mariani et al., J. Biol. Chem., 137:221-229 (1997)). Crystallographic studies of soluble Apo2L/TRAIL reflect 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, it was found that, in the best of the e from other members of the TNF family, protein Apo2L/TRAIL has a unique structural feature, namely, that three cysteine residue (230 th position of each subunit of homotrimer) jointly coordinate the zinc atom and the binding of zinc is important for the stability and biological activity of the trimer (Hymowitz et al., above; Bodmer et al., J. Biol. Chem., 275:20632-20637 (2000)).

In the literature published information that Apo2L/TRAIL may play a role in modulating 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. Exp. Med., 189:1343-1353 (1999); Song et al., J. Exp. Med., 191:1095-1103 (2000)].

It was also reported that a soluble form of Apo2L/TRAIL induce apoptosis in many types of 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, issued February 29, 2000; U.S. patent 6746668, issued 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)). In addition, in vivo studies in models of tumors in mice suggest that Apo2L/RAIL alone or in combination with chemotherapy or radiation therapy may run a significant antitumor effects (see, for example, 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); PCT publication US/00/15512; publication PCT US/01/23691. Unlike many types of cancer cells, most normal cells types in the body are resistant to induction of apoptosis specific recombinant forms of Apo2L/TRAIL (Ashkenazi et al., above; Walzcak et al., above). Jo et al. reported that the labeled polyhistidine soluble form of Apo2L/TRAIL induces apoptosis in vitro in normal dedicated human hepatocytes, but not in hepatocytes another biological origin (Jo et al., Nature Med., 6:564-567 (2000); see also Nagata, Nature Med., 6:502-503 (2000)). I think that some of recombinant drugs Apo2L/TRAIL can vary in terms of biochemical properties and biological activity relative to the normal and pathologically altered cells depending, for example, from the presence or absence of molecules tags on the content of zinc and the percentage of 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)).

It was found that Apo2L/TRAIL may be contacted at least five different receptors. At least two of the receptors that communicate with Apo2L/TRAIL, contain a functional cytoplasmic domain of cell death. One of the receptors is called "DR4" (alternative what he called 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 2003/0036168, published February 20, 2003; US 6433147, issued August 13, 2002; US 6461823, issued October 8, 2002, and US 6342383, issued January 29, 2002).

Another receptor for Apo2L/TRAIL is called DR5 (alternative he called Apo-2; TRAIL-R or TRAIL-R2, TR6, Tango-63, hAP8, 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 published on September 24, 1998; Screaton et al.,Curr. Biol., 2:693-696 (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; EP870827, 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 March 11, 1999; WO 03/042367, published on may 22, 2003; WO 02/097033 published 5 December 2002; WO 03/038043, published may 8, 2003; US 2002/0072091, published August 13, 2002; US 2002/0098550, published 7 December 2001; US 6313269, issued December 6, 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 2004/0141952, published July 22, 2004; US 2005/0129699 published 16 June 2005; US 2005/0129616 published 16 June 2005; US 6342369, issued in February 2002; US 6569642, issued may 27, 2003, US 6072047, issued June 6, 2000, US 6642358, issued November 4, 2003; US 6743625, issued June 1, 2004). It was reported that, like DR4, DR5 it contains a cytoplasmic domain of cell death and is able to transmit a signal apoptosis upon binding with the ligand (or binding to this molecule as an agonistic antibody that mimics the activity of the ligand). Crystal structure of the complex formed between the Apo-2L/TRAIL and DR5 described in Hymowitz et al.,Molecular Cell,4:563-571 (1999).

Upon binding with the ligand as DR4 and DR5 can independently run apoptosis, attracting and activating factor in the initiation of apoptosis, caspase-8, through adapting molecule containing the domain of cell death and is known as FADD/Mort1 [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 information that Apo2L/TRAIL also binds to receptors known as DcR1, DcR2 and OPG, which, according to the researchers, primarily act as inhibitors, but not transmitters of signals (see, for example, DCR1 (also called 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); and Mongkolspaya 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)], and OPG [Simonet et al.,above]). In contrast to the DR4 and DR5 receptors DcR1 and DcR2 not transmit signals apoptosis.

The literature describes some antibodies bind to the receptors DR4 and/or DR5. For example, anti-DR4 antibodies directed to the receptor DR4 and exhibiting agonistic or apoptotic activity in some mammalian cells, described in documents 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 on may 22, 2003; WO 03/038043, published may 8, 2003; WO 03/037913, published may 8, 2003; US 2003/0073187 published 17 April 2003; US 2003/0108516, published June 12, 2003, Similarly described some anti-DR5 antibodies, see, for example, 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., "Antibody to DR5 (TRAIL receptor-2) inhibits the growth obtained from diseased cells of the gastro-intestinal tumors in SCID mice", book of abstracts, 2nd international Congress on monoclonal antibodies in cancer, 2 August - September 1, 2002, Banff, Alberta, Canada; WO 03/038043, published may 8, 2003; WO 03/037913, published may 8, 2003; US 2003/0180296, published on 25 September 2003 In addition, the literature describes some antibodies exhibiting cross-reactivity with receptors DR4 and DR5 receptors (see, for example, U.S. patent 6252050, issued June 26, 2001).

The INVENTION

The open invention relates to methods and analytical approaches to the study of the expression of one or more biomarkers in samples of tissue or mammalian cells, and the expression of one or more of these biomarkers is predictive indicator of the sensitivity of these samples of cells and tissues to such agents as Apo2L/TRAIL or agonistic anti-DR5 antibodies. In various embodiments of the invention these methods and analytical approaches allow us to study the expression of molecules in the family of proteins GalNac-T, in particular, GalNAc-T14 or GalNAc-T3.

As discussed above, the majority of healthy cells in the body are resistant to induction of apoptosis specific recombinant forms of Apo2L/TRAIL (Ashkenazi et al., above; Walzcak et al., above; Walzcak et al., above). Were also made observations about the fact that some populations of human cells affected by disease (for example, some populations of cancer cells), touchily 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, studying a sample of tissue or cells on the expression of selected biomarkers using this analysis, it is convenient and efficient way to obtain useful information for assessment/selection of appropriate and effective therapies. For example, information obtained from analysis to identify the expression of GalNac-T14 in a sample of tissue or cells of a mammal, can give the doctor valuable information that you can use when choosing the optimal regimen (with the use of Apo2L/TRAIL or agonistic antibodies, receptors, cell death) for patients suffering from cancer or a disease associated with impaired immunity, such as autoimmune disease.

The invention relates to methods for predicting the sensitivity of samples of tissue or mammalian cells (e.g. cancer cells to Apo2L/TRAIL or agonistic antibody receptor cell death. In some embodiments of the invention these methods include obtaining a sample of tissue or cells of a mammal and the study of this sample of tissue or cells on the expression of GalNac-T14. These methods can be implemented in a variety of formats analysis, including tests to detect expression of mRNA and/or protein analyses on enzymatic activity and other types of analysis is s, discussion in the present description. Determination of expression of GalNac-T14 in the above tissues or cells has implications for predictions of the sensitivity of these tissues or cells by inducing apoptosis activity of Apo2L/TRAIL and/or antibodies to receptor cell death. In some optional embodiments of the invention the tissue or cells can also be examined on the expression of 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, comprising the stage of obtaining a sample of tissue or cells, the study of tissues or cells on the expression of GalNac-T14, and, when detecting that a specific tissue or cell expresses GalNac-T14, stage effects on the sample an effective amount of Apo2L/TRAIL or agonistic antibodies of cell death receptors. These stages are ways to study the expression of GalNac-T14 can be done in a variety of formats analysis, including tests to detect expression of mRNA and/or protein analyses on enzymatic activity and other types of analyses discussed in the present description. In some optional embodiments of the invention these methods also include the study of a sample of tissue or cells for the expression of the receptors DR4, DR5, DcR1 or DcR2. Optionally, the tissue sample and the cell contains cancerous tissue or cells. Optionally, a tissue sample or cells contains cells non-small cell lung cancer cells, pancreatic cancer, breast cancer cells or cells of non-Hodgkin's lymphoma.

Another series of methods according to the invention includes methods of treating diseases in a mammal, for example, cancer or disease associated with impaired immunity, and these methods include the steps of obtaining a tissue sample or cells, the study of tissues or cells on the expression of GalNac-T14, and, when detecting that a specific tissue or cell expresses GalNac-T14, the introduction of the specified mammal an effective amount of Apo2L/TRAIL or agonistic antibodies of cell death receptors. These stages are ways to study the expression of one or more biomarkers can be done in a variety of formats analysis, including tests to detect expression of mRNA and/or protein analyses on enzymatic activity and other types of analyses discussed in the present description. In some optional embodiments of the invention these methods also include the study of a sample of tissue or cells for the expression of the receptors DR4, DR5, DcR1 or DcR2. Optionally, the methods include treatment of a cancer of a mammal. Optional methods, in addition to conducting an effective amount of Apo2L/TRAIL and/or agonistic anti-Christ. ate receptors of cell death includes the introduction of a specified mammal a chemotherapeutic drug (funds) or radiation therapy.

In other embodiments of the invention, the above methods may include the study of tissues or cells of mammals in the expression of other molecules GalNac-T, for example, GalNac-T3.

Other embodiments of the invention for example can be illustrated in the following patent 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 in order to detect the expression of GalNac-T14, and the expression specified GalNac-T14 allows to predict that the sample of tissue or cells susceptible to inducing apoptosis activity of Apo2L/TRAIL.

2. The method according to claim 1, in which the specified expression of GalNac-T14 investigating, determining the expression of mRNA of GalNac-T14.

3. The method according to claim 1, in which the specified expression of GalNac-T14 explore methods of immunohistochemistry.

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, in which a sample of tissue or cells contains cancerous tissue or cells.

6. The method according to claim 5, in which these cancer CL the weave or fabric represent material from cancers of the pancreas, lymphoma or non-small cell lung cancer.

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 in order to detect the expression of GalNac-T14 and, in case such expression, subsequent exposure to a specified sample of tissue or cells an effective amount of Apo2L/TRAIL.

8. The method according to claim 7, in which the specified expression of GalNac-T14 investigating, determining the expression of mRNA of GalNac-T14.

9. The method according to claim 7, in which the specified expression of GalNac-T14 explore methods of immunohistochemistry.

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, wherein said tissue sample or cells contains cancerous tissue or cells.

12. The method according to claim 11, in which these cancer cells or tissue are the material of cancerous tumors of the pancreas, lymphoma or non-small cell lung cancer.

13. The method according to claim 7, in which these cells are exposed to an effective amount of the polypeptide Apo2L/TRAIL containing amino acids 114-281 in accordance with figure 1.

14. A method of treating disease in a mammal, for example, is associated with impaired immunity or cancer, VK is uchumi stages:

a sample of tissue or cells from the mammal;

the research sample of tissue or cells in order to detect the expression of GalNac-T14 and, in case such expression, the introduction of the specified mammal an effective amount of Apo2L/TRAIL.

15. The method according to 14, in which the specified expression of GalNac-T14 investigating, determining the expression of mRNA of GalNac-T14.

16. The method according to 14, in which the specified expression of GalNac-T14 explore methods of immunohistochemistry.

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 cells.

18. The method according to 14, in which a sample of tissue or cells contains cancerous tissue or cells.

19. The method according to p in which these cancer cells or tissue are the material of cancerous tumors of the pancreas, lymphoma or non-small cell lung cancer.

20. The method according to 14, in which the specified mammal is administered an effective amount of the polypeptide Apo2L/TRAIL containing amino acids 114-281 in accordance with figure 1.

21. The method according to 14, in which the specified mammal also introduce chemotherapeutic agent (funds) or treated with radiation therapy.

22. The method according to 14, in which the specified mammal also introduce a cytokine, a cytotoxic agent or an inhibitor of cell growth.

23. The method according to the .7, wherein said polypeptide Apo2L/TRAIL connected with one molecule of polyethylene glycol.

24. The method according to 14, wherein said polypeptide Apo2L/TRAIL connected with one molecule of polyethylene glycol.

25. A method of predicting the sensitivity of a tissue sample or cells of the mammal antibody receptor cell death, which includes stages:

a sample of tissue or cells of a mammal;

the research sample of tissue or cells in order to detect the expression of GalNac-T14, and the expression specified GalNac-T14 allows to predict that the sample of tissue or cells susceptible to inducing apoptosis activity of the antibody receptor cell death.

26. The method according A.25, which indicated the expression of GalNac-T14 investigating, determining the expression of mRNA of GalNac-T14.

27. The method according A.25, which indicated the expression of GalNac-T14 explore methods of immunohistochemistry.

28. The method according A.25 additionally includes the stage of study the expression of receptors DR4, DR5, DcR1 or DcR2 in a specified sample of tissue or cells.

29. The method according A.25, in which a sample of tissue or cells contains cancerous tissue or cells.

30. The method according to clause 29, in which these cancer cells or tissue are the material of cancerous tumors of the pancreas, lymphoma or non-small cell lung cancer.

31. The method according A.25 in which these antibodies Retz is Perov cell death are agonistic anti-DR4 and anti-DR5 antibodies.

32. 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 in order to detect the expression of GalNac-T14 and, in case such expression, subsequent exposure to a specified sample of tissue or cells an effective amount of an antibody receptor cell death.

33. The method according to p, which indicated the expression of GalNac-T14 investigating, determining the expression of mRNA of GalNac-T14.

34. The method according to p, which indicated the expression of GalNac-T14 explore methods of immunohistochemistry.

35. The method according to p additionally includes the stage of study the expression of receptors DR4, DR5, DcR1 or DcR2 in a specified sample of tissue or cells.

36. The method according to p, wherein said tissue sample or cells contains cancerous tissue or cells.

37. The method according to p in which these cancer cells are cancer cells or tissue of the pancreas, lymphoma or non-small cell lung cancer.

38. The method according to p in which these cells are exposed to an effective amount of agonistic antibodies to DR4 or DR5.

39. The method according to § 38, in which these cells are exposed to an effective amount of agonistic DR5 antibodies that bind to the receptor DR5, presented at IgA.

40. A method of treating disease in a mammal, for example, is associated with impaired immunity or cancer, including stage:

a sample of tissue or cells from the mammal;

the research sample of tissue or cells in order to detect the expression of GalNac-T14 and, in case such expression, the subsequent introduction of the specified mammal an effective amount of an antibody receptor cell death.

41. The method according to p, which indicated the expression of GalNac-T14 investigating, determining the expression of mRNA of GalNac-T14.

42. The method according to p, which indicated the expression of GalNac-T14 explore methods of immunohistochemistry.

43. The method according to p additionally includes the stage of study the expression of receptors DR4, DR5, DcR1 or DcR2 in a specified tissue or cells.

44. The method according to p, in which a sample of tissue or cells contains cancerous tissue or cells.

45. The method according to item 44, in which these cancer cells or tissue are the material of cancerous tumors of the pancreas, lymphoma or non-small cell lung cancer.

46. The method according to p in which the specified mammal is administered an effective amount of anti-DR4 or anti-DR5 antibodies.

47. The method according to p in which the specified mammal also introduce chemotherapeutic agent (funds) or treated with radiation therapy.

48. The method according to p, inwhich the specified mammal also introduce a cytokine, the cytotoxic agent or inhibitor of cell growth.

BRIEF DESCRIPTION of FIGURES

Figure 1 presents the nucleotide sequence of cDNA ligand Apo-2 (SEQ ID NO:2) and its derived amino acid sequence (SEQ ID NO:1). The symbol "N" in nucleotide position 447 is used to indicate that the nucleotide base may be "T" (thymine) or G (guanine).

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

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

On fig.3D-1, 3D-2 and 3D-3 presents the nucleotide sequence of the cDNA (SEQ ID NO:7) for DcR1 human full length and its derived amino acid sequence (SEQ ID NO:8). The corresponding nucleotide and s is nakikita sequence for human DcR1 (and their specific domains) are also shown and described in WO 98/58062.

On file (3E-1, 3E-2) presents the nucleotide sequence of the cDNA (SEQ ID NO:9) for DcR2 human full length and its derived amino acid sequence (SEQ ID NO:10). The corresponding nucleotide and amino acid sequences for human DcR2 (and their specific domains) are also shown in WO 99/10484.

On figa (4A-1 to 4A-4) presents the nucleotide sequence GalNac-T14 human (SEQ ID NO:11) and its derived amino acid sequence (SEQ ID NO:12). These sequences are also described by Wang et al., BBRC, 300:738-744 (2003).

On FIGU (4B-1 to 4B-4) presents the nucleotide sequence GalNac-T3 human (SEQ ID NO:13) and its derived amino acid sequence (SEQ ID NO:14). These sequences are also described (Bennett et al., J. Biol. Chem., 271:17006-17012 (1996).

Figure 5 presents a summary diagram IC50 data obtained in the analysis of cell lines of non-small cell lung cancer ("NSCLC") on the sensitivity or resistance to apoptotic activity of Apo2L (a+0.5% fetal bovine serum "FBS" or 10% FBS) or monoclonal antibodies DR5 "DR5 ab"cross-linked "XL" or unbound (+0,5% fetal bovine serum "FBS" or 10% FBS) according to the measurement results in MTT assays for cytotoxicity.

Figure 6 presents a summary diagram IC50 data obtained in the analysis of cancer cell lines podgeludognoi cancer susceptibility or the resistance to apoptotic activity of Apo2L (a+0.5% fetal bovine serum "FBS" or 10% FBS) or monoclonal antibodies DR5 "DR5 ab", cross linked "XL" or unbound (+0,5% fetal bovine serum "FBS" or 10% FBS) according to the measurement results in MTT assays for cytotoxicity.

Figure 7 presents a summary diagram IC50 according to the data obtained in the analysis of cancer cell lines of non-Hodgkin's lymphoma ("NHL") on the sensitivity or resistance to apoptotic activity of Apo2L (+10% fetal bovine serum "FBS") or monoclonal antibodies DR5 "DR5 ab"cross-linked "XL" or not connected (+10% fetal bovine serum "FBS") to the measurement results in MTT assays for cytotoxicity.

On Fig presents a comparison of the sensitivity (sen) or sustainability ("RES") of selected lines of cancer cells NSCLC, pancreatic cancer and NHL to DR5 antibodies and correlation with expression of GalNac-T14 in the measurement of mRNA expression GalNac-T14.

Figure 9 presents in the form of a bar chart graph showing the different cell line NSCLC, pancreatic cancer and NHL ranked (in descending order) at the level of mRNA expression GalNac-T14.

On figa-D illustrates the differential expression of specific enzymes of O-glycosylation in the lines of cancer cells sensitive and resistant to Apo2L/TRAIL: (A) cell Viability was measured after incubation with different doses of Apo2L/TRAIL. Figure IC50 for each cell line was calculated as the concentration is the situation of Apo2L/TRAIL, that reduces cell viability by 50%. Each experiment to analyze the viability of the cells was repeated at least three times in the presence of low (0.5%) and high (10%) concentrations of fetal bovine serum. Black, grey or unfilled symbols represent cell lines highly sensitive, moderately sensitive or resistant to Apo2L/TRAIL, respectively. (B) the Level of expression of mRNA ppGalNAcT-14 (a set of probes 219271_at) in cell lines of pancreatic cancer and malignant melanoma. Cell lines are ordered according to the tissue type and sensitivity to Apo2L/TRAIL. Black, grey or unfilled symbols represent cell lines in accordance with a fragment of A. (C) the Level of expression of mRNA Fut-6 (the upper region, the set of probes 211885_x_at) and ppGalNAcT-3 (lower section, a set of probes 203397_s_at) in cell lines of colorectal (colon and rectal) cancer. Cell lines are ordered according to fragment B. P Values in areas B and C based on criteria correlation Fisher sensitivity of cell lines (including high and moderate rates), and the mRNA expression above the cut-off points. (D) the Effect of Apo2L/TRAIL on the proven growth of tumor xenografts. Nude naked mouse with a mutation in the gene nude), bearing GalNAcT-3/Fut-6-positive (left panel) or GalNAcT-3/Fut-6-negative (right panel) tumors that received the empty vector or Apo2L/TRAIL (60 mg/kg/day administered intraperitoneally with 0-th to 4-th day) monitoring of tumor volume (mean±standard deviation, N=10 mice per group).

Figure 11 illustrates the modulation of specific enzymes of O-glycosylation sites, altering the sensitivity to Apo2L/TRAIL. (A) Colo205 Cells were pre-incubated with a universal inhibitor of the enzymes of O-glycosylation benzyl-GalNAc (bGalNAc) and processed Apo2L/TRAIL within 24 hours, after which determined the cell viability (DMSO = control with empty vector). (B) Cells PSN-1 (carcinoma of the pancreas) and Hs294T (melanoma) were transliterowany siRNAs (small interfering RNA) caspase-8 or ppGalNAcT-14 for 48 hours, then incubated with Apo2L/TRAIL for 24 hours after which determined the cell viability. As a control (NTC) were used siRNAs duplexes against non-target sequence (Dharmacon). (C) Cells of colorectal cancer DLD-1 were transliterowany siRNAs ppGalNAcT-3 or Fut-6 and investigated in accordance with paragraph B. (D) HEK293 Cells were cotransfection plasmids encoding indicator genes in combination with ppGalNAcT-14 or vector control. Apoptosis was assessed after 24 hours by staining Annexin V (left panel). Melanoma cells H1569 were transpulmonary a retrovirus that controls the expression of ppGalNAcT-14, or a control retrovirus; the resulting cell pools were treated with Apo2L/TRAIL within 24 hours with subsequent determination of the viability of CL is current (right panel). To verify the expression of ppGalNAcT-14 labeled epitope was used for Western blotting with anti-FLAG antibodies.

On Fig illustrated by (A) analysis of caspase cascade induced by Apo2L/TRAIL. Cells PSN-1 and DLD-1 were transliterowany siRNAs ppGalNAcT-14 or Fut-6, respectively, within 48 hours. Then the cells were treated with Apo2L/TRAIL for 4 or 8 hours, and cell lysates were analyzed by Western blot turns with antibodies specific for caspase-8, Bid, caspase-9, caspase-3 or actin, used as a boot control. (B) Cells PSN-1 were transliterowany siRNAs ppGalNAcT-14 in accordance with paragraph A, then processed Apo2L/TRAIL for 4 hours, after which cell lysates were determined by the enzymatic activity of caspase-3/7. (C) Analysis of Apo2L/TRAIL on the DISC method. Cells PSN-1 were transliterowany siRNAs ppGalNAcT-14 in accordance with paragraph A. Then these cells were added FLAG-Apo2L/TRAIL (1 mg/ml) for 0-60 min, the cells were literally and was thus subjected to anti-FLAG antibodies. Using Western blot turns was determined by DISC-associated FADD, caspase-8, DR4. (D) Cells PSN-1 were transliterowany, processed and subjected to thus by the method DISC in accordance with paragraph C, after which the DISC-associated enzymatic activity of caspase-8 was measured as described previously (Sharp et al.,J. Biol. Chem.,280:19401 (2005)).

On Fig illustrated(A) monosaccharide analysis of recombinant human DR5 (long variant splicing), produced in CHO cells, which was conducted by the method of HPAEC-PAD (anion-exchange chromatography high-resolution pulsed amperometric detection). (B) comparison of sequences of the receptors Apo2L/TRAIL man (human DR5 length of 440 amino acids - form "hDR5L", human DR5 length of 411 amino acids - short form "hDR5S" and hDR4), mouse DR5 (mDR5), human Fas (hFas) and TNFR1 person (hTNFR1). The rectangles indicate the presumed sites of O-glycosylation. (C) Analysis by the method of Western blot turns all cell lysates corresponding to point D. DR5L-5T and DR5S-5T is a construct that contains 5 replacement of threonine by alanine, and DR5L-5T3S and DR5S-5T3S is a construct that contains 5 replacement of threonine by alanine and three replacement of serine by alanine, respectively, in those amino acid residues which, presumably, are the sites of O-glycosylation. (D) HEK293 Cells were cotransfection indicator designs DR5 together with the vector or plasmid ppGalNAcT-14 for 48 hours, after which apoptosis was determined by staining with Annexin V. (E) the Level of expression of mRNA for ppGalNAcT-14 (Affymetrix chip, a set of probes 219271_at) in primary samples of human cancers: skin cancer (SCC = squamous cell carcinoma), lung cancer, pancreatic cancer (Panc), breast cancer, ovarian cancer (Ov), cancer of the endometrium (Endo), bladder cancer (Bla, TCC=perekhodnocletocny arcinoma) and NHL (FL=follicular lymphoma, DLBCL=diffuse large B-cell lymphoma). The average expression in the samples for each class is shown by the grey horizontal stripes. Shown off at the level of 500 and 200 (melanoma) in accordance with data on cell lines from figv.

On Fig illustrated by (a) reducing the mRNA expression ppGalNAcT-14 or ppGalNAcT-3 in cells PSN-1 and DLD-1 after 48-hour siRNAs knockdown assessed in the analysis of TaqMan. (B) Expression of GalNAcT-14 cells PSN-1 is restored by transfection with empty plasmid (Empty), GalNAcT-14 wild-type (GalNAcT-14) or GalNAcT-14 containing silent mutations siRNAs (GalNAcT-14 si(1)Mut) after indirect siGalNAcT-14 (1) knockdown ppGalNAcT-14. (C) Inhibition of ppGalNAcT-3 or Fut-6 through interfering RNA suppresses induced by Apo2L/TRAIL and cell death in cells C170 (colorectal cancer). Experimental procedure corresponds to the item 11C. (Table 1) A) Summary of phenotypes resulting from knockdown of siRNAs. Cell line, in which inhibition of GalNAcT-14 or ppGalNAcT-3 and Fut-6 resulted in protection against Apo2L/TRAIL, marked with an indication of protection of less than (+) or more than 50% (++) testing at least one of the oligonucleotide siRNAs. (0) indicates no protection against Apo2L/TRAIL. (D), (E) After the 48-hour knockdown indicator siRNAs cells were treated with increasing doses of etoposide or staurosporine (STS) for 24 hours and analyzed for life is osobnosti. (F) Pools of cells with retroviral ppGalNAcT-14 (with excessive expression of the PA-TU-8902) and cell lines PL-45 analyzed for cell viability after treatment Apo2L/TRAIL. Analysis by the method of Western blotting with anti-FLAG antibodies indicates that retroviral expression of ppGalNAcT-14 in these cells.

Fig(A). Analysis by the method of Western blotting with the definition induced by Apo2L/TRAIL cascade activation of caspase in Colo205 cells, sensitive to Apo2L/TRAIL, and colorectal cancer cells that are resistant to Apo2L/TRAIL, as well as in cell lines RKO and SW1417. Cells were treated with 1000 ng/ml Apo2L/TRAIL for 8 and 24 hours, and the resulting cell lysates were analyzed using Western blotting with antibodies specific against caspase-8, Bid, caspase-9, caspase-3 and actin as a boot control. (B) Knockdown with limited involvement Fut-6 and activation of caspase-8 in cells DLD-1 by the method of Apo2L/TRAIL DISC. Experimental procedure corresponds to the item 12D. (C) Expression of DR4 and DR5 on the cell surface was measured by FACS analysis on a method in cells that were subjected to knockdown siRNAs with indicator genes.

DETAILED description of the INVENTION

Methods and procedures described herein or referred to as links, in General, is quite clear, and in most cases are applied in the framework of the traditional methodology experts given in the second area, such as, for example, a widely used methodology molecular cloning described by Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of marketed kits and reagents are typically performed in accordance with defined their manufacturers protocols and/or parameters, unless specifically stated otherwise.

Before detailing the invention of methods and analytical techniques, it should be noted that the invention is not limited to the particular methodology, protocols, cell lines, species or genera of animals, structures and described reagents, since they are all, of course, can change. It should also be noted that in this case the terminology is used only to describe specific embodiments of the invention and should not limit the scope of the present invention, which is limited only represented by the patent claims.

It should be noted that when used in this description and in the attached claims grammatical forms of the singular include references to the plural, unless the context clearly requires otherwise. So, for example, reference to "a gene is a practical alteration" means the set of all such alterations, and the reference to "a probe" includes reference to one or more probes and cash equivalents known to the specialist, etc.

All cited publications are included as references to the disclosure of the description of the methods and/or materials in connection with which cited these publications. Cited in the present description publication known from the prior art before the filing date of this application. Nothing in this document should not be construed as admissions that the applicants have no right to date of publication retroactively based on the earliest priority date or the earlier date of invention. Next, the actual date of publication may differ from those presented in the present invention and require independent verification.

I. DEFINITIONS

The terms "Apo2L/TRAIL", "Apo-2L" and "TRAIL" are used in this description to refer to polynucleotide sequence which includes amino acid residues 114-281, inclusive, 95-281, inclusive, residues 92-281, inclusive, residues 91-281, inclusive, residues 41-281 inclusive, residues 15-281, inclusive, or residues 1-281, inclusive, of the amino acid sequence represented in figure 1, as well as biologically active fragments and variants of these sequences with deletions, insertions or substitutions. In one embodiment, the implementation of izobreteny the polypeptide sequence contains residues 114-281, presented in figure 1, and, optionally, consists of residues 114-281 presented in figure 1. Optionally, the polypeptide sequence contains residues 92-281 or residues 91-281, presented in figure 1. Polypeptides Apo-2L can be encoded natural nucleotide sequence represented in figure 1. Optionally, the codon, which is encoded amino acid residue Pro119 (figure 1)can have the structure of "CCT" or "CCG". In other embodiments of the invention are fragments or variants are biologically active and have at least about 80% amino acid sequence identity, more preferably at least about 90% identity, or, 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 is encoded by a nucleotide sequence, which in severe conditions hybridizes with the encoding polynucleotide sequence presented in figure 1. This definition encompasses variants of Apo2L/TRAIL with substitutions, in which at least one of the natural amino acids substituted by alanine residue. Special variants of Apo2L/TRAIL with substitutions include those in which at least one substituted amino acid residue is alanine. This option is with the substitutions include, which are identified as "D203A"; "D218A" and "D269A." This nomenclature is used to identify variants of Apo2L/TRAIL in which the aspartic acid residues at positions 203, 218, and/or 269 (in accordance with the numbering presented in figure 1) is substituted by alanine residues. Optional, options Apo2L can contain one or more substitutions involving alanine, listed in table I published patent application PCT WO 01/00832. Variants with substitutions include one or more substitutions of amino acid residues identified in table I of 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 manufactured by recombinant or synthetic methods. Apo2L/TRAIL according to the invention includes polypeptides, called Apo2L/TRAIL or TRAIL, which are disclosed in PCT publications under the numbers WO 97/01633 and WO 97/25428. The terms "Apo2L/TRAIL or Apo2L" is used to generally designate the forms of Apo2L/TRAIL, which include Monomeric, dimeric or trimeric forms of the polypeptide. All numbering of amino acid residues having the sequence of Apo2L, based on the numbering shown in figure 1, unless specifically stated otherwise. For example, "D203" or "Asp203" refers to the residue of aspartic acid at position 203 sequence represented is a figure 1.

The term "extracellular domain Apo2L/TRAIL or Apo2L/TRAIL ECD" refers to a form of Apo2L/TRAIL, which is substantially free of transmembrane and cytoplasmic domains. Usually ECD has less than 1% of the transmembrane and cytoplasmic domains, preferably, less than 0.5% of such domains. It will be understood that any transmembrane domain (domains)identified in relation to polypeptide invention is identified according to those criteria, which are usually used in this field to identify the hydrophobic domains of this type. The exact boundaries of the transmembrane domain may change, but most likely not more than 5 amino acids at either end of the domain that was originally identified. In preferred variants of the invention, the ECD consists of soluble sequence of the extracellular domain of the polypeptide, which is free of the transmembrane and cytoplasmic or intracellular domains (and is not associated with the cellular membrane). A special sequence of the extracellular domain of Apo-2L/TRAIL described in PCT publications under the numbers WO 97/01633 and WO 97/25428.

The term "monomer Apo2L/TRAIL" or "monomer Apo2L" means a covalent chain sequence of the extracellular domain Apo2L.

The term "dimer Apo2L/TRAIL" or "dimer Apo2L" means two monomer Apo-2L, connected by covalent the ligature through a disulfide bridge. When used in this specification, the term includes freely spaced dimers Apo2L, as well as dimers Apo2L included in the trimeric form of Apo2L (i.e. paired with another, a third monomer Apo2L).

The term "trimer Apo2L/TRAIL" or "trimer Apo2L" means three monomer Apo2L, ecovalence related to each other.

The term "unit Apo2L/TRAIL" is used to denote samoassotsiiruyutsya higher oligomeric forms of Apo2L/TRAIL, for example, trimers Apo2L/TRAIL, which form, for example, review or nanoscale forms of Apo2L/TRAIL. The presence and quantity of monomer, dimer or trimer of Apo2L/TRAIL (or other units) can be made by the methods and analytical techniques known in the field (using commercially available materials, for example, HPLC with the exception of the natural size ("SEC"), denaturing the size using sodium dodecyl sulfate ("SDS " SEC"), reversed-phase HPLC and capillary electrophoresis.

The term "receptor ligand Apo-2 includes receptors that are referred to this area as "DR4 and DR5", polynucleotide and polypeptide sequence of which is presented in figure 2 and 3 respectively. Pan et al. described member of the family of TNF receptors, called "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, 199,; WO 00/73349, published 7 December 2000; US 6433147, issued August 13, 2002; US 6461823, issued October 8, 2002, and US 6342383, issued 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 is called DR5 (the receptor is also mentioned under alternative names 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; EP870827, 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 002/0098550, published 7 December 2001; US 6313269, issued December 6, 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, issued may 27, 2003, US 6072047, issued June 6, 2000, US 6642358, issued November 4, 2003). As described above, other receptors for Apo-2L include DcR1, DcR2 and OPG (see, Sheridan et al.,above; Marsters et al.,above; and Simonet et al.,above). The term "receptor Apo-2L" when used in this description covers Rotz is a torus with the natural sequence and variants of the receptor. These terms cover the receptor for Apo-2L, expressed in a number of mammals, including humans. The receptor for Apo-2L can be expressed endogenous, which occurs naturally in many lines of human tissue, or can be expressed recombinant or synthetic methods. The term "receptor Apo-2L with the natural sequence" refers to a polypeptide having the same sequence as the receptor for Apo-2L of natural origin. Thus, the receptor for Apo-2L with the natural sequence can have the amino acid sequence of the receptor Apo-2L of natural origin from any mammal. This receptor Apo-2L with the natural sequence can be obtained naturally or produced recombinant or synthetic means. The term "receptor Apo-2L with the natural sequence" specifically encompasses natural truncated or secreted forms of the receptor (for example, soluble form containing, for example, the sequence of the extracellular domain), variant forms of natural origin (for example, resulting from alternative splicing), as well as allelic variants of natural origin. Variants of the receptor may include fragments or to be deletion mutants of the natural sequence of the receptor Apo-2L. The piano is GA presents the amino acid sequence of human DR5 of 411 amino acids, as it was published in WO 98/51793 dated November 19, 1998 In this area known transcriptional splicing variant human DR5. This alternative splicing encodes the DR5 DR5 sequence of a person of 440 amino acids presented on figv and 3C, as it was published in WO 98/35986 dated August 20, 1998

The term "antibody receptor cell death" is used in this description to denote, in General, the antibodies or antibodies directed to the receptor of the superfamily of receptors of the factor of tumor necrosis and containing a death domain that is able to transmit the signal apoptosis. Such antibodies include antibody DR5 and DR4 antibody.

The term "antibody receptor DR5, DR5 antibody" or "anti-DR5 antibody" is used broadly to refer to antibodies that bind at least one form of the receptor DR5, for example, the sequence 1-411 presented on figa, or sequence 1-440 presented on FIGU-3C, or their extracellular domain. Optionally, the DR5 antibody is fused or linked to a heterologous sequence or molecule. Preferably heterologous sequence allows or helps the antibody to form a higher order or oligomeric complexes. Optionally, the DR5 antibody binds to the receptor DR5, but not linked or not cross react with any l the Bo additional receptor Apo-2L (for example, with DR4, DcR1 or DcR2). Optionally, the antibody is an agonist signaling activity of DR5.

Optionally, the DR5 antibody of the invention binds to a receptor DR5 in the concentration range from approximately 0.1 nm to approximately 20 mm when measured by the analytical method binding BIAcore). Optional, DR5 antibodies of the invention show an Ic value 50 in the concentration range from approximately 0.6 nm to approximately 18 mm (measured on the analytical methodology BIAcore binding).

The term "antibody receptor DR4, DR4 antibody" or "anti-DR4 antibody" is used broadly to refer to antibodies that bind at least one form of the receptor DR4 or his (their) extracellular domain. Optionally, the DR4 antibody is fused or linked to a heterologous sequence or molecule. Preferably heterologous sequence allows or helps the antibody to form a higher order or oligomeric complexes. Optionally, the DR4 antibody binds to the receptor DR4, but not linked or not cross react with any additional receptor Apo-2L (for example, DR5, DcR1 or DcR2). Optionally, the antibody is an agonist signaling activity of DR4.

Optionally, the DR4 antibody of the invention binds to a receptor DR4 in the concentration range from about 0.1 nm d is approximately 20 mm when measured by the analytical method binding BIAcore). Optional, DR4 antibodies of the invention have a value of Ic 50 in the concentration range from approximately 0.6 nm to approximately 18 mm (measured on the analytical methodology BIAcore binding).

The term "agonist" is used 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 DR5 in vitro, in situ or in vivo. Examples of such biological activities include binding of Apo2L/TRAIL to DR4 or DR5, including apoptosis, as well as other options in the published literature. The agonist may function directly or indirectly. For example, the agonist may function to partially or fully enhancing, stimulating or activating one or more of the biological activities of DR4 or DR5 in vitro, in situ or in vivo, as a result of its direct binding to DR4 or DR5, which causes activation of the receptor or signal transduction. The agonist can also function indirectly, partially or fully enhancing, stimulating or activating one or more of the biological activities of DR4 or DR5 in vitro, in situ or in vivo, for example, as a result of stimulation other effector molecules, which, in turn, causes activation of DR4 or DR5 or signal transduction. It is assumed that the agonist can act as a molecule enhancer, the which operates indirectly, enhancing or increasing 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 achieved, for example, the preliminary formation of the complex DR4 or DR5 or stabilization of the complexes of the corresponding ligand with the receptor DR4 or DR5 (for example, stabilisation of the nature of the complex formed between the Apo-2L and DR4 or DR5).

The term "biomarker"as used in this application means, in General, a molecule (including gene, protein, carbohydrate structure, or glycolipid), the expression of which in the tissues or cells of a mammal can be identified by standard methods (or methods disclosed in this application and in which it is possible to predict the sensitivity of cells or tissues of a mammal to Apo2L/TRAIL or the antibody receptor cell death. Such biomarkers of the present invention include, but are not limited to, molecules of the protein family GalNac-T. In the literature described the members of the family of genes N-acetylgalactosaminyltransferase life (GalNac-T) and coded for proteins (see, for example, Hang et al., "The chemistry and biology of mucin-type O-linked glycosylation initiated by the polypeptide N-acetyl- -galactosaminyltransferases", Bioorganic & Medicinal Chemistry (material available from may 2005 atwww.sciensedirect.com) and references cited therein; Wang et al., BBRC, 300:738-744 (2003) and references cited therein), and researchers believe, Thu whom they act, determining the number and position of O-linked sugar chains in proteins. Optionally, expression of such a biomarker is defined at a higher level than observed in the control sample of tissue or cells. For example, optionally, the expression of such biomarkers can be identified using the microarray gene expression, quantitative PCR or immunohistochemical tissues analysis. Optionally, the expression of a biomarker, GalNac-T, for example, GalNac-T14 or GalNac-T3 in the experimental sample of tissue or cells can be determined in the analysis with the Affymetrix microarray U133P at least 750 times the level or 500-fold, or, preferably, at least 1000-fold compared with control tissue samples or cells in the detection of expression of the biomarker using quantitative PCR.

"UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-T14", "pp-GalNac-T14", "GalNac-T14", "GALNT14" are used in this case to denote membrane protein type II, with characteristic features of a family of molecules GalNac-T, containing N-terminal cytoplasmic domain, a transmembrane domain, a stem region and catalytic domain. Optionally, in one of the embodiments of the invention, the molecule of GalNac-T14 person contains 1659 base pairs encoding a protein of 552 amino acids, as shown in figa. Full-size cDNA is the ne was deposited in GenBank under inventory number AB078144. As disclosed by Wang et al., BBRC, 300:738-744 (2003), were identified splanirowannya isoforms GalNac-T14, which include (or not include) special exons, for example, exons 2, 3 and/or 4. The present invention considers the study of the expression of such changing isoforms of GalNac-T14, and the expression of any such isoforms can predict the sensitivity of a sample of tissue or mammalian cells to Apo2L/TRAIL or the antibody receptor cell death.

"UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-T3", "pp-GalNac-T3", "GalNac-T3", "GALNT3" are used in this case to denote membrane protein type II, with characteristic features of a family of molecules GalNac-T, containing N-terminal cytoplasmic domain, a transmembrane domain, a stem region and catalytic domain. Optionally, in one of the embodiments of the invention the polypeptide GalNac-T3 contain the amino acid sequence represented by figv. GalNac-T3 is additionally described by Bennett et al., J. Biol. Chem., 271:17006-17012 (1996).

The terms "individual" or "patient" refers to any single individual, including humans, in need of treatment. In addition, as individuals are referred to any individuals involved in clinical trials, but does not exhibit any signs of disease, or individuals involved in clinics is their tests, or individuals, used as a control.

The term "mammal"as used herein refers to any animal classified as a mammal, including humans, cows, horses, dogs and cats. In a preferred embodiment of the invention under mammals implies man.

The term "tissue sample or cells" means the collected material of similar cells obtained from a tissue of an individual or patient. The source of the tissue sample or cells can be dense tissue in the form of fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood components; biological fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells obtained in any stage of pregnancy or at any stage of development of the individual. The tissue sample can be primary or can be a result of the cultivation of cells or cell lines. Optionally, a sample of tissue or cells can be obtained from primary or metastatic tumors. The tissue sample may contain compounds, which under natural conditions is not mixed with a cloth, for example preservatives, anticoagulants, buffers, latches, nutrients (nutrients), antibio the IKI etc.

In the context of the present description "slice" of tissue sample means single piece or a piece of a tissue sample, for example, a thin layer of cut tissue or cells from the tissue sample. Clearly, the present invention can be obtained and analyzed multiple sections of tissue samples based on the fact that the present invention covers such a way, when one and the same slice of the tissue sample is analyzed both at morphological and molecular level or analyzed as proteins and nucleic acids.

The terms "correlate" or "conduct correlation" refers to the comparison of the implementation and/or results of the first analysis or Protocol with performance and/or results of the second analysis or Protocol. For example, the person skilled in the art can use the results of the first analysis or Protocol the following protocols and/or use the results of the first analysis or Protocol for evaluating whether to conduct a second review or Protocol. With regard to different embodiments of the present invention, it is possible to use the results of analytical samples, for example, the expression of mRNA or tissues to assess whether you want to apply special treatment scheme with the use of Apo2L/TRAIL or antibody receptor cell death.

By "nucleic acid" units is eveda any kind of DNA or RNA. For example, it may be chromosomal, mitochondrial, viral and/or bacterial nucleic acid present in the tissue sample. The term "nucleic acid" encompasses one or filament strand, or both strands of double-strand molecules of nucleic acid, including any fragment or any part of the intact molecules of nucleic acids.

The term "gene" means any nucleic acid sequence or part thereof having a functional role in coding or transcription of the protein, or in the regulation of expression of another gene. Gene can consist of all nucleic acids that are responsible for encoding a functional protein or part of nucleic acids, responsible for encoding or the expression of the protein. The sequence of the nucleic acid may contain a genetic anomaly within exons, introns, regions of the initiation or termination, promoter sequences, other regulatory sequences or unique region adjacent to the gene.

As used in the description, the term "label" in this document means a compound or composition, which directly or indirectly involves or merged with a reagent, for example, a probe nucleic acid or antibody and facilitates the identification of the reagent with which it is associated or merged. The label can be viable the mine itself (for example, radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of the substrate compound or composition to be revealing.

The term "antibody" is used in this document in its broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, polyspecific antibodies (for example, bespecifically antibodies)formed from at least two intact antibodies, and antibody fragments (as long as they exhibit the desired biological activity).

"Fragments of antibodies are part of an intact antibody, preferably containing antigennegative or variable region. Examples of fragments of antibodies include Fab fragments, Fab', F(ab')2and Fv; dyatel; linear antibodies; single-chain molecule antibodies; and polyspecific antibodies formed from fragments of antibodies.

"Native antibodies" are usually heterotetrameric glycoproteins with a mass of approximately 150000 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 in the heavy chains of different immunoglobulin isotypes variable is. Each heavy and light chain also has nutritive disulfide bridges are spaced from each other at a certain distance. Each heavy chain has at one end has a variable domain (VH), followed by a number of constant domains. Each light chain is at one end has a variable domain (VL), and the other end is a constant domain; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the variable domain of the light chain is linked to the c variable domain of the heavy chain. It is believed that the surface boundary between the variable domains of the light chain and heavy chain formed by particular amino acid residues.

The term "variable" refers to the fact that some areas of the variable domains largely differ in the sequence of the antibody and are used for specific binding of each particular antibody to its specific antigen. However, the variability is not evenly distributed across the variable domains of antibodies. It is concentrated in three segments called hypervariable or complementary determining regions within the boundaries of the variable regions of both light and heavy chains. More safe or conservative part of the variable domains are called the frame regions (FR). Each var is abelly domain of native heavy and light chains contain four FR, largely the receiving structure of beta-sheet, connected by three hypervariable regions, which form the hinge connection, and in some cases form part of the structure of beta-sheet. Hypervariable region of each chain are held together in close proximity with FR and hypervariable regions of the other circuit, contributing to the formation of 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, for example, participate in activity-dependent antibodies cell-mediated cytotoxicity (ADCC).

Cleavage of antibodies with papain leads to the appearance of two identical antigenspecific fragments, called fragments "Fab", and each of them has only antigennegative website, as well as residual fragment "Fc", whose name reflects its ability to crystallize. Treatment with pepsin results in the appearance of fragment F(ab')2having two antigenspecific site and still capable of crosslinking with the antigen.

"Fv" is the minimum antibody fragment that contains a complete website recognition and binding of the antigen. This region sostoi is from dimer variable domains of one heavy chain and one light chain, in strong non-covalent linkages. It is in this configuration, the three hypervariable region of each variable domain interact with each other, defining antigennegative site on the surface of the dimer VH-VL. All together six hypervariable regions give the antibody antigennegative specificity. However, even a single variable domain (or half of an Fv, containing only three hypervariable region specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity compared with the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fragments, Fab' differ from Fab fragments by the fact that at the carboxyl end of the domain of their heavy chains added several residues, including one or more cysteine residues of the hinge region of the antibody. Fab'-SH in the present description is used to denote Fab'in which the residue (remainder) of cysteine constant domain carries at least one free thiol group. Fragments of antibodies F(ab')2originally produced as pairs of Fab fragments', between which are the hinges of cysteine. There are also other chemical compounds fragments of antibodies.

The "light chains" of antibodies (them is noglobulins), belonging to any kind of vertebrate, can be attributed to one of two clearly distinct types, referred to as Kappa (κ) and lambda (λ), and this division is based on the amino acid sequences of their constant domains.

Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be divided into different classes. Famous 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., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The constant domains of the heavy chain corresponding to the different classes of antibodies are called α, δ, ε, γ and µ, respectively. Well-known structures of subunits and three-dimensional configurations of different classes of immunoglobulins.

Fragments of the antibodies of the type "single-chain Fv" or "scFv" contain domains of the antibody VHand VLmoreover , these domains is presented in a single polypeptide chains. In a preferred embodiment, 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 according to Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 259-315 (1994).

The term "diately" refers to small fragments of antibodies with two antihistamine what AIT, moreover, these 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). Through the use of a linker that is too short for mating (conjugation) of the two domains on the same chain, the domains are pushed forcibly to mate with complementary domains of another chain and create two antigenspecific site. Diately more fully described, for example, in the references EP 404097, WO 93/11161 and Hollinger et al., Proc. Natl. Acad. Sex. USA, 90:6444-6448 (1993).

The term "monoclonal antibody" when used in the present description means an antibody obtained from a population of substantially homogeneous antibodies, i.e, the individual antibodies comprising the population are almost identical, except for the possible natural mutations that may be present in minor amounts. Monoclonal antibodies of vysokospetsifichnymi, i.e. directed against a single antigenic site. In addition, unlike traditional drugs (polyclonal) antibodies, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single antigen determinants. In addition to the high specificity of monoclonal antibodies having the t the advantage of they are synthesized in the culture of hybridoma, not contaminated by the presence of other immunoglobulins. The attribute "monoclonal" indicates the sign that the antibody obtained from an almost homogeneous population of antibodies, and should not be constructed taking into account the requirements of the production of antibody by any particular method. For example, monoclonal antibodies are used in accordance with the present invention, can be obtained by way of hybridoma, first described by Kohler et al., Nature, 256:495 (1975), or by methods of recombinant DNA (see, for example, U.S. patent No. 4816567). "Monoclonal antibodies" you can also select from a library of phage antibodies using techniques described, for example, Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

Monoclonal antibodies referred to in this document, 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, and the remainder of the chain (chain) identical with or homologous to corresponding sequences in antibodies derived from other species or belonging to another class or subclass antibodies as well as fragments of such antibodies, with the proviso that they PR what are the desired biological activity (U.S. patent No. 4816567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest in this context include "primaryservername" antibodies containing antigennegative sequence of the variable domain obtained from nonhuman primates (e.g., from the Old world monkeys, such as baboons (baboons), rhesus monkeys or Griboedov (Svan)), and sequences of the constant regions of human (U.S. patent No. 5693780).

"Humanized" forms of antibodies mechelovskogo origin (e.g., mouse) are chimeric antibodies that contain minimal sequence derived from the immunoglobulin inhuman origin. Humanized antibodies are mostly human immunoglobulins (recipient antibody)in which amino acid residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of the other species (donor antibody)such as mouse, rat, rabbit or nonhuman primates, and they have the desired specificity, affinity and functional activity. In some cases, remnants of the framework region (FR) of a human immunoglobulin replaced by the corresponding residues of other species. Furthermore, humanized antibodies may contain residues that are missing in the antibody-recipient or antibody-donor. These modifications contribute to the further improvement of the performance of the antibodies. In General, humanitariannet antibody contains essentially all of at least one or typically two, variable domains, in which all or nearly all of the hypervariable loops correspond to the loops of immunoglobulins nonhuman origin, and all or nearly all FR correspond to the sequence of human immunoglobulin. Optional, humanitariannet antibody also comprises at least a portion of constant region of immunoglobulin (Fc), typically of a human immunoglobulin. For more information, see the references 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" when used in this description refers to the amino acid residues of the antibody, which is responsible for binding to the antigen. Hypervariable region contains amino acid residues from a "region that defines complementarity" 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 residues from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the variable domain of the light chain I-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 those variable domain residues that differ from the given in the present description detection of hypervariable region.

An antibody "which binds" an 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.

In order that haunts the present invention, "immunotherapy" refers to a method of treatment of a mammal (preferably, a sick person) antibodies and specific antibody can be unconjugated or naked antibody or antibody may be conjugated or fused to a heterologous molecule (molecules) or agent (agents), for example, with one or more cytotoxic agents, which leads to the creation of "immunoconjugate".

"Isolated" antibody is a human antibody that has been identified and separated and/or extracted from the components of its natural environment. Polluting components of the environment are materials that are capable of being in order to interfere with diagnostic or therapeutic uses of the antibody, these include some enzymes, hormones and other relcovaptan or nebulophone solute. In preferred embodiments of the invention the antibody is purified (1) to more than 95% by weight with determination by the method of Lowry, most preferred more than 99% by weight, (2) and sufficient to obtain at least 15 N-terminal or internal amino acid residue sequence using sequencing machine with rotating cups, or (3) to homogeneity by the method of SDS-PAGE in reducing or nereguliruemyi conditions using the dye Kumasi blue or, preferably, color silver. The selected antibody includes the antibody in situ within recombinant cells in cases when there is no at least one component of the natural environment antibodies. However, as a rule, the selected antibody receive at least one stage of the treatment.

The expression "effective amount" means such amount of the agent (for example, Apo2L/TRAIL, anti-DR4 antibodies or DR5 and so on), which is effective for prevention, improvement of symptoms or treatment of a particular disease or condition.

The terms "treatment" and "therapy", as used in this document, means curative therapy, prophylactic therapy, and preventative therapy. Serial is Uchenie or introduction means treatment at least on a daily basis without interruption in treatment for one or more days. Alternating treatment or the introduction of either the treatment or the introduction of alternating fashion means such treatment, which in its essence is cyclical rather than sequential.

The term "cytokine" is a generic term used to refer to proteins released by one cell population which act on another cell as intercellular mediators. Examples of cytokines are lymphokines, Monokini and traditional polypeptide hormones. The group of cytokines are growth hormones, such as human growth hormone, N-methionyl-human growth hormone and growth hormone in cattle; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prolactin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogenic; alpha-factor and beta factor tumor necrosis; mullerova inhibiting substance; mouse peptide associated with gonadotropin; inhibin; activin; factor vascular endothelial growth; integrin; thrombopoietin (TPO); nerve growth factors; platelet-derived growth factor; trasformarsi growth factors (TGF)such as TGF-b and TGF-b; insulin-like growth factor I and II; erythropoietin (EPO); osteoid aktivnye factors; interferons such as alpha-, beta-, and gamma-interferon; colony stimulating factors (CSF), for example, CSF macrophages (M-CSF), CSF granulocyte macrophage (GM-CSF) and CSF granulocyte (G-CSF); interleukins (IL)such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17; and other polypeptide factors including LIF and kit ligand (KL). When used in this document, the term cytokine includes proteins from natural sources or from a culture of recombinant cells and biologically active equivalents of the natural sequences of cytokines.

The term "cytotoxic agent" as used in this document means a compound that inhibits or prevents the function of cells and/or causes destruction of cells. This term is intended to include radioactive isotopes (e.g., I131I125, Y90and Re186), chemotherapeutic agents, and toxins, for example, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.

"Chemotherapeutic agent" is a chemical compound suitable for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents, for example, thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulphonates such as busulfan, improsulfan and piposulfan; aziridines such the AK benzodepa, carboquone, matureup and uredepa; ethylenimines and methylmelamine, including altretamin, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and triethylenemelamine; acetogenins (especially bullatacin, bullatacin); camptothecin (including the synthetic analogue topotecan); bryostatin; callistemon; CC-1065 (including synthetic analogs of adozelesin, carzelesin and bizelesin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictyin; spongistatin; derivatives of nitrogen mustard such as chlorambucil, chlornaphazine chlorpropamide, estramustine, ifosfamide, mechlorethamine, hydrochloride oxide mechlorethamine, melphalan, novemberin, finestein, prednimustine, trofosfamide, uracil-mustard; nitrosoanatabine, for example, carmustin, chlorozotocin, fotemustine, lomustin, nimustine, ranimustine; antibiotics, for example, enediyne antibiotics (for example, calicheamicin, especially calicheamicin gamma 1I and calicheamicin Phi 1I (see, e.g., Agnew, Chem Intl. Ed. Engl., 33:183-186, 1994); dynemicin, including dynemicin a; biophosphonate, for example, clodronate; spiramycin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotics chromophores), aclacinomycin, actinomycin, autralian, azaserine, bleomycin, actinomycin, karab the Qing, karminomitsin, calcination, chromomycin, dactinomycin, daunorubicin, demoralizing, 6-diazo-5-oxo-L-norleucine, doxorubicin (Adriamycin™) (including morpholino doxorubicin, cyanomethane doxorubicin, 2-pyrroline doxorubicin and desoxycortisol), epirubicin, zorubicin, idarubitsin, marsellaise, mitomycin, for example, mitomycin C, mikofenolna 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, for example, deeperin, methotrexate, peripherin, trimetrexate; purine analogues, such as fludarabine, 6-mercaptopurine, timipre, tioguanin; pyrimidine analogues, for example, ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antagonists of the adrenal glands, for example, aminoglutethimide, mitotane, trilostane; folic acid supplements, for example, prolinnova acid; Eagleton; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; astroball; bisantrene; edatrexate; defaming; demecolcine; dietician; alternity; slipline acetate; epothilone; e is glucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoid, for example, maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitrogen; pentostatin; penomet; pirarubicin; losoxantrone; podofillina acid; 2-acylhydrazides; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogermanium; tinoisamoa acid; tradicion, 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, for example, paclitaxel (TAXOL®Bristol-Myers Squibb Oncology, Princeton, new Jersey) and docetaxel (TAXOTERE®, Rhône-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, for example, retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids and derivatives of any of the above substances. This definition also includes protivokomarinye means of regulating or talauma hormone action on tumors, in particular, antiestrogens and selective estrogen receptor modulators (SERM), including, for example, tamoxifen (including Nolvadex™), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (Fareston™); aromatase inhibitors, i.e. substances that suppress the activity of the enzyme aromatase, which regulates the production of estrogen in the adrenal glands, for example, 4(5)-imidazoles, aminoglutetimid, megestrol acetate (Megace™), exemestane, formestane, fadrozole, vorozole (Rivisor™), letrozole (Femara™) and anastrozole (Arimidex™); and antiandrogens, such as flutamide, nilutamide, bikalutamid, leuprolide, goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above substances.

The term "inhibitory agent of growth", as used in this document, refers to the compound or compositions that inhibit the growth of cells, particularly cancer cells, showing a surplus expression of any of the identified genes here, either in vitro or in vivo. Thus, the inhibitory agent of growth represents the agent that significantly reduces the percentage of cells exhibiting excessive expression of these genes in S-phase. Examples of inhibitory agents growth include agents that block the progression of the cell cycle (other than S phase), estiality, inducing a delay in G1 - and M-phase. Classic blockers M-phase include Vincas (vincristine and vinblastine), Taxol and inhibitors of topo II, for example, doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that delay G1, also are expanding their effect on the delay of the S-phase is, for example, DNA alkylating agents, in particular, tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. for More information, see "The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogens, and antineoplastic drugs"by the authors Murakami et al. (WB Saunders: Philadelphia, 1995), especially page 13.

The terms "apoptosis" and "apoptotic activity" used in the present description in a broad sense and refer to the orderly or controlled form of cell death in mammals, usually accompanied by one or more characteristic cell changes, including condensation of cytoplasm, loss of microvision membrane, segmentation of the nucleus, the destruction of chromosomal DNA or loss of mitochondrial function. This activity can be defined and measured, for example, by tests on the survival of cells (in particular, the analysis with the dye Alamar blue or MTT analysis, FACS analysis, activation of caspase, fragmentation of DNA (see, for example, Nicoletti et al.,J. Immunol. Methods,139:271-279 (1991)), and the e polymerase poly-ADP ribose, "PARP", analyses on splitting, known in this field.

When used in the present description, the term "disorder" in General refers to any condition that can be improved as a result of treatment described in the present description compositions, including any disease or disorder that can be treated with an effective amount of Apo2L/TRAIL, anti-DR4 antibodies and/or anti-DR5 antibodies. These include acute and chronic disorders, as well as those pathological conditions that create the mammal, the susceptibility to discuss disorders. Non-limiting examples of disorders amenable to treatment in accordance with the present invention, are benign and malignant tumors; inflammatory, angiogenic and immunologic disorders, autoimmune disorders, arthritis (including rheumatoid arthritis), multiple sclerosis and HIV/AIDS.

The terms "cancer", "cancer" or "cancerous" refer to the physiological conditions in mammals that is typically characterized by uncontrolled growth of cells (or describe such States). Examples of cancer include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma and sarcoma. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, Nemacolin CNY lung cancer, the glioma, Hodgkins lymphoma, nahodkinskuju lymphoma, cancer of the gastrointestinal tract, kidney, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, cancer of the colon and rectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, carcinoma shell colon, and head and neck cancer.

The term "immunosupresive disease" means a disease in which component of the immune system of a mammal induces or mediates the incidence of the specified mammal or makes any contribution to this incidence. This group includes such diseases in which stimulation of the immune response or interference in the immune response have a positive effect in relation to the course/progression of the disease. This term covers autoimmune diseases, immune inflammatory diseases, nimmanoradee inflammatory diseases, infectious diseases and diseases caused by immunodeficiency. Examples of immune and inflammatory diseases, some of which is immune in nature or mediated by T-cells and who can be treated by the methods of the present invention, include systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), syndrome of Segren (Sjogren), systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal night hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purple, immunopositive thrombocytopenia), thyroiditis (graves ' disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immunopositive kidney disease (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the Central and peripheral nervous system, such as multiple sclerosis, idiopathic demyelinizing polyneuropathy or Guillain-Barre syndrome (Guillain-Barré) and chronic inflammatory demyelinizing polyneuropathy, hepatobiliary diseases such as infectious hepatitis (hepatitis A, B, C, D, E and other nagapattanam viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory and fibrations lung diseases, such as inflammatory bowel disease (ulcer is hydrated colitis: Crohn's disease), patentability enteropathy, and Whipple's disease (Whipple), autoimmune or immune skin diseases, in particular, bullous skin lesions, erythema multiforme and contact dermatitis, psoriasis, allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, food Allergy and urticaria, immunologic diseases of the lung, for example, eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitive pneumonitis, diseases associated with transplantation, including graft rejection and disease "graft versus host". Infectious diseases include AIDS (HIV), hepatitis A, B, C, D and E, bacterial infections, fungal infections, protozoal infections and parasitic infections.

"Autoimmune disease" is used in the present description in a broad and General sense to refer to diseases or conditions in mammals, when the destruction of normal or healthy tissue is the result of a humoral or cellular immune response specific to the mammal at his/her own tissue components. Examples include, but are not limited to, lupus erythematosus, thyroiditis, rheumatoid arthritis, psoriasis, multiple sclerosis, autoimmune diabetes, and inflammatory bowel disease (IBD).

The term "labeled" in the line who communicate in this document refers to a chimeric molecule, containing the antibody or polypeptide fused with a polypeptide-tagged". Polypeptide-label has a sufficient number of amino acid residues to cover the epitope against which may be generated antibody, or to give a labeled molecule some additional features, for example, the ability to oligomerization (as occurs, for example, peptides having domains with latinboy lightning), but it remains quite short and usually does not affect the activity of the antibody or polypeptide. Polypeptide-label, preferably, also so unique that specific on the label the antibody does not pronounced cross-react with other epitopes. Suitable polypeptides tags often have at least six amino acid residues, usually from about 8 to about 50 amino acid residues (preferably, from about 10 to about 20 amino acid residues).

The term "divalent metal ion" refers to a metal ion having two positive charge. Examples of divalent metal ions include, but are not limited to, ions of zinc, cobalt, Nickel, cadmium, magnesium and manganese. Deserving special attention such metals, which can be used in the present invention include a salt form (e.g., pharmaceutically acceptable Olevia form), in particular, chloride, acetate, carbonate, citrate and sulfate forms of the above-mentioned divalent metal ions. Optional, divalent metal ion for use in the framework of the present invention may be of the zinc ion, preferably in salt form, i.e. in the form of zinc sulfate or zinc chloride.

The term "isolated" is used in this description to describe the different peptides or proteins disclosed in the present invention, means a peptide or protein, has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that can interfere with diagnostic or therapeutic uses of peptide or protein, and these include enzymes, hormones and other relcovaptan or nebulophone solute. In preferred variants of the invention, the peptide or protein is purified (1) sufficient to obtain at least 15 N-terminal or internal amino acid residue sequence using sequencing machine with rotating glass, or (2) to homogeneity by the method of SDS-PAGE in nereguliruemyi or reducing conditions using dye Kumasi blue or, preferably, painting sulfur is rhenium, or (3) to homogeneity, as determined by the methods of mass spectrometry or mapping of peptides. The selected material includes a peptide or protein in situ within recombinant cells, in those cases where at least one component of its natural environment is missing. However, as a rule, the selected peptide or protein get at measures, with one stage of the treatment.

The definition of "percentage (%) amino acid sequence identity" with respect to the sequences identified in this document, means the percentage of amino acid residues in the sequence of the candidate that are identical with amino acid residues of the reference sequence after alignment of the two sequences and, if necessary, inserting gaps to achieve the maximum percent identity, and any conservative substitutions are not considered as part of the identity sequence. Reconciliation in order to establish the percentage identity of amino acid sequences can be carried out in various ways known in this area and determine appropriate parameters for measuring compliance, including assignment algorithms needed to achieve maximal alignment over the entire length of the compared sequences. For the purposes of this PA is entei applications percentage of amino acid identity can be obtained, using a computer program to compare sequences ALIGN-2, developed by Genentech Inc., the original text of which was made together with the user documentation to the Registrar of copyrights, U.S. Copyright Office, Washington, DC, 20559) and registered under number TXU510087. The program ALIGN-2 publicly available through Genentech, Inc. (South San Francisco, CA). All parameters comparison of the sequences specified by the program ALIGN-2 and not subject to change.

"Rigidity" of hybridization reactions is well understandable to the average person skilled in the art, typically representing the result of the empirical calculation based on the length of the probe, the temperature laundering and concentration of the salt solution. In General, longer probes require higher temperatures for proper annealing, whereas for relatively short probes the desired lower temperature. Hybridization generally depends on the ability of denatured DNA re-annealing, if the complementary strands in the environment presents at a temperature below the melting point. The higher the desirable degree of identity between the probe and hybridizing sequence, the higher the relative temperature that can be used. It follows that to achieve the more stringent reaction conditions need higher temperatures, while at lower temp is the temperature of the reaction conditions will be less hard. More detailed information and explanations regarding the stringency of hybridization reactions, see Ausubel et al.,Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

"Conditions of high stringency"as used in this description are defined as follows: (1) use to launder low ionic strength and high temperature: of 0.015 M sodium chloride/0,0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization following denaturing agent: 50% (vol/vol) formamide with 0.1% albumin bovine serum/0.1% environment ficoll/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 mmol of sodium phosphate (pH of 6.8), 0.1% sodium pyrophosphate, 5× denhardt's solution, processed by the ultrasound DNA salmon scramble sperm (50 µg/ml), 0.1% of SDS and 10% dextran sulfate at 42°C laundering at 42°C in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C with the subsequent laundering of high rigidity, consisting of 0.1×SSC containing EDTA at 55°C.

"Conditions of medium hardness" can be identified in accordance with the description Sambrook et al.,Molecular Cloning: A Laboratory ManualNew York: Cold Spring Harbor Press, 1989 - these conditions include incubation over night at 37°C in a solution comprising: 20% formamide, 5×SC (150 mm NaCl, 15 mm triacrylate), 50 mm sodium phosphate (pH of 7.6), 5× denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured DNA salmon scramble sperm (sheared salmon sperm), with the subsequent laundering of the filters in 1×SSC at a temperature of about 37-50°C. the Skilled expert should know how to properly select the temperature, ionic strength, etc., taking into account factors such as probe length and the like

The term "primer" or "primer" refers to an oligonucleotide sequences that hybridize with polynucleotides target DNA or RNA and serve as the starting point for step-by-step synthesis of polynucleotide of mononucleotides under the influence nucleotidyltransferase, as it happens, for example, polymerase chain reaction.

The term "regulatory sequence" refers to DNA sequences necessary for the expression of the operatively linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes include, for example, 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 functionally linked"when it is in functional relationship with another placentas is lacking nucleic acid. For example, DNA for predpolagavshegosja or secretory leader is functionally linked to DNA for a polypeptide if it is expressed as preblock (preprotein), which participates in the secretion of the polypeptide. The promoter or enhancer functionally linked to the coding sequence if it affects the transcription of the sequence, and the binding site of the ribosome is functionally linked to the coding sequence if it is so easy translation. In General the concept of "functional connectivity" means that such DNA sequences are located next to each other, and in the case of a secretory leader is not only related but are in the phase of reading. However, enhancers need not be contiguous. Pair accompanied blended in suitable restriction sites. If such sites do not, then in accordance with the accepted practices used synthetic oligonucleotide adaptors or linkers.

The terms "dependent antibodies cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells that Express Fc receptors (FcR) (e.g., natural killer cells (NK), neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cells. The basic cell for OPOS is adowanie ADCC, the NK cells, Express FcγRIII only, whereas monocytes Express FcγRI, FcγRII and FcγRIII. Summary data on the expression of FcR hematopoietic cells are shown in table 3 on page 464 in the work of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). In order to assess the activity type ADCC in the interest of the molecule, it is possible to carry out analysis of ADCC in vitro, for example, described in U.S. patent No. 5500362 or 5821337. Useful effector cells for such assays include mononuclear cells of peripheral blood (PBMC) and natural killer cells (NK). Alternative or additionally, ADCC activity of interest molecules can be assessed in vivo, e.g., in an animal model, described Clynes et al. PNAS (USA) 95:652-656 (1998).

"Effector cells" are cells, expressing one or more FcR and acting as effectors. Preferably, the cells expressed at least FcγRIII and performed the function of an ADCC effectors. Examples of human leukocytes mediating ADCC include mononuclear cells of peripheral blood (PBMC), natural killer cells (NK), monocytes, cytotoxic T cells and neutrophils, with preference being given to cells PBMC and NK.

The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to the Fc region of antibodies. Preferred FcR is a natural placentas is required FcR person. Moreover, a preferred FcR binds an IgG antibody (a gamma receptor) and includes receptors of the subclasses of the FcγRI, FcγRII and FcγRIII, including allelic variants and forms of the receptor resulting from alternative splicing. The FcγRII receptors include varieties of FcγRIIA (an"activating receptor") and FcγRIIB (an"inhibiting receptor"), which have almost identical amino acid sequences that differ primarily in the cytoplasmic domains. Activating receptor FcγRIIA in its cytoplasmic domain contains immunoreceptor motive activation based on tyrosine (ITAM). Inhibiting receptor FcγRIIB in its cytoplasmic domain contains immunoreceptor motive inhibition based on tyrosine (ITIM) (see Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). Review FcR presented 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). In this text the term FcR covers and other FcR, including those that can be identified in the future. This term also includes the receptor newborn (FcRn), which is responsible for the transfer of maternal IgG antibodies to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). In this document FcR also include polymorphic variants, in particular, genetic dimorphism in gene, encoding a FcγRIII, the result of which is the presence of the Lee phenylalanine (F), or valine (V) at 158 position, which falls in the region of the receptor, sviazyvalsia with IgG1. It was found that homozygous valine FcγIIIa (FcγIIIa-158V) has a higher affinity to human IgG1 and mediates increased level of ADCC in vitro compared with homozygous phenylalanine FcγIIIa (FcγIIIa-158F) or heterozygous receptors (FcγIIIa-158F/V).

The term "dependent complement cytotoxicity" or "CDC" refers to the ability of the molecule to lyse the target cell in the presence of complement. Path activation of complement is initiated by the binding of the first component of the complement system (C1q) with a molecule (e.g. antibody) in complex with cognate antigen. In order to assess activation of the complement, it is possible to analyze the CDC, for example, as described in Gazzano-three-bet et al., J. Immunol. Methods 202:163 (1996).

II. EXEMPLARY METHODS AND MATERIALS of INVENTION

Disclosed in this description of the methods and analytical techniques aimed to study the expression of one or more biomarkers in samples of tissues or cells of the mammal, and detecting the expression of one or more of these biomarkers is predictive or diagnostic indicator of the sensitivity of these samples of cells and tissues to such agents as Apo2L/TRAIL and/or antibodies of cell death receptors, such as agonistic anti-DR5 the antibodies is a or agonistic anti-DR4 antibodies. Methods and analytical techniques include, by which it is possible to investigate the expression of members of a family of molecules GalNac-T, including GalNac-T14 and GalNac-T3.

As discussed above, there are some populations of human cells of the patient type (for example, certain populations of cancer cells), which are immune to signals cell death, including the effects of Apo2L/TRAIL or agonistic antibodies of cell death receptors. Therefore, we must assume that disclosed in the present description, the methods and analytical techniques can provide a convenient, effective and possibly cost-effective means to obtain data and information useful in assessing the applicability and the effectiveness of specific approaches to the treatment of patients. For example, a patient who has been diagnosed with cancer or IMMUNOSUPRESSIVE disease should undergo biopsy to obtain a tissue sample or cells, and the resulting sample should be investigated by different analytical techniques in vitro to determine whether sensitive cells of the patient to the effects of a therapeutic agent, for example, Apo2L/TRAIL or antibody receptor cell death.

The invention provides methods for predicting the sensitivity of samples of tissue or mammalian cells (e.g. cancer cells to Apo2L/TRAIL or agonistic antibodies, nab is allonym receptors on cell death. Not necessarily, obtained a sample of tissue or cells of a mammal is examined for the expression of GalNac-T14. The methods of the invention can be implemented in many formats analysis, including identification of the mRNA expression, protein expression (for example, immunohistochemical analysis), as well as biochemical analyses that determine the enzymatic activity of UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. Determination of the expression of such biomarkers GalNac-T14 in (or on) the above tissues or cells has implications for predictions of the sensitivity of these tissues or cells by inducing apoptosis activity of Apo2L/TRAIL and/or an agonistic antibody receptor cell death. Applicants unexpectedly discovered that the expression of GalNac-T14 is correlated with the sensitivity of such tissues and cells to Apo2L/TRAIL and agonistic antibodies to the receptor cell death.

As discussed above, the expression of different biomarkers, e.g., GalNac-T14, the sample can be analyzed based on multiple methodologies, most of which are known in this field and understandable experienced, including, but not limited to, immunohistochemical and/or Western analysis, quantitative analysis blood samples (e.g. serum ELISA method) (for example, to study the expression level of the protein), biochemical analyzephasefive activity the in situ hybridization, Northern analysis and/or PCR analysis of mRNA and genomic southern analysis (for example, for studies of gene deletion or amplification), as well as any other of a wide range of analyses that can be performed in a set of genes and/or tissues. Typical protocols for evaluating the status of genes and gene products can be found, for example, Ausubel et al. eds., 1995, Current Protocols In Molecular Biology, sections 2 (Northern blotting), 4 (southern blotting), 15 (Western blot turns) and 18 (PCR analysis).

The following protocols related to the detection of GalNac-T14 in the sample presented in this description for illustrative purposes.

Optional methods of the invention include protocols which examine or test the presence of GalNac-T14 in a sample of tissue or cells of a mammal. For detection of GalNac-T14 can be used many ways, including, for example, immunohistochemical analysis, immunoprecipitation, analysis Western blot analysis, molecular binding, ELISA, ELIFA, sorting of fluorescently activated cells (FACS), as well as immunoprecipitation with subsequent mass spectrometry (MS) and the analysis of monosaccharides. For example, optional method for detecting the expression of GalNac-T14 in the tissue or sample includes contact of the sample with anti-GalNac-T14 antibody and then detecting the binding of antibodies with GalNac-T14 in the sample.

In some variant of the tenth embodiment of the invention the expression of GalNac-T14 in the sample examined, using immunohistochemistry protocols with staining. It was demonstrated that immunohistochemical staining of tissue sections is a reliable way to assess or identify the presence of proteins in the sample. Methods immunohistochemistry ("IHC") use antibody for sensing and visualization of cellular antigens in situ, usually chromogenic or fluorescent methods.

For sample preparation, you can use a sample of tissue or cells from a mammal (typically from a sick person). Examples of samples include, but are not limited to, cancer cells, for example, tumors of the colon, breast, prostate, ovarian, lung, stomach, pancreas, and cancer cell lymphoma and leukemia. Optionally, the samples include cells of non-small cell lung cancer cells, pancreatic cancer or cell non-Hodgkin lymphoma. The sample can be obtained by many methods known in this field, including, but not limited to, surgical excision, aspiration and biopsy. The fabric can be fresh or frozen. In one of the embodiments of the invention the sample is fixed and pour paraffin or subjected to any such process.

The tissue sample may be fixed (i.e. preserved) by traditionally the 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, D.C.). The expert will understand that the choice of the retainer depends on the purpose for which a particular specimen must be histologically stained or analyzed in some other way. The specialist will also understand that the duration of fixation depends on the size of the tissue sample and from used fixer. As an example, for fixing the sample, you can use neutral buffered formalin, the latch Buena or paraformaldehyde.

Typically, the sample is first fixed, and then dehydrated in an ascending series of alcohols, infiltrate and fill with paraffin or other medium for receiving the slices, so that the tissue sample can be cut thin layers. Alternatively, you can make a thin tissue section and record the resulting slice. As an example, a tissue sample can be poured paraffin and processed according to traditional methodology (see, for example, "Manual of Histological Staining Method of the Armed Forces Institute of Pathology", above). Examples of the wax, suitable for use include, but are not limited to, Paraplast, Broloid and Tissuemay. After pouring the tissue sample, a paraffin can be cut with a microtome Il is a similar tool (see, for example, "Manual of Histological Staining Method of the Armed Forces Institute of Pathology", above). As an example for this procedure, the sections can vary in thickness from about three to about five microns. After receiving sections can be mounted on slides to various standard ways. Examples of adhesive substances for fixing sections on slides include, but are not limited to, silane, gelatin, poly-L-lysine, etc. In the example sections, paraffin embedded, can be attached to the subject glasses with positive electric charge and/or glass coated with poly-L-lysine.

If the filling material was used in paraffin, tissue sections are usually deparaffinizing and rehydrating water. The tissue sections can be deparaffinizing several traditional standard methodologies. For example, you can use xylene and gradually descending series of alcohols (see, for example, "Manual of Histological Staining Method of the Armed Forces Institute of Pathology", above). Alternatively, you can use coming out of inorganic deparaffinizing agents, for example, Hemo-De7 (CMS, Houston, Texas).

Not necessarily, after receiving the drug tissue slice can be analyzed by IHC method. Research method IHC can be done in combination with additional techniques, for example, which are morphologically the practical coating and/or fluorescent in situ hybridization. There are two General methods IHC: direct and indirect analysis. According to the first assay, binding of antibody to the target antigen (e.g., GalNac-T14) is carried out in a direct way. This direct analysis uses a labeled reagent, for example a fluorescent label or enzyme-labeled primary antibody that can be visualized without further interaction of the antibodies. In a typical indirect analysis of unconjugated primary antibody binds to the antigen, and then labeled secondary antibody binds to primary antibody. If a secondary antibody conjugated with an enzyme label, for visualization of the antigen add chromogenic or fluorogenic substrate. The amplification of the signal occurs because some of the secondary antibodies can interact with different epitopes on the primary antibody.

Primary and/or secondary antibody used in immunohistochemical analysis, as a rule, mark detectable component. Available numerous labels, which generally can be grouped into the following categories:

(a) Radioisotopes, such as35S14C,125I3H and131I. Antibody can be observed with the radioisotope using the techniques described, for example, Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, New York, Pubs. (1991), and radioactiv the spine can be measured with a scintillation counter.

(b) Particles of colloidal gold.

(c) Fluorescent labels, including, but not limited to, rare earth chelates (europium chelates), theheavy (Texas red, rhodamine, fluorescein, dansyl, lissamine, umbelliferone, phycoerythrin, phycocyanin, or commercially fluorophores, for example, SPECTRUM ORANGE7 and SPECTRUM GREEN7 and/or derivatives of any of the above substances. Fluorescent labels can be konjugierte with antibodies, using techniques disclosed, for example, Current Protocols in Immunology, see above. Fluorescence can be quantified by fluorimetry.

(d) Available different label type enzyme substrates, and U.S. patent No. 4275149 gives an overview on some of them. The enzyme generally catalyzes a chemical alteration of the chromogenic substrate, which can be measured by using different methods. For example, the enzyme may catalyze the substrate color change, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence substrate. Methods of quantitative determination of fluorescence changes described above. Chemiluminescent substrate becomes electronically excited by chemical reaction, after which it can emit light that is measurable (for example, using chemiluminometer), or transmits the energy of the fluorescent acceptor. Note the factors of enzymatic labels include luciferase (e.g., the 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, beta-galactosidase, glucoamylase, lysozyme, SharedAccess (for example, glucoseoxidase, galactosidase and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microbiocides etc. Methods of conjugating 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 the substrate, and the hydrogen peroxide oxidizes a dye precursor (e.g., orthophenylene (OPD) or 3,3',5,5'-tetramethylbenzidine (TMB));

(ii) Alkaline phosphatase (AP) with paranitrophenylphosphate as a chromogenic substrate, and

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

Specialists in this area are also known and available for many other combinations of enzymes with substrates. Overview this is a poll, see in U.S. patent No. 4275149 and 4318980. Sometimes the label is conjugated with the antibody indirectly. An experienced specialist should know about the different methods by which you can achieve this. For example, the antibody can be conjugated with Biotin and any of the four broad categories of labels mentioned above can be conjugated with Avidya or Vice versa. Biotin selectively binds with Avidya, therefore, the label can be conjugated with the antibody of this indirect way. Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody is conjugated with a small hapten, and one of the above-mentioned various types of labels conjugated with protivohrapovym antibody. Thus, it can be achieved indirect conjugation of the label with the antibody.

In addition to the procedures of sample preparation, discussed above, may require further processing of the tissue slice before, during or after IHC, for example, the means of restoring epitope, such as heating the tissue sample in citrate buffer (see, e.g., Leong et al. Appl. Immunohistochem. 4(3):201 (1996)).

After the optional step of blocking tissue slice is introduced into contact with the primary antibody for a sufficient duration of time and in appropriate conditions, due to which the primary antibody binds to the target protein antigen in a tissue sample. Rela is eastwoodiae conditions to achieve this result, you can define the usual experimentation. The degree of binding of the antibody with the sample determined using any of the detectable labels discussed above. Preferably, the label was enzyme (for example, HRPO) and catalyze chemical alteration of the chromogenic substrate, for example, 3,3'-diaminobenzidine. Preferably, the enzyme label was conjugated with an antibody that specifically binds to the primary antibody (for example, the primary antibody is a polyclonal antibody rabbit, and the secondary antibody is an antibody goat against rabbit).

Optionally, the antibodies involved in the IHC analysis to determine the expression of GalNac-T14, are anti-GalNac-T14 antibody. Alternatively, you can use antibodies to other antigens GalNac-T, which cross-react with GalNac-T14. Optional anti-GalNac-T14 antibody is a monoclonal antibody.

Samples prepared in this way, you can mount and close the cover glass. Then conduct the evaluation study slides, for example, under the microscope, and at this stage it is possible to apply the criteria in the intensity of staining, usually used in this field. Criteria of staining intensity can be estimated as follows:

TABLE 1
Character coloringScore
Staining in cells not observed0
Faint/barely perceptible staining is defined in more than 10% of cells1+
Staining from weak to moderate is defined in more than 10% of cells2+
Staining moderate to strong is defined in more than 10% of cells3+

Generally, it is considered that the scoring nature of the staining approximately 2+ or more in this IHC analysis is a predictor or indicator of the sensitivity of mammalian cells (e.g. cancer cells of a mammal) to Apo2L/TRAIL or agonistic antibodies of death receptors.

In alternative methods, the sample can be introduced into contact with the antibody, specific for a given biomarker, and under such conditions sufficient for the formation of the complex of antibody-biomarker, as well as for detection of the specified property. The presence of the biomarker can be achieved in many ways, for example, Western blotting (immunoprecipitate or without it) and procedures for ELSA analytical study of a wide range of fabrics and designs, including plasma and serum. Known and available to a wide range of immunoassay techniques using such an analytical format (see, for example, U.S. patent No. 4016043, 4424279 and 4018653). These include both single-site, dvuhsimovyiy or "sandwich" assays non-competitive types, as well as the traditional options analysis of competitive binding. These analyses also include direct binding of the labeled antibody with the target biomarker.

Sandwich assays are the most useful and frequently used types of analysis. There are a variety of methods sandwich-analysis, and they can all be used in the present invention. Briefly, in a typical direct analysis of unlabeled antibody immobilized on a solid substrate and the sample to be tested is introduced into contact with the bound molecule. After an appropriate incubation period, during this time, which is sufficient for the formation of complex antigen-antibody to the sample add a second antibody specific to the antigen, and labeled reporter group that can produce a detectable signal, and then incubated for a time sufficient for the formation of another complex of antibody-antigen-labeled antibody. Any unreacted material is washed, and the presence of the antigen is determined by observing the signal, produced the first reporter group. The results can be either qualitative, in the form of a simple observation of the visible signal, or quantitative, providing a comparison with a control sample containing a known quantity of the biomarker.

Options direct analysis include simultaneous analysis in which the sample and labeled antibody are simultaneously added to the linked antibody. These techniques are well known experienced, including how you can easily understand their slight variations. In a typical direct composite analysis, the first antibody is specific for the biomarker, or covalently or passively linked to a solid surface. Typical solid surface is a glass or polymer, and the most commonly used polymers are cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride and polypropylene. The solid support can be in the form of tubes, beads, discs or microplastic, as well as any other surface suitable for conducting immunoassay. The binding process is well known in this area and generally consist of cross-covalent binding or physical adsorption, and the complex antigen-antibody washed in the preparation of the test sample. Then aliquot portion of the sample to be tested is added to the solid phase complex and cuberoot for a certain time (for example, from 2 to 40 minutes, or during the night or any other suitable period), and in certain conditions (for example, from room temperature to 40°C or 25°C to 32°C inclusive), which allows you to connect any subunits presented in antibodies. After an incubation period subunit antibody in the solid phase is washed and dried, and then incubated with the second antibody is specific to a part of the biomarker. The second antibody is associated with a reporter group, which is used as an indicator of binding of the second antibody with a molecular marker.

An alternative method involves the immobilization of target biomarkers in the sample and subsequent contact immobilized target-specific antibody labeled or its reporter molecule. Depending on the number of targets and signal intensity of the reporter group linked objective (target) may be determinable by direct labeling of the antibody. In an alternative method, the second labeled antibody specific for the first antibody is introduced into contact with the complex goal (target)is the first antibody to obtain a tertiary complex target (target)-first antibody-second antibody. This complex is determined by the signal emitted by the reporter group. Under "reporter group", as used in the present description, refers to a molecule which, by its chemical nature generates an analytically identifiable signal which allows to identify the antibody bound to the antigen. The most commonly used in this type of analysis reporter groups are either enzymes, fluorophores, or molecules containing radionuclides (for example, radioactive isotopes) and chemiluminescent molecules.

In the case of enzyme immunoassay enzyme kongugiruut with the secondary antibody, generally by means of glutaraldehyde or periodate. However, as can be easily understood, there is a wide range of different methods of conjugation, who easily accessible to experienced specialist. Commonly used enzymes include, among others, horseradish peroxidase, glucose oxidase galactosidase and alkaline phosphatase. The substrates that are used with the specific enzymes are usually chosen to develop a measurable color change under the influence of the corresponding enzyme. Examples of suitable enzymes include alkaline phosphatase and peroxidase. In addition, you can use fluorogenic substrates that produce fluorescent product and not a chromogenic substrates noted above. In all cases, the enzyme-labeled antibody is added to the complex of the first antibody with a molecular marker on the I binding and subsequent laundering of the excess reagent. Then the complex of antibody-antigen-antibody add the solution containing the appropriate substrate. This substrate will interact with the enzyme linked to the second antibody, resulting in a qualitative visual signal, which can be quantified, usually by means of spectrophotometry, for indicating the quantity of the biomarker represented in the sample. Alternatively, fluorescent compounds such as fluorescein and rhodamine, can chemically combine with antibodies without altering their binding capacity. When activated by light irradiation with a specific wavelength antibody labeled with fluorochrome, absorbs light energy inducyruya state of excitability in the molecule, which is accompanied by light emission characteristic color tones that visually determined under a light microscope. As well as EIA (enzyme immunoassay analysis), a fluorescently labeled antibody is allowed to communicate with the complex of the first antibody-molecular marker. After washing off the unbound reagent, the remaining tertiary complex is subjected to irradiation with light of appropriate wavelength, and the observed fluorescence indicates the presence information of interest to the researcher molecular marker. Both techniques (immunofluorescence assay and EIA) is firmly rooted in Dan the Oh region. However, you can use other reporter groups, for example, radioisotopes, chemiluminescent or bioluminescent molecule.

Implied in the question that the above techniques can also be used to detect the expression of GalNac-T14.

The methods of the present invention include protocols which examine the presence and/or expression of GalNac-T14 in a sample of tissue or cells. Evaluation methods mRNAs in cells are well known and include, for example, hybridization assays using probes complementary DNA (in particular, in situ hybridization using labeled ribosomal GalNac-T14, Northern blot and related techniques)and various analyses amplification of nucleic acids (e.g., RT-PCR [polymerase chain reaction with reverse transcription] using complementary primers specific GalNac-T14, and other methods of determining amplification type, such as a method of amplification, branched DNA, SISBA, TMA and the like).

Samples of tissue or mammalian cells can be easily analyzed, for example, mRNA GalNac-T14, using options analysis, Northern blot, dot blot or PCR. For example, analyses of the type RT-PCR, such as quantitative PCR, well known in this field. In the illustrative embodiment of the invention a method of detecting mRNA of GalNac-T14 in bio is ogicheskom the sample includes the production of cDNA from the sample by reverse transcription using at least one primer; the thus obtained amplification of cDNA using polynucleotide GalNac-T14 as sense and antisense primers to amplify cDNA GalNac-T14 in the sample; and detecting the presence of amplified cDNA GalNac-T14. In addition, such methods can include one or more steps that allow one to determine the mRNA level of GalNac-T14 in a biological sample (for example, by simultaneous research of a comparative control mRNA sequence of this gene is "household"as a member of a family of actin genes). Optionally, you can define the sequence of the amplified cDNA GalNac-T14.

The tangible embodiments of this aspect of the invention include primers GalNac-T14 and a pair of primers that provide specific amplification according to the invention of polynucleotides or any specific parts of polynucleotides, and probes that selectively or specifically hybridize with nucleic acid molecules considered in the present invention, or with any parts of amino acids. The probes may be in the state of detectable marker such as a radioisotope, fluorescent compound, bioluminescent compound, chemiluminescent compound, a chelator of metals or enzyme. Such probes and primers can be used clavioline presence of polynucleotides GalNac-T14 in the sample, and also as a means to identify the cellular protein expression GalNac-T14. As will be clear to an experienced specialist, on the basis presented in the present description of the sequences can be made so many different primers and probes that can be used effectively to amplify, clone and/or determination of the presence and/or level of mRNA GalNac-T14.

Optional methods of the invention include protocols that in a sample of tissue or cells on the basis of microarray technology to investigate or detect the mRNA, for example, mRNA GalNac-T14. When using a microarray of nucleic acids subjects and control mRNA samples from subjects and control tissue samples are subjected to reverse transcription and tagging to generate cDNA probes. Then the probes hybridizing matrix of nucleic acids immobilized on solid support. The matrix is configured as a sequence, and position of each member of this matrix is known. For example, the selection of genes that have the potential expression under certain conditions, can be built on a solid support. Hybridization of the labeled probe with a specific member of the matrix indicates that the sample from which the probe expresses this gene. Analysis of the diseased tissue on the expression of various genes can give valuable the information. The microarray technology uses techniques of hybridization of nucleic acids and computer technology to assess the profile of mRNA expression of thousands of genes within a single experiment (see, for example, WO 01/75166, published on October 11, 2001, 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) for more detailed information on the manufacture of matrices). The DNA microarray are miniature matrix containing fragments of genes that are either directly synthesized on glass or other substrates, or transferred to them. A single matrix usually contains thousands of genes. A typical experiment with a microchip includes the following stages: 1) preparation of fluorescently labeled target RNA extracted from the sample; 2) hybridization of the labeled target with a microchip; 3) washing, staining and scanning of the matrix; 4) analysis of the scanned image, and 5) generation of gene expression profiles. Currently there are two main types of microarrays DNA oligonucleotide matrix (usually from 25 to 70 monomers) and matrix gene expression, containing the PCR products derived from cDNA. When forming the matrix oligonucleotides or can be made in advance and transported to the surface, or directly synthesized on the surface in situ).

System Affymetrix GeneChip®is a selling system microarray, which contains the matrix obtained by direct synthesis of oligonucleotides on a glass surface. Matrix probe/gene:oligonucleotides, typically from 25 monomers are synthesized directly on the glass substrate by combining technologies of photolithography on the semiconductor base and solid-phase chemical synthesis. Each matrix contains up to 400000 various oligomers, and each oligomer represented by the millions of copies. Because oligonucleotide probes are synthesized on the matrix in known positions, the nature of hybridization and signal intensity can be interpreted in terms of the identity of genes and levels of relative expression using the software Affymetrix Microarray Suite. Each gene is represented on the matrix series of different oligonucleotide probes. Each pair of probes consists of the oligonucleotide with the ideal line and the oligonucleotide with poor compliance. Probe with perfect match is a sequence completely complementary to a specific gene, so that it can measure gene expression. Probe with lack of compliance differs from the probe with perfect match replacement of one base in a Central position, due to which naruse is by linking the transcript of the target gene. This helps to determine the background and non-specific hybridization, which gives the contribution to the signal measured by the oligonucleotide with a perfect match. Software Microarray Suite subtracts the intensity of hybridization of the probes with a lack of conformity of the intensity of hybridization of the probe with perfect match, determining the absolute or specific intensity for each set of probes. The probe selection based on current information from gene Bank (Genbank), and other repositories of oligonucleotides. I believe that these sequences recognize a unique region on the 3'end of the gene. Hybridization oven GeneChip (oven "rotisserie") is used for the simultaneous hybridization to 64 matrices. The fluidics station produces laundering and dyeing matrix probes. This station is fully automated and contains four modules, each module processes one matrix probes. Each module is independently controlled by software Microarray Suite using pre-programmed protocols inkjet automation. As the scanner is used confocal laser fluorescent scanner, measuring the intensity of fluorescence emitted from labeled crnc associated with a matrix of probes. The fluidics station and scanner controls the slave computer is tea station with software Microarray Suite. Running software Microarray Suite can be up to eight stations inkjet technique that uses pre-programmed protocols hybridization, washing and staining of the matrix probes. This software also captures data on the intensity of hybridization and converts them into a signal presence/absence for each gene, using appropriate algorithms. And finally, this software can identify changes in gene expression in a series of experiments by means of comparative analysis, and formats the output as text files (with .txt)that can be used in other programs for further analysis.

To detect the mRNA expression of the biomarker using labeled probes can also be used fluorescent in situ hybridization (FISH). Analyses of this type are well known in the art (see, e.g., Kallioniemi et al., 1992, U.S. patent 6358682).

The expression of selected biomarkers can be assessed by examining the deletion of the gene or gene amplification. A deletion or amplification of the gene can be measured on the basis of any of the many protocols that are known in this field, for example, traditional techniques southern blotting, Northern blotting to quantify the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)), the dot-nl is Tinga (DNA) or in situ hybridization (e.g., FISH) using respectively labeled probe, cytogenetic methods or comparative genomic hybridization (CGH) using respectively labeled probe. As an example, these methods can be used to identify deletions or gene amplification GalNac-T14.

Additionally, you can examine the methylation status of a biomarker, for example, a gene of GalNac-T14 in a sample of tissue or cells. In immortalized and transformed cells often occurs abnormal demethylation and/or hypermethylation of the Islands in the 5'-regulatory regions of the gene that causes changes in the expression of different genes. In this area there are many tests to study the methylation status of the gene. For example, in the approaches of the type southern hybridization to assess the methylation status of CpG Islands can be used is sensitive to methylation of restriction enzymes, which are able to cleave sequences that contain methylated CpG sites. In addition, to quickly outline the status of methylation of all CpG sites represented in the CpG Islands of this gene may MSP (PCR specific for methylation). This procedure includes an initial modification of DNA by sodium bisulfite (which converts all neetilirovannye cytosine in uracil) and subsequent amplification using primers specific for methylated (unlike demetilirovanny) DNA. Protocols, including intervention in 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 Are Prev., 1998, 7:531-536); and Lethe et al., Int. J. Cancer 76(6): 903-908 (1998).

The expression of GalNac-T14 in a tissue sample or cells could also be investigated using functional or based on activity analysis. For example, you can spend well known in this field of analysis for determining or identifying the presence of the activity of a specific enzyme (N-acetylgalactosaminyltransferase) in a tissue sample or cells (see, for example, Bennett et al., J. Biol. Chem., 271:17006-17012 (1996); Wang et al., BBRC, 300:738-744 (2003); Hang et al., above, access from may 2005 to address www.sciencedirect.com).

In the methods of the present invention assumes that a sample of tissue or cells can also be examined for the expression of Apo2L/TRAIL or receptor in the sample that binds to Apo2L/TRAIL or agonistic antibodies to receptors of cell death. As described above, and other sources of information on this field of knowledge, at the present time believe that Apo2L/TRAIL binds at least five different receptors: DR4, DR5, DcR1, DcR2 and OPG. The expression of Apo2L/TRAIL, DR4, DR5, DcR1, DcR2 and/or OPG at the mRNA level and the protein level can be investigated using techniques known in this area, including those described in the present description. As the example to detect the presence in the sample of one or more of these molecules is possible to apply the above methods IHC. It is assumed that the ways in which a sample of tissue or cells are exploring not only the presence of the marker GalNac-T14, but also on the presence of, for example, DR4, DR5 or DcR1, from the same sample of tissue is possible to prepare different microscopic preparations, each of these drugs can be analyzed with the reagent that is specific for a particular biomarker or receptor. In an alternative embodiment of a tissue sample or cells can be prepared one microscopic preparation, using antibodies directed to each of the biomarker or the receptor according to the Protocol multicolor staining that allows you to visualize and identify relevant biomarkers or receptors.

After identifying that a sample of tissue or cell expresses GalNac-T14, suggesting that this tissue or cells sensitive to Apo2L/TRAIL or agonistic antibodies, receptors, cell death, it is assumed that the mammal is possible to introduce an effective amount of Apo2L/TRAIL or antibody receptor cell death for the treatment of diseases that suffering is a mammal, such as cancer or immunosensing disease. Diagnosis in mammals of different pathological conditions described in the present description, can be done an experienced practitioner. In this area is swesty and available diagnostic methods, which allow, for example, to diagnose or identify the mammal has cancer or IMMUNOSUPRESSIVE disease. For example, cancer can be identified by methods including, but not limited to, palpation, blood test, x-ray examination, magnetic resonance imaging and other Immune diseases also can be identified without too much difficulty.

Apo2L/TRAIL or antibody receptor cell death can be entered in accordance with known methods, for example, intravenous bolus (inkjet) or continuous drip infusion over a certain period of time, and through intramuscular, intraperitoneal, (inside)cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical (local) or inhalation route. Optional, drug administration can be performed through a mini-pump infusion, using a variety of marketed devices.

Effective dosages and schemes Apo2L/TRAIL or antibody receptor cell death can be determined empirically, based skills and experience in this field. You can use both single and multiple dosages of medicines. Currently, it is believed that an effective dosage or amount of Apo2L/TRAIL in isolated when is Anenii (without other drugs) can vary from about 1:g/kg to about 100 mg/kg of body weight per day. Interspecies conversion of dosages can be performed by a method known in this field, for example, as described Mordenti et al.,Pharmaceut. Res., 8:1351 (1991).

If you use the introduction of Apo2L/TRAIL in vivo, the normal dose can vary from about 10 ng/kg to 100 mg/kg body weight of the mammal per day, preferably from about 1 μg/kg/day to 10 mg/kg/day (depending on route of administration). Guidelines for specific dosages and methods of delivery in the body found in the literature (see, for example, U.S. patent No. 4657760, 5206344 or 5225212). It is expected that for different drug compounds and in various diseases will be effective in different formulations, and that the introduction aimed at a specific organ or tissue, will be insufficient to another organ or tissue, i.e. will require a different shipping method.

It is assumed that the treatments considered in this invention may be used in combination with additional options of therapy. One or more other therapies may include, but are not limited to, radiation therapy, cytokines, growth inhibitors, chemotherapeutic agents, cytotoxic agents, tyrosine kinase inhibitors, inhibitors of ras farnesyltransferase, angiogenesis inhibitors and inhibitors of cyclin-dependent kinases, which are known in the data is the second region and further defined with the above details. It is assumed that these additional treatments can be applied in the form of funds, independent of Apo2L/TRAIL or antibody receptor cell death. In addition, you can apply treatment options based on therapeutic antibodies that target tumor antigens, for example, Rituxan™ or Herceptin™and protivougonnye antibodies, for example anti-VEGF.

Preparation and dosing schedules for chemotherapeutic agents can be found on the manufacturers ' instructions or to determine empirically that it is available to experienced practitioner. Preparation 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 applied to Apo2L/TRAIL or agonistic antibodies, receptors, cell death, after, or simultaneously with them.

It may also be desirable antibodies directed against other antigens, such as antibodies that bind to CD20, CD11a, CD18, CD40, ErbB2, EGFR, ErbB3, ErbB4, vascular endothelial factor (VEGF) or other members of the TNFR family (such as OPG, TNFR1, TNFR2, GITR, Apo-3, TACI, BCMA, BR3).

Alternative or in addition the patient can be simultaneously enter two or more antibodies that bind the same or two or more different antigens, discussed in n the building description. Sometimes it may be helpful to further the introduction to the patient one or more cytokines. After the introduction of these tools, you can analyze the treated cells in vitro. If the treatment was carried out in vivo, the cells are subjected to the treatment of a mammal can be monitored in various ways well known to the skilled practitioner. For example, Pato(morpho)logical to investigate tumor cells by necrosis or analyze serum for signs of a response of the immune system.

For use in the patent claims described or outlined above, the present invention also provides ready-made kits or products. Such kits may contain drugs and carriers, divided into compartments and enclosed in one or more containers such as vials, tubes and the like, and each container contains one of the elements used in a particular way. For example, one container may contain a probe that has already Machen or may be mechen for subsequent detection. Such probe can be an antibody or polynucleotide, is specific to the protein of GalNac-T14 or gene GalNac-T14, or to genetic message, respectively. If the set uses the hybridization of the nucleic acid for detection of target nucleic acid is you, the set can also have containers containing nucleotide(s) for amplifying the sequence of the target nucleic acid, and/or the container containing the reporter tool, for example, a protein binding to Biotin (avidin or streptavidin), which is associated with a reporter group (enzymatic, fluorescent or radioisotopic label).

Set according to the invention typically will contain the above container, and one or more other containers with materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for use. The container can include a label indicating that the composition should be used for specific treatment or not for medical purposes, and on this label may also contain instructions for use in vivo or in vitro, for example, as described above.

Kits according to the invention have many applications. Typical usage is a kit including a container, a label on the specified container and a composition contained within the container, and the composition includes a primary antibody that binds to the polypeptide sequence GalNac-T14, and the label is and the specified container contains information about this composition can be used to assess the presence of proteins GalNac-T14 at least one type of mammalian cells, along with instructions for use the antibody GalNac-T14 for evaluating the presence of these proteins in at least one type of mammalian cells. In addition, the kit may contain a set of instructions and materials for obtaining a tissue sample, and using antibodies and probes on the same slice of the tissue sample. The set can include both primary and secondary antibody, and secondary antibody conjugated to a labeled, for example with an enzyme label.

Another possible application is a kit including a container, a label on the specified container and a composition contained within the container, and the composition includes polynucleotide that's hybrid with complement polynucleotide GalNac-T14 in harsh conditions, and the label on the specified container contains information about what this song can be used to assess the presence of GalNac-T14 in at least one type of mammalian cells, along with instructions for use of polynucleotide GalNac-T14 for evaluating the presence of DNA or RNA GalNac-T14 at least one type of mammalian cells.

Other components of the kit optionally include one or more buffers (e.g., Blo is yuushi buffer, a wash buffer, substrate buffer, and so on), other reagents, such as the substrate (for example, a Chromogen, which are susceptible to chemical change with the introduction of an enzymatic label, a solution to restore epitope, control samples (positive and/or negative control), control slides, etc.

EXAMPLES

Various aspects of the invention will be further described and illustrated by the following examples, none of which should not be construed as limiting the scope of the invention.

METHODS AND MATERIALS

Cell culture and cell line

The following line of human cells: line non-small cell lung cancer (NSCLC): H2122, A427, H647, SK-MES-1, H838, H358, H2126, H460, H1703, H2405, H650, H1568, H1666, H322T, SW1573, H292, H1650, H522, EKVX, H661, H23, LXFL 529, H226, A549, H1781, H1299, HOP 62, H2009, HOP-92, H1793, H1975, H1651, calu-1, H1435, HOP 18, H520, H441, H2030, H1155, H1838, H596, HLFa; cell line cancer of the pancreas: Panc 05.04, BxPC3, HPAC, SU.86.86, HuP-T3, PSN1, Panc 08.13, MiaPaCa-2, PA-TU-8988T, Panc 03.27, Capan-1, SW 1990, CFPAC-1, PA-TU-8902, Panc 02.03, Panc 04.03, PL45, Aspc-1, Hs766T, Panc 10.05, Panc1, Capan-2, HPAF-II and NHL: JEKO-1, SU-DHL-4, OCI-LY-19, SR, Farage, DOHH-2, Toledo, WSU-NHL, KARPAS-422, GRANTA-519, Pfeiffer, HT, SC-1, DB. Cell lines were obtained from Depository 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 under the Ana in medium RPMI-1640 with the addition of 10% heat-inactivated fetal bovine serum, 2 mmol L-glutamine and 10 mm HEPES.

Tests for cytotoxicity

To determine the number of living cells after treatment with Apo2L/TRAIL or DR5 antibody was used for analysis of MTT (CellTiter 96®- non-radioactive analysis of cell proliferation from Promega), which is a colorimetric analysis, based on the ability of living cells to restore soluble yellow salt tetrazole (MTT) to a blue crystals formazan. The MTT analysis was carried out by adding pre-mixed optimized dye solution to the cell culture in the wells of 96-hole of tablet containing different concentration of Apo2L/TRAIL or DR5 antibodies (from 0 to 1000 ng/ml). During a 4-hour incubation of living cells convert tetrazolium component of the dye solution in formosanus product. Then in wells with cell culture solution was added to the solubilization/stop reagent for dissolving formisano product and perform the recording of the absorption of light with a wavelength of 570 nm using a reader for 96-well plates (SpectraMax). Read the absorbance of light with a wavelength of 570 nm is directly proportional to the number of cells that are commonly used in analyses of proliferation. Although the maximum absorption for formisano product falls at 570 nm and its pure solutions look blue, the color of the sample to the CE analysis may not be blue, that depends on the number of formazan in the sample in relation to other components in the culture medium, including serum oxidized phenol red and unrestored MTT.

The number of cells was optimized by titration to obtain the analytical signal, approaching the upper limit of the linear range in this analysis. Because different types of cells have different levels of metabolic activity, the above procedure was carried out separately for each cell line. For most of the investigated tumor cells was used concentration of 5000 cells per well up to 20,000 cells per well.

Below is a step-by-step description of the tests:

1. Cells used for bioanalysis, were taken from the original (pure) cultures.

2. Determination of cell number and viability by staining Trifanova blue with subsequent suspendirovanie cells to a finite amount of from 5,000 to 20,000 cells per well.

3. The distribution of 50 μl of cell suspension in a 96-well plate.

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

5. Adding to the samples in the 96-hole tablet 50 μl of culture medium containing different concentration of Apo2L/TRAIL or DR5 antibodies (ranging from 0 to 1000 ng/ml). As control were used 50mkl culture medium (without Apo2L/TRAIL or DR5 antibodies) and 100 µl of culture medium (without cells).

Each experiment was conducted three times (in three sets of holes and on three different days). The total volume of material in the wells was 100 μl per well.

6. Incubation tablets at 37°C in humidified atmosphere with 5% CO2within 72 hours.

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

8. Incubation tablets at 37°C in humidified atmosphere with 5% CO2up to 4 hours.

9. Add 100 ál of the solution for solubilization/stop solution to each well.

10. Incubation tablets at 37°C during the night.

11. Record the absorbance of light with a wavelength of 570 nm using a reader for 96-well plate. To reduce the background created by the debris of cells, fingerprints and other non-specific sources, absorption, was used a reference wavelength of 750 nm.

12. As an empty value (blank) for the negative control was used the average values of absorption, which was subtracted from all other indices are read. To calculate the number of living cells (in percent) average values of absorption for each concentration of Apo2L/TRAIL or DR5 antibody divided by the average value of the values of absorption in the positive control (100% unprocessed [untreated] living cells).

13. Were constructed graphs of the percentage of living cells(Y-axis) depending on the concentration of Apo2L/TRAIL or DR5 antibodies (the X-axis, a logarithmic scale) with subsequent determination of IC50 values for a point on the axis (ng/ml), corresponding to 50% of living cells.

The Protocol of Affymetrix labeling

For all samples was taken reading OD260/280, and the samples were passed through a Bioanalyzer. Only been used 5 μg of total RNA of high quality.

A. Synthesis of the first cDNA strands:

1. Hybridization of the primer

DEPC-H2Aboutx álMixed vortex motion. Quickly rotate
RNA (5 µg)y álIncubated at 70°C for 10 minutes
Emissions (dilution 1:4 5 µg of the initial solution)1 álQuickly rotated and put on ice
Primer T7-(dT)241 ál
Volume12 ál

2. Temperature control

5x buffer of the first cDNA strands4 ál
Added 7 μl of a mixture left) to each sample
0.1 M DTT2 álMixed vortex motion. Quickly rotate
10 mm dNTP mix1 álIncubated at 42°C for 2 minutes
Volume7 ál

3. The synthesis of the first thread

Was added to each sample, 1 μl of SSII RT
SSII RT1 álMixed by pipetting up or down OR easy vortex motion. Quickly rotate
Total20 álIncubated at 42°C for 1 hour

B. Synthesis of the second cDNA strands

1. Set the reaction of the first threads on the ice. Spent a short centrifugation to knock down the condensation on the side walls of the tube.

2. Received the following master mix to the second thread:

H2Oh, DEPC treated91 ál
5x PE clanny buffer to the second thread 30 ál
10 mm dNTP mix3 ál
10 U/µl DNA ligase1 ál
10 U/µl DNA polymerase I4 ál
2 U/ál P-called H1 ál
Total130 ál

3. Added 130 μl of the second master mix 20 ál of the first cDNA strands (Final volume = 150 ál)

4. Mixed by pipetting up or down OR easy vortex motion. Quickly rotated.

5. Incubated at 16°C for 2 hours in a cooling water bath.

6. Added 2 μl of [10] T4 DNA polymerase. Mixed by pipetting up or down OR easy vortex motion. Quickly rotated.

7. Were incubated for 5 minutes at 16°C.

8. Added 10 μl of 0.5 M EDTA. Mixed easy vortex motion. Quickly rotated.

9. Started cleaning procedure cDNA OR stored material at -20°C for further use.

Purification of double-strand cDNA (cleaning module samples GeneChip)

1. Added 600 μl of binding buffer cDNA to 162 ál final preparation of the synthesis of double-strand cDNA.

Mixed vortex motion within 3 seconds.

2. Was bilalis in fact, h is about the color of the mixture is yellow (similar to binding buffer cDNA without cDNA synthesis rxn.)

If the mixture had orange or purple color, was added 10 μl of 3 M sodium acetate (pH 5.0) and stirred.

The mixture had to buy a yellow color.

3. Brought to 500 μl of the sample to the spin column for purification of cDNA that is inserted into the collecting tube with a capacity of 2 ml, and centrifuged for 1 minute with a velocity ≥8000 x g (≥10,000 rpm). Dumped the flowing fluid in *hazardous waste.

4. Have rebooted the spin column with the remainder of the mixture (262 μl) and centrifuged as above.

Dumped the flowing fluid in *hazardous waste and disposed collectively tube.

5. Transferred spin column in a new collecting tube with a capacity of 2 ml (included in the delivery). He pietravalle 750 ál of wash buffer cDNA in the spin column. Centrifuged for 1 minute with a velocity ≥8000 x g (≥10,000 rpm). Dumped the flowing liquid.

6. He opened the lid of the spin column and centrifuged the sample for 5 minutes at maximum speed (≤25000×g). Put the column in a centrifuge, using every second slot. Positioned caps adjacent nests, guiding them in the opposite rotation direction (i.e., if the rotation was clockwise, the caps should have been sent counterclockwise). This technique made it possible to avoid damage to the of lackow. Dumped the flowing fluid and the collecting tube in the waste.

7. Transferred spin column in a collecting tube with a capacity of 1.5 ml was Pietravalle 10 μl of an eluting buffer cDNA on the membrane of the spin column. Was convinced that an eluting buffer cDNA deposited directly on the membrane. Were incubated for 1 minute at room temperature and centrifuged for 1 minute at maximum speed (≤25000×g) for elution.

Configuring and running the IVT reaction Enzo: Kit for labeling of RNA transcript Bioarray HighYield (component No. 900182)

1. Used 10 μl of purified double-strand cDNA.

2. Received the following master mix IVT:

Distilled or deionized water12 ál
10x reaction buffer HY4 ál
A 10-fold the number of ribonucleotides labeled with Biotin4 ál
10x DTT4 ál
A 10-fold amount of the mixture with the inhibitor P-called4 ál
20-fold quantity of RNA polymerase T72 ál
Total30 ál

3. Added 30 μl of master mix IVT to 10 ál of double-strand cDNA (Total volume = 40 ml).

4. Mixed by pipetting up and down OR easy vortex motion. Quickly rotated.

5. Immediately put the tube in a water bath with a temperature of 37°C. cells were then Incubated for 5 hours.

6. If not carried out immediately purification RNA was stored material at -20°C.

Cleaning crnc labeled with Biotin (cleaning module samples GeneChip)

1. Added 60 µl of water to the IVT reaction and stirred vortex motion within 3 seconds.

2. Added 350 μl of binding buffer for IVT RNA to the sample and mixed vortex motion within 3 seconds.

3. Was added to the lysate 250 ál of ethanol (96-100%) and well mixed by pipetting. Not centrifuged.

4. Made the sample (700 ál) to the spin column IVT for cleaning crnc inserted into the collecting tube with a capacity of 2 ml were Centrifuged for 15 seconds at speeds ≥8000 x g (≥10,000 rpm).

5. Missed the eluate through the column again.

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

Dumped the flowing fluid in the **hazardous waste and disposed collectively tube.

6. Transferred spin column in a new collecting tube with a volume of 2 ml (included in the complex is the delivery).

7. Added 500 ál of wash buffer IVT for crnc and centrifuged for 15 seconds at speeds ≥8000 x g (≥10,000 rpm) to launder.

Dumped the flowing liquid.

8. He pietravalle 500 ál of 80% ethanol (volume/volume) in the spin column and centrifuged for 15 seconds at speeds ≥8000 x g (≥10,000 rpm). Dumped the flowing liquid.

9. He opened the lid of the spin column and centrifuged the sample for 5 minutes at maximum speed (≤25000×g).

Dumped the flowing fluid and the collecting tube in the waste.

10. Transferred spin column in a new collecting tube of 1.5 ml.

11. He pietravalle 11 μl of water without R is called directly on the membrane of the spin column. Gave the material to stand for 1 minute. Centrifuged for 1 minute at maximum speed (≤25000×g) for elution.

12. He pietravalle 10 ál of water without R is called directly on the membrane of the spin column. Gave the material to stand for 1 minute.

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

Quantitative determination crnc (product IVT)

In order to determine the yield of RNA was used spectrophotometric analysis. Took the condition that 1 OD at a wavelength of light 260 nm corresponds to 40 ág/ml RNA.

To determine the concentration of the purpose and purity of the sample was checked by OD at 260 nm and 280 nm.

Maintained the ratio of A260/A280 for pure RNA as close as possible to 2.0 (acceptable range is from 1.9 to 2.1).

For the quantitative determination crnc when using as starting material the total RNA was necessary to calculate a correction for the output crnc, reflecting the release of unlabeled total RNA. Applying the emission of 100%, used the following formula to determine the corrected output crnc:

Fixed output crnc = RNA (total RNK)(y)

RNA = number crnc measured after IVT (µg)

total RNK = starting amount of total RNA (ág)

y = fraction of cDNA reaction used in IVT

Fragmenting cDNA for the target drug

To slice used a fixed concentration crnc.

1. Added 2 μl of 5 × buffer to slice for every 8 ál of RNA with water.

20 µg crnc1-32 ál
5x buffer for framing8 ál
Water without R-callup to 40 ál

Total40 ál

2. Were incubated for 30 minutes pri°C. Immediately after incubation, put the material on the ice.

Getting hybridization of the target

1. Heated 20-fold the number of eukaryotic hybridization controls and oligo-B2 for 5 minutes at 65°C.

Set eukaryotic control hybridization of Affymetrix GeneChip, component No. 900362 (150 reactions)

2. Mixed easy vortex motion, dumped the speed of rotation.

3. Master mix (assuming the concentration of fragmented crnc 0.5 μg/μl):

td align="left"> Hu cot-1 DNA (1 mg/ml)
A standard set(ál)The final concentration
Fragmented crnc 15 mcg300.05 µg/µl
Oligo-B2 (3 nm)550 PM
20-fold emission control15of 1.5, 5, 25, 100 PM
(Bio B, C, D, Cre)
Sperm DNA herring30.1 mg/ml
Acetylated BSA30.5 mg/ml
300.1 mg/ml
2-fold MES buffer Hyb150
H2O64
Finite volume300

4. Distributed master mix aliquot portions (270 ml) in test tubes and added to each tube and 30 ál of fragmented crnc. Received hybridization mixture.

5. Immediately prior to use balanced probe matrix at room temperature.

6. Filled probe matrix 1 × MES buffer Hyb and incubated for 10 minutes in the oven with rotation (60 rpm) at 45°C.

7. Heated hybridization mixture for 5 minutes in a water bath with a temperature of 99°C.

8. Transferred hybridization mixture for 5 minutes in a water bath with a temperature of 45°C.

9. Centrifuged the hybridization mixture for 5 minutes at maximum speed.

10. Removed 1-fold MES buffer Hyb from the matrix probes.

11. Filled probe matrix 200 μl of hybridization mixture (from the upper layer).

12. To summarize partitions sealant Tough-Spots.

13. Was hybridisable probe matrix at 45°C and 60 rpm minute hours.

14. Washed, stained and scanned probe matrix according to Affymetrix protocols.

Materials Affymetrix

PositionProvidercatalog No.
Primer T7-(dT)24Biosearch Technolgiescustom
Control peaksin-house-
Superscript II/5× buffer first thread/0.1 M DTTInvitrogen18064-014
5× buffer to the second threadInvitrogen10812-014
10 mm dNTPInvitrogen18427-088
10 U/µl DNA ligase E. coliInvitrogen18052-019
10 U/µl DNA polymerase I of E. coliInvitrogen18010-025
2 U/ál R-co HInvitrogen18021-071
10 U/µl T4 DNA polymeraseInvitrogen18005-025
0.5 M EDTASigmaE-7889
Set ENZO for tagging transcript RNA with high yieldAffymetrix or ENZO900182 (ENZO)
The module for cleaning GeneChip sampleAffymetrix900371
Acetylated serum albumin bovineInvitrogen15561-020
Goat IgG - class reagentsSigmaI-5256
Antibodies against streptavidin (goat), biomineralogy vectorLabsBA-0500
R-phycoerythrin streptavidinMolecular ProbesS-866
20×SSPEBioWhittaker51214
Set eukaryotic controlAffymetrix900362
Water, class of molecular biologyAmbion9934
DNA is the ne Cot-1 Roche1-581-074
5 M NaCl without R-call and On-callAmbion9760
The non 0-30SigmaA-8082
10% tween-20Pierce Chemical28320
Acid monohydrate without MESSigmaM5287
Sodium salt MESSigmaM3885
Disodium salt EDTA, 0.5 mol solutionSigmaE7889
Tough Spots, stickers (labels)USA Scientific9902
Bake for GeneChip hybridization 640Affymetrix800139
The GeneChip scanner 3000 workstationAffymetrix00-0074
The fluidics stationAffymetrix00-0081
The autoloader with barcode reader Affymetrix00-0129

Quantitative PCR

Synthesis of cDNA:

ComponentVolume (μl)
10× buffer RT10
25× dNTP mixture4
10× random primers10
MultiScribe RT (50 U/µl)5
H2About without R-call21
RNA (100 ng)50
Finite volume100

Conditions of incubation:

25° for 10 minutes

37° for 2 hours

Reaction with TaqMan sequanorum detector ABI Prism 7700:

ComponentVolume (μl)
Universal master mix TaqMan PCR (2×)25
The TaqMan probe (20×) (Assays-on-Demand™)2,5
cDNA (100 ng)2
2About20,5
Finite volume50

Conditions of temperature cycles:

95° for 10 minutes

40 cycles: 95° for 15 seconds

60° for 1 minute

The TaqMan probes: Assays-on-Demand™ (TaqMan® MGB probes, FAM™ dye-labeled)

- Amplification of the endogenous control, GAPDH (at a concentration of 100 nm probe, and the concentration of forward and reverse primers and 200 nm) was performed to standardize the amount of RNA in the sample (cDNA)added to each reaction.

Relative quantification (quantitative determination) was performed using the standard curves. For quantification, normalized to the endogenous control, standard curves deduced for both the target and the endogenous reference. In each experimental sample, the amount of target and endogenous reference was determined by the corresponding normal curve. Then the number of the target divided by the number of endogenous benchmark, resulting in the normalized value for the target. One of the experimental samples served as a calibrator or sample of 1×. Then each normalized value of the target divided by the normalized value of the calibrator getting in the relative level of expression.

Experimental results:

The former is eriment were conducted using the above-described materials and methods. The results of the experiments are illustrated in figure 5-9, as discussed below.

Figure 5 is a brief diagram of the IC50 data obtained in the analysis of cell lines of non-small cell lung cancer ("NSCLC") on the sensitivity or resistance to apoptotic activity of Apo2L (a+0.5% fetal bovine serum "FBS" or 10% FBS) or monoclonal antibodies DR5 mab"cross-linked "XL" or unbound (+0,5% fetal bovine serum "FBS" or 10% FBS) according to the measurement results in MTT assays for cytotoxicity.

6 represents a short circuit IC50 data obtained in the analysis of cell lines of pancreatic cancer susceptibility or resistance to apoptotic activity of Apo2L (a+0.5% fetal bovine serum "FBS" or 10% FBS) or monoclonal antibodies DR5 mab"cross-linked "XL" or unbound (+0,5% fetal bovine serum "FBS" or 10% FBS) according to the measurement results in MTT assays for cytotoxicity.

Fig.7 is a brief diagram IC50 according to the data obtained in the analysis of cancer cell lines of non-Hodgkin's lymphoma ("NHL") on the sensitivity or resistance to apoptotic activity of Apo2L (+10% fetal bovine serum "FBS") or monoclonal antibodies DR5 mab"cross-linked "XL" or unbound (+0,5% fetal bovine serum "FBS" or 10% FBS) according to the measurement results in MTT assays on cyto is oxicet.

Fig is the comparison of the sensitivity (sen) or sustainability ("RES") of selected lines of cancer cells NSCLC, pancreatic cancer and NHL to DR5 antibodies and correlation with the expression of GalNac-T14 in the measurement of mRNA expression GalNac-T14.

Fig.9 is in the form of a bar chart graph showing the different cell line NSCLC, pancreatic cancer and NHL ranked (in descending order) at the level of mRNA expression GalNac-T14.

Apoptotic program in cell death plays an important role in the development and homeostasis of multicellular organisms (Danial et al., Cell, 116:205 (2004)). Intracellular stimuli can initiate apoptosis through inherent in the cells on the nature of the path that is associated with members of the superfamily of genes Bcl-2 and activation of apoptotic biomechanics caspase (Cory et al., Nat. Rev. Cancer, 2:647 (2002)). Some cytokines belonging to the superfamily of factor tumor necrosis (TNF), are able to activate apoptosis through not inherent in the cells of the way, interacting with certain receptors that contain functional “death domain” (DD), inducing apoptosis (Ashkenazi et al., Science, 281:1305 (1998)). The Fas ligand (FasL) stimulates apoptosis through Fas (Apo1/CD95), while the ligand Apo-2/associated with the TNF ligand, inducing apoptosis (Apo2L/TRAIL) triggers apoptosis through DR4 (TRAIL-R1) and/or DR5 (TRAIL-R2) (LeBlanc et al., Cell Death Differ., 10:66 (2003)). When linking your cognat the first ligand, these receptors bind adapting molecule FADD (associated with Fas death domain), and this molecule is attracted to the action of initiating apoptosis caspase-8 with the formation of inducing cell death signaling complex (DISC) (see, for example, Kischkel et al., EMBO J. 14:5579 (1995); Kischkel et al., Immunity, 12:611 (2000)). The complex DISC stimulates caspase-8, which, in turn, cleaves and activates the effector protease, for example, caspase-3, 6 and 7, which is of apoptotic program in cell death. In many types of cells crossed with natural cell by may further amplify the incoming cells from outside the signal of death (Scaffidi et al., J. Biol. Chem., 274:1541 (1999)). Apo2L/TRAIL induces apoptosis in many types of tumor cells, without affecting or little impact on normal tissue, and this allows you to think about the fact that this factor may be useful in cancer therapy (see, for example, Ashkenazi, Nat. Rev. Cancer, 2:420 (2002); Kelley et al., Curr. Opin. Pharmacol., 4:333 (2004)). The alteration of various components of the apoptotic pathways can reduce the sensitivity of the specific lines of cancer cells to Apo2L/TRAIL (Igney et al., Nat. Rev. Cancer, 2:277 (2002)).

In accordance with the methods and protocols described below, conducted various experiments and their data are presented on figure 10-15.

In order to investigate the sensitivity to activation of the receptor, the survival of cells was analyzed as a function of the concentration of Apo2L/TRAIL in the panel lines cancerous glue is OK man, including 23 adenocarcinoma of the pancreas, 18 malignant melanomas and 36 colorectal adenocarcinomas (figa, data not shown). The results of this analysis 29/77 (38%) cell lines were regarded as highly or moderately sensitive to Apo2L/TRAIL. The concentration of Apo2L/TRAIL needed to achieve the death of 50% of the cells in 29 cell lines were changed from 3 to 800 ng/ml, with an average value of 250 ng/ml

The panel of cell lines was also investigated on the profile of gene expression using a microchip with a set of 54613 gene probes. Although with a few exceptions, cell lines of pancreatic cancer and melanoma, which showed sharp or intermediate sensitivity to Apo2L/TRAIL, expressed significantly higher levels of mRNA of the enzyme O-glycosylation of ppGalNAcT-14 than the corresponding, but resistant cell lines (p=0.5×10-4for Fisher's exact test, iterative cutoff 750 for carcinoma of the pancreas and 300 for melanoma) (pigv). Most cell lines of colorectal cancer that is sensitive to Apo2L/TRAIL, showed high expression of mRNA related enzyme O-glycosylation of ppGalNAcT-3, although some resistant lines also expressed the gene that was expressed in a weaker, but still statistically significant difference (p=0,026 when atsec the e at the level of 2000) (figs, below). Exceptions in the entire panel were: (a) 5/29 (17%) cell lines that were sensitive, but expressed ppGalNAcT-14 or ppGalNAcT-3 below cutoff level; (b) 16/48 (33%) of resistant cell lines, which, however, showed the level of ppGalNAcT-14 or ppGalNAcT-3 above the cutoff. In the study the mRNA expression of other enzymes of O-glycosylation in colorectal cell lines increased level Fut-6 was detected in 10/12 (83%) sensitive and 6/24 (25%) of resistant cell lines (p=0,013, cutoff 200) (figs, above). Combined expression of ppGalNAcT-14 cells, pancreatic cancer and melanoma with Fut-6 cell lines of colorectal cancer showed a very strong correlation with sensitivity to Apo2L/TRAIL (p=1,83×10-7N=77). This set of genes correctly predicted sensitivity or resistance to 23/32 (72%) positive and 39/45 (87%) negative marker of cell lines, respectively.

Sensitivity to Apo2L/TRAIL also investigated in vivo in xenografts tumors. Five-day treatment with Apo2L/TRAIL mice bearing grafted tumors that were derived from Fut-6-positive cell lines of colorectal cancer Colo205 and DLD-1, resulted in regression of tumors and strong delay further progression (fig.10D). In contrast, tumors derived from Fut-6-negative cell lines colorectal the CSOs Colo320 cancer and RKO did not respond to such treatment.

Pre-incubation ppGalNAcT-3/Fut-6-positive cell line Colo205 with pan-inhibitor of O-glycosyltransferases benzyl-GalNAc (Delannoy et al., Glycoconj., 13:717 (1996)) significantly decreased the sensitivity to Apo2L/TRAIL (figa)that allows you to think about the functional relationship between O-glycosylation and signal Apo2L/TRAIL. For further investigation of this assumption as the target mRNA ppGalNacT-14, ppGalNacT-3 or Fut-6 small interfering RNA oligonucleotides (siRNAs). To avoid vaclavik effects for each gene were synthesized non-overlapping siRNAs followed by verification in quantitative RT-PCR of their ability to reduce the target expression (figa). Applicants additionally confirmed the specificity of siRNAs with mutant plasmid ppGalNacT-14, containing 6 “silent” nucleotide substitutions in the target area siRNAs (Editorial, Nat. Cell. Biol., 5:489 (2003)) (pigv). Transfection of cell lines ppGalNAcT-14-positive carcinoma of the pancreas PSN-1 and Hs294T melanoma material siRNAs ppGalNAcT-14 significantly reduced sensitivity to Apo2L/TRAIL, while siRNAs caspase-8, in accordance with expectations, provide almost complete protection (pigv). Similarly, transfection of cells of colorectal cancer DLD-1 or C170 material siRNAs GalNAcT-3 or Fut-6 significantly reduced the sensitivity to Apo2L/TRAIL (figs and 14C). In General, siRNAs GalAcT-14 reduced sensitivity to Apo2L/TRAIL in 4 of the 5 cell lines of pancreatic cancer and in 2 of the 2 cell lines of melanoma, whereas siRNAs ppGalNAcT-3 or Fut-6 (each) reduced sensitivity in the 2 cell lines of colorectal cancer 3. In contrast, transfection of cells PSN-1 or Hs294T material siRNAs GalNAcT-14 did not change the sensitivity to etoposide, a topoisomerase II inhibitor (fig.14D). Similarly, transfection of cells PSN-1 or C170 material siRNAs GalNAcT-14 or GalNAcT-3 did not affect sensitivity to staurosporine, the inhibitor of protein kinase a wide range (fige). Since etoposide, and staurosporin stimulate apoptosis through inherent in the cells the natural way (Wei et al., Science, 292:727 (2001)), these studies suggest that the enzymes of O-glycosylation is able to modulate the signals apoptosis through a pathway signals the cells from the outside.

Transfection of HEK293 cells material ppGalNAcT-14 led to cell death by cotransfection with DR4 or DR5, but not with the closely related receptors Fas and TNFR1 or agonist of the natural way of apoptosis Bax (fig.11D). Moreover, transfection of ppGalNAcT-14 increased sensitivity to Apo2L/TRAIL-resistant cell lines of melanoma H1568 (fige), and carcinoma of the pancreas PA-TU-8902 and PL-45 (fig.14F), but did not change the sensitivity to etoposide (data not shown). In General, overexpression of GalNAcT-14 was senzibilizirana to Apo2L/TRAIL 4 cell lines from 7.

The influence of the "knockdown" siRNAs ppGalNacT-14 or Fut-6 studies dovana on the processing of caspase, induced by Apo2L/TRAIL. In cells PSN-1 and DLD-1, transfected with control siRNAs, Apo2L/TRAIL induced almost complete processing of caspase-8, leading to the cleavage of Bid, caspase-9 and caspase-3 (fega). Transfection of siRNAs caspase-8 was warned of these phenomena. Knockdown ppGalNAcT-14 cells PSN-1 or Fut-6 in cells DLD-1 also significantly weakened induced by Apo2L/TRAIL processing of caspase-8, Bid, caspase-9 and caspase-3 (fega) and stimulation of the activity of caspase-3/7 (pigv). Resistant to Apo2L/TRAIL colon cancer cell lines RKO and SW1417, expressing ppGalNAcT-3 and Fut-6 at a low level, showed a similar block processing of caspase-8 (figa). Thus, the enzymes of O-glycosylation can modulate the way Apo2L/TRAIL above (earlier) phenomena, leading to activation of caspase-8.

Activation of caspase-8 requires complex DISC (Ashkenazi et al., Science, 281:1305 (1998)). The analysis DISC Apo2L/TRAIL (Kischkel et al., Immunity, 12:611 (2000)) in cells PSN-1 and DLD-1 indicates that knockdown ppGalNAcT-14 or Fut-6 reduces the involvement of FADD and caspase-8 in the complex DISC, the processing of caspase-8 associated with the DISC, and the stimulation of the enzymatic activity of caspase-8 associated with the DISC (Sharp et al., J. Biol. Chem., 280:19401 (2005)) (figs, 12D and 15B). Neither siRNAs ppGalNacT-14 or siRNAs Fut-6 did not significantly change the number of DR4 and DR5 in the complex DISC, and a dose-dependent binding of Apo2L/TRAIL with cells PSN-1 and DLD-1 expressing both DR4 and DR5 (figs and 15B, the data is not pre is presented). Thus, ppGalNAcT-14 and Fut-6, apparently, not modulate apoptosis through the damage of cell surface receptors or binding Apo2L/TRAIL. In line with this, the sensitivity to Apo2L/TRAIL in a panel of 77 cell lines did not show significant correlation with the expression of the cell surface cognatic signaling receptors DR4 and DR5 or fake receptors DcR1 and DcR2 (data not shown). Moreover, most of siRNAs against ppGalNAcT-14, ppGalNacT-3 or Fut-6 does not change the level of DR4 and DR5 on the cell surface PSN-1, C170 or DLD-1 (figs). Two siRNAs do not cause detectable reduction of DR4 and DR5 in certain cell lines (figs). However, other siRNAs against the same enzymes inhibit the apoptosis induced by Apo2L/TRAIL, with no impact on the level of receptors.

The extracellular domain (ECD) of the receptor DR5 man was expressed in cells of the ovary of the Chinese hamster, secreted protein was purified and subjected to acid hydrolysis, and the associated monosaccharides were analyzed (figa). In full compliance with the absence of the predicted sites of N-glycosylation in the ECD of the receptor DR5 applicants have not identified N-linked glycans. However, two samples from two independent experiments showed 3 mol GalNAc and 3 mol Gal per mol DR5 ECD (figa)that allows you to think about O-linked modifications of the three sites of the receptor DR5 erased newim Picanol GalNAc-Gal.

It is known that O-glycosylation of the protein is associated with a modification of Surinov or threonine. Using previously established a bioinformatics tool to predict potential sites of O-glycosylation (http://www.cbs.dtu.dk/services/NetOGlyc) (Julenius et al., Glycobiology, 15:153 (2005)), applicants have identified two such areas in the total sequence ECD long (DR5-L) and short (DR5-S) variants of splicing human DR5, and a third site within the area of alternative splicing (pigv). The first amino acid segment (74-77) contains three serine residue, the second (130-144) five residues threonine, and the third four residue threonine and three serine residue. Murine DR5 has a sequence similar to the first two segments, with two Suriname and four threonine respectively, whereas DR4 person also has two similar sequences, contains one serine and five threonines. To test whether these sites for posttranslational modification of DR5 was obtained a number of mutants DR5L and DR5S, with replacement by alanine or five threonines segment 130-144 (DR5L-5T, DR5S-5T), or the same five threonines and three Surinov segment 74-77 (DR5L-5T3S, DR5S-5T3S). The immunoblot DR5 antibodies from lysates of HEK293 cells, transfected DR5L or DR5S, reflects the presence of the expected ligaments DR5L or DR5S (figs). The antibody also revealed ligament DR5 higher molecular weight of the (MW), which become more abundant when cotransfection DR5L or DR5S material ppGalNAcT-14 compared to control (figs, asterisks). The abundance of such bundles with higher MW and growth of cotransfection ppGalNAcT-14 was significantly decreased when using DR5L-5T or DR5S-5T and almost eliminated when using DR5L-5T3S or DR5S-5T3S (compared with the structures of wild type). These results allow us to think about what chords with higher MW are O-glycosylated forms of DR5: ppGalNAcT-14 contributes to their education, and the progressive elimination of the predicted sites of O-glycosylation by substitutions by alanine gradually reverses this effect in the opposite direction. Transfection of HEK293 cells murine DR5 or DR4, DR5L, DR5S of man leads to cell death (fig.13D), with each DR5 mutant shows less activity compared with the corresponding structure of wild-type and DR5S-5T3S (which lost all three sites) have the weakest activity. Cotransfected material ppGalNAcT-14 significantly enhances the phenomenon of cell death using all designs DR4 and DR5, except DR5S-5T3S, which show much lower activity.

Most samples of normal tissues and tumor cells from cancers of skin, lung, pancreas, breast, ovary, endometrium, and bladder PU is Ira or from non-Hodgkin's lymphoma are the expression of mRNA ppGalNAcT-14 values below the cutoff defined at the level of 500 for most cancers and 200 for skin cancers (fige). However, a significant subset of tumor samples, varying from 10% for lobular (lobular) breast cancer up to 30% for lung cancer and diffuse large B-cell lymphoma, demonstrate excessive expression of ppGalNAcT-14. Some cancer samples show the level of mRNA expression, more than 1000 times greater than that of the corresponding normal tissues. Dynamic expression of ppGalNAcT-14 in cancer cells allows you to think about what this gene, and possibly other related enzymes could serve as useful biomarkers for the identification of tumors with high sensitivity to Apo2L/TRAIL.

O-linked glikana show considerable structural diversity and modulate various aspects of the biology of membrane proteins, including conformation, aggregation, directed migration, half-life, as well as cell adhesion and signaling activity (Hang et al., Bioorg. Med. Chem., 13:5021 (2005); Hanisch, Biol. Chem., 382:143 (2001)). Cancer cells often exhibit a sharp change in the profile of O-glycans, creating a unique carbohydrate antigens associated with tumors (Brockhausen, Biochim. Biophys. Acta, 1473:67 (1999); Dube et al., Nat. Rev. Drug Discovery, 4:477 (2005); Fuster et al., Nat. Rev. Cancer, 5:526 (2005)). O-glycosylation also plays an important role in homing tumor cells in the specification the definition foci of metastasis (Fuster et al., Cancer Res., 63:2775 (2003); Ohyama et al., EMBO J. 18:1516 (1999); Takada et al., Cancer Res., 53:354 (1993)). A significant subset of samples of primary tumors in many cancer diseases, including colon cancer and colorectal tumor samples of melanoma cells and chondrosarcoma demonstrate increased expression of the enzyme O-glycosylation of ppGalNAcT-14.

WAYS

Materials

Reagents for cell culture were purchased in the company Gibco (Invitrogen/Gibco, carlsbad, California), unlabeled soluble Apo2L/TRAIL was obtained as previously described (Lawrence et al., Nat. Mad., 7:383 (2001)), inhibitor of O-linked glycosylation benzyl-a-GalNAc was purchased in the company Calbiochem, and all other chemicals, including etoposide and staurosporine, Sigma Aldrich (St. Louis, Missouri).

Cell culture and cell line

All 119 cell lines of human carcinoma (name and directory are listed in supplementary data) were obtained from ATCC or DSMZ (Braunschweig, Germany) and cultivated at 37°C in an atmosphere with 5% CO2in RPMI1640 with the addition of 10% heat-inactivated fetal bovine serum, 2 mm L-glutamine and 10 mm HEPES without antibiotics such as penicillin/streptomycin. 293 sample embryonic kidney cells (catalog number CRL-1573) were also obtained from ATCC and cultured in 100% medium Needle (modification of Dulbecco) with the addition of 10% FBS. M is Tantra for O-glycosylation cell line CHO, CHO ldlD, was licensed by Dr. Monty Kreiger, company MIT (Boston, Massachusetts).

Tests for the assessment of survival and apoptosis

In order to determine the rate of IC50 with respect to Apo2L/TRAIL, cells carried with triple duplicate on 96-well tablets and gave them to adhere to the wells for 24 hours and then treated with recombinant Apo2L/TRAIL of the man in increasing concentrations (up to 1000 ng/ml). After 72-h incubation, they were subjected to the analysis of survival (MTT analysis [Pierce] or fluorescent survival analysis cells CellTiter-Glo [Promega]) in accordance with the Protocol of the manufacturer. Each experiment to estimate survival of the cells was repeated at least three times in serum with low (0.5%) and high (10%) content FBS, and cell lines with intermediate sensitivity was determined by the variability between the values of IC50 in independent experiments or between sera with high and low content of FBS. Applicants have identified a cell line as a sensitive, based on the induction of apoptosis by at least 50% of the cells at a concentration of Apo2L/TRAIL 1 μg/ml, and an intermediate sensitivity quantitative variability of apoptosis induced in independent experiments, or by differences between the low (0.5%) and high (10%) content FBS serum. Quantitative assessment of AP is ptosis was performed by the method of flow cytometrical analysis, determining the average percentage of the collected cells (sticky + floating in the environment), stained with annexin V (BD Pharmingen).

Hybridization microchip and data analysis

Total cellular RNA was obtained from untreated cells (3×106) using the RNeasy kit (Quiagen). Labeled crnc was obtained and hybridized with nucleotide microarrays (U133P GeneChip; Affymetrix Incorporated, Santa Clara, California)as previously described (Hoffman et al., Nat. Rev. Genetics, 5:229 (2004); Yauch et al., Clin. Cancer Res., 11:8686 (2005)). Scanned image files were analyzed using computer programs GENECHIP 3.1 (Affymetrix), Spotfire, GenePattern and Cluster/TreeView. In order to identify genes with the most differential expression in sensitive and resistant cell lines, the applicants were subjected values of gene expression analysis in variational filter, through which it was possible to exclude genes with minimal variation in the series of analyzed samples, testing them on multiple changes and absolute variability, comparing the ratio of maximum and minimum (max/min) and the difference between the maximum and minimum (max-min) with pre-calculated values and excluding genes that do not meet both conditions.

Design expression and retroviral transduction

The DNA fragment encoding ppGalNAcT-14, was cloned from cDNA collected in the form of a pool of cell lines, sensitive to Apo2L/TRAIL, and inserted in expressing plasmid pcDNA3.1 (Invitrogen) with an N-terminal FLAG label. Then this design was subjected to site-specific mutagenic effects (set for mutagenesis Quikchange, Stratagene) to generate silent mutant siRNAs having 4-6 swing changes base pairs in the sequence, homologous siRNAs, without changing the protein sequence. Mutations covers the area length of 10 base pairs in the center of the binding sequences of siRNAs from 19 base pairs. The DNA sequence for DR5Long and DR5Short, DR4, murine receptor for TRAIL, DR4, Fas (option 1), TNFR1 and Bax (beta version) were cloned from cDNA pools and inserted into the expression vector pRK (Genentech). Mutants of O-glycosylation DR5L and DR5S were generated by site-specific mutation that replaces four residue of threonine by alanine, Mut4×TA (T130, T131, T132, T135) or five residues of threonine by alanine, Mut5×TA (T130, T131, T132, T135, T143). Transient transfection of HEK293 cells with expression constructs of proapoptotic molecules was carried out in 6-hole tablets at a concentration of proapoptotic molecules 0.5 μg per well and the concentration of ppGalNAcT-14 or vector control 2.0 µg. For transfection cells were used lipofectamine 2000 in accordance with the Protocol of the manufacturer. After 48-hour incubation, the cells were analyzed for apoptosis.

To generate pet is virusnyh structures ppGalNAcT-14 and mutants were cloned into the retroviral vector pQCXIP (Clontech). The retroviral supernatant were generated using line helper cells NX-Ampho. Transfection of packaging cells was performed using calcium phosphate (Invitrogen). Supernatant were isolated 48 hours after transfection and added to the target cells along with 10 µg/ml polybrene. Then there was the stage centrifugation for 1 hour at a speed of 2700 rpm to enhance infection. After transduction, the cells were subjected to selection with 2 μg/ml puromycin.

Design of siRNAs and transfection protocols

Design of siRNAs against ppGalNAcT-14, ppGalNAcT-3, caspase-8 and DR5 were created by Dharmacon (Lafayette, Colorado) using proprietary criteria. We have selected the following sequence:

siGalNAcT-14 (1): 5' GACCATCCGCAGTGTATTA-dTdT 3' (=14-4) (SEQ ID NO:15)

siGalNAcT-14 (2): 5' ATACAGATATGTTCGGTGA-dTdT 3' (=14-6) (SEQ ID NO:16)

siGalNAcT-3 (1): 5' CCATAGATCTGAACACGTT-dTdT 3' (=3-2) (SEQ ID NO:17)

siGalNAcT-3 (2): 5' GCAAGGATATTATACAGCA-dTdT 3' (=3-7) (SEQ ID NO:18)

siFut-6 (1): 5' GUACCAGACACGCGGCAUA-dTdT 3' (=6-1) (SEQ ID NO:19)

siFut-6 (2): 5' ACCGAGAGGUCAUGUACAA-dTdT 3' (=6-2) (SEQ ID NO:20)

siCaspase-8: 5' GGACAAAGTTTACCAAATG-dTdT 3' (SEQ ID NO:21)

All siRNAs were purchased as a double-strand oligonucleotides RNA and transliterowany in the appropriate cell line with a final concentration of 25 nm siRNAs for each. As control were used siRNAs duplexes against non-target sequence (Dharmacon). Cells were transliterowany at the same time lipofectamine 2000 (Invitrogen) by reverse transfection, when these cells were added to the suspension with pre-made in tablets complexes lipid-siRNAs. The concentration of lipofectamine 2000 corresponded to the Protocol of the manufacturer. After 48-hour incubation, the cells were collected for mRNA analysis or incubated further with recombinant Apo2L/TRAIL man, etoposide or staurosporine in 24-72 hours for analysis on the survival or for 4, 8 or 24 hours for analysis by the method of Western blotting.

Inhibition of O-glycosylation using benzyl-a-GalNAc

Cell line Colo205 were grown in the presence of benzyl-a-GalNAc (2 mm or 4 mm) for 72 hours. After that, they were re-posted on the wells of 96-well plates and left there for 24 hours to attach with preservation of the inhibitor. Then, stimulated by increasing concentrations of Apo2L/TRAIL, as described and used in the analyses for the survival of cells.

Quantitative PCR

The expression level of transcripts GalNacT-14 and GalNacT-3 was assessed by quantitative RT-PCR carried out by standard techniques of TaqMan. The level of transcript were normalized gene ' household', GAPDH, and the results were expressed as normalized values in the expression (=2-DCt). Sets of primers/probes for GalNacT-14 (catalog No.: Hs00226180_m1_GT14), GalNacT-3 (catalog No.: Hs00237084_m1_GT3) and GAPDH (catalog No.: 402869) were zamapleny company Applied Biosystems (foster city, California.

Immunoprecipitate, analysis, Western blotting and antibodies

Immunoprecipitate: Monoclonal antibodies type anti-Apo2L (2E11; No. of access ATCC HB-12256), anti-DR4 (3G1 and 4G7, no access ATCC PTA-99) and anti-DR5 (3H3, no access ATCC 12534 and 5C7) were generated in the company Genentech Inc., using as antigens the drain proteins receptor Fc. Monoclonal anti-DR4 antibody (4G7) and anti-DR5 (5C7), used for thus Apo2L/TRAIL DISC, were conjugated with agarose using orientational set ImmunoPure Protein G IgG Plus (catalog No. 44990) from Pierce. Monoclonal anti-DR4 (3G1) and anti-DR5 (3H3) antibodies used for immunodetection DR4/5 immunoprecipitated DISC were biktimirova using the kit biotinidase EZ-link Sulfo-NHS-LC (catalog No. 21217) from Pierce. Apo2L/TRAIL with a label FLAG has been received and transversely stitched with the antibody anti-FLAG M2 (Sigma), as described in the literature (Kischkel, Immunity, 12:611 (2000)). These experiments were conducted as described previously for the analysis of Apo2L/TRAIL-FLAG + anti-FLAG DISC (Kischkel, see above). The experiments thus DR4/5 DISC was also conducted in accordance with the description, except that the monoclonal anti-DR4 (4G7) and anti-DR5 (5C7) antibodies were directly conjugated with agarose for thus (Sharp et al., J. Biol. Chem., 280:19401 (2005)).

Western blotting: 6-hole tablets were seeded cells and the rate of 5×l0 5cells per well. For knockdown experiments with RNK cells were treated with different siRNAs for 48 hours followed by treatment of the Apo2L/TRAIL for 4, 8 or 24 hours. After the indicated time intervals, cells were washed in chilled PBS on ice and literally in 1% solution of Triton X-100 containing hypotonic litany buffer (20 mm HEPES pH 7.5, 10 mm KCL, 1.5 mm MgCl2, 1 mm EDTA and 1 mm DTT). For each sample, 40 µg of protein were isolated in reducing conditions using 10% or 10-20% gradient SDS-polyacrylamide gels. After the transfer material on nitrocellulose membrane (Schleicher and Schuell) these membranes were incubated for 1 hour in 10% fat-free milk powder with a further 1 hour incubation with the following primary antibodies: goat antibody anti-caspase-3 (1:1000, R&D), rabbit antibody anti-caspase-8 (1:1000, Pharmingen), mouse antibody anti-caspase-9 5B4 (1:1000, MBL), rabbit antibody anti-Bid (1:1000, Pharmingen), rabbit antibody anti-DR5 (1:500, Cayman) or goat antibody anti-actin (1:200, Santa Cruz Biotechnology). Membranes are washed five times TBS/0,05% Tween, and then incubated with the appropriate conjugated with peroxidase and affinity purified secondary antibody (1:5000, Biorad) for 30 minutes. After that, the membrane five times washed, and developed using enhanced chemiluminescence (ECL, Amersham) and exposed to film Kodak Biomax.

Flow cytometry analysis of FAS

Surface expression of receptors of the TNF family (DR4 and DR5) was determined by the method of sorting of fluorescently activated cells (FACS), using flow cytometer FACS Calibur (Becton Dickinson Immunocytometry System, San Jose, California). Cell line C170 and PSN-1, transfetsirovannyh indicated siRNAs for 48 hours, stained with 10 μg/ml primary antibody 4G7 (anti-DR4) or 3H3 (anti-DR5), or mouse control IgG antibody for 1 hour at 4°C. Then cells were washed by PBS, and incubated for 30 minutes at 4°C with secondary goat protivolednym antibody conjugated with fluorescein (FITC) (Jackson Laboratories). After that, cells were analyzed by flow cytometry using flow cytometer FACS Calibur.

Analyses of caspase

The activity of caspase-3/-7 analyzed at 37°C in 40 μl Casanova buffer (50 mm HEPES [pH to 7.4], 100 mm NaCl, 10% sucrose, 1 mm EDTA, 0,1% CHAPS and 10 mm DTT)containing 100 μm fluorogenic peptide Ac-DEVD-AFC. Activity was measured continuously over a specified time for the release of the AFC DEVD-AFC, using the device type of molecular fluorimetry in the kinetic mode and with a pair of filters 405-510. To evaluate the activity of caspase 20 µg of total cell protein extracts Triton X-100) was used in 40 µl Casanova buffer (containing 100 μm DEVD-AFC).

Analysis of carbohydrates in DR5 obtained from CHO

Monosaccharide composition R5 from CHO cells was obtained after hydrolysis with 4 N. TN. Analysis of released monosaccharides were performed in the system HPLC Dionex BioLC, using anion-exchange chromatography high resolution in combination with pulse amperometric detector.

Animal studies and subcutaneous xenografts

Female Nude "Nude" mice (Jackson Laboratory, Bar Harbor, Maine, USA) before use in the experimental study were acclimatized for at least 1 week in vivarium company Genentech. All experimental procedures were approved by the departmental Committee of Genentech by the animal care and use (IACAUC). Mice were inoculable subcutaneously with 5×106cells at the animal Colo205 cells, DLD-1 and RKO individual or 20×106cells in animal cell carcinoma human colon Colo320HSR (culture collection of the American type). The dimension of the tumors was performed with a caliper with digital display, and the volume of tumor was calculated by the formula D~G (A= length B=width)2. After tumors had reached a volume of approximately 150-200 mm3mice are randomly divided into groups and treated by intraperitoneal injection or blank media (control)or Apo2L/TRAIL (60 mg/kg/day) with the 0-th to 4-th day of the study.

1. A method for predicting the sensitivity of a tissue sample or cells of a malignant tumor of a mammal to AGON is displayed antibody receptor DR4 or an agonistic antibody receptor DR5, including the stage of the research sample of tissue or cells in immunohistochemical analysis to detect the expression of GalNac-T14, where specified immunohistochemical analysis performed using antibodies against GalNac-TH, which bind the polypeptide GalNac-T14 person containing amino acids 1-552 or amino acids 39-552 listed in figure 4A (SEQ ID NO:12), and where the expression specified GalNac-T14 is a prognostic sign of the sensitivity of a specified sample of tissue or cells to the apoptosis-inducing activity of the antibody receptor DR4 or antibody receptor DR5.

2. The method according to claim 1, in which the indicated antibody against GalNac-T14 is a monoclonal antibody that binds to the polypeptide GalNac-T14 person containing amino acids 1-552 listed in figure 4A (SEQ ID NO:12).

3. 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.

4. The method according to claim 1, in which these malignant tumors are cells or tissue of pancreatic cancer, lymphoma, non-small cell lung cancer, colon cancer, colorectal cancer, melanoma or chondrosarcoma.

5. Method of inducing apoptosis in a tissue sample or cells of a malignant tumor mammals, including successive stages:
stage of the research sample cloth is or cells in immunohistochemical analysis for the detection of polypeptide GalNac-T14, where specified immunohistochemical analysis performed using antibodies against GalNac-T14 person that bind the polypeptide GalNac-T14 containing amino acids 1-552 or amino acids 39-552 listed in figure 4A (SEQ ID NO:12), and where the expression of the specified polypeptide GalNac-T14 is a prognostic sign of the sensitivity of a specified sample of tissue or cells to the apoptosis-inducing activity of agonistic antibody receptor DR4 or agonistic antibodies receptor DR5;
and after detection of the expression of the specified polypeptide GalNac-T14 stage of impacts on specified sample of tissue or cells an effective amount of an antibody receptor DR4 and antibody receptor DR5.

6. The method according to claim 5, further comprising a stage of study the expression of receptors DR4, DR5, DcR1 or DcR2 in a specified sample of tissue or cells.

7. The method according to claim 5, in which these malignant tumors are cells or tissue of pancreatic cancer, lymphoma, non-small cell lung cancer, colon cancer, colorectal cancer, melanoma or chondrosarcoma.

8. The method according to claim 5, in which these cells are treated with an effective amount of agonistic DR5 antibody.

9. The method according to claim 5, in which these cells are also exposed to chemotherapy(CSO) facilities(a) or radiation therapy.

10. The method according to claim 5, inwhich these cells are also treated with a cytokine, cytotoxic agent or inhibitor of cell growth.

11. The method of claim 8, where the said receptor antibodies bind DR5 receptor DR5, as indicated on figa (SEQ ID NO:5).

12. The method according to claim 5, where these cells are treated with an effective amount of antibody to DR4.

13. The method according to claim 5, where the cells of malignant tumor cells are non-small cell lung cancer.

14. The method according to claim 11, where the cells of malignant tumor cells are non-small cell lung cancer.

15. The use of antibodies or polynucleotide to detect the expression of protein GalNac-T1-4 or mRNA in a tissue sample or cells of a malignant tumor of a mammal to obtain a set or articles for predicting the sensitivity of a tissue sample or cells of a mammal to an agonistic antibody receptor DR4 or an agonistic antibody receptor DR5, where the expression specified GalNac-T14 is a prognostic sign of the sensitivity of a specified sample of tissue or cells to the apoptosis-inducing activity of the antibody receptor DR4 or antibody receptor DR5.

16. The application of clause 15, wherein the specified expression of GalNac-T14 evaluate the mRNA expression GalNac-T14.

17. Application of clause 15, wherein the specified expression of GalNac-T14 examined in immunohistochemical analysis.

18. The application indicated in paragraph 15, in which additional the stage but are study the expression of receptors DR4, DR5, DcR1 or DcR2 in a specified sample of tissue or cells.

19. The application of clause 15, wherein the specified malignant tumors are cells or tissue of pancreatic cancer, lymphoma, non-small cell lung cancer, colon cancer, colorectal cancer, melanoma or chondrosarcoma.

20. Application of a set or articles in the method for predicting the sensitivity of a tissue sample or mammalian cells to apoptosis induced by agonistic antibodies against DR4 or agonistic antibodies against DR5, where the set or the product includes a container with a label on the specified container with the specified container is composition, which includes the primary antibody, binding to GalNac-T14, and on the label of the specified container indicates that the composition can be used for detection of GalNac-T14 in at least one type of mammalian cell, and instructions for use the antibody GalNac-T14 to assess the presence of GalNac-T14 in at least one type of mammalian cell.

21. Application of a set or articles in claim 20, where the aforementioned antibody is a monoclonal antibody GalNac-T14.

22. Use set method for predicting the sensitivity of a tissue sample or cells of a mammal in apoptosis induced agonistic DR4 antibody or agonistic DR5 antibody, where nab is R contains a container with a label on the specified container, moreover, in the specified container holds a composition which includes polynucleotide that hybridizes with complementary polynucleotides GalNac-T14 in harsh conditions, and on the label of the specified container indicates that the composition can be used for detection of GalNac-T14 in at least one type of mammalian cell, and instructions for use of polynucleotide to assess the presence of RNA or DNA GalNac-T14 at least one type of mammalian cells.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, in particular toxicology and resuscitation science and can be used for early prognosis of course of acute poisoning with psychotropic drugs. At the time of patient's admission to hospital albumin fraction of blood serum is isolated. After that, general level of reduced thiols is determined, and if its value is lower than 220 mcmol/l development of negative disease dynamics is predicted.

EFFECT: method allows to increase efficiency of performed treatment in said category of patients.

1 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention refers to chlitin derivative photoproteins and to application thereof both as intracellular calcium indicators, and in cellular studies. Said proteins are produced by mutagenesis of a coding sequence of chlitin. Also, there are offered nucleic acids coding said protein, a vector containing said nucleic acids and a host cell carrying the vector. They can find application in genetic communication technologies for monitoring the cellular events associated with signal transmission and gene expression. Besides, photoproteins of the present invention can be used as intracellular calcium indicators in diagnostic techniques based on calcium concentration measurement in response to the various effects.

EFFECT: produced proteins exhibit enhanced bioluminescence, high affinity to calcium and prolonged light emission.

19 cl, 16 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: in order to realise the method level of ferritin in blood serum and bile bladder tissue, rang point is defined, and ferritin values in serum from 0 to 10 ng/ml, and bile bladder from 0 to 0.25 mg/l - are taken for 1 point, respectively, level of ferritin in serum 70 ng/ml is taken as 7 points, and in bile bladder tissue 0.75 mg/l is taken as 3 point, and if sum is 10 points, conclusion is made about non-destructive cholecystitis, and if sum is from 10 points conclusion about destructive cholecystitis is made.

EFFECT: application of invention allows to increase accuracy of diagnostics of bile bladder tissue destruction in acute cholecystitis.

3 ex

FIELD: medicine.

SUBSTANCE: invention relates to pharmacology. Medication for brain cell protection contains dominant protein extract from placenta tissue, obtained by chromatographic separation of protein fraction of 8-10 components with total molecular weight from 15 to 200 kDa, 70-80% of fraction consisting of protein with molecular weight about 70 kDa. In order to obtain said medication powdered placenta is extracted by means of alkalescent buffer in presence of inhibitors of proteolytic protein cleavage and centrifuged. Centrifugate is passed through chromatographic column with anion-exchange carriers balanced by means of the same buffer, column is washed from unbound material, proteins are eluted with neutral salt solution. Collected material is diluted with water bringing pH to subacid value and diluted substrate is successively passed through column with anion-exchange carrier and column with cation-exchange carrier, balanced with buffer solutions to pH about 6.0. Final product is obtained after sterilisation.

EFFECT: method application allows to obtain efficient medication of high purity, normalising nervous system functions.

3 cl, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, namely to surgery. Blood is sampled from cubital vein in patients with acute pancreatitis on 1-3 and 7-10 day from disease beginning. Level of blood serum myoglobin is determined by method of performing reaction of passive hemagglutination. If level of blood serum myoglobin on 1-3 day increase from 95 to 128 ng/ml, acute fatty pancreatic necrosis is diagnosed, if myoglobin level in blood serum is higher than 128 ng/ml, hemorrhagic pancreatic necrosis is diagnosed, and if level of myoglobin in blood serum on 7-10 day increases higher than 256 ng/ml, hemorrhagic pancreatic necrosis in stage of infection of pancreatic necrosis nidus is diagnosed.

EFFECT: method allows to correct drug therapy and individually ground tactics of treating patients with acute pancreatitis depending on severity of disease form and presence of infection.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: there is offered an arteriosclerosis diagnostic technique based on the detection of various combinations of specific polypeptide markers. To detect the presence or absence of polypeptide marker, capillary electrophoresis, gaseous-phase ion spectrometry and/or mass spectrometry are used.

EFFECT: method allows early high-probability minimally invasive arteriosclerosis detection.

13 cl, 1 ex, 5 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to leprology, and can be used particularly for the detection of specific liver disease in the leprosy patients. Blood serum of the leprosy patients is analysed to determine the level of US-M.leprae and DIS-BSA leprosy mycobacteria antigen antibodies, and also the concentration of alpha-1-antitrypsin. If observing the level of antibodies to two antigens increased as compared to a norm and the concentration of alpha-1-antitrypsin increased higher than 346.7 mg/dl, liver disease is detected in the leprosy patients.

EFFECT: method provides higher accuracy.

4 ex

FIELD: medicine.

SUBSTANCE: essence of invention includes selection of sample for testing from area of malignant affection in patient, suffering from cancer of large intestine, selection of control sample, measurement of the level of one or several genetic markers, selected from the group, comparison of measured levels in experimental samples with control ones. Reducing of levels of one or several genetic markers in comparison with control sample, indicates increased resistance to docetaxel. Also described are sets which allow to predict or realise monitoring of patient's response to docetaxel.

EFFECT: identification of genetic markers, by which it is possible to realise patient's response to chemical therapy by docetaxel.

18 cl, 9 ex, 3 tbl, 16 dwg

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention relates to molecular biology and can be used in designing agent and methods of modulating body functions associated with HGF/c-met signalling pathway. The invention discloses HGF/c-met polypeptide-antagonists which are mutant forms of HGF which contain a mutation in the N-terminal part of the β-chain and/or in its dimerisation part. The disclosed polypeptides have lower biological activity compared to wild type HGG and can be used in modulating activity of c-met, cell proliferation, cell migration and angiogenic cell activity.

EFFECT: invention describes a method of obtaining HGF muteins using DNA recombinant technology and agents which are necessary for its existence.

22 cl, 8 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: in order to predict insufficiency of saturation with oxygen of peripheral blood of pregnant women who have herpes-virus infection, content of glucose-6-phosphat-dehydrogenase and TNFα in peripheral blood is determined. Discriminant equation D=-0.589·G-6-PDG+{+1.23·TNFα} is solved. If value of discriminant function is equal or greater than 89.95, predicted is development of oxygen insufficiency, accompanied with reduction in peripheral blood of pregnant women of pO2 with titre of antibodies to Herpes simplex virus 1:12800 to 27.63±0.7 mm Hg, and of HbO2 to 90.2±0.47% (control: pO2 - 39.22±0.5 mm Hg; HbO2 - 95.3±0.27%).

EFFECT: increased accuracy of determining possibility of development of oxygen insufficiency in pregnant woman with herpes-virus infection.

3 tbl

FIELD: medicine, ophthalmology.

SUBSTANCE: in lacrimal liquid one should detect the content of interleukin 8 (IL-8) and that of interleukin 1 beta (IL-1β) to calculate prognostic coefficient (PC) due to dividing the first value by the second one by the following formula: At PC value being below 10.0 one should predict favorable disease flow, and at PC value being above 10.0 - unfavorable flow.

EFFECT: higher accuracy of prediction.

2 ex

FIELD: forensic medicine.

SUBSTANCE: for the purpose to detect the sequence of applied lesions at availability of several wounds, scratches and ecchymoses on a cadaver one should study the activity of alkaline peptides isolated out of affected tissue by the impact of blood neutrophils of healthy donors upon phagocytosis. Moreover, the highest stimulating effect belongs to the peptides isolated out of the lesion applied earlier. The method enables to detect the sequence of applied lesions more accurately and differentiate the repeated lesion applied 5 min later, or more.

EFFECT: higher efficiency and accuracy of detection.

2 ex, 2 tbl

FIELD: medicine, biochemistry.

SUBSTANCE: in blood serum one should detect the level of lactoferrin and biliary acids. At their ratio being equal to 5-17 it is necessary to detect chronic hepatitis of high activity.

EFFECT: higher accuracy of detection.

3 ex

FIELD: medicine.

SUBSTANCE: method involves determining cathepsin D activity in endometrium bioptate. The value being equal to or less than 0.1 units of enzymatic activity per hour, external genital endometriosis is diagnosed.

EFFECT: high accuracy of diagnosis.

1 tbl

FIELD: medicine.

SUBSTANCE: method involves studying lactoferrin content in blood serum and peritoneal exudates in postoperative period every day during the first three days. Lactoferrin concentration in blood serum being concurrently reduced by 0.02 mcmole/l or less and increasing lactoferrin concentration in peritoneal exudates by 0.04 mcmole/l or more, enteric detoxication is considered to be effective.

EFFECT: high quality of estimation.

2 tbl

FIELD: medicine.

SUBSTANCE: method involves determining plasminogen/plasmin, α2-macro-globulin, α1-antitripsin content at the first, third, fifth and tenth day. The plasminogen/plasmin level being equal to 66-74 mcmole/l or 100-120 mcmole/l, α2-macro-globulin level of 2.7-3.0 mcmole/l, α1-antitripsin content of 2.38-3.2 mcmole/l, systemic inflammatory response to purulent infection, light severity degree endotoxicosis is diagnosed and favorable disease outcome is predicted. The plasminogen/plasmin level being equal to 50-65 mcmole/l or 125-160 mcmole/l, α2-macro-globulin level of 2.3-2.6 mcmole/l, α1-antitripsin content of 3.3-4.0 mcmole/l, sepsis with organ and system dysfunction, moderate severity degree endotoxicosis is diagnosed and septic complication availability and lingering disease development course is predicted. The plasminogen/plasmin level being equal to 39-40 mcmole/l, α2-macro-globulin level of 1.58-2.08 mcmole/l, α1-antitripsin content of 5.0-6.2 mcmole/l, severe sepsis, septic shock, severe degree endotoxicosis is diagnosed and unfavorable disease outcome is predicted.

EFFECT: high accuracy of diagnosis.

5 tbl

FIELD: medicine, biochemistry.

SUBSTANCE: at testing one should precipitate high-molecular compounds with acetonitrile and register supernatant's spectral characteristics. Supernatant should be applied onto a paper filter, dried and put into solution containing aromatic aldehyde, acetone and concentrated hydrochloric acid taken at weight ratio of 70:5:1 to be kept for 2-3 min. Then it should be once again dried up to detect qualitative and semiquantitative content of oxidized tryptophan metabolites by intensity and chromatic shades. Moreover, by chromatic shades of yellow dyeing it is possible to detect the content of hydroxylated metabolites and by chromatic shades of violet dyeing - that of unhydroxylated ones.

EFFECT: higher significance of detection.

3 ex

FIELD: medicine, anesthesiology, resuscitation.

SUBSTANCE: in patients one should study the content of lactoferrin in peritoneal exudates during the 1st d of postoperational period and at decreased value being below 3500 ng/ml on should predict unfavorable result. The suggested method provides correction of possible postoperational complications that deteriorate the flow of peritonitis and lead to lethal result.

EFFECT: higher accuracy of prediction.

3 ex

FIELD: veterinary medicine.

SUBSTANCE: method involves determining low and middle molecular mass substances content in blood plasma and erythrocytes and general blood plasma albumin concentration. Integral index is calculated on basis of obtained values using formula II=100*S238-298(plasma)/S238-298(erythrocytes)*GAC, where S238-298(plasma) and S238-298(erythrocytes) are the low and middle molecular mass substances content in blood plasma and erythrocytes, respectively, determined from area of figures restricted by spectral curves in wavelength range of 238-298 nm and abscissa axis (conditional units2); GAC is the general blood plasma albumin concentration (g/l). The value being from 2.1 to 3.0, the first endotoxicosis degree is diagnosed. The value being from 3.1 to 4.5, the second endotoxicosis degree is diagnosed. The value being from 4.5 to 6.0, the third endotoxicosis degree is diagnosed. The value being greater than 6.0, the fourth endotoxicosis degree is diagnosed. The normal value is equal to 0.5-2.0.

EFFECT: high accuracy of diagnosis.

1 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: method involves separating blood serum proteins into fractions, determining albumins and alpha-2-globulins content and controlling their content changes during the disease development process. Gamma-globulin content is determined in per cent ratio with respect to total protein quantity. Then, changes in the fractions content are controlled from the first to the third week. Albumin content being in norm and alpha-2-globulins content becoming greater to the end of the first week by 30-50% when compared to normal value and dropping to norm at the second week end and gamma-globulin content increasing from norm by 10-30% to the second or the third week, high inflammatory process activity is to be diagnosed. Albumin content dropping by 10-30% from normal value at the second week, alpha-2-globulins content growing by 10-20% of norm and gamma-globulin content dropping by 30-50% at the second or the third week when compared to norm, low inflammatory process activity is to be diagnosed.

EFFECT: high accuracy and reliability of diagnosis.

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