Apoptotic apo2l/trail sensitivity by testing galnac-t14 expression in cells/tissues
SUBSTANCE: inventions concern methods and analyses for studying expression of one or more biomarkers in a mammal's tissue or cell sample; study kits and products are presented also. Identifying expression of GalNac-T14 molecules predicts sensitivity or specifies that the tissue or cell sample is supposed to be sensitive to apoptosis inducing agents, such as Apo2L/TRAIL.
EFFECT: information obtained by the analysis aiming at identifying GalNac-T14 expression in the tissue or the cell sample in a mammal can provide an attending physician with the information which can be used for prescribing an optimum treatment regimen for the patients suffering such diseases as pancreatic cancer, lymphoma, non-small cell carcinoma of lung, colon cancer, rectal cancer, melanoma or chondrosarcoma.
22 cl, 50 dwg, 6 tbl
This application claims the priority of provisional application U.S. number 60/708677, filed August 16, 2005, and on the provisional application U.S. number 60/808076, filed may 24, 2006, the contents of which is incorporated herein by reference.
The SCOPE TO WHICH the INVENTION RELATES.
Described here, the invention relates to methods and assays for identifying biomarkers that predict sensitivity of mammalian cells to Apo2L/TRAIL and/or antibody-receptor agonists death. More specifically, the present invention relates to methods and assays that detect molecules associated with the family of proteins GalNac-T, which predict the sensitivity of malignant mammalian cells to Apo2L/TRAIL or the antibody-receptor agonists death, such as antibodies, agonists DR4 or DR5.
PREREQUISITES TO the CREATION of INVENTIONS
In the prior art have been identified by various ligands and receptors belonging to the superfamily of factors tumor necrosis (TNF). These ligands included tumor necrosis factor alpha ("TNF-alpha), tumor necrosis factor beta ("TNF-β" or "lymphotoxin-alpha"), lymphotoxin beta ("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 CD95 ligand), the ligand Apo-2 (also called Apo2L or TRAIL), leagues who nd Apo-3 (also called TWEAK), APRIL, OPG ligand (also known as 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, published January 16, 1997; WO 97/25428, published July 17, 1997; Marsters et al., Curr. Biol., 8:525-528 (1998); Chicheportiche et al., Biol. Chem., 272:32401-32410 (1997); " Hahne " et al., J. Exp. Med., 188:1185-1190 (1998); WO 98/28426, published on 2 July 1998; WO 98/46751, published October 22, 1998; WO 98/18921, published may 7, 1998; Moore et al., Science, 285:260-263 (1999); Shu et al., J. Leukocyte Biol., 65:680 (1999); Schneider et al., J. Exp. Med., 189:1747-1756 (1999); Mukhopadhyay et al., J. Biol. Chem., 274:15978-15981 (1999)).
Induction of various cellular responses mediated by such ligands of the TNF family, usually begins with their binding to specific cell receptors. Some, but not all, of the ligands of the TNF family are associated with the "death receptors" cell surface and thereby induce different biological activity, activation of caspases, or enzymes that carry out the processes of cell death or apoptosis (Salvesen et al., Cell 91:443-446 (1997). Among the representatives of superselect the TNF receptors, identified so far included 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., 87:3127-3131 (1990); EP 417563 published March 20, 1991; Loetscher et al., Cell, 61:351 (1990); Schall et al., Cell 61:361 (1990); Smith et al., Science, 248:1019-1023 (1990); Lewis et al., Proc. Natl. Acad. Sci., 88:2830-2834 (1991); Goodwin et al., Mol. Cell. Biol., 11:3020-3026 (1991); Stamenkovic et al., EMBO J. 8:1403-1410 (1989); Mallett et al., EMBO J. 9:1063-1068 (1990); Anderson et al., Nature, 390:175-179 (1997); Chicheportiche et al., J. Biol. Chem., 272:32401-32410 (1997); Pan et al., Science, 276:111-113 (1997); Pan et al., Science, 277:815-818 (1997); Sheridan et al., Science, 277:818-821 (1997); Degli-Esposti et al., J. Exp. Med., 186:1165-1170 (1997); Marsters et al., Curr. Biol., 7:1003-1006 (1997); Tsuda et al., BBRC, 234:137-142 (1997); Nocentini et al., Proc. Natl. Acad. Sci., 94:6216-6221 (1997); vonBulow et al., Science 278:138-141 (1997)).
Most of these representatives of the family of TNF receptors have a common characteristic structure of cell surface receptors, including extracellular, transmembrane and intracellular region, while others are found in nature in the form of soluble proteins lacking transmembrane and intracellular who Omena. The extracellular part of the characteristic TNFR contains a repeating structure of the amino acid sequences of multiple domains, cysteine-rich (CRD), starting with NH2-end.
The ligand, called Apo-2L) or TRAIL, was discovered a few years ago as the representative of the TNF family of cytokines (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 native sequence Apo2L/TRAIL polypeptide is in the length of 281 amino acid type II transmembrane protein. Some cells can produce a natural soluble form of the polypeptide by enzymatic cleavage of the extracellular region of the polypeptide (Mariani et al., J. Cell. Biol., 137:221-229 (1997)). Crystallographic studies of soluble Apo2L/TRAIL demonstrate 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 Apo2L/TRAIL, unlike other members of the TNF family, has a unique structural feature in that three cysteine residue (at position 230 of each subunit in homotrimer) together coordinate the zinc atom and the binding of zinc I what is important for the stability of the trimmer and biological activity (Hymowitz et al., supra; Bodmer et al., J. Biol. Chem., 275:20632-20637 (2000)).
In the literature it was reported that Apo2L/TRAIL may play a role in modulation of the immune system, including autoimmune diseases such as rheumatoid arthritis [see, for example, Thomas et al., J. Immunol., 161:2195-2200 (1998); Johnsen et al., Cytokine, 11:664-672 (1999); Griffith et al., J. 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 various malignant cells, including tumors of the bowel, lung, breast, prostate, bladder, kidney, ovary and brain, as well as melanoma, leukemia, and multiple myeloma (see, e.g., Wiley et al., supra; Pitti et al., supra; 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 vivostudies on mouse models of tumors additionally suggest that Apo2L/TRAIL, alone or in combination with chemotherapy or radiation therapy can have a significant antitumor effect (see, e.g., Ashkenazi et al., supra; Walzcak et al., supra; Gliniak et al., Cancer Res., 59:6153-6158 (1999); Chinnaiyan et al., supra; Roth et al., Biochem. Biophys. Res. Comm., 265:1999 (1999); PCT application US/00/15512; stated the ku PCT US/01/23691). Unlike many types of malignant cells, most normal cell types are resistant to induction of apoptosis under certain recombinant forms of Apo2L/TRAIL (Ashkenazi et al., supra; Walzcak et al., supra). Jo et al. reported that the soluble form of Apo2L/TRAIL, marked polyhistidine, induced apoptosisin vitroin normal dedicated human hepatocytes, but not in hepatocytes, not owned by the person (Jo et al., Nature Med., 6:564-567 (2000); see also Nagata, Nature Med., 6:502-503 (2000)). It is believed that some of recombinant drugs Apo2L/TRAIL can vary on the basis of biological properties and biological activities in relation to pathological cells compared to normal, depending on, for example, from the presence or absence of tag-molecules, zinc content and the % content of the trimer (See, Lawrence et al., Nature Med., Letter to the Editor, 7:383-385 (2001); Qin et al., Nature Med., Letter to the Editor, 7:385-386 (2001)).
It was found that Apo2L/TRAIL associated with at least five different receptors. At least two of the receptors that bind Apo2L/TRAIL, contain a functional cytoplasmic domain of death. One such receptor was named "DR4" (and, alternatively, 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 December 7, 2000; US 2003/0036168 published 20 fevralya; US 6433147, issued August 13, 2002; US 6461823, issued October 8, 2002, and US 6342383, issued January 29, 2002).
Another receptor for Apo2L/TRAIL was named DR5 (alternative he was also named as Apo-2; TRAIL-R or TRAIL-R2, TR6, Tango-63, hAPO8, TRICK2 or KILLER) (see, e.g., Sheridan et al., Science, 277:818-821 (1997), Pan et al., Science, 277:815-818 (1997), WO 98/51793, published November 19, 1998; WO 98/41629 published on September 24, 1998; Screaton et al., Curr. Biol., 7:693-696 (1997); Walczak et al., EMBO J. 16:5386-5387 (1997); Wu et al., Nature Genetics, 17:141-143 (1997); WO 98/35986, published August 20, 1998; EP 870827, published on October 14, 1998; WO 98/46643, published October 22, 1998; WO 99/02653 published January 21, 1999; WO 99/09165, published on 25 February 1999; WO 99/11791, published March 11, 1999; WO 03/042367, published 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 August 2, 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). Like DR4, it was reported that DR5 contains a cytoplasmic death domain and is capable of passing the te signal apoptosis upon binding with the ligand (or binding molecule, such as antibody agonist that mimics the activity of this ligand). Crystal structure of the complex formed between the Apo-2L/TRAIL and DR5, described by Hymowitz et al., Molecular Cell, 4:563-571 (1999).
Upon binding with the ligand as DR4 and DR5 can trigger apoptosis, independently engaging and activating the initiator of apoptosis, caspase-8 molecules through the adaptor containing a death domain, called 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)].
It was reported that Apo2L/TRAIL also binds to receptors called DcR1, DcR2 and OPG, which presumably function as inhibitors, but not transductor signal (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); Mongkolsapaya et al., J. Immunol., 160:3-6 (1998); DCR2 (also called TRUNDD or TRAIL-R4) [Marsters et al., Curr. Biol., 7:1003-1006 (1997); Pan et al., FEBS Letters 424:41-45 (1998); Degli-Esposti et al., Immunity, 7:813-820 (1997)], and OPG [Simonet et al., supra]. In contrast to DR4 and DR5, receptors DcR1 and DcR2 not give the signal for apoptosis.
In the literature it was reported about specific antibodies, which bind to receptors DR4 and/or DR5. For example, anti-DR4 antibodies to receptor DR4 and having agonistic or apoptotic activity in some mammalian cells description the us, for example, in WO 99/37684, published on July 29, 1999; WO 00/73349, published July 12, 2000; WO 03/066661, published 14 August 2003. Also, see, e.g., Griffith et al., J. Immunol., 162:2597-2605 (1999); Chuntharapai et al., J. Immunol., 166:4891-4898 (2001); WO 02/097033, published December 2, 2002; WO 03/042367, published may 22, 2003; WO 03/038043, published may 8, 2003; WO 03/037913, published may 8, 2003; US 2003/0073187 published 17 April 2003; US 2003/0108516 published 12 June 2003. Also 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., “An Antibody to DR5 (TRAIL-Receptor 2) Suppresses the Growth of Patient Derived Gastrointestinal Tumors Grown in SCID mice”, Abstract, 2d International Congress on Monoclonal Antibodies in Cancers, Aug. 29-Sept. 1, 2002, Banff, Alberta, Canada; WO 03/038043, published may 8, 2003; WO 03/037913, published may 8, 2003; US 2003/0180296, published on 25 September 2003. In addition, they described some antibodies with cross-reactivity against both DR4 and DR5 receptors (see, for example, U.S. patent 6252050, issued June 26, 2001).
SUMMARY of the INVENTION
This invention relates to methods and analyses to study the expression of one or more biomarkers in a sample of mammalian tissue or cell sample, in which expression of one or more such biomarkers predicts the sensitivity of the tissue sample or cleto the aqueous sample to such agents, as Apo2L/TRAIL or antibody-agonists to DR5. In various embodiments, the implementation of this invention, methods and analyses study the expression of molecules in GalNac-T family of proteins, in particular GalNAc-T14 or GalNAc-T3.
As discussed above, most normal cell types develops resistance to the induction of apoptosis under the influence of some recombinant forms of Apo2L/TRAIL (Ashkenazi et al., supra; Walzcak et al., supra). It was also noted that some populations of pathological types of human cells (for example, some populations of malignant cells are resistant to induction of apoptosis under the influence of some recombinant forms of Apo2L/TRAIL (Ashkenazi et al., J. Clin. Invest., 1999, supra; Walczak et al., Nature Med., 1999, supra). Therefore, examining a sample of tissue of a mammal or cell sample on the expression of selected biomarkers by analyzing can be conveniently and effectively to obtain information useful for assessing appropriate or effective ways of treating patients. For example, information obtained as a result of analysis, aimed at identifying the expression of GalNac-T14 in a tissue sample or cell sample of a mammal, can provide a doctor with useful information that can be used to determine the optimal treatment regimen (with the use of Apo2L/TRAIL or antibody-receptor agonists death) for patients is now, suffering from such a disease as cancer, or disease associated with the immune system such as autoimmune disease.
The invention relates to methods for predicting the sensitivity of a tissue sample or mammalian cells (such as malignant cells to Apo2L/TRAIL or the antibody-receptor agonist death. In some embodiments, the implementation of the methods include obtaining a sample of tissue or cells of a mammal and the study of tissues or cells on the expression of GalNac-T14. The methods can be performed in a variety of formats, including analyses that determine the expression of mRNA and/or protein assays of enzyme activity and other methods discussed here. Determination of expression of GalNac-T14 in the above tissues or cells would predict that such tissues or cells are sensitive to apoptosis-induced activity of Apo2L/TRAIL and/or the antibody of receptor of death. In optional embodiments, the implementation of the tissues 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 of a mammal, the study of tissues or cells on the expression of GalNac-T14, and when determining that the specified pattern cloth is or cells expresses GalNac-T14, the impact on the specified sample of tissue or cells an effective amount of Apo2L/TRAIL or the antibody is an agonist of the receptor of death. Steps in how to study the expression of GalNac-T14 may be performed in various analytical formats, including studies to determine the expression of mRNA and/or protein, enzyme activity and other methods discussed here. In optional embodiments, the implementation of the methods also include the study of a sample of tissue or cells for the expression of the receptors DR4, DR5, DcR1 or DcR2. Optional sample of tissue or cells contain malignant tissue or cells. Optionally, a tissue sample or cells contains cells non-small cell lung cancer, malignant tumors of the pancreas, breast cancer cells or cells of non-Hodgkin's lymphoma.
Other methods of the invention include methods of treating diseases in a mammal, such as a disease associated with the immune system, or cancer stage providing a sample of tissue or cells of the mammal, the study of tissues or cells on the expression of GalNac-T14, and when determining that the given sample of tissue or cell expresses GalNac-T14, the introduction of the specified mammal an effective amount of Apo2L/TRAIL or antibody-receptor agonist death. Stage in these methods, the EC research is pressey one or more biomarkers can be performed in different analytical formats, including the study determine the expression of mRNA and/or protein, enzyme activity and other methods described here. In optional embodiments, the implementation of the methods also include the study of a sample of tissue or cells for the expression of the receptors DR4, DR5, DcR1, or DcR2. Optional methods include the treatment of malignant disease of a mammal. These optional methods in addition to the introduction of an effective amount of Apo2L/TRAIL and/or antibody-receptor agonist death include the introduction of a given mammal chemotherapeutic agents(a) or radiation therapy.
In additional embodiments, the implementation of the above-mentioned methods may include examination of tissue or cells of a mammal on the expression of other GalNac-T molecules, such as GalNac-T3.
Other variants of implementation are illustrated as an example, the following items:
1. A method for predicting the sensitivity of a tissue sample 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 of the mammal to detect the expression of GalNac-T14, where the expression specified GalNac-T14 predicts the sensitivity of a specified sample of tissue or cells to the apoptosis-inducing activity of Apo2L/TRAIL.
2. The method according to item 1, which indicated the expression of GalNac-T14 examined by determining the mRNA expression GalNac-T14.
3. The method according to item 1, which indicated the expression of GalNac-T14 examined using immunohistochemistry.
4. The method according to item 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 item 1, in which a sample of tissue or cells contain malignant tissue or cells.
6. The method according to item 5, in which these malignant cells are cells or tissue of pancreatic cancer, lymphoma, or non-small cell lung cancer.
7. Method of inducing apoptosis in a tissue sample or a mammalian cell, comprising the stage of:
a sample of tissue or cells of a mammal;
the research sample of tissue or cells to detect expression of GalNac-T14, and
after the detection of the expression specified GalNac-T14 impact on the specified sample of tissue or cells an effective amount of Apo2L/TRAIL.
8. The method according to item 7, which indicated the expression of GalNac-T14 examined by testing the expression of mRNA of GalNac-T14.
9. The method according to item 7, which indicated the expression of GalNac-T14 examined using immunohistochemistry.
10. The method according to item 7, further comprising a stage of research expression re is atarov DR4, DR5, DcR1 or DcR2 in a specified sample of tissue or cells.
11. The method according to item 7, wherein said tissue sample or cells contain malignant tissue or cells.
12. The method according to item 11, in which these malignant cells are malignant cells or tissue of pancreatic cancer, lymphoma, or non-small cell lung cancer.
13. The method according to item 7, in which these cells influence the effective amount of the polypeptide Apo2L/TRAIL containing amino acids 114-281 presented in figure 1.
14. A method of treating disease in a mammal, such as diseases associated with the immune system, or malignant disease, including stage:
a sample of tissue or cells specified mammal;
the research sample of tissue or cells to determine the expression of GalNac-T14, and
after determining the expression specified GalNac-T14 introduction to the specified mammal an effective amount of Apo2L/TRAIL.
15. The method according to item 14, which indicated the expression of GalNac-T14 examined by determining the mRNA expression GalNac-T14.
16. The method according to item 14, which indicated the expression of GalNac-T14 examined using immunohistochemistry.
17. The method according to item 14, further comprising a stage of study the expression of receptors DR4, DR5, DcR1 or DcR2 specified in TKA and or cell.
18. The method according to item 14, in which a sample of tissue or cells contain malignant tissue or cells.
19. The method according to paragraph 18, in which these malignant cells or tissue containing cells or tissue of pancreatic cancer, lymphoma, or non-small cell lung cancer.
20. The method according to item 14, in which the effective amount of the polypeptide Apo2L/TRAIL containing amino acids 114-281 presented in figure 1, enter the specified mammal.
21. The method according to item 14, in which the specified mammal also introduce chemotherapeutic agent (assets) or radiation therapy is also used.
22. The method according to item 14, in which the specified mammal also introduce a cytokine, a cytotoxic agent or a growth inhibitor.
23. The method according to item 7, wherein said polypeptide Apo2L/TRAIL associated with a molecule of polyethylene glycol.
24. The method according to paragraph 14, where the specified polypeptide Apo2L/TRAIL associated with a molecule of polyethylene glycol.
25. A method for predicting the sensitivity of a tissue sample or cells of a mammal antibodies to death receptor, comprising the stage of:
a sample of tissue or cells of a mammal;
the research sample of tissue or cells to determine the expression of GalNac-T14, where the expression specified GalNac-T14 predicts the sensitivity of a specified sample of tissue or cells and to optos-inducing activity of antibodies to receptor of death.
26. The method according to item 25, which indicated the expression of GalNac-T14 examined by determining the mRNA expression GalNac-T14.
27. The method according to item 25, which indicated the expression of GalNac-T14 examined using immunohistochemistry.
28. The method according to item 25, further comprising a stage of study the expression of receptors DR4, DR5, DcR1 or DcR2 in a specified sample of tissue or cells.
29. The method according to item 25, in which a sample of tissue or cells includes tissue malignant tumor or malignant cells.
30. The method according to paragraph 29, in which these malignant cells are malignant cells or malignant tissue tumors of the pancreas, lymphoma or non-small cell lung cancer.
31. The method according to item 25, in which antibodies to death receptor are anti-DR4 or anti-DR5 antibodies.
32. Method of inducing apoptosis in a tissue sample or a mammalian cell, comprising the stage of:
a sample of tissue or cells of a mammal;
the research sample of tissue or cells to detect expression of GalNac-T14, and
after the detection of the expression specified GalNac-T14 impact on the specified sample of tissue or cells an effective amount of antibodies to receptor of death.
33. The method according to item 32, which indicated the expression of GalNac-T14 examined by testing exp is ASCII mRNA GalNac-T14.
34. The method according to item 32, which indicated the expression of GalNac-T14 examined using immunohistochemistry.
35. The method according to item 32, further comprising a 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 item 32, wherein said tissue sample or cells contain malignant tissue or cells.
37. The method according to item 36, in which these malignant cells are malignant cells or tissue of pancreatic cancer, lymphoma, or non-small cell lung cancer.
38. The method according to item 32, in which these cells influence the effective amount of the antibody-agonist DR4 or DR5.
39. The method according to paragraph 38, in which these cells influence the effective amount of the antibody-DR5 agonist that binds to a receptor DR5, depicted on Figa.
40. The method of treatment of a mammal, for example, diseases associated with the immune system, or malignant disease, including stage:
a sample of tissue or cells specified mammal;
the research sample of tissue or cells to detect expression of GalNac-T14, and
after determining the expression specified GalNac-T14 introduction to the specified mammal an effective amount of antibodies to receptor of death.
41. SPO is about to paragraph 40, in which the specified expression of GalNac-T14 examine by identifying the mRNA expression GalNac-T14.
42. The method according to clause 40, which indicated the expression of GalNac-T14 examined using immunohistochemistry.
43. The method according to paragraph 40, further comprising the stage of studying the expression of receptors DR4, DR5, DcR1 or DcR2 in a specified tissue or cell.
44. The method according to item 40, in which a sample of tissue or cells contain malignant tissue or malignant cells.
45. The method according to item 44, in which these malignant cells or malignant tissue contains cells or tissue malignant pancreatic tumor, lymphoma, or non-small cell lung cancer.
46. The method according to paragraph 40, where the specified mammal is administered an effective amount of anti-DR4 or DR5 antibodies.
47. The method according to paragraph 40, where the specified mammal also introduce chemotherapeutic agent or radiation therapy is also used.
48. The method according to item 40, in which the specified mammal also introduce a cytokine, a cytotoxic agent or an inhibitor of growth factor.
BRIEF DESCRIPTION of DRAWINGS
Figure 1 shows the nucleotide sequence of cDNA ligand Apo-2 (SEQ ID NO:2) and derive from it the amino acid sequence (SEQ ID NO:1). "N" in 447 the position of the nucleotide sequence used for the hereafter the treatment nucleotide bases, which may be "T" or "G".
On Figa and 2B shows the nucleotide sequence of the cDNA (SEQ ID NO:4) full-length human DR4 and derive from it the amino acid sequence (SEQ ID NO:3). The corresponding nucleotide and amino acid sequence of human DR4 also described by Pan et al.,Science,276:111 (1997).
On Figa shows the sequence of 411 amino acids (SEQ ID NO:5) DR5 man, published in WO 98/51793 19 November 1998. In the prior art variant transcription of splicing human DR5. This alternative splicing encodes the DR5 sequence of 440 amino acids (SEQ ID NO:6) DR5 people shown on Figv and 3C, published in WO 98/35986 20 August 1998.
On Fig.3D shows the nucleotide sequence of the cDNA (SEQ ID NO:7) full-length human DcR1 and deriving from them the amino acid sequence (SEQ ID NO:8). The corresponding nucleotide and amino acid sequence of human DcR1 (and its separate domains) are also shown and described in WO 98/58062.
On File shows the nucleotide sequence of the cDNA (SEQ ID NO:9) full-length human DcR2 and deriving from them the amino acid sequence (SEQ ID NO:10). The corresponding nucleotide and amino acid sequence of human DcR2 (and its separate domains) are also presented in WO 99/10484.
On Figa shows the nucleotide sequence GalNac-T14 h the rights (SEQ ID NO:11) and derive from it the amino acid sequence (SEQ ID NO:12). These sequences are also described by Wang et al., BBRC, 300:738-744 (2003).
On FIGU shows the nucleotide sequence GalNac-T3 human (SEQ ID NO:13) and derive from it the amino acid sequence (SEQ ID NO:14). These sequences are also described in Bennett et al., J. Biol. Chem., 271:17006-17012 (1996).
Figure 5 presents summary data table on the IC50 obtained in the analysis of cell lines of non-small cell lung cancer ("NSCLC") on the sensitivity or resistance to apoptotic activity of Apo2L (+ 0,5% fetal calf serum "FBS" or 10% FBS) and monoclonal antibody to DR5 "DR5 ab, cross-linked "XL" or not cross-linked, to + 0.5% fetal calf serum "FBS" or 10% FBS), measured in MTT assays of cytotoxicity.
Figure 6 presents summary data table on the IC50 obtained in the analysis of cell lines of pancreatic cancer susceptibility or resistance to apoptotic activity of Apo2L (+ 0,5% fetal calf serum "FBS" or 10% FBS) and monoclonal antibody to DR5 "DR5 ab, cross-linked "XL" or not cross-linked, to + 0.5% fetal calf serum "FBS" or 10% FBS), measured in MTT assays of cytotoxicity.
Figure 7 presents summary data table on the IC50 obtained in the analysis of cell lines of non-Hodgkin's lymphoma ("NHL") sensitivity and resistance to apoptotic activity of Apo2L (+ 10% fetal calf serum "FBS") and monoclonal antibody to DR5 "DR5 ab", cross-linked "XL" or not cross-linked (+ 10% fetal calf serum "FBS"), measured in MTT assays of cytotoxicity.
On Fig presents a comparison of the sensitivity (sen) or resistance ("RES") of the selected cell lines NSCLC, pancreatic cancer and NHL to DR5 antibody and correlation with expression of GalNac-T14, measured by expression of mRNA GalNac-T14.
Figure 9 presents the histogram of different NSCLC cell lines, pancreatic cancer and NHL, located (in descending order) according to the levels of expression of mRNA samples GalNac-T14.
Figa-D illustrate the differential expression of specific enzymes of O-glycosylation in Apo2L/TRAIL-sensitive and-resistant malignant cell lines: (A) the variability of the cells was measured after incubation with different doses of Apo2L/TRAIL. IC50 for each cell line was calculated as the concentration of Apo2L/TRAIL, which gives a 50% loss of viability. Each experiment on the viability was repeated at least three times in the presence of low (0.5%) and high (10%) concentrations of fetal calf serum. Black, grey or unfilled symbols represent cell lines, which are the most sensitive, moderately sensitive or resistant to Apo2L/TRAIL, respectively. (B) levels of expression of mRNA ppGalNAcT-14 (a set of probes 219271_at) in cleocin the x lines of the pancreas and malignant melanoma. Cell lines distributed according to tissue type and sensitivity to Apo2L/TRAIL. Black, grey or where there's no shading columns indicate cell lines as in A. (C) the levels of expression of mRNA Fut-6 (top panel, a set of probes 211885_x_at) and ppGalNAcT-3 (bottom panel, a set of probes 203397_s_at) in cell lines of colorectal cancer. Cell lines are distributed as in B. P Values in panels B and C based on criteria Fisher correlation between the sensitivity of cell lines (including high and moderate) and mRNA expression above the threshold. (D) the Effect of Apo2L/TRAIL on the growth of the implanted tumor xenografts. Nude mice nude, posing GalNAcT-3/Fut-6-positive (left panel) or GalNAcT-3/Fut-6-negative (right panel) tumors were injected carrier or Apo2L/TRAIL (60 mg/kg/day intraperitoneally on days 0-4) and watched the tumor size (mean±SE, N=10 mice per group).
11 illustrates the modulation of specific enzymes of O-glycosylation modifies the sensitivity to Apo2L/TRAIL. (A) Colo205 cells pre-incubated with the enzyme inhibitor pan O-glycosylation benzyl-GalNAc (bGalNAc), was treated with Apo2L/TRAIL for 24 h and determined cell viability (DMSO=media-control). (B) Cells PSN-1 (carcinoma of the pancreas) and Hs294T (melanoma) was transfectional by caspase-8 or ppGalNAcT-14 siRNAs for 48 h, incubated with Apo2L/TRAIL for another 24 h and Oprah is elali cell viability. Duplex siRNAs against non-target sequence (Dharmacon) was used as control (NTC). (C) cells DLD-1 colorectal carcinoma was transfectional ppGalNAcT-3 or Fut-6 siRNAs and tested as in B. (D) HEK293 Cells co-transfectional plasmids encoding these genes in combination with ppGalNAcT-14 or vector control. Apoptosis was measured after 24 h by staining with Annexin V (left panel). Cells H1569 melanoma was transducible the retrovirus directing the expression of ppGalNAcT-14 or a control retrovirus; the resulting pools of cell lines was treated with Apo2L/TRAIL for 24 h and determined cell viability (right panel). Western blot analysis using anti-FLAG antibody was used to verify the expression labeled epitope ppGalNAcT-14.
Fig illustrates (A) analysis Casanova cascade induced by the action of Apo2L/TRAIL. Cells PSN-1 and DLD-1 were transfectional siRNAs against ppGalNAcT-14 or Fut-6, respectively, within 48 hours the Cells were treated with Apo2L/TRAIL for 4 or 8 h, and cell lysates were analyzed using Western blot turns with antibodies specific to caspase-8, Bid, caspase-9, caspase-3, or actin as a reference load. (B) Cells PSN-1 was transfectional ppGalNAcT-14 siRNAs as in A, were treated with Apo2L/TRAIL for 4 h and determined the enzymatic activity of caspase-3/7 in cell lysates. (C) Analysis of Apo2L/TRAIL DIS. Cells PSN-1 was transfectional ppGalNAcT-14 siRNAs as in A. Added FLAG-Apo2L/TRAIL (1 mg/ml) for 0-60 min, the cells were literally and subjected to thus anti-FLAG antibody. DISC-associated FADD, caspase-8, DR4, and was identified by Western blot turns. (D) Cells PSN-1 was transfectional, processed and subjected DISC thus as in C, and DISC-associated enzymatic activity of caspase-8 was measured as described previously (Sharp et al.,J. Biol. Chem.,280:19401 (2005).
Fig illustrates (A) monosaccharide analysis of recombinant human DR5 (long variant splicing)produced in CHO cells made using HPAEC-PAD (high performance anion exchange chromatography with pulsed amperometric detection). (B) Comparison of sequences of Apo2L/TRAIL receptors man (human DR5 length 440 A.K. form hDR5L", DR5 human short form 411 A.K. "hDR5S" and hDR4), DR5 mouse or rat (mDR5), human Fas (hFas) human TNFR1 (hTNFR1). The boxes indicate putative sites of O-glycosylation. (C) Western blot turns total cell lysates corresponding to D. DR5L-5T and DR5S-5T is a construct that contains 5 replacement threonine-to-alanine and DR5L-5T3S and DR5S-5T3S is a construct that contains 5 replacement threonine-to-alanine and three replacement of serine-to-alanine, respectively, in residues that are potential sites of O-glycosylation. (D) HEK293 cells co-who was respectively specified DR5 designs together with a vector or plasmid ppGalNAcT-14 for 48 h and apoptosis was measured by staining with Annexin V. (E) the levels of expression of mRNA for ppGalNAcT-14 (Affymetrix chip, a set of probes 219271_at) in samples of primary human tumors derived from malignant tumors of the skin (SCC=this form may cell carcinoma), lung, pancreas (Panc), breast, ovary (Ov), endometrium (Endo), bladder (Bla, TCC= perekhodnocletocny carcinoma) and NHL (FL=follicular lymphoma, DLBCL=diffuse both b-cell lymphoma). The average values of the expression in the samples indicated the grey horizontal stripe for each class. Shows the boundary values of 500 and 200 (melanoma), the corresponding data cell lines with Figv.
Fig illustrates (A) a decrease in the mRNA expression ppGalNAcT-14 or ppGalNAcT-3 in cells PSN-1 and DLD-1 after 48 h siRNAs knockdown using Taqman analysis. (B) expression of GalNAcT-14 restore in cells PSN-1 by transfection with the empty plasmid (Empty), GalNAcT-14 wild-type (GalNAcT-14) or GalNAcT-14 containing silent mutations siRNAs (GalNAcT-14 si(1)Mut) after siGalNAcT-14 (1) - mediated knockdown ppGalNAcT-14. (C) Negative regulation of ppGalNAcT-3 or Fut-6 under the action of interfering RNA inhibits induced by Apo2L/TRAIL cell death in cells C170 (colorectal cancer). Experimental procedure as in 11C. (Table 1) A) summary table of knockdown phenotypes under the action of siRNAs. Cell lines in which negative regulation of GalNAcT-14 or ppGalNAcT-3 and Fut-6, as a result, leads to the aside from the effect of Apo2L/TRAIL, marked showing less than (+) or more than 50% (++) protection of at least one of the test oligonucleotide siRNAs. (0) indicates no protection from Apo2L/TRAIL. (D), (E) After 48 h knockdown indicated siRNAs, cells were treated with increasing doses of etoposide or staurosporine (STS) for 24 h and tested the viability of the cells. (F) Retroviral ppGalNAcT-14, do overexpress PA-TU-8902 and pools of cell lines PL-45 were subjected to analysis of cell viability after treatment Apo2L/TRAIL. Western blotting using anti-FLAG antibody shows retroviral downregulation of ppGalNAcT-14 in these cells.
Fig (A) Western blotting induced by Apo2L/TRAIL cascade activation of caspase cell lines of colorectal cancer Colo205, sensitive to Apo2L/TRAIL, and resistant, RKO and SW1417. Cells were treated with 1000 ng/ml Apo2L/TRAIL for 8 and 24 h, and total cell lysates were subjected to Western-blotting using antibodies specific to caspase-8, Bid, caspase-9, caspase-3 and actin as a reference load. (B) Knockdown Fut-6 reduced the update pool and the activation of caspase-8 in Apo2L/TRAIL DISC in cells DLD-1. Experimental procedure corresponds to 12D. (C) expression of cell surface DR4 and DR5 was measured using FACS analysis in cells that were subjected to knockdown siRNAs against the indicated genes.
A DETAILED DESCRIPTION of IZABERETE THE OIA
The methods and techniques described here or on that link, mostly well understood and widely applied using conventional methodology by experts in the field, such as shirokopolosnye methods molecular cloning, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Accordingly, treatments, including the use of commercially available kits and reagents are typically performed in accordance with protocols established by the manufacturers and/or parameters, if not marked otherwise.
Before, as will be described in the methods and assays of the present invention, it should be clear that this invention is not limited to the particular methodology, protocols, cell lines, species or genera of animals, constructs, and reagents described as such, of course, may vary. Should also be understood that the terminology used in the description only for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention, which will be limited only by the attached claims.
It should be noted that here and in the accompanying claims forms in the singular include references to the plural number is a, unless the context expressly requires otherwise. Thus, for example, reference to "genetic alteration" includes many of these alterations and the reference to "a probe" includes reference to one or more probes and cash equivalents, known to specialists in this field, and so on.
All publications mentioned herein are included in the present description by reference to disclose and describe the methods and/or substances for which the publications are cited. Mentioned here are the publications cited in connection with their disclosure prior to the filing date of this application. Nothing here should be interpreted as an admission that the inventors are not entitled to refer to an earlier date of publication on the basis of an earlier priority date or the priority date of the invention. In the future, the effective date may differ from the dates presented here, and require independent verification.
Used herein, the terms "Apo2L/TRAIL", "Apo-2L" and "TRAIL" refers to a polypeptide 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 shown in Fig, as well as biologically active fragments, deletion variants with insertions or substitutions of the above sequences. In one embodiment, the polypeptide sequence comprises residues 114-281 Figure 1, and, optionally, consists of residues 114-281 Figure 1. Optionally, the polypeptide sequence contains residues 92-281 or residues 91-281 Figure 1. Polypeptides Apo-2L can be encoded native nucleotide sequence represented in figure 1. Optionally, the codon that encodes the remainder Pro119 (Figure 1), can be "CCT" or "CCG". In other embodiments, implementation of the fragments or variants are biologically active and have at least 80% amino acid sequence identity, more preferably at least 90% sequence identity, and even more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity to any of the above sequences Apo2L/TRAIL. Optionally, the polypeptide Apo2L/TRAIL is encoded by a nucleotide sequence that's hybrid in terms of severity with the encoding polynucleotide sequence presented in figure 1. This definition encompasses variants of Apo2L/TRAIL with substitutions, in which at least one natural amino acid is replaced by an alanine residue is. Specific options for Apo2L/TRAIL with substitutions include those in which at least one amino acid is replaced by an alanine residue. These variants with substitutions include options that are marked 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 (using the numbering shown in figure 1) is substituted by alanine residues. Optional, options Apo2L can contain one or more substitutions by alanine, which are referred to in Table I published PCT application WO 01/00832. Variants with substitutions include one or more substitutions of residues listed in Table I application WO 01/00832, published on 4 January 2001. This definition also encompasses native sequence Apo2L/TRAIL, isolated from the source of Apo2L/TRAIL or received recombinant or synthetic methods. Apo2L/TRAIL according to the invention includes polypeptides, referred to Apo2L/TRAIL or TRAIL that is described in the publication nos WO 97/01633 and WO 97/25428. The terms "Apo2L/TRAIL or Apo2L" are used to identify the main forms of Apo2L/TRAIL, which include Monomeric, dimeric or trimeric forms of the polypeptide. In all of numbering amino acid residues indicated in the sequence Apo2L, use the numbering according to Figure 1, if special education is ω unless otherwise noted. For example, "D203" or "Asp203" refers to the residue of aspartic acid at position 203 in the sequence presented in figure 1.
The term "extracellular domain Apo2L/TRAIL or Apo2L/TRAIL ECD" refers to a form of Apo2L/TRAIL, which is essentially free of the transmembrane and cytoplasmic domains. As a rule, ECD will have less than 1% of such transmembrane and cytoplasmic domains and preferably, will have less than 0.5% of such domains. It will be understood that any transmembrane domain(s)identified for the polypeptides of the present invention, identified in accordance with the criteria used in this field for identifying that type of hydrophobic domain. The exact boundaries of the transmembrane domain may change, but most likely not more than 5 amino acids at either end of the domain, installed originally. In preferred embodiments, the implementation of the ECD will comprise a sequence of the polypeptide is soluble extracellular domain, free of the transmembrane and cytoplasmic domains (and not associated with the membrane). The specific sequence of the extracellular domain of Apo-2L/TRAIL described in PCT publications No. WO 97/01633 and WO 97/25428.
The term "monomer Apo2L/TRAIL" or "monomer Apo2L" refers to covalent chain sequence of the extracellular domain Apo2L.
The term "dime the Apo2L/TRAIL" or "dimer Apo2L" refers to two molecules of Apo-2L, United covalent bond via a disulfide bond. Used here, the term includes stand-alone dimmers Apo2L and dimers Apo2L that are in the trimeric forms of Apo2L (i.e. associated with another, the third monomer Apo2L).
The term "trimer Apo2L/TRAIL" or "trimer Apo2L" refers to the three monomers Apo2L that are not associated ecovalence.
The term "aggregate Apo2L/TRAIL" is used to denote samoassotsiiruyutsya oligomeric forms of Apo2L/TRAIL of a higher order, such as trimers Apo2L/TRAIL, for example, review and nanoscale forms of Apo2L/TRAIL. The presence and quantity of monomer, dimer or trimer of Apo2L/TRAIL (or other aggregated forms) can be performed using the methods and assays known in the prior art (and using commercially available materials, for example, native exclusion HPLC (SEC), denaturing exclusion using sodium dodecyl sulfate ("SDS " SEC"), HPLC with reversed phase and capillary electrophoresis.
"Receptor ligand Apo-2" includes receptors known in the prior art "DR4 and DR5", polynucleotide and polypeptide sequence of which is shown in figure 2 and 3 respectively. Pan et al. described representative of the family of TNF receptors, named "DR4" (Pan et al., Science, 276:111-113 (1997); see also WO 98/32856, published July 30, 1998; WO 99/37684, published on July 29, 1999; WO 00/73349,published 7 December 2000; US 6433147, 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 November 19, 1998; WO 98/41629 published on September 24, 1998). This receptor called DR5 (this alternative receptor was named 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 March 11, 1999; US 2002/0072091, published August 13, 2002; US 2002/0098550, published 7 December 2001; US 6313269, issued December 6, 2001; US 2001/0010924, published August 2, 2001; US 2003/01255540, published 3 July 2003; US 2002/0160446, published on 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.,supra;Marsters et al.,supra; Simonet et al.,supra). The term "receptor Apo-2L" when used here covers the receptor with the native sequence and variants of the receptor. These terms cover the receptor for Apo-2L, expressed in a variety of mammals, including the people. The receptor for Apo-2L can be expressed endogenous, as occurs in nature in various tissues of origin of the person, or may be expressed by recombinant or synthetic methods. "The receptor for Apo-2L with native sequence" includes a polypeptide having the same amino acid sequence as the receptor for Apo-2L of natural origin. Therefore, the receptor for Apo-2L with a native sequence can have the amino acid sequence of the natural receptor for Apo-2L of any mammal. This receptor Apo-2L with a native sequence can be isolated from a natural source or may be obtained by recombinant or synthetic means. The term "receptor Apo-2L with native sequence" especially covers natural truncated or secreted forms of the receptor (for example, a soluble form containing, for example, the sequence of the extracellular domain), variants of natural forms (e.g. forms as the result of alternative splicing) and natural allelic variants. Variants of the receptor may include fragments or deletion mutants of the receptor Apo-2L with the native sequence. On Figa shows the sequence of 411 amino acids DR5 person, as published in WO 98/51793 19 November 1998. Variant transcription of splicing DR5 person from the local to the prior art. This alternative splicing encodes the DR5 sequence 440 amino acids DR5 people shown on Figv and 3C, as published in WO 98/35986 20 August 1998.
"The antibody to the receptor of death" in the context of the present invention applies mainly to the antibody or antibodies directed to the receptor superfamily receptor tumor necrosis factor, and containing a death domain that is able to transmit the signal apoptosis, and such antibodies include antibody to DR5 and antibody to DR4.
"The antibody to the receptor DR5 or 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, 1-411 sequence shown in Figa, or 1-440 sequence shown in FIGU-3C, or its extracellular domain. Optional antibody to DR5 fused or linked to a heterologous sequence or molecule. Preferably heterologous sequence allows the antibody to form a higher order oligomeric complexes, or contributes to this. Optional antibody to DR5 binds to a receptor DR5, but not bind or not cross-react with any additional receptor for Apo-2L (for example, DR4, DcR1 or DcR2). Optionally, the antibody is an agonist signaling activity of DR5.
Neobyazatel is, antibody to DR5 according to the invention binds to DR5 receptor in a concentration range from about 0.1 nm to about 20 mm, measured in the BIAcore analysis of binding. Optional antibodies against DR5 according to the invention demonstrate the value of Ic 50 of about 0.6 nm to about 18 mm, measured in the BIAcore analysis of binding.
"The antibody to the receptor DR4", "antibody to DR4 or anti-DR4 antibody" is used in a broad sense to refer to antibodies that bind at least one form of the receptor DR4 or its extracellular domain. Optional antibody to DR4 fused or linked to a heterologous sequence or molecule. Preferably heterologous sequence allows the antibody to form a higher order oligomeric complexes, or contributes to this. Optional antibody to DR4 is associated with the receptor DR4, but not linked or not cross-react with any additional receptor for Apo-2L (for example, DR5, DcR1 or DcR2). Optionally, the antibody is an agonist signaling activity of DR4.
Optional antibody to DR4 according to the invention binds to a receptor DR4 in the concentration range from about 0.1 nm to about 20 mm, measured in the BIAcore analysis of binding. Optional antibodies to DR4 according to the invention demonstrate the value of Ic 50 of about 0.6 nm to about 18 mm, measured in the analysis of St the statements BIAcore.
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 situorin vivo. Examples of such biological activities include binding of Apo2L/TRAIL to DR4 or DR5, including apoptosis, as well as those which are additionally reported in the literature. The agonist may act directly or indirectly. For example, the agonist may act for partial or full enhance, stimulate or activate one or more of the biological activities of DR4 or DR5,in vitro,in situorin vivoas a result of its direct binding to DR4 or DR5, which causes activation of the receptor or signal transduction. The agonist may also act indirectly, for partial or full enhance, stimulate or activate one or more of the biological activities of DR4 or DR5,in vitro,in situorin vivoas a result, for example, stimulation of other effector molecules, which then causes activation of DR4 or DR5 or signal transmission. It is assumed that the agonist can act as a molecule enhancer that acts indirectly to enhance or increase the activation or activity of DR4 or DR5. For example, the agonist may enhance the activity of Hohenlohe Apo-2L in a mammal. This can be accomplished, for example, by pre-complexing DR4 or DR5 or by stabilizing the complexes of the corresponding ligand with the receptor DR4 or DR5 (for example, stabilizing the native complex formed between the Apo-2L and DR4 or DR5).
The term "biomarker"as used in this application, refers mainly to the molecule, including gene, protein, carbohydrate structure or glycolipid expression in the tissue or tissues of a mammal, or in the cell, or the cell can be determined by standard methods (or methods described herein) and which predicts the sensitivity of cells or tissue from mammals to Apo2L/TRAIL or the antibody of receptor of death. Such biomarkers considered in this invention include, but are not limited to, molecules of the protein family GalNac-t Were described members of the family of genes and proteins N-acetylgalactosaminyltransferase ("GalNac-T") of a person (see, for example, Hang et al., “The chemistry and biology of mucin-type O-linked glycosylation initiated by the polypeptide N-acetylgalactosaminyltransferases”, Bioorganic & Medicinal Chemistry (available in may 2005 on www.sciencedirect.com) and references cited therein; Wang et al., BBRC, 300:738-744 (2003) and references cited therein), and it is believed that they function to determine the number and position of the O-linked chains of sugars to proteins. Optional is determined that the expression of such a biomarker is use expression, observed in the control sample of tissue or cells. Optional, for example, expression of this biomarker is determined using a microarray chip for gene expression, quantitative PCR or immunohistochemical (IHC) analysis. Optionally, the expression of the biomarker GalNac-T, such as GalNac-T14 or GalNac-T3, will be determined at the level of at least 750, measured using microcapsule research Affymetrix U133P, or 500 times, or preferably at least 1000 times higher in the sample under test tissue or a cell by detecting expression of the biomarker using quantitative PCR.
"UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-T14", "pp-GalNac-T14", "GalNac-T14", "GALNT14" is used here to denote a membrane protein type II, have characteristics of a family of molecules GalNac-T, containing N-terminal cytoplasmic domain, a transmembrane domain, a stem region and catalytic domain. In an additional embodiment, the molecule of GalNac-T14 person contains 1659 base pairs encoding a protein 552 amino acids, as shown in Figa. cDNA human full length was deposited in GenBank with the access number No. AB078144. As described by Wang et al., BBRC, 300:738-744 (2003), were identified splanirowannya isoforms GalNac-T14, which include (or not include) specific exons, so is e as exons 2, 3 and/or 4. The present invention addresses the study of the expression of any of these various isoforms of GalNac-T14, and that any expression of these isoforms predicts the sensitivity of the tissue sample, or mammalian cells to Apo2L/TRAIL or the antibody of receptor of death.
"UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-T3", "pp-GalNac-T3", "GalNac-T3", "GALNT3" are used in this description to denote membrane protein type II, have characteristics of a family of molecules GalNac-T, containing N-terminal cytoplasmic domain, a transmembrane domain, a stem region and catalytic domain. In an additional embodiment, GalNac-T3 polypeptide person contains the amino acid sequence shown in Figv. GalNac-T3 is additionally described by Bennett et al., J. Biol. Chemistry, 271:17006-17012 (1996).
Under the "individual" or "patient" refers to any individual who needs treatment, including people. Also designed to enable individual any individuals participating in clinical trials have not shown any signs of disease, or individuals involved in epidemiological studies, or individuals who are control patients.
Used in the description, the term "mammal" refers to any mammal, against whom asempa to the mammal, including humans, cows, horses, dogs and cats. In a preferred variant of the invention, the mammal is man.
Under "sample of tissue or cells" refers to a cluster of similar cells obtained from a tissue of the subject or patient. The source of the tissue sample or cells may be solid tissue as from a newly obtained, frozen and/or fixed organ or tissue sample or biopsy or aspirate; blood or any blood constituents; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells with any period of gestation or development of the organism. The tissue sample may also be a primary or cultured cells or cell lines. Optionally, a sample of tissue or cells obtained from primary or metastatic tumors. The tissue sample may contain compounds that are naturally blended with cloth in nature, such as preservatives, anticoagulants, buffers, latches, nutrients, antibiotics and the like.
For the purposes of the present invention "slice" of tissue sample means a separate part or fragment of a tissue sample, for example a thin layer of tissue or cells cut from the tissue sample. Clearly, what can be done with many sliced samples of cloth, they can be subject to investigation in accordance with the present invention, provided that it is implied that the present invention includes a method by which the same tissue sample analyzed both at morphological and molecular levels, or it is being researched in relation to proteins and nucleic acids.
Under the "correlated" or "correlating" implied comparison, in any way, holding and/or the results of the first analysis or Protocol, and/or the results of the second analysis or Protocol. For example, one can use the results of the first analysis or Protocol with other protocols and/or may use the results of the first analysis or Protocol to determine whether to perform another analysis or Protocol. With respect to the various embodiments in the context of the present invention one may use the results of the analytical study, for example, the expression of mRNA or IHC to determine whether to hold a special treatment with the use of Apo2L/TRAIL or antibodies to receptor of death.
By "nucleic acid" refers to the inclusion of any DNA or RNA. For example, chromosomal, mitochondrial, viral and/or bacterial nucleic acids present in the sample tissue. The term "nucleic acid" encompasses any or both double-stranded helix of the Molek is s nucleic acid and includes any fragment or part of intact molecules of nucleic acids.
By "gene" is meant any nucleic acid sequence or part thereof with a functional role in coding or transcription of a protein, or regulation of expression of other genes. 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 abnormality in exons, introns, regions of initiation or termination, promoter sequences and other regulatory sequences or unique areas adjacent to this gene.
When used in the description, the word "label" refers to a compound or composition that is conjugated or fused directly or indirectly with a reagent, such as a probe nucleic acid or antibody, and facilitates the identification of the reagent to which it is conjugated or condensed. The label itself may be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration detected connection substrate or composition.
The term "antibody" is used in the description in the broadest sense and mainly refers to intact monoclonal antibodies, polyclonal who antibodies, multispecific antibodies (for example, bespecifically antibodies)formed from at least two intact antibodies or fragments of antibodies, provided that they exhibit the desired biological activity.
"Antibody fragments" comprise a portion of an intact antibody, preferably containing antigennegative or variable region. Examples of fragments of antibodies include Fab, Fab', F(ab')2and Fv fragments; dyatel; linear antibodies; single-chain molecule antibodies; and multispecific antibodies formed from fragments of antibodies.
"Native antibodies" are usually heterotetrameric glycoproteins approximately 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different isotypes of immunoglobulins. Each heavy and light chain also has a properly spaced intrachain disulfide bridges. Each heavy chain has at one end has a variable domain (VH), followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at the other end; the constant domain of the light CE and coincides with the first constant domain of the heavy chain, and the variable domain light chain coincides with the variable domain of the heavy chain. It is believed that certain amino acid residues forming the interface between the variable domains of light and heavy chains.
The term "variable" refers to the fact that the sequence of some sections of the variable domains differ greatly among antibodies and are used for binding and specificity of each particular antibody with its specific antigen. However, the variability is not evenly distributed on all variable domains of antibodies. It is concentrated in three segments called or hypervariable regions defining complementarity, as in the light chain and heavy chain variable domains. More highly conserved areas of variable domains are called the frame regions (FR). Each variable domain of native heavy and light chains contain four FR, mainly with β-folded configuration, connected by three hypervariable sites, which form loops connecting, and in some cases forming part of β-folded structure. Hypervariable sites in each chain are held together in close proximity with FR and hypervariable parts of the other circuit, participating in education antigennegative site of antibodies (the m Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not directly involved in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).
Cleavage of antibodies with papain produces two identical antigenspecific fragment, called "Fab"fragments, each with one antigennegative site, and a residual "Fc"fragment, whose name reflects its ability to easy crystallization. Treatment with pepsin yields F(ab')2the fragment that has two antigenspecific site and still capable of cross-contact with the antigen.
"Fv" is the minimum antibody fragment which contains a complete antipersonnel and antigennegative website. This site consists of a dimer variable domain of one heavy chain and one light chain in close, non-covalent linkages. It is in this configuration that the three hypervariable section of each variable domain interact with the formation antigennegative site on the surface of the dimer VH-VL. Together the six hypervariable sites give the antibody antigennegative specificity. However, even a single variable domain (or half of an Fv, include the traveler only three hypervariable area, specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the binding site in its entirety.
Fab-fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab'fragments differ from Fab fragments by the addition of a few residues at the carboxyl end of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of the antibody. In this case, Fab'-SH is the designation for Fab'in which the cysteine residue(s) of the constant domains bear at least one free Tilney group. F(ab')2fragments of the antibodies were initially obtained in the form of pairs of Fab'-fragments, which have a hinged cysteine. Also known other chemical compounds fragments of antibodies.
The "light chains" of antibodies (immunoglobulins) spine of any kind can be attributed to one of two clearly differentiated types, called Kappa (κ) and lambda (λ), 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 assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and several of them can additionally be divided into subclasses (isotypes),for example,IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The constant domains of the heavy chains, which correspond to the different classes of antibodies are called α, δ, ε, γ and μ, respectively. Structures of subunits and three-dimensional configurations of different classes of immunoglobulins are well known.
"Single-chain Fv" or "scFv fragments of antibodies contain VHand VLdomains of antibodies, where these domains are in the same polypeptide chain. Preferably, the Fv polypeptide additionally contained a polypeptide linker between the VHand VLdomains that enables scFv to form the desired structure for antigen binding. A review of scFv see Plűckthun inThe Pharmacology of Monoclonal Antibodiesvol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
The term "diately" refers to small fragments of the antibodies with two antihistamine sites, these fragments contain the variable domain of the heavy chain (VH)associated with the variable domain (VL) light chain in a single polypeptide chain (VH- VL). Using a linker that is too short for pairing between the two domains on the same chain, the domains are forced to form with complementary domains of another chain and create two antigenspecific site. More full of diately described, for example, in EP 404097; WO 93/11161; and Hollingeret al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
Used specterman "monoclonal antibody" refers to an antibody obtained from the population, essentially homogeneous antibodies,iethe individual antibodies comprising the population are identical except for possible naturally mutations that may be present in small quantities. Monoclonal antibodies are highly specific, being directed to one antigenic site. In addition, unlike conventional drugs (polyclonal) antibodies, which typically include different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single antigenic determinant. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by the hybridoma culture, not contaminated by other immunoglobulins. The definition of "monoclonal" indicates the character of the antibody as being obtained essentially of a homogeneous population of antibodies, and is not considered requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present isoberlinia can be obtained hybridoma method first described by Kohleret al.,Nature, 256:495 (1975), or may be obtained by methods of recombinant DNA (see, for example, U.S. Patent No. 4816567). "Monoclonal antibodies" can also be distinguished from ragovoy library of antibodies, COI is lsua techniques described, for example, Clacksonet al., Nature,352:624-628 (1991) and Markset al.,J. Mol. Biol., 222:581-597 (1991).
In the context of this invention, monoclonal antibodies are particularly 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 specific class or subclass of antibody, while the remainder of the chain (chain) is identical with or homologous to corresponding sequences in antibodies derived from other species, or belongs to another class or subclass antibodies as well as fragments of such antibodies, provided that they exhibit the desired biological activity (U.S. patent No. 4816567; Morrisonet al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In the context of the invention of interest chimeric antibodies include "primaryservername" antibodies containing the sequence antigennegative variable domain obtained from nonhuman primates (e.g. Monkeys of the Old world, such as baboon, macaque-rhesus or cynomolgus macaque), and sequences of constant regions that belong to someone (U.S. patent No. 5693780).
"Humanized" forms of the antibody, non-antibody-human (e.g. murine)are chimeric antibodies to the e contain minimal sequence, derived from immunoglobulin non-human immunoglobulin. Basically, humanized antibodies are human immunoglobulins (recipient antibody)in which residues from a hypervariable area of the recipient replaced by residues from a hypervariable site, species, non-human (donor antibody)such as mouse, rat, rabbit and nonhuman Primate, with the desired specificity, affinity and activity. In some cases, remnants of the framework region (FR) of a human immunoglobulin are replaced by corresponding residues, non-human immunoglobulin. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. These modifications carried out for the further improvement of the functional activity of the antibodies. Basically, humanitariannet antibody will comprise substantially all of at least one, and typically two, variable domains, in which all, or substantially all of the hypervariable loops correspond to those of immunoglobulin, non-human, and all, or essentially all, FR are those with the sequence of human immunoglobulin. Humanitariannet antibody also necessarily contain at least frequent the constant region (Fc) of an immunoglobulin, usually immunoglobulin. For more details see Joneset al., Nature321:522-525 (1986); Riechmannet al., Nature332:323-329 (1988); and Presta,Curr. Op. Struct. Biol.2:593-596 (1992).
When used in this invention, the term "hypervariable area" refers to amino acid residues of an antibody which are responsible for binding to the antigen. Hypervariable the site comprises 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; Kabatet 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 light chain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and LeskJ. Mol. Biol.196:901-917 (1987)). "Framework" or "FR" residues are variable domain residues other than residues from a hypervariable area defined in the present description.
An antibody "which binds" an antigen is an antibody capable of binding that antigen with sufficient affinity and/or avidity such that this antibody is applicable as a therapeutic or diagnostic tools for natalian the I to the cell, expressing this antigen.
For the purposes of this invention "immunotherapy" will refer to a method of treatment of a mammal (preferably a human patient human) antibody, where the antibody can be unconjugated or naked antibody or antibody may be conjugated or fused to a heterologous molecule (molecules) or agent (agents), for example, one or more cytotoxic agents, thereby reproducing "immunoconjugate".
"Isolated antibody" is an antibody that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are substances which would hinder its diagnostic or therapeutic use of this antibody, and may include enzymes, hormones and other relcovaptan and non-protein solute. In preferred embodiments, the implementation of the antibody will be purified (1) to more than 95% by weight of antibody as determined by the method of Lowry, and most preferably more than 99% by weight of (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by using a sequencing machine with rotating cups, or (3) to homogeneity by SDS-PAGE in the ed is arousih or nereguliruemyi conditions using Kumasi blue or preferably, staining with silver. The selected antibody includes antibodyin situwithin recombinant cells, since at least one component of the natural environment antibodies will not be present. As a rule, however, isolated antibody is obtained by at least one purification step.
The expression "effective amount" refers to the amount (for example, Apo2L/TRAIL, anti-DR4 or DR5 antibody and so on), which is effective to prevent, alleviate or treat the disease or condition.
Used herein, the terms "treatment", "therapeutic effect" and "therapy" refer to curative therapy, prophylactic therapy, and preventative therapy. Consistent therapeutic effect or the introduction refers at least to therapeutic effect on a daily basis without interruption of treatment on one or more days. Fractional therapeutic effect, or the introduction, or therapeutic effect, or the introduction to the periodic mode refers to treatment that is not consistent, but rather periodic in nature.
The term "cytokine" is a generic term for proteins secreted by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, mono the ins and conventional polypeptide hormones. Among the cytokines include growth hormone such as human growth hormone, N-methionyl of human growth hormone and the growth hormone bull; 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; tumor necrosis factor-α and-β; müller inhibiting substance; mouse peptide associated with gonadotropin; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors; platelet-growth factor; transforming growth factors (TGF)such as TGF-α and TGF-β; insulin-like growth factor-I and-II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β and-γ; colony stimulating factors (CSFs)such as macrophage CSF (M-CSF); granulocytemacrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF); interleukins (IL)such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17; and other polypeptide factors including LIF and kit ligand (KL). Used in the description, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence citoc is new.
Used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. This term includes radioactive isotopes (e.g.,I131I125, Y90and Re186), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or toxins of animal origin, or fragments thereof.
"Chemotherapeutic agent" is a chemical compound suitable for the treatment of tumors. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulphonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzodepa, carboquone, matureup and uredepa; ethylenimines and methylmelamine, including altretamin, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and triethylenemelamine; acetogenin (mostly bullatacin, bullatacin); camptothecin (including the synthetic analogue topotecan); bryostatin; callistemon; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; Pankrat the statin; sarcodictyin; spongistatin; nitrogen mustard such as chlorambucil, chlornaphazine, chlorpropamide, estramustine, ifosfamide, mechlorethamine, hydrochloride oxide mechlorethamine, melphalan, novemberin, finestein, prednimustine, trofosfamide, uramustine; nitrosoanatabine, such as carmustine, chlorozotocin, fotemustine, lomustin, nimustine, ranimustine; antibiotics such as andinavia antibiotics (for example, calicheamicin, mainly calicheamicin gamma and calicheamicin phiI1, see, e.g., Agnew, Chem Intl. Ed. Engl., 33:183-186 (1994); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; spiramycin; and neocarzinostatin chromophore and related chromoprotein andinomys antibiotics chromophores), aclacinomycin, actinomycin, autralian, azaserine, bleomycin, actinomycin, carubicin, karminomitsin, casinopolis, chromomycin, dactinomycin, daunorubicin, demoralizing, 6-diazo-5-oxo-L-norleucine, doxorubicin (AdriamycinTM) (including morpholino doxorubicin, cyanomethane doxorubicin, 2-pyrroline doxorubicin and desoxidation), epirubicin, zorubicin, idarubitsin, marsellaise, mitomycin, such as mitomycin C, mycofenolate acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, colomycin, radiobeacon, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; intimate is ality, such as methotrexate and 5-fluorouracil (5-FU); analogs of folic acid, such as deeperin, methotrexate, peripherin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, timipre, tioguanin; pyrimidine analogues such as ancitabine, azacytidine, 6-azauridine, Carmarthen, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antagonists of adrenal hormones, such as aminoglutetimid, mitotane, trilostane; metabolite of folic acid, such as folinovaya acid; Eagleton; aldophosphamide; aminolevulinic acid; eniluracil; amsacrine; astroball; bisantrene; edatrexate; defaming; demecolcine; diazinon; alternity; slipline acetate; epothilone; etoposide; gallium nitrate; oxytocin; lentinan; lonidamine; maytansinoid, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitrogen; pentostatin; penomet; pirarubicin; losoxantrone; podophyllin acid; 2-acylhydrazides; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogermanium; tinoisamoa acid; triaziquone; 2, 2',2"-trihlortrietilamin; trichothecenes (especially T-2 toxin, verrucarin a, roridin A and unguided); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipop the man; Galitsin; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoid, for example, paclitaxel (TAXOL®Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Rhône-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GemzarTM); 6-tioguanin; mercaptopurine; methotrexate; platinum analogues, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NavelbineTM); Novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; deformational (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts or derivatives of any of the above. This definition also includes antihormone tools that act for the regulation or suppression of hormone action on tumors such as antiestrogens and selective estrogen receptor modulators (SERM), including, for example, tamoxifen (including NolvadexTM), raloxifene, tomoxifen, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (FarestonTM); aromatase inhibitors that inhibit the enzyme aromatase, which regulates the synthesis of estrogen in the adrenal glands, such as 4(5)-imidazoles, aminoglutethimide, megestrol acetate (MegaceTM), exemestane, formestane, fadrozole, vorozole (Rivsor TM), letrozole (FemaraTM), and anastrozole (ArimidexTM); and anti-androgens such as flutamide, nilutamide, bikalutamid, leuprolide and goserelin; and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.
"Growth inhibitor" when used in the context of the present invention refers to a compound or composition which inhibits growth of the cell, mainly malignant cells that do overexpress any of these genes, orin vitroorin vivo.Thus, the growth inhibitor is a tool that significantly reduces the percentage of cells that do overexpress these genes in S phase. Examples of growth inhibitors include tools that block the development of the cell cycle (not in S phase), such as tools, inducing a delay in G1 and M phases. Classic blockers M-phase include Vincas (vincristine and vinblastine), Taxol and inhibitors of topo II, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Means that delay G1, also fall within the delayed S-phase, for example, means alkylating DNA, such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and Ara-C. further information can be found inThe Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "ell cycle regulation, oncogens, and antineoplastic drugs" authors Murakamiet al.(WB Saunders: Philadelphia, 1995), in the main, page 13.
The terms "apoptosis" and "apoptotic activity" are used in a broad sense and refer to the orderly or controlled form of cell death in mammals, that is usually accompanied by one or more characteristic cellular changes, including condensation of cytoplasm, loss of cytoplasmic membrane microvilli, segmentation of the nucleus, degradation of chromosomal DNA or loss of mitochondrial function. This activity can be defined and measured, for example, studies of the viability of the cells (such as research with Alamar blue, MTT), FACS analysis, activation of caspase, DNA fragmentation (see, for example, Nicoletti et al., J. Immunol. Methods, 139:271-279 (1991) and polymerase poly-ADP ribose, "PARP", analyses cleavage, known in the prior art.
Used in the description of the term "breach" generally refers to any condition that could have improved as a result of treatment compositions described herein, including any disease or disorder that can be treated with effective amounts of Apo2L/TRAIL or anti-DR4 antibodies and/or anti-DR5 antibodies. It includes chronic and acute disorders, as well as pathophysiological conditions which predispose the mammal to the disorder. Neo the limits violations that are treated in the present invention include 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 status of mammals that is typically characterized by unregulated cell growth or describe this condition. Examples of malignant tumors include, but not limited to, carcinoma, lymphoma, leukemia, blastoma and sarcoma. More specific examples of such malignancies include squamocellular carcinoma, myeloma, small cell lung cancer, glioma, Hodgkins lymphoma, Nakhodkinskoye lymphoma, cancer of the gastrointestinal tract, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, polymorphic glioblastoma, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, carcinoma of the intestine and cancer tissues of the head and neck.
The term "disease associated with the immune system", means a disease in which the immune component is the first system of a mammal calls, mediates or otherwise contributes to the incidence of a mammal. Also included are diseases in which stimulation or interference with the immune response has a beneficial effect on the disease. In this term included autoimmune diseases, immuno-mediated inflammatory diseases, inflammatory diseases, not mediated by the immune system, infectious diseases and immunodeficiency diseases. Examples of diseases associated with the immune system, and inflammatory diseases, some of which are immunologically mediated or mediated by T-cells, which can be treated in accordance with this invention include systemic lupus erythematosus, rheumatoid arthritis, chronic juvenile arthritis, spondyloarthropathy, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjogren syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (disease Graves ' disease, thyroiditis Hashimoto, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, is mediated immunologic disease of the kidneys (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the Central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelinizing polyneuropathy or syndrome Jillian-Barr and chronic demieliniziruuchaya polyneuropathy, hepatobiliary diseases such as infectious hepatitis (hepatitis A, B, C, D, E and other nagapattanam viruses), autoimmune chronic active hepatitis, primary billiary cirrhosis, granulomatosis hepatitis, and sclerosing cholangitis, inflammatory and fibrotic lung diseases, such as inflammatory bowel disease (ulcerative colitis: Crohn's disease), gluten enteropathy and disease wipe, or immunologically mediated autoimmune diseases of the skin including bullous skin diseases, polymorphic erythema, and contact dermatitis, psoriasis, allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, food sensitization and urticaria, immunologic diseases of the lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitive pneumonitis, diseases associated with transplantation, including graft rejection and disease graft-versus-host. Infectious diseases include AIDS (HIV), hepatitis A, B, C, D and E, Baker the real infection, fungal infections, protozoal infections and parasitic infections.
"Autoimmune disease" is used in the description in a broad General sense to refer to disorders or conditions in mammals, in which the destruction of normal or healthy tissue is the result of a humoral or cellular immune response of the individual mammal of the components of his or her own tissues. Examples include, but not limited to, systemic lupus erythematosus, thyroiditis, rheumatoid arthritis, psoriasis, multiple sclerosis, autoimmune diabetes, and inflammatory bowel disease (IBD).
The term "labeled" for use in the present invention refers to a chimeric molecule containing the antibody or polypeptide fused to a "tag-polypeptide". Tag-polypeptide has enough residues to provide epitope in respect of which may be obtained antibody, or to provide some other features such as the ability to oligomerization (e.g., as occurs with peptides having domains latinovich clasps), yet short enough so that basically does not inhibit the activity of the antibody or polypeptide. Tag-polypeptide preferably is somewhat unique, so the tag-specific antibody, essentially not cross reagiruete other epitopes. Suitable tag-polypeptides generally have at least six amino acid residues and usually from about 8 to about 50 amino acid residues (preferably, from about 10 to about 20 residues).
The term "divalent metal ion" refers to a metal ion having two positive charge. Examples of divalent metal ions include, but not limited to, zinc, cobalt, Nickel, cadmium, magnesium and manganese. Special forms of such metals, which can be used include salt forms (e.g., pharmaceutically acceptable salt forms such as the form of the chloride, acetate, carbonate, citrate and sulfate above-mentioned divalent metal ions. Optional, divalent metal ion for use in the present invention is a zinc and preferably in salt form zinc sulfate or zinc chloride.
"Isolated" when used to describe the various peptides or proteins disclosed in this invention, means a peptide or protein that has been identified or separated and/or removed from a component of its natural environment. Contaminant components of its natural environment are substances that normally would prevent its diagnostic or therapeutic applications of the peptide or protein, and may include EN zymes is s, hormones and other relcovaptan or non-protein solute. In preferred embodiments, the implementation of this peptide or protein will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by using a sequencing machine with rotating glass, or (2) to homogeneity by SDS-PAGE in reducing or nereguliruemyi conditions using Kumasi blue or, preferably, silver staining, or (3) to homogeneity by using mass spectroscopic methods or techniques for peptide mapping. The selected substance includes a peptide or proteinin situwithin recombinant cells, since at least one component of the natural environment will not be present. Typically, however, the selected peptide or protein is obtained by at least one purification step.
"Percent (%) amino acid sequence identity" with respect to the sequences identified in the present invention, is defined as the percentage of amino acid residues in the candidate sequence that are identical with amino acid residues in the reference sequence, after aligning the sequences and, if necessary, the introduction of gaps to achieve maximum is Noah percent identical sequences, and not considering any conservative substitutions as part of the identity of the sequence. Alignment to determine the percentage of amino acid identity can be achieved in various ways, which are a special knowledge in this field, you can determine appropriate parameters for determining the alignment, including setting algorithms needed to achieve maximal alignment over the entire length of the compared sequences. For the purposes of the present invention the percentage of amino acid identity can be obtained using a computer program to compare sequences, ALIGN-2, which is developed by Genentech, Inc. and the original program which was declared user documentation in the office for the protection of copyrights of the United States, Washington, DC, 20559, registered in accordance with the register of copyrights U.S. No. TXU510087. The program ALIGN-2 publicly available through Genentech, Inc., South San Francisco, CA. All parameters comparison of the sequences set by the program ALIGN-2 and do not change.
"Stringency" of hybridization reactions is easily determined by a person skilled in the art and generally is an empirical calculation depending on the length of the probe, the temperature of the wash and the salt concentration. Usually the longer probes require Bo is its high temperatures for proper annealing, whereas for shorter probes need lower temperatures. Hybridization mainly depends on the ability of denatured DNA re-annealing, when complementaria chains are in an environment below their melting temperature. The higher the desired degree of identity between the probe and hybridomas sequence, the higher the relative temperature which can be used. As a result, it follows that the higher the relative temperature would tend to make the reaction conditions more stringent, whereas lower temperatures or less. Additional details and explanation of stringency of hybridization conditions, see Ausubelet al.,Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
"Conditions of high stringency"defined in the present invention, are defined so that: (1) low ionic strength and high temperature for washing; of 0.015 M sodium chloride/0,0015 M sodium citrate/0.1%, dodecyl sodium sulfate at 50oC; (2) during the hybridization is used denaturing agent; 50% (vol./about.) formamide with 0.1% bovine serum albumin/0.1%, Ficoll/0.1% polyvinylpyrrolidone/50 mm phosphate-sodium chloride buffer at pH 6.5 with 750 mm sodium chloride, 75 mm sodium citrate at 42oC; or (3) is used 50% of formamide, 5 × SSC (0,75 M NaCl, of 0.075 M sodium citrate), 50 mm sodium phosphate (pH of 6.8), 0.1% sodium pyrophosphate, 5 × of rastkogelhutte, DNA salmon sperm subjected to ultrasound (50 µg/ml), 0.1% of SDS and 10% dextran sulfate at 42oC, with washes at 42oC in 0.2 × SSC (sodium chloride/sodium citrate) and 50% of formamide when 55oC with subsequent washing of high severity, consisting of 0.1 × SSC containing EDTA at 55oC.
"Conditions of moderate severity" can be identified as described by Sambrooket al.,Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the incubation over night at 37oC in a solution comprising: 20% formamide, 5 × SSC (150 mm NaCl, 15 mm chinatravel citrate), 50 mm sodium phosphate (pH of 7.6), 5 × denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured DNA salmon sperm, followed by washing the filters in 1 × SSC at about 37-50oC. the person skilled in the art will understand how to set the temperature, ionic strength, etc. required to conform to factors such as probe length and the like.
The term "primer" or "primer" refers to an oligonucleotide sequences that hybridize with the complementary target polynucleotide RNA or DNA and serve as starting points for the stepwise synthesis of polynucleotide of mononucleotides under the action of nucleotidyltransferase, for example, as occurs in the polymerase chain reaction.
The term "control sequences" Rel which relates to DNA sequences, necessary for the expression of the operatively linked coding sequence in a particular organism, the host. Control sequences that are suitable for prokaryotes, for example, include a promoter, an optional operator sequence and the site of binding to the ribosome. It is known that eukaryotic cells utilize promoters, polyadenylation signals, and enhancers.
Nucleic acid is operatively linked"when it is in functional relationship with another nucleic acid sequence. For example, DNA for predpolagavshegosja or secretory leader is operatively linked to DNA for a polypeptide if it is expressed in the form of preprally that participates in the secretion of the polypeptide; a promoter or enhancer is operatively linked with a coding sequence if it affects the transcription of the sequence; or a binding site with the ribosome is operatively linked with a coding sequence if it is so easy translation. Basically, "operatively linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and at the stage of reading. However, the enhancers should not be continuous. The binding is established leagues is the formation of suitable restriction sites. If such sites are not present, use synthetic oligonucleotide adapters or linkers, in accordance with common practice.
"Antibody-dependent mediated cell cytotoxicity" or "ADCC" refers 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 the target cell and subsequently cause lysis of the target cells. The primary cells for mediating ADCC, NK cells, Express FcγRIII only, whereas monocytes Express FcγRI, FcγRII and FcγRIII. The FcR expression on hematopoietic cells is summarized in the form is a Table 3 on page 464 publication Ravetch and Kinet,Annu. Rev. Immunol.9:457-92 (1991). To assess ADCC activity of interest molecules, it is possible to analyze ADCCin vitrofor example, described in U.S. patent No. 5500362 or 5821337. Suitable effector cells for such studies include the mononuclear cells of peripheral blood (PBMC) cells and natural killer cells (NK). Alternative or additionally, ADCC activity of the molecule of interest can be estimated activityin vivofor example, in animal models, for example, described in Clyneset al. PNAS (USA)95:652-656 (1998).
"Effector cells" represents the t of a leukocyte, which Express one or more FcR and perform effector functions. Preferably, cells that Express at least FcγRIII and perform effector function is ADCC. Examples of human leukocytes which mediate ADCC include mononuclear cells of peripheral blood (PBMC)cells, natural killer cells (NK), monocytes, cytotoxic T cells and neutrophils; with the most preferred PBMC and NK cells.
The terms "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc site antibodies. Preferred FcR is a native sequence FcR person. Moreover, a preferred FcR is a receptor that binds to IgG antibody (a gamma receptor) and includes receptors of the subclasses of the FcγRI, FcγRII and FcγRIII, including allelic variants and forms of alternative splicing of these receptres. FcγRII receptors include FcγRIIA (an"activating receptor") and FcγRIIB (an"inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains a motif that activates immunoreceptor based on tyrosine (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB in its cytoplasmic domain contains a motif, ingibiruet the rd immunoreceptor, based on tyrosine (ITIM). (See Daëron, Annu. Rev. Immunol.15:203-234 (1997)). The review of FcR are presented in Ravetch and Kinet,Annu. Rev. Immunol.9:457-92 (1991); Capelet al., Immunomethods4:25-34 (1994); and de Haaset al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcR, including those that will be installed in the future, here are covered by the term "FcR". This term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyeret al., J. Immunol. 117:587 (1976) and Kimet al., J. Immunol. 24:249 (1994)). In the present invention FcR include polymorphisms, such as genetic dimorphism in the gene that encodes FcγRIIIa, in which, or phenylalanine (F)or valine (V) at amino acid position 158, located in the region of the receptor, which binds to IgG1. It has been shown that homozygous FcγRIIIa with valine (FcγRIIIa-158V) have a higher affinity to human IgG1 and mediate increased ADCCin vitrocompared with homozygous FcγRIIIa with phenylalanine (FcγRIIIa-158F) or heterozygous (FcγRIIIa-158F/V) receptors.
"Complement-dependent cytotoxicity" or "CDC" refers to the ability of the molecule to lyse target in the presence of complement. Path activation of complement is triggered by binding of the first component of the complement system (C1q) with a molecule (e.g. antibody), formed a complex with cognate antigen. To assess activation of the comp is ementa you can analyze CDC, for example, as described in Gazzano-three-betet al.,J. Immunol. Methods202:163 (1996).
II. The METHODS AND MATERIALS of the INVENTION CITED AS an EXAMPLE
Methods and studies described in the present description, aimed at studying the expression of one or more biomarkers in the tissue sample of a mammal or sample of cells, where determining the expression of one or more such biomarkers predicts or indicates whether a sample of tissue or cells sensitive to such agents as Apo2L/TRAIL and/or antibodies of death receptors, such as antibodies, agonists to or DR5 antibody agonists to DR4. These methods and research include methods and studies that examine the expression of members of the family of molecules GalNac-T, including GalNac-T14 and GalNac-T3.
As discussed above, there are several populations of pathological types of human cells (for example, certain populations of malignant cells), which are resistant to the actions of Apo2L/TRAIL induces cell death, including the actions of antibody receptors death. Therefore, it is believed that the described methods and research can provide appropriate, effective and potentially cost-effective means of obtaining data and information used to assess adequate or effective schemes for the treatment of patients. For example, in a patient, in which the CSOs diagnosed malignant disease or pathological condition associated with the immune system, it would be possible to take a biopsy to obtain a tissue sample or cells, and this sample can be verified through various testsin vitroto determine whether cells of a patient sensitive to a therapeutic agent, such as Apo2L/TRAIL or the antibody of receptor of death.
The invention relates to methods of predicting the sensitivity of a tissue sample or cells of a mammal (for example, malignant cells to Apo2L/TRAIL or the antibody-receptor agonist death. Not necessarily, get a sample of tissue or mammalian cells and examined for the expression of GalNac-T14. These methods can be performed in a variety of formats research, including studies that determine the expression of mRNA, protein expression (for example, immunohistochemical studies and biochemical assays, detecting the enzymatic activity of UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. Detection of expression of such GalNac-T14 biomarkers in (or on) the above tissues or cells, is the prediction that such tissues or cells will be sensitive to the biological effects of Apo2L/TRAIL and/or antibody of receptor of death. Applicants unexpectedly discovered that the expression of GalNac-T14 correlates with the sensitivity of these tissues and cells to Apo2L/TRAIL and the antibody-receptor agonists death.
As RA is considered below, the expression of various biomarkers, such as GalNac-T14 in the sample, can be analyzed using several techniques, many of which are known in the prior art and friendly professionals, including, but not limited to, immunohistochemical and/or Western analysis, quantitative blood tests (as for example Serum ELISA) (e.g., for the study of expression levels of the protein), and biochemical studies of the enzymatic activity, hybridizationin situ, Northern analysis and/or PCR analysis of mRNA and genomic southern analysis (for example, to investigate deletions or gene amplification), as well as any of a variety of methods of analysis that can be performed using matrix analysis of genes and/or tissues. Standard protocols for evaluating the status of genes or gene products are, for example, Ausubel et al. eds., 1995, Current Protocols In Molecular Biology, Units 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 below to illustrate.
Optional methods of the invention include protocols which examine or test the presence of GalNac-T14 in the sample tissue or mammalian cells. For detection of GalNac-T14 can be used in a variety of ways, and they include, for example, immunohistochemical the cue analysis, immunoprecipitation, Western blot analysis, the study of molecular binding, ELISA, ELIFA, fluorescence-activated sorting of cells (FACS) and immunoprecipitation with subsequent MS analysis of monosaccharides. For example, an optional method of detecting the expression of GalNac-T14 in the tissue or sample includes contacting the sample with anti-GalNac-T14 antibody and then detecting the binding of antibodies with GalNac-T14 in the sample.
In specific embodiments of the invention the expression of GalNac-T14 in the sample examined using immunohistochemistry staining protocols. It was shown that immunohistochemical staining of tissue section is a reliable way to measure or detect the presence of proteins in the sample. In immunohistochemical techniques ("IHC") using antibodies for sensing and visualization of cellular antigensin situmainly using chromogenic or fluorescent methods.
To obtain the sample can be a tissue sample or cells of a mammal (typically a person). Examples of samples include, but not limited to, cancer cells, such as malignant cells of the bowel, breast, prostate, ovary, lung, stomach, pancreas, lymphoma and leukemia cells. Optional designs include malignant cells amalgalite the aqueous lung cancer, malignant cells of the pancreas or malignant cell non-Hodgkin lymphoma. The sample can be obtained by using a number of techniques known in the art, including, but not limited to, surgery, aspiration or biopsy. The fabric can be fresh or frozen. In one embodiment, the sample is fixed and enclosed in paraffin, or the like.
The tissue sample may be fixed (i.e. conserved) using standard techniques (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 person skilled in the art it will be clear that the choice of the retainer depends on the purpose of histological staining or other studies of this sample. The person skilled in the art also will understand that the duration of fixation depends on the size of the tissue sample and used the lock. As an example, for fixing the sample can be used neutral buffered formalin, the latch Buena or paraformaldehyde.
Basically, first the sample is fixed, and then digitalout through ascending series of alcohols, impregnate and conclude in paraffin or other environment for the manufacture of the population slices thus, so from the tissue sample, it was possible to get cut. Alternatively, you can make the cut tissue and to record the received slices. As an example, a tissue sample may be entered into and processed in paraffin using standard techniques (see, for example, “Manual of Histological Staining Method of the Armed Forces Institute of Pathology",supra). Examples of paraffin, which can be used include, but are not limited to, Paraplast, Broloid and Tissuemay. Once the tissue sample is enclosed in paraffin, you can make slices of the sample by using a microtome or the like (see, for example, “Manual of Histological Staining Method of the Armed Forces Institute of Pathology”,supra). As an example of this technique, the thickness of the slices may vary from about three microns to about five microns. After receiving sections can be attached to subject the glass several standard ways. Examples of adhesives for glass slides include, but not limited to, silane, gelatin, poly-L-lysine and the like. As an example, the sections enclosed in paraffin, can be attached to the positively charged subject to the glass and/or subject to slides coated with poly-L-lysine.
When using wax as a substance for the conclusion of the preparation of the tissue sections, as a rule, free from paraffin and registryroot in the water. Tissue samples can be removed from paraffin using a few about aprimatic standard techniques. For example, can be used xylene and gradient decreasing series of alcohols (see, for example, “Manual of Histological Staining Method of the Armed Forces Institute of Pathology”,supra). Alternatively, you can use commercially available deparaffinizing inorganic agents, such as Hemo-De7 (CMS, Houston, Texas).
Optional, after preparation of the sample, a tissue section can be analyzed using IHC. IHC can be performed in combination with additional techniques, such as morphological staining and/or fluorescent in situ hybridization. There are two main ways IHC; direct and indirect analysis. In accordance with the first assay, binding of antibody to the target antigen (e.g., GalNac-T14) is determined directly. This direct analysis uses a labeled reagent, such as a fluorescent label or enzyme labeled primary antibody, which can be visualized without further interaction of the antibody. In the standard indirect analysis of unconjugated primary antibody binds to the antigen, and then labeled secondary antibody binds to primary antibody. Where a secondary antibody conjugated with an enzyme label, add chromogenic or fluorogenic substrate to provide visualization of the antigen. Is the amplification of the signal, as several secondary antibodies can vzaimodejstvovatx different epitopes on the primary antibody.
Primary and/or secondary antibody used in immunohistochemistry, there will typically be observed detectable molecule. There are numerous labels that can mainly be grouped into the following categories:
(a) Radioisotopes, such as35S14C,125I3H and131I. Antibody can be observed with the radioisotope using the techniques described in theCurrent Protocols in Immunology, Volumes 1 and 2, Coligenet al.,Ed. Wiley-Interscience, New York, New York, Pubs. (1991)for example and radioactivity can be measured using scintillation counting.
(b) Particles of colloidal gold.
(c) Fluorescent labels including, but not limited to, rare earth chelates metals (europium chelates), Texas red, rhodamine, fluorescein, dansyl, lissamine, umbelliferone, phycoerythrin, phycocyanin, or commercially available fluorophores such as SPECTRUM ORANGE7 and SPECTRUM GREEN7 and/or derivatives of any one or more specified above. Fluorescent labels can be conjugated to the antibody using the techniques described, for example, inCurrent Protocols in Immunology,supra. Fluorescence can be measured quantitatively using fluorimetry.
(d) There are various enzyme-substrate labels, and in U.S. patent No. 4275149 presents an overview of some of them. The enzyme mainly catalyzes a chemical alteration of the chromogenic substrate to the / establishment, which can be measured using various techniques. For example, the enzyme may catalyze a color change in the substrate which can be quantitatively measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence substrate. Methods of quantitative determination of change of fluorescence described above. Is the electronic excitation of the chemiluminescent substrate under the action of a chemical reaction and may then emit light which can be quantified (for example, using chemiluminometer) or transfers energy to the acceptor fluorescence. Examples of enzymatic labels include luciferase (e.g., of Firefly luciferase and bacterial luciferase; U.S. patent No. 4737456), luciferin, 2,3-dihydropteridine, malatdegidrogenaza, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, secrete lysozyme, oxidase saccharides (for example, glucoseoxidase, galactosidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microbiocides, and the like. Methods of conjugation of enzymes to antibodies are described in O'sullivanet al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, inMethods in Enzym.(ed. J. Langone &H. Van Vunakis), Academic press, New York, 73:147-166 (1981).
Examples of enzyme-substrate combinations include, nab the emer:
(i) horseradish peroxidase (HRPO) with hydrogen peroxide as a substrate, where the hydrogen peroxide oxidizes a dye precursor (e.g., orthophenylene (OPD) or 3,3',5,5'-tetramethylbenzidine (TMB));
(ii) alkaline phosphatase (AP) with para-nitrophenylphosphate as chromogenic substrate; and
(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate (for example, 4-methylumbelliferyl-β-D-galactosidase).
Numerous other enzyme-substrate combinations are available to professionals in this field. For a General overview see U.S. patent No. 4275149 and 4318980. Sometimes the label is indirectly conjugated with the antibody. Different methods to achieve this are in the competence of the specialist. 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, orvice versa. Biotin specifically binds with Avidya, and, therefore, this label can be conjugated with the antibody in such an indirect way. Alternatively, to achieve indirect conjugation of the label with the antibody, antibody kongugiruut with a small hapten, and one of the different types of labels mentioned above, kongugiruut with an antibody to the hapten. Thus, it is possible to achieve the nepr is my conjugation of the label with the antibody.
In addition to the methods of obtaining the sample, described above, may be appropriate additional processing of the tissue section prior to, during or after IHC, for example, methods of play epitopes, for example, it is possible to carry out heating of the tissue sample in citrate buffer (see, for example, Leonget al. Appl. Immunohistochem. 4(3):201 (1996)).
After an optional phase lock the tissue section is subjected to the action of the primary antibody for a sufficient period of time and under suitable conditions, so that the primary antibody is contacted with the target protein antigen in a tissue sample. The appropriate conditions to achieve this, you can define the standard experiments. The degree of binding of the antibody with the sample is determined using any of the detectable labels discussed above. Preferably, the label is an enzyme label (e.g., HRPO), which catalyzes a chemical alteration of the chromogenic substrate such as 3,3'-diaminobenzidine. Preferably, the enzyme label kongugiruut with an antibody that specifically binds to the primary antibody (for example, the primary antibody is a rabbit polyclonal antibody and the secondary antibody is a goat anti-rabbit antibody).
Optionally, the antibodies used in IHC analysis to detect the expression of alNac-T14, are anti-GalNac-T14 antibody. Alternatively, it may be used antibodies to other GalNac-T antigens that cross-react with GalNac-T14. Optional anti-GalNac-T14 antibody is a monoclonal antibody.
Thus prepared samples can be concluded on Wednesday and taped cover glass. Then you can evaluate histological preparation, for example, using a microscope, and criteria of staining intensity, usually used in this field. Criteria of staining intensity can be estimated as follows:
|In cells staining is not observed||0|
|Unclear/barely perceptible staining is detected in more than 10% of cells||1+|
|Weak to moderate staining is observed in more than 10% of cells||2+|
|Moderate to intense staining is observed in more than 10% of cells||3+|
It is usually assumed that the point staining specimen is approximately 2+ or higher in this IHC study is to predict or measure the sensitivity of mammalian cells (for example, malignant mammalian cells to Apo2L/TRAIL antibody-receptor agonist death.
In alternative methods, the sample may be contacted with an antibody specific to a given biomarker, under conditions sufficient for the formation of a complex of antibody-biomarker, and then identifying the specified property. The presence of a biomarker can be defined in a number of ways, such as Western blotting (immunoprecipitate or not) and ELISA to evaluate a large variety of tissues and samples, including plasma or serum. There are many methods of immunological analysis using this type of research, see, for example, U.S. patent No. 4016043, 4424279 and 4018653. They include both single-site and dvuhsimovyiy or "sandwich" analysis of non-competitive types, as well as traditional competitive analysis link. These studies also include direct binding of the labeled antibody with the target biomarker.
Among the most suitable and commonly used are the sandwich assays. There are several different methods of holding the sandwich-analysis, and they are all covered by the present invention. Briefly, in a typical direct analysis of unlabeled antibody immobilizing on a solid substrate, and the test sample is brought into contact with the bound molecule. After a suitable period of incubate the period of time sufficient for the formation of a complex of antibody-antigen, then add the second antibody specific to this antigen, labeled reporter molecule capable of giving the detected signal, and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labeled antibody. Any unreacted substance is washed off, and the presence of the antigen is determined by monitoring the signal generated by the reporter molecule. The results may either be qualitative, simple observation of the visible signal, or may be quantitative by comparing 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 added simultaneously to the bound antibody. These techniques are well known to specialists in this field, including any minimal changes, which will be quite obvious. In the standard direct sandwich the analysis of the first antibody having specificity for the biomarker, is either covalently or passively bound to a solid surface. Solid surface is typically a glass or polymer, the polymers most commonly used are cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. Solid under which Oki can be in the form of tubes, beads, disks, or microplates, or any other surface suitable for conducting immunoassay. The binding process is well known in the prior art and generally consist of cross-covalent binding or physical adsorption, complex polymer-antibody is washed in preparation for sample testing. Then add an aliquot of the test sample to the solid phase complex and incubated for a period of time sufficient (e.g., 2-40 minutes or overnight if more convenient)and under appropriate conditions (for example, from room temperature to 40oC, for example, from the 25oC to 32oC inclusive) to allow binding of any subunit present in the antibody. After an incubation period subunit antibody in the solid phase is washed and dried and incubated with a second antibody specific to the part of the biomarker. The second antibody binds to the reporter molecule, which is used to indicate the binding of the second antibody with a molecular marker.
An alternative method involves the immobilization of target biomarkers in the sample, and then the impact on immobilized target-specific antibodies, which can be observed or may not be observed reporter molecule. Depending on the number of targets and signal strength of reporter molecules tie is nnow target can be identified by direct labelling of the antibody. An alternative to the second labeled antibody specific for the first antibody, the complex of the target-the first antibody with the formation of a tertiary complex target-first antibody-second antibody. This complex is detected using the signal emitted by the reporter molecule. By "reporter molecule" in the present invention refers to molecules which its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. The most commonly used reporter molecules in this type of research are either enzymes, fluorophores or molecules containing radionuclides (i.e. radioisotopes) and chemiluminescent molecules.
In the case of enzyme immunoassay enzyme kongugiruut with the second antibody, generally by means of glutaraldehyde or periodate. However, no work will be clear that there is a wide variety of different conjugation techniques that are easily accessible to specialists. Commonly used enzymes, among others, include horseradish peroxidase, glucoseoxidase, galactosidase and alkaline phosphatase. The substrates used with the specific enzymes, mainly choose to obtain hydrolysis under the action of the corresponding enzyme detected color change. The example is of suitable enzymes include alkaline phosphatase and peroxidase. You can also use fluorogenic substrates that yield fluorescent product and not a chromogenic substrates noted above. In all cases, the antibody labeled with an enzyme, is added to the first antibody complex is a molecular marker to allow for binding, and then wash off the excess reagent. Then add a solution containing the appropriate substrate, the complex of antibody-antigen-antibody. The substrate interacts with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantified, usually spectrophotometrically, to obtain guidance on the number of biomarkers that are present in the sample. Alternatively, fluorescent compounds such as fluorescein and rhodamine, may be chemically linked to antibodies without altering their binding capacity. When activated by illumination with light of a specific wavelength, the antibody labeled with fluorochrome, absorbs light energy, causing the molecule in the state of excitation, followed by light emission characteristic colors visually detected in the light microscope. As in the EIA, a fluorescently labeled antibody provide binding with the first antibody complex is a molecular marker. After washing off the unbound reagent, the remaining tertiary complexate is exposed to light of an appropriate wavelength, the observed fluorescence indicates the presence of a molecular marker of interest. As methods immunofluorescence assay and EIA are common in this area. However, other reporter molecules, such as radioisotopes, chemiluminescent or bioluminescent molecules can also be used.
It is assumed that the above-described methods can also be used to detect the expression of GalNac-T14.
The methods according to the invention additionally include protocols which examine the presence and/or expression of mRNA 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 complementary DNA probes (such asin situhybridization using labeled robopro GalNac-T14, Northern blot and related techniques) and various studies of nucleic acid amplification (e.g., RT-PCR using complementary primers specific for GalNac-T14, and other ways of detecting amplification type, such as branched DNA, SISBA, TMA and the like).
Samples of tissue or mammalian cells can be conveniently examined, for example, mRNA GalNac-T14 using Northern, dot blot or PCR analysis. For example, RT-PCR analysis, such as quantitative PCR analysis, well zwesten in the prior art. In the illustrative embodiment of this invention, a method of detecting mRNA of GalNac-T14 in a biological sample involves obtaining cDNA from the sample by reverse transcription using at least one primer; amplifying cDNA thus obtained, using polynucleotide GalNac-T14 as sense and antisense primers to amplify cDNA GalNac-T14; and detecting the presence of amplified cDNA GalNac-T14. In addition, such methods can include one or several stages, which provide the ability to determine the mRNA level of GalNac-T14 in a biological sample (for example, by simultaneous study of levels comparative control mRNA sequence "obligate" genes, for example, the representative of a family of actin). Optionally, you can define the sequence of the amplified cDNA GalNac-T14.
Practical embodiments of this aspect of the invention include GalNac-T14 primers and a pair of primers, which allow the specific amplification of polynucleotides according to the invention or of any specific parts, and probes that selectively or specifically hybridize with nucleic acid molecules according to the invention or any part of them. The probes may be in the state of detektivami marker such as a radioisotope, free is the can connection, bioluminescent compound, chemiluminescent compound, metal chelator or enzyme. Such probes and primers can be used to detect the presence of polynucleotide GalNac-T14 in the sample, and as a means of identifying cells expressing GalNac-T14 proteins. The person skilled in the art it will be clear that a very large number of different primers and probes can be obtained on the basis of the presented sequences and effectively used for amplification, cloning, and/or determine the presence and/or levels of mRNA GalNac-T14.
Optional methods of the invention include protocols to study or detect mRNA, such as mRNA GalNac-T14, in a sample of tissue or cells using microarray technologies. Using microarrays of nucleic acids, study and control sample of mRNA from the test and control tissue samples back transcribers and mark to obtain cDNA probes. The probes then hybridizing matrix of nucleic acids immobilized on a solid substrate. The configuration matrix create in such a way that the sequence and position of each member of the matrix is known. For example, the set of genes that can be expressed under certain pathological conditions, may be arranged in a certain order on a solid substrate. Hybridization of the labeled probe is specific member of the matrix indicates, a sample of this probe, expresses this gene. Analysis of differential gene expression pathological tissues can provide valuable information. In the technology of microchips used methods of hybridization of nucleic acid and computational technique for the evaluation of the profile of mRNA expression of thousands of genes in one experiment (see, for example, WO 01/75166, published on October 11, 2001; (See, for example, US 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) to consider the manufacture of matrices). The DNA microarray are miniature matrix containing fragments of genes that are either synthesized directly or on glass or other substrates, or cause spots. Thousands of genes are usually represented in a single matrix. The standard experiment using 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) creating profiles of gene expression. Currently use two main types of DNA microarray: oligonucleotide (usually from 25 to 70 monomer unit) of a matrix and matrix gene expression, containing the PCR products derived from cDNA. In the PE the new matrix oligonucleotides can either be made in advance and deposited on the surface in the form of spots, either synthesized directly on this surface (in situ).
System Affymetrix GeneChip® is a commercially available system microarray, which contains the matrix obtained by direct synthesis of oligonucleotides on a glass surface. Matrix probe/gene: oligonucleotides, typically 25 Monomeric units, directly synthesized on a glass plate by combining the technologies of photolithography on the semiconductor base and solid-phase chemical synthesis. Each matrix contains up to 400000 various oligomers, and each oligomer are presented in millions of copies. Because oligonucleotide probes are synthesized in known positions on the matrix, especially hybridization and signal intensity can be interpreted in terms of gene identity and relative levels of expression using software Affymetrix Microarray Suite. Each gene is represented on the matrix series of different oligonucleotide probes. Each pair of probes consists of a complementary oligonucleotide and complementario of the oligonucleotide. Complementary probe has a sequence fully complementary to a specific gene and, thus, determines the expression of a gene. Complementary probe differs from the complementary probe by single nucleotide substitution in the Central position of the base of the Oia, disrupting the binding of a target gene transcript. This helps to determine the background and non-specific hybridization, which contributes to the signal, defined in relation to the complementary oligonucleotide. Software Microarray Suite intensity of hybridization complementary probes are subtracted from the intensities of hybridization of complementary probes to determine the magnitude of the absolute or specific intensity for each set of probes. The probes are chosen based on current information from Genbank and other nucleotide databases. It is believed that the sequence recognize the unique sites on the 3' end of the gene. Hybridization oven GeneChip (oven "grilled") is used for hybridization up to 64 matrices at one time. Spray plant performs washing and staining arrays of probes. It is fully automated and contains four modules, each module holds one matrix probes. Each module can be independently controlled by software Microarray Suite using pre-programmed protocols for inkjet units. The scanner is a confocal laser fluorescent scanner, which measures the fluorescence intensity emitted by the labeled crnc associated with matrices probes. Computer workstation with software is mnim software Microarray Suite controls inkjet installation and scanner. Software Microarray Suite can control up to eight inkjet units, using pre-programmed protocols for hybridization, washing and staining for each matrix probes. This software also receives information and converts the data on the intensity of the hybridization signal presence/absence for each gene, using appropriate algorithms. Finally, this software will detect changes in gene expression between experiments by comparing the analysis and formats the output in .txt files that you can use with other programs for further analysis of the data.
Fluorescent in situ hybridization (FISH) can also be used to detect the mRNA expression of the biomarker, using labeled probes. Such methods are known in the prior art (see, e.g., Kallioniemi et al., 1992; U.S. patent 6358682).
The expression of selected biomarkers can be assessed by studying the deletion of the gene or gene amplification. The deletion of the gene or amplification can be determined by any of a wide variety of protocols known in the prior art, such as the southern blot, Northern blot testing to quantify the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)), dot-blotting (DNA analysis), or by hybridization of thein situ(for example, FISH), is using the respectively labeled probe, cytogenetic methods or comparative genomic hybridization (CGH)using respectively labeled probe. For example, these methods can be used to identify deletions or gene amplification GalNac-T14.
In addition, you can examine the methylation status of a biomarker, such as a gene GalNac-T14, in a sample of tissue or cells. Aberrant demethylation and/or hypermethylation of the CpG Islands in the 5' regulatory regions of the gene occurs frequently in immortalized and transformed cells, and as a result may lead to the altered expression of different genes. In the prior art a number of analyses to study the methylation status of the gene. For example, you can use the approaches southern hybridization, restriction enzymes that are sensitive to methylation, which cannot cleave sequences that contain methylated CpG sites to assess the status of methylation of CpG-Islands. In addition, using MSP (PCR specific for methylation) it is possible to depict the profile of the methylation status of all CpG sites present in the CpG island of a given gene. This method includes the initial modification of DNA by the action of sodium bisulfite (which will turn all of demetilirovanny cytosine to uracil), followed by amplification using primers specific to me is pilirovanny DNA unlike demetilirovanny DNA. The protocols involve meteorology interference, 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 can also be examined using analyses of a function or activity. For example, you can conduct research, known in the prior art, to determine or detect the presence of a given enzymatic activity of 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., supra, available from may 2005 to www.sciencedirect.com.
In the methods of the present invention, it is assumed that the sample of tissue or cells can also be examined on the expression of Apo2L/TRAIL or receptors in the sample that bind Apo2L/TRAIL or the antibody to the receptor of death. As described above and in the prior art at the present time it is believed that Apo2L/TRAIL connects at least five different receptors: DR4, DR5, DcR1, DcR2 and OPG. Using methods known in the prior art, including those described in this invention, the expression of Apo2L/TRAIL, DR4, DR5, DcR1, DcR2 and/or OPG possible to determine the mRNA level and protein level. For example, IHC methodology described above can be used to identified the I presence of one or more of such molecules in the sample. It is assumed that the ways in which the tissue or sample exploring not only the presence of the marker GalNac-T14, and whether, for example, DR4, DR5 or DcR1, separate histological preparations can be obtained from the same tissue or sample, and each histological drug testing with reagent specific for a particular biomarker or receptor. Alternatively, the individual histological preparation may be obtained from a tissue sample or cells, and antibodies directed to each of the biomarker or the receptor can be applied to multi-color staining Protocol for visualization and detection of relevant biomarkers or receptors.
After determining that the sample of tissue or cells is expressed GalNac-T14, indicating that this sample of tissue or cells to be sensitive to Apo2L/TRAIL or the antibody to the receptor of death, it is assumed that the effective amount of Apo2L/TRAIL or antibodies to receptor of death, you can enter the mammal to treat such violation as a malignant disease or a disease associated with the immune system that affects the mammal. Diagnosis in mammals of different pathological conditions described herein, may conduct an experienced practitioner. In the prior art there are ways to diagnose, which is s allow, for example, diagnose or identify the mammal malignant diseases or diseases associated with the immune system. For example, malignant disease can be identified by methods including, but not limited to, palpation, blood test, x-ray, NMR and the like. Diseases associated with the immune system, can also be easily identified.
Apo2L/TRAIL or the antibody to the receptor of death can be entered in accordance with known methods, such as intravenous bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebral, by subcutaneous, intra-articular, intrasynovial, into the cavity of the spinal canal, oral, local, or by inhalation. Optionally, the introduction can be performed by infusion mini-pump, using a variety of commercially available devices.
Effective doses and schemes Apo2L/TRAIL or antibodies to receptor of death can be determined empirically, and such definitions are in the competence of the specialist in this field. You can use single or multiple doses. Currently, it is believed that an effective dosage or amount of Apo2L/TRAIL, used separately, may be varied PR is from about 1 μg/kg to about 100 mg/kg of body weight or more per day. Interspecific transfer of doses can be performed by a method known in the prior art, for example, as described by Mordenti et al., Pharmaceut. Res., 8:1351 (1991).
When usingin vivothe introduction of Apo2L/TRAIL standard dose can vary from about 10 ng/kg to 100 mg/kg of body weight of the mammal or more per day, preferably from about 1 μg/kg/day to 10 mg/kg/day depending on the route of administration. Guidance on specific doses and delivery methods presented in the literature; see, for example, U.S. patent No. 4657760; 5206344; or 5225212. It is expected that various compositions will be effective for a variety of medicinal compounds and various diseases that introduction, focus on one organ or tissue, for example, it may be necessary shipping, is different from that to another organ or tissue.
It is assumed that the additional therapeutic effects can be used in these ways. One or more other therapeutic effects may include, but not limited to, the use of radiation therapy, cytokines (cytokines), inhibitor (inhibitors) growth, chemotherapeutic agents(a), cytotoxic agents(a), tyrosine kinase inhibitors, inhibitors farnesyltransferase ras, angiogenesis inhibitors and inhibitors of cyclin-dependent kinases, which are known in the prior art, and additionally characterized in asanoi above feature. It is assumed that similar therapeutic effects can be used as a means, separate from Apo2L/TRAIL or antibodies to receptor of death. In addition, therapeutic effects, based on therapeutic antibodies that target tumor antigens, such as RituxanTMor HerceptinTMand antiangiogenic antibodies such as anti-VEGF.
Drug and schemes chemotherapeutic agents can be used in accordance with manufacturer's instructions or obtained empirically experienced practitioner. Drug and schemes for such chemotherapy are also described inChemotherapy ServiceEd., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992). Chemotherapeutic agent may precede the introduction or follow the introduction of Apo2L/TRAIL, or antibodies to receptor of death, or it can be entered at the same time.
It may be desirable to introduce antibodies to 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). Alternatively, or additionally, two or more antibodies binding the same or two or more different antigens described here, you can enter the patient together. Sometimes it may be effective that the same introduction to the patient one or more cytokines. After the introduction, you can analyze the treated cellsin vitro. When exposed toin vivomammal, which carried out the treatment can be monitored in various ways well known to the skilled practitioner. For example, tumor cells can be investigated in pathological analyses to assess necrosis or you can investigate the serum responses of the immune system.
For use as described and suggested above appointments invention also relates to kits or products. Such kits can contain a vehicle, compartmentalization to obtain strict isolation of one or more containers such as vials, tubes and the like, each of the containers contains one of the individual elements used in this way. For example, one container may contain a probe that is or may be detektirano Machen. Such probe may be an antibody or polynucleotide, specific protein GalNac-T14 or GalNac-T14 gene, or unit of the genetic code, respectively. In those cases, when the set is used, the hybridization of the nucleic acid for detection of target nucleic acid in the set can also be containers containing nucleotide(s) for amplification of the target nucleic acid sequence, abordage may include containers, containing nucleotide(s) for amplification of the target nucleic acid sequence and/or the container containing the reporters, the Biotin-binding protein such as avidin or streptavidin, bound with a reporter molecule such as an enzyme, fluorescent or radioisotope label.
Set according to the invention will usually contain the container described above and one or more other containers containing the materials necessary from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. The label may be in the container to indicate that the composition is used for a specific therapy or non-therapeutic application, and may also specify how to apply orin vivoorin vitroas explained above.
Kits according to the invention have a number of options for implementation. The standard of the embodiment is a kit containing a container, a label on the specified container and a composition contained within the specified container; where the composition includes a primary antibody that binds to the polypeptide sequence GalNac-T14, the label on the specified container indicates that the composition can be used to assess the presence of proteins GalNac-T14 at least one type of glue is OK mammals and instructions for using the antibody to GalNac-T14 to assess the presence of proteins in at least one type of mammalian cells. Optionally, the kit may contain a set of instructions and materials for obtaining a tissue sample and application of antibody and probe on the aforementioned slice of a tissue sample. The set may include both primary and secondary antibody, the secondary antibody conjugated with a label such as an enzyme label.
Another variant implementation is a set containing a container, a label on the specified container and a composition contained within the container; where the composition includes polynucleotide that's hybrid with complement polynucleotide GalNac-T14 in stringent conditions, the label on the container indicates that the composition can be used to assess the presence of GalNac-T14 in at least one type of mammalian cell, and instructions for use of polynucleotide GalNac-T14 to assess the presence of RNA or DNA GalNac-T14 at least one type of mammalian cell.
Other optional components in the kit include one or more buffers (e.g., blocking buffer, wash buffer, substrate buffer, and so on), other reagents, such as the substrate (for example, a Chromogen), which is chemically modified enzyme label, a solution to restore epitopes, control samples (positive and/or negative controls), control histological preparation(s) and d
Various aspects of the invention is additionally described and illustrated in the following examples, none of which is intended to limit 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; line pancreatic cancer: 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. These lines were obtained from ATCC Depository (Manassas, Virginia), DSMZ (German collection of microorganisms and cell cultures), JCRB (Japanese Bank for cellular resources) or ECACC (European collection of cell cultures), and were cultured in medium RPMI-1640, supplemented with 10% V / V heat inactivated fetal calf serum, 2 mm L-glutamine and 10 mm HEPES.
Studies of cytotoxicity
MTT assay (CellTiter96® non-radioactive analysis of cell proliferation from Promega), which is a colorimetric analysis, based on the ability of viable cells to recover soluble salt tetrazolium (MTT) to a blue crystals formosana), used to determine the number of viable cells after treatment Apo2L/TRAIL or antibody to DR5. MTT analysis was carried out by adding pre-mixed optimized solution of the dye in the culture wells of 96-hole of tablet containing different concentrations (0 to 1000 ng/ml Apo2L/TRAIL or antibody to DR5. During a 4-hour incubation of living cells turn tetrazolium component of the dye solution in formosanus product. Then to culture wells were added to the solubilization/stop solution for dissolving formisano product and record the absorbance at 570 nm using a 96-hole tablet reader (SpectraMax). Reading the absorption at 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 is 570 nm, and clean solution looks blue, the color at the end of the study may not be blue and depends on the number of the present formazan relative to other components (including serum oxidized phenol red and unrestored MTT in culture medium.
The number of cells was optimized by conducting tests on cells to obtain the analytical signal, which is close to the upper limit of the linear region of this study. Because different types of cells have different levels of metabolic activity for each cell line did separately. For most of the investigated tumor cells used from 5000 cells per well up to 20,000 cells per well.
The following is step-by-step description of the used research:
1. Cells used for biological analysis were from pure cultures.
2. Determination of cell number and viability by Trypanosoma blue and the suspension of the cells to a final number from 5000 to 20000 cells per well.
3. The distribution of 50 ál of cell suspension in a 96-well plate.
4. Incubation tablets at 37°C in a humidified atmosphere of 5% CO2throughout the night.
5. Add 50 ál of culture medium containing different concentrations in the range from 0 to 1000 mg/ml Apo2L/TRAIL or antibody to DR5 to the samples in 96-well pad. Controls were 50 µl of culture medium (without Apo2L/TRAIL or antibody to DR5) and 100 µl of culture medium (without cells).
Each experiment was performed in a triple series of holes in three separate days. The total volume of the holes was 100 µl/well.
6. Incubation tablets at 37°C for 72 hours in a humidified atmosphere of 5% CO2.
7. Add 15 μl of dye solution to each well.
8. Incubation tablets at 37°C over a period of time up to 4 hours in a humidified atmosphere of 5% CO2.
9. Add 100 ál of solubi siraudeau/stop solution to each well.
10. Incubation of the plates overnight at 37°C.
11. Registration absorption at a wavelength of 570 nm using a 96-hole tablet reader. The reference wavelength of 750 nm was used to reduce background, which was made of cellular debris, fingerprints and other nonspecific absorption.
12. The average absorbance for the negative control was used as blank values and subtracted from all other readings. The average absorption values for each concentration of Apo2L/TRAIL or antibody to DR5 divided by the average absorption values of the positive control (100% viable cells untreated) to calculate the number of viable cells (%).
13. The percentage of viable cells (Y-axis) depending on the concentration of Apo2L/TRAIL or antibody to DR5 (the X-axis, logarithmic scale) noted on the chart, and the IC50 values were determined by the location of the values on the X-axis (ng/ml), corresponding to 50% of viable cells.
The Protocol introducing labels from Affymetrix
For all samples took indicators OD260/280, and the samples were driven through the BioAnalyzer. Used 5 µg total RNA of high quality.
A. Synthesis of the first chain cDNA:
1. Hybridization of the primer
DEPC-H2O x ál Mixing by shaking. Quickly precipitate by centrifugation.
RNA (5 μg) y ál Incubation at 70°C for 10 minutes.
Con the roll mortar (1:4 dilution of the initial solution for 5 ág) 1 ál High speed and location on the ice
T7-(dT)24 primer 1 ál
Volume of 12 ál
2. The temperature setting
5X buffer for the 1st chain cDNA 4 ál
Add 7 ál (to the mixture specified on the left) to each sample.
0.1 M DTT 2 ál Mixing by shaking. Rapid rotation.
10 mm dNTP mix 1 ál of Incubation at 42°C for 2 minutes.
volume of 7 ál
3. The synthesis of the first chain
Add 1 ál of SSII RT to each sample.
SSII RT 1 ál Mixing by pipetting up and down OR gentle agitation.
Total volume of 20 µl Incubation at 42°C for 1 hour.
B. Synthesis of the second chain cDNA
1. Place the reaction mixture for the first circuit on the ice. Short centrifugation to reduce condensation on the sides of the tubes.
2. The following mixture for the second chain.
DEPC-treated H2O 91 ál
5X Reaction buffer for the second circuit 30 ál
10 mm dNTP mix 3 ál
10 U/µl DNA ligase 1 ál
10 U/µl DNA polymerase I 4 ál
2 U/μl RNase H 1 ál
Total volume of 130 μl
3. Add 130 µl of the working mixture for the second circuit to 20 ál of the first chain cDNA. (Final volume = 150 ál)
4. Mix by pipetting up and down OR light shaking. Rapid rotation.
5. Incubation at 16°C for 2 hours in a cooling water bath.
6. Add 2 ál [10 Units] T4 DNA polymerase. Mix by pipetting VVER is down OR light shaking. Rapid rotation.
7. Incubate for 5 minutes at 16°C.
8. Add 10 ál of 0.5 M EDTA. Weak shaking. Rapid rotation.
9. Go to the procedure cDNA purification OR stored at -20°C for later use.
Clearing double helix cDNA (Module for cleaning samples GeneChip)
1. Add 600 ál of cDNA binding buffer to 162 ál final preparation of the synthesis of the double helix cDNA.
Mixing by shaking for 3 seconds.
2. Check that the color of the mixture is yellow (similar to cDNA binding buffer w/o the cDNA synthesis rxn.)
If the color of the mixture is orange or violet, add 10 ál of 3 M sodium acetate, pH 5.0, and mix.
The color of the mixture will go into yellow.
3. Apply 500 ál of the sample on the column for chromatography by centrifugation for purification of cDNA, 2 ml collection tube, and centrifuged for 1 min at ≥8000 x g (≥10,000 rpm). Dispose of breakthrough as hazardous waste.
4. To restart the spin column with the remaining mixture (262 μl) and centrifuged as above.
Dispose of slippage and the collection tube. *hazardous waste.
5. Transfer the spin column in a new 2 ml collecting tube (included). Transfer by pipette 750 ál of cDNA wash buffer to the spin column. Centrifuged for 1 min at ≥8000 x g (≥10,000 rpm).
Drop n is a discontinuous stream.
6. Open the lid of the spin column and centrifuged for 5 minutes at maximum speed (≤ 25000 x g). To place the column in a centrifuge, using every second slot of the rotor, positioning cap tubes above the adjacent slots of the rotor so that they are oriented in the opposite direction of rotation, i.e. if the rotation is clockwise, cap oriented in the counterclockwise direction. This avoids damage to the covers.
Throw a continuous stream and remove the collector tube.
7. Transfer the spin column in a 1.5-ml collecting tube. Move the pipette 10 ál of buffer for elution cDNA directly on the membrane of the spin column. Make sure that the buffer for elution cDNA distributed directly on the membrane.
Incubate for 1 minute at room temperature and centrifuged for 1 minute at maximum speed (≤ 25000 x g) for the elution.
Preparing and conducting the reaction of IVT
Enzo: Set for labeling RNA transcript Bioarray HighYield (Batch No. 900182)
1. Use 10 μl of purified double helix cDNA.
2. Prepare the following mixture for IVT:
Distilled or deionized H2O 12 ál
10X HY reaction buffer 4 ál
10x ribonucleotides labeled with Biotin 4 ál
10X DTT 4 ál
10X mix, inhibiting RNase 4 ál
2X T7 RNA polymerase 2 ál
Total volume: 30 ál
3. Add 30 ál of the working mixture IVT to 10 ál of double helix cDNA (Total volume = 40 ml).
4. Mix by pipetting up and down OR light shaking. Rapid rotation.
5. Immediately place the test tube in a water bath at 37°C. Incubate for 5 hours.
6. Store at -20°C if not to make immediate purification of RNA.
Cleaning crnc labeled with Biotin (GeneChip Sample Cleanup Module).
1. Add 60 ál of H2O to the IVT reaction and mix by shaking for 3 seconds.
2. Add 350 ál of binding buffer IVT crnc to the sample, mix by shaking for 3 seconds.
3. Add 250 ál ethanol (96-100%) to this lysate and mix thoroughly by pipetting. Not centrifuged.
4. Apply the sample (700 ál) to the cleansing of the spin column for IVT crnc installed in 2 ml of reservoir tube.
Centrifuged for 15 seconds at ≥8000 x g (≥10,000 rpm).
5. Skip the eluate through the column again.
Centrifuged for 15 seconds at ≥8000 x g (≥10,000 rpm).
Throw a continuous stream as **hazardous waste and remove the collector tube.
6. Transfer the spin column in a new 2 ml collecting tube (included).
7. Add 500 ál of wash buffer to IVT crnc and centrifuged for 15 seconds at ≥8000 x g (≥10,000 rpm) to wash.
Discard continuous the stream.
8. Apply a pipette 500 ál of 80% (vol./about.) ethanol to the spin column and centrifuged for 15 seconds at ≥8000 x g (≥10,000 rpm). Drop a steady stream.
9. Open the lid of the spin column and centrifuged for 5 minutes at maximum speed (≤25000 x g).
Throw a continuous stream and remove the collector tube.
10. Transfer spin column into a new 1.5-ml collecting tube.
11. Apply the pipette 11 μl of water without RNase directly on the membrane of the spin column. Leave on for 1 minute.
Centrifuged for 1 minute at maximum speed (≤25000 x g) for elution.
12. Apply the pipette 10 ál of water without RNase directly on the membrane of the spin column. Leave on for 1 minute.
Centrifuged for 1 minute at maximum speed (≤25000 x g) for elution.
Determining the number crnc (IVT product).
To determine whether the RNA using spectrophotometric analysis. Accept the condition that 1 OD at 260 nm equals 40 μg/ml RNA.
Check the OD at 260 nm and 280 nm to determine the concentration of the sample and purity.
To maintain the ratio A260/A280 close to 2.0 for pure RNA (acceptable ratio of from 1.9 to 2.1).
For the quantitative determination crnc when using total RNA as starting material adjusted output crnc should calc the drain for the expression of the rest of unlabeled total RNA. Using assessment 100% transfer use the following formula to determine the adjusted output crnc:
The adjusted output crnc = Rncm - (General RNC)(y)
Rncm = number crnc measured after IVT (µg)
General RNK = initial amount of total RNA (ág)
y = fraction of cDNA reaction used in IVT
Fragmentation crnc to obtain the target
For fragmentation, use the corrected concentration crnc.
1. Add 2 ál of 5x buffer for fragmentation for each 8 ál of RNA plus H2O.
20 µg crnc 1 to 32 ál
5X buffer for fragmentation 8 ál
Water without RNase to 40 ál
Total volume: 40 ál
2. Incubate at 94°C for 30 minutes. After incubation, immediately put on ice.
Receiving target for hybridization
1. Heat 20X hybridization controls for eukaryotes and oligo B2 for 5 minutes at 65°C.
Set for hybridization control eukaryotes Affymetrix GeneChip, Party #900362 (150 reactions)
2. Light shaking, slow rotation.
3. The working mixture (assuming that the concentration of fragmented crnc is 0.5 ág/ál):
Standard matrix (ál) Final concentration
Fragmented crnc 15 ág 30 0.05 µg/µl
Oligo B2 (3 nm) 5 50 PM
20x control Spike 15 is 1.5, 5, 25, 100 PM
(Bio B, C, D, Cre)
Sperm DNA herring 3 0.1 mg/ml
Acetiminophen the initial BSA 3 0.5 mg/ml
Hu cot-1 DNA (1 mg/ml) 30 0.1 mg/ml
2X MES died. the buffer 150 1X
The final volume 300
4. Pour an aliquot of the working mixture 270 ál in test tubes and to each add 30 ál of fragmented crnc. It is the hybridization mixture.
5. Immediately prior to use of a matrix of probes to be brought into equilibrium with room temperature.
6. Fill the matrix probes 1x MES hybridization buffer and incubate in the oven grill for 10 minutes at 45°C, 60 rpm
7. Heat the hybridization mixture on the water bath at 99°C for 5 minutes.
8. To transfer the hybridization mixture in a water bath at 45°C for 5 minutes.
9. Centrifuged hybridization mixture for 5 minutes at maximum speed.
10. Remove 1x MES hybridization buffer from the matrix probes.
11. To fill a matrix of probes to the brim with 200 μl hybridization mixture.
12. Sealing the cell with Tough-Spots.
13. To hybreed probe arrays at 45°C, 60 RPM for 19 hours.
14. Wash, stain and scan the probe arrays according to the Affymetrix protocols.
Name supplier number directory
T7-(dT)24 primer Biosearch Technolgies to order
Control spikes produced -
Superscript II/5X Buffer
for the synthesis of the first chain/0.1 M DTT Invitrogen 18064-014
5X buffer for the synthesis of the second chain Invitrogen 10812-014
10 mm dNTP Invtrogen 18427-088
10 U/µl DNA ligase E. coli Invitrogen 18052-019
10 U/µl DNA polymerase I of E. coli Invitrogen 18010-025
2 U/ál RNase H Invitrogen 18021-071
10 U/µl DNA polymerase T4 Invitrogen 18005-025
of 0.5 M EDTA Sigma E-7889
ENZO High Yield RNA Transcript
labeling kit, Affymetrix or ENZO 900182 (ENZO)
GeneChip Sample Cleanup Module, Affymetrix 900371
serum albumin Invitrogen 15561-020
Goat IgG - chemically pure Sigma I-5256
Antibodies to streptavidin (goat),
biotinylated Vector Labs BA-0500
R-icaritin streptavidin Molecular Probes S-866
20X SSPE BioWhittaker 51214
Eukaryotic Control Kit (Affymetrix 900362
Water, Molecular Biology Grade Ambion 9934
DNA human Cot-1 Roche 1-581-074
5 M NaCl without RNase, without DNase Ambion 9760
Protivovspenivayushchie substance 0-30 Sigma A-8082
10% tween-20 Pierce Chemical 28320
The monohydrate of the free acid MES Sigma M5287
Sodium salt MES Sigma M3885
The disodium salt of EDTA, 0.5 M solution Sigma E7889
Tough Spots, Label Dots USA Scientific 9902
GeneChip hybridization oven 640 Affymetrix 800139
GeneChip scanner 3000 w/workstation Affymetrix 00-0074
Spray plant Affymetrix 00-0081
Autoloader w/External Barcode Reader Affymetrix 00-0129
Synthesis of cDNA:
|10X buffer for reverse transcription||10|
|25X dNTP mix||4|
|10X random primers||10|
|Multiplex (50 U/µl)||5|
|H2O without RNase||21|
|RNA (100 ng)||50|
Conditions of incubation:
25° for 10 minutes
37° for 2 hours
TaqMan reaction using the ABI Prism 7700 Sequencing Detector:
|Universal fuel mixture TaqMan PCR (2X)||25|
|The TaqMan probe (20X)|
|cDNA (100 ng)||2|
Thermal conditions cycles:
95° for 10 minutes
40 cycles: 95° for 15 seconds
60° for 1 minute
TaqMan probes: Assays-on-Demand™ (TaqMan® MGB probes, FAM™ dye-labeled)
Amplification of the internal control, GAPDH (to ncentrate probe 100 nm, concentrations of forward and reverse primers and 200 nm) was performed to standardize the amount of RNA sample (cDNA)added to each reaction.
Comparative quantitative analysis was performed using a calibration curve. For quantitative analysis, normalized to internal control, calibration curves were built for both the target and internal standard. For each experimental sample, the amount of target and internal standard to determine the appropriate calibration curve. Then the number of the target divided by the number of internal control to obtain the normalized desired. One of the experimental samples serves as a calibrator or 1x sample. Each of the normalized desired values and then divided by the normalized desired value for the calibrator to obtain the relative levels of expression.
The experimental results.
The experiments were conducted using the methods and materials described above. The results of these experiments are shown in Fig. 5-9, discussed below.
Figure 5 presents a summary table of data for IC50 obtained in the analysis of sensitivity or resistance cell lines of non-small cell lung cancer ("NSCLC") to the apoptotic activity of Apo2L (+ 0,5% fetal calf serum "FBS" or 10% FS) or monoclonal antibody to DR5 mab", cross-linked "XL" or not cross linked, to + 0.5% fetal calf serum "FBS" or 10% FBS), as defined in MTT assays of cytotoxicity.
Figure 6 presents a summary table of data for IC50 obtained in the analysis of sensitivity or resistance lines malignant pancreatic cells to the apoptotic activity of Apo2L (+ 0,5% fetal calf serum "FBS" or 10% FBS) or monoclonal antibody to DR5 mab"cross-linked "XL" or not cross linked, to + 0.5% fetal calf serum "FBS" or 10% FBS), as defined in MTT assays of cytotoxicity.
Figure 7 presents a summary table of data for IC50 obtained in the analysis of the sensitivity or resistance of malignant cell lines of non-Hodgkin's lymphoma ("NHL") to the apoptotic activity of Apo2L (+ 10% fetal calf serum "FBS") or monoclonal antibody to DR5 mab"cross-linked "XL" or not cross linked, to + 0.5% fetal calf serum "FBS" or 10% FBS), as defined in MTT assays of cytotoxicity.
On Fig comparison of sensitivity (sen) or resistance ("RES") of selected lines of malignant cells NSCLC, pancreatic cancer and NHL for the antibody to DR5 and correlation with expression of GalNac-T14, measured by mRNA expression GalNac-T14.
Figure 9 presents the histogram p is slichnih NSCLC cell lines, pancreas and NHL cell located (in descending order) by the levels of mRNA expression.
The program apoptotic cell death plays an important role in the development and homeostasis of multicellular organisms (Danial et al., Cell, 116:205 (2004)). Intracellular stimuli can trigger apoptosis via the endogenous pathway, which is caused by the representatives of the superfamily of genes Bcl-2, activation of apoptotic Casanova mechanism (Cory et al., Nat. Rev. Cancer, 2:647 (2002)). Certain cytokines that belong to the superfamily of tumor necrosis factor (TNF), can activate apoptosis through an external pathway by interacting with certain receptors that contain functional inducing apoptosis "death domain" (DD) (Ashkenazi et al., Science, 281:1305 (1998)). Fas ligand (FasL) stimulates apoptosis through Fas (Apo1/CD95), whereas Apo-2 ligand/TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) triggers apoptosis through DR4 (TRAIL-R1) and/or DR5 (TRAIL-R2) (LeBlanc et al., Cell Death Differ., 10:66 (2003)). When binding a cognate ligand, these receptors bind the adapter molecule FADD (associated with Fas death domain), which attracts the initiator of apoptosis caspase-8 to the formation of inducing death signaling complex (DISC) (see, for example, Kischkel et al., EMBO J. 14:5579 (1995); Kischkel et al., Immunity, 12:611 (2000)). DISC Association stimulates caspase-8, which in itsturn, activates effectiye proteases, such as caspase-3, 6 and 7, for the execution of apoptotic program death. In many cell types, a reciprocal influence on the internal pathway may further amplify the external cell death signals (Scaffidi et al., J. Biol. Chem., 274:1541 (1999)). Apo2L/TRAIL induces apoptosis in various types of tumor cells with little impact or no impact on normal tissue, indicating that Apo2L/TRAIL can be used for the treatment of malignant diseases (see, for example, Ashkenazi, Nat. Rev. Cancer, 2:420 (2002); Kelley et al., Curr. Opin. Pharmacol., 4:333 (2004)). Changes in various components of the pathways of apoptosis can reduce sensitivity to Apo2L/TRAIL in certain malignant cell lines (Igney et al., Nat. Rev. Cancer, 2:277 (2002)).
Performed various experiments in accordance with the methods and protocols described below, and the data presented on Figure 10-15.
To study the sensitivity to activation of the receptor tested the survival of cells depending on the concentration of Apo2L/TRAIL in a group of malignant cell lines, including 23 adenocarcinoma of the pancreas, 18 malignant melanomas and 36 colorectal adenocarcinomas (Figa and data not shown). As a result of this analysis 29/77 (38%) cell lines were classified as highly or moderately sensitive to Apo2L/TRAIL. The concentration of the Apo2L/TRAIL, required to achieve 50% cell death in these 29 cell types, ranged from 3 to 800 ng/ml with an average value of 250 ng/ml
The group of cell lines was also investigated on the profile of gene expression using microarray sets 54613 gennych probes. Though with some exceptions, cell lines of pancreatic cancer and melanoma, which showed strong or moderate sensitivity to Apo2L/TRAIL, expressed significantly higher mRNA levels of the enzyme O-glycosylation of ppGalNAcT-14 than the corresponding drug-resistant cell lines (p=0.5×10-4by exact Fisher test, the limit on a defined multiple times at 750 for carcinoma of the pancreas and 300 for melanoma) (Pigv). Most of Apo2L/TRAIL-sensitive cell lines of colorectal cancer showed high expression of mRNA related O-glycosylated enzyme ppGalNAcT-3, although some resistant cell lines also expressed the gene, thus resulting in a weak, yet significant difference (p=0,026, the threshold value defined in 2000) (Figs, below). Exceptions to the whole group were: (a) 5/29 (17%) cell lines that were sensitive, yet expressed ppGalNAcT-14 or ppGalNAcT-3 below threshold levels; (b) 16/48 (33%) of resistant cell lines, which, however, had ur the attention ppGalNAcT-14 or ppGalNAcT-3 above the threshold values. Examining the mRNA expression of other enzymes of O-glycosylation in colorectal cell lines, higher levels Fut-6 were detected in 10/12 (83%) sensitive compared to 6/24 (25%) of resistant cell lines (p=0,013; the threshold value determined at 200) (Figs, above). Combined expression of ppGalNAcT-14 with pancreas cancer and melanoma with Fut-6 cell lines of colorectal cancer is very strongly correlated with sensitivity to Apo2L/TRAIL (p = 1,83×10-7N=77). This gene set correctly predicted sensitivity or resistance to 23/32 (72%) marker-positive and 39/45 (87%) marker-negative cell lines, respectively.
Apo2L/TRAIL sensitivity was studied alsoin vivousing tumor xenografts. 5-day treatment Apo2L/TRAIL of mice with tumors originating from Fut-6-positive cell lines of colorectal cancer Colo205 and DLD-1 caused regression of tumors after a significant delay of tumor progression (Fig.10D). In contrast, tumors originating Fut-6-negative cell lines of colorectal cancer Colo320 and RKO did not respond to this treatment.
Pre-incubation ppGalNAcT-3/Fut-6-positive cell lines Colo205 with inhibitor pan O-glycosyltransferases benzyl-GalNAc (Delannoy et al., Glycoconj., 13:717 (1996)) was significantly reduced sensitivity to Apo2L/TRAIL (Figa), indicating a functional link to the e between O-glycosylation and signaling by Apo2L/TRAIL. For additional research on specific small interfering (si)RNA oligonucleotides used in such a way that they are aimed at mRNA ppGalNacT-14, ppGalNacT-3 or Fut-6. To exclude non-target effects have synthesized numerous tile display siRNAs for each gene and tested their ability to reduce the target expression using quantitative RT-PCR (Figa). The specificity of the siRNAs was additionally confirmed using mutant plasmids ppGalNacT-14, containing 6 "silent" nucleotide changes within the target area for siRNAs (Editorial, Nat. Cell. Biol., 5:489 (2003)) (Pigv). Transfection of cell lines ppGalNAcT-14-positive PSN-1 carcinoma of the pancreas and Hs294T melanoma using ppGalNAcT-14 siRNAs significantly reduced the sensitivity to Apo2L/TRAIL, whereas siRNAs caspase-8, as expected, provided essentially complete protection (Pigv). Similarly, transfection of DLD-1 or C170 cells of colorectal cancer using GalNAcT-3 or Fut-6 siRNAs significantly reduced the sensitivity to Apo2L/TRAIL (Figs and Figs). In sum, GalNAcT-14 siRNAs decreased the sensitivity to Apo2L/TRAIL in 4/5 cell lines pancreatic cancer and 2/2 melanoma cell lines, whereas ppGalNAcT-3 or Fut-6 siRNAs each reduced sensitivity 2/3 cell lines of colorectal cancer. In contrast, transfection of PSN-1 or Hs294T cells with use the of GalNAcT-14 siRNAs did not change the sensitivity to topoisomerase II inhibitor to etoposide (Fig.14D). Similarly, transfection of PSN-1 or C170 cells using GalNAcT-14 or GalNAcT-3 siRNAs did not affect sensitivity to the inhibitor of protein kinases a wide range of actions staurosporine (Fige). Since etoposide, and staurosporin stimulate apoptosis through the inner cell path (Wei et al., Science, 292:727 (2001), these studies indicate that the enzymes of O-glycosylation can modulate the transmission of the apoptotic signal through an external pathway.
Transfection of HEK293 cells with the use of ppGalNAcT-14 was detected cell death when combined transfection with DR4 or DR5, but not related receptors Fas and TNFR1 or agonist Bax inner cell path (Fig.11D). Moreover, ppGalNAcT-14 transfection increased sensitivity to Apo2L/TRAIL-resistant cell lines H1568 melanoma (Fige) and PA-TU-8902 and PL-45 carcinoma of the pancreas (Fig.14F), but did not change the sensitivity to etoposide (data not shown). Finally, overexpression of GalNAcT-14 was senzibilizirana 4/7 cell lines to the effect of Apo2L/TRAIL.
The effect of siRNAs knockdown ppGalNacT-14 or Fut-6 were investigated on Apo2L/TRAIL-induced processing of caspase. In cells PSN-1 and DLD-1 cells were transfectional control siRNAs, Apo2L/TRAIL induced essentially complete processing of caspase-8, leading to the cleavage of Bid, caspase-9 and caspase-3 (Fega). Transfection using siRNAs cusp is s-8 prevented these events. Knockdown ppGalNAcT-14 cells PSN-1 or Fut-6 in cells DLD-1 also significantly weakened Apo2L/TRAIL-induced processing of caspase-8, Bid, caspase-9 and caspase-3 (Fega) and stimulation of the activity of caspase-3/7 (Pigv). Apo2L/TRAIL-resistant colon cancer cell lines RKO and SW1417, which Express low levels of ppGalNAcT-3 and Fut-6, showed similar blockade at the level of processing of caspase-8 (Figa). Thus, the enzymes of O-glycosylation can modulate the events preceding Apo2L/TRAIL cascade, leading to activation of caspase-8.
To activate caspase-8 requires the DISC Assembly (Ashkenazi et al., Science, 281:1305 (1998)). Analysis of Apo2L/TRAIL DISC (Kischkel et al., Immunity, 12:611 (2000)) in cells PSN-1 and DLD-1 was indicated that knockdown ppGalNAcT-14 or Fut-6 reduced recruiting FADD caspase-8 to the DISC, the processing DISC-associated caspase-8 and stimulation associated with the DISC and the enzymatic activity of caspase-8 (Sharp et al., J. Biol. Chem., 280:19401 (2005)) (Figs, 12D and Figv). No ppGalNacT-14 or Fut-6 siRNAs essentially did not change the number of DR4 and DR5 in DISC or dose-dependent binding of Apo2L/TRAIL with cells PSN-1 and DLD-1, which Express both DR4 and DR5 (Figs, Figv and data not shown). Thus, ppGalNAcT-14 and Fut-6, apparently, not modulate apoptosis by influencing the levels of receptors on the cell surface or Apo2L/TRAIL linking. In line with this, Apo2L/TRAIL sensitivity in the group of 77 cell lines did not show C is uchitelnoj correlation with expression on the cell surface are recognized signaling receptors DR4 and DR5 or false receptors DcR1 and DcR2 (data not shown). Moreover, most of siRNAs against ppGalNAcT-14, ppGalNacT-3 or Fut-6 did not change the levels of DR4 and DR5 on the cell PSN-1, C170 or DLD-1 (Figs). Two siRNAs did not cause detectable reduction of DR4 and DR5 in certain cell lines (Figs). However, other siRNAs against the same enzyme inhibited Apo2L/TRAIL-induced apoptosis without affecting the levels of receptors.
The extracellular domain (ECD) of human DR5 expressed in Chinese hamster oocytes, secreted protein was purified, subjected to acid hydrolysis and analyzed the corresponding monosaccharides (Figa). In accordance with the absence of the expected sites of N-glycosylation in DR5 ECD, the authors did not find N-linked glycans. However, in two samples of 2 independent experiments was identified 3 mol GalNAc and 3 mol Gal per mole DR5 ECD (Figa), indicating O-linked modification of the three sites to DR5 with crustal Picanol GalNAc-Gal.
O-glycosylation of the protein modifies serine or threonine. Using previously established the bioinformatics tool for predetermining possible sites of O-glycosylation (http://www.cbs.dtu.dk/services/NetOGlyc) (Julenius et al., Glycobiology, 15:153 (2005)), the authors identified two such areas in the total sequence ECD long (DR5-L) and short (DR5-S) variants of splicing human DR5, and the third site is within the area of alternative splicing (Pigv). The first amino acid segment (74-77) content is it 3 serine; the second (130-144) has 5 threonine, while the third has a 4-threonine and 3 serine. DR5 mouse has a sequence similar to the first 2 segments 2 Suriname and 4 threonine respectively, whereas DR4 person also has 2 similar sequence containing 1 serine and 5 threonine. To test whether these sites can be important for post-translational modification DR5, was a series of DR5L and DR5S mutants with substitution on alanine or 5 threonine segment 130-144 (DR5L-5T, DR5S-5T), or the same 5 threonine, and 3 Surinov segment 74-77 (DR5L-5T3S, DR5S-5T3S). DR5 or antibody-based test, the Western blot turns lysates of HEK293 cells, transfection DR5L or DR5S has revealed the presence of the expected bands DR5L or DR5S (Figs). This antibody also revealed bands DR5 higher molecular mass (MM), which is becoming more common in co-transfection DR5L or DR5S using ppGalNAcT-14 compared to control (Figs, asterisks). The distribution of these bands higher MM and their rise under the action of ppGalNAcT-14 decreased significantly with DR5L-5T or DR5S-5T and almost disappeared with DR5L-5T3S or DR5S-5T3S compared with the structures of the wild type. These results indicate that band higher MM are O-glycosylated forms of DR5: ppGalNAcT-14 contributes to their education, and the gradual destruction of the alleged sites of O-glycosylation by replacing the alanine after avatele modifies this effect. Transfection of HEK293 cells using mouse DR5 or DR4 person, DR5L or DR5S showed the death of cells (Fig.13D); each DR5 mutant showed less activity than the corresponding structure of the wild type, with DR5S-5T3S (which lacks all three sites), with the weakest activity. Co-transfection with ppGalNAcT-14 markedly increased death of cells under the action of all structures DR4 and DR5, except DR5S-5T3S, which showed significantly less activity.
Most samples of normal tissue and tumor samples of skin cancer, lung, pancreas, breast, ovary, endometrium, and bladder or non-Hodgkin's lymphoma demonstrated the expression of mRNA ppGalNAcT-14 below the threshold values were set at 500 for most malignant tumors and 200 for skin cancer, File). However, a significant subset of tumor samples obtained in the range from 10% for lobular breast cancer up to 30% for lung cancer and both diffuse b-cell lymphoma showed overexpression of ppGalNAcT-14. Levels of mRNA expression of some samples of malignant cells were more than 1000 times higher than the values in the corresponding normal tissues. Dynamic expression of ppGalNAcT-14 in malignant tumors indicates that this gene, and possibly other related enzymes may provide useful biomarkers on what I identify tumors with greater sensitivity to Apo2L/TRAIL.
O-linked glikana show considerable structural diversity, and they modulate various aspects of the biology of plasma membrane proteins, including conformation, aggregation, directed migration, the half-life, as well as cell adhesion and activity of signal transduction pathways (Hang et al., Bioorg. Med. Chem., 13:5021 (2005); Hanisch, Biol. Chem., 382:143 (2001)). In malignant cells often appear dramatic changes profiles of O-glycans, creating a unique associated with carbohydrate tumor antigens (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 the return of tumor cells to specific sites 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)). Significant subgroup samples of primary tumors of various human malignant tumors demonstrates increased expression of the enzyme O-glycosylation of ppGalNAcT-14, including the sample of cells of the large intestine and the colon, rectum, samples of melanoma cells and the sample cells chondrosarcoma.
Reagents for cell culture were purchased by the company Gibco (Invitrogen/Gibco, Carlsbad, CA), unlabeled soluble Apo2L/TRAIL were prepared as previously described (Lawrence et al., Nat. Med., 7:383 (2001)), inhibitor of O-linked glycosylation benzyl-a-GalNAc. what do Calbiochem, all other chemicals (including etoposide and staurosporine) were from Sigma Aldrich (St. Louis, MO).
Cell culture and cell line
All 119 cell lines carcinoma person (name and catalog numbers, see supplementary information) were obtained from ATCC or DSMZ (Braunschweig, Germany) and were cultured at 37°C and 5% CO2in RPMI1640, supplemented with 10% V / V heat inactivated fetal calf serum, 2 mm L-glutamine and 10 mm HEPES without antibiotics, such as penicillin/streptomycin. 293 embryonic kidney cells (catalog number CRL-1573) were also obtained from ATCC and cultured in 100% modified by Dulbecco environment, Needle, supplemented with 10% FBS. On O-glycosylated mutant cell line, CHO, CHO ldlD permission was obtained from Dr. Monty Kreiger, MIT (Boston MA).
Analyses of cell viability and analysis of apoptosis
To determine the IC50 for Apo2L/TRAIL cells were sown in three repetitions in 96-well plates, were left for adhesion for 24 hours and then treated with recombinant Apo2L/TRAIL of the man in increasing concentrations up to 1000 ng/ml After incubation for 72 h and then subjected to the study on the viability - MTT analysis (Pierce) or fluorescence analysis of cell viability CellTiter-Glo (Promega) in accordance with the Protocol of the manufacturer. Each experiment, cell viability repeat the whether at least three times at low (0.5%) and high (10% FBS) concentrations of serum and cell lines with transition sensitivity was characterized by fluctuations between the IC50 values of independent experiments or between low and high serum concentrations. The cell line was characterized as sensitive, based on the induction of apoptosis by at least 50% of the cells at a concentration of Apo2L/TRAIL 1 MGL/ml and as transition sensitivity on the basis of the extent of apoptosis induced in independent experiments or in the presence of low serum concentrations (0,5%) compared with high serum (10%) concentration. Apoptosis was quantitatively assessed using flow cytometry average percentage of collected cells adhered + floating in the environment) stained with annexin V (BD Pharmingen).
Hybridization on the microarray and data analysis
Total cellular RNA was obtained from untreated cells (3 x 106using a set RNeasy Kit (Quiagen). Labeled crnc received and hybridisable with nucleotide microarrays (U133P GeneChip; Affymetrix Incorporated, Santa Clara, CA)as described previously (Hoffman et al., Nat. Rev. Genetics, 5:229 (2004); Yauch et al., Clin. Cancer Res., 11:8686 (2005)). Files scanned images were analyzed using GENECHIP 3.1 (Affymetrix), Spotfire, GenePattern and Cluster/TreeView. To identify the most differently expressed genes between sensitive and resistant cell lines, the authors subjected the values of g the authorized expression changes are selected, which was excluded genes with minimal change in all the analyzed samples, testing on the rate of change and absolute change in all samples, comparing the ratio of maximum and minimum (max/min) and the difference between the maximum and the minimum min (max-min) with pre-defined values and excluding genes that do not satisfy both conditions.
Expression constructs and retroviral transduction
The DNA fragment encoding ppGalNAcT-14, cloned from cDNA pooled from Apo2L/TRAIL-sensitive cell lines, and inserted into the expression plasmid pcDNA3.1 (Invitrogen) with an N-terminal Flag tag. This design was then subjected to site-directed mutagenesis (Quikchange Mutagenesis kit, Stratagene) to generate siRNAs silent mutants that had 4-6 “wobble” change of base pairs in the sequence, homologous siRNAs, without changing the protein sequence. These mutations were blocked region 10 nucleotide pairs in the center of the binding sequences of siRNAs length 19 i.e. the DNA Sequence for DR5Long and DR5Short, DR4, TRAIL receptor mouse, DR4, Fas (option 1), TNFR1 and Bax (beta version) was cloned from cDNA pools and inserted into the expression vector pRK (Genentech). O-glycosylated mutants DR5L and DR5S received site-specific mutation of four threonines on residues of alanine, Mut4xTA (T130, T131, T132, T135) or five threonines on residues alanine is Mut5xTA (T130, T131, T132, T135, T143). Transient transfection in HEK293 cells expression constructs proapoptotic molecules was carried out in 6-hole tablets with a concentration of 0.5 μg/well of proapoptotic molecules and 2.0 µg ppGalNAcT-14 or vector control. The cells were transfectional using lipofectamine 2000 in accordance with the Protocol of the manufacturer. After 48 h incubation, cells were analyzed for apoptosis.
To obtain retroviral constructs ppGalNAcT-14 and mutants were cloned into the retroviral vector pQCXIP (Clontech). High titer retroviral supernatants received with use of line helper cells ΦNX-Ampho. Packaging cells were transfectional using calcium phosphate (Invitrogen). Supernatant was isolated 48 hours after transfection and added to the target cells along with 10 µg/ml polybrene, followed by stage 1 h centrifugation at 2700 rpm for amplification of infection. After transduction, the cells were subjected to selection with 2 μg/ml puromycin.
The design of siRNAs and transfection protocols
siRNAs against ppGalNAcT-14, ppGalNAcT-3, caspase-8 and DR5 were designed Dharmacon (Lafayette, CO) using their own selection criteria. The selected sequences were:
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) (SQ 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)
si-8: 5' GGACAAAGTTTACCAAATG-dTdT 3' (SEQ ID NO:21)
siRNAs were purchased in the form of a double helix RNA oligonucleotides and transfectional in the appropriate cell line with a final concentration of 25 nm siRNAs for each. Duplex siRNAs against non-target sequence (Dharmacon) was used as control. The cells were transfectional using lipofectamine 2000 (Invitrogen) by reverse drasticly, where cells are added to the suspension to pre-printed complexes lipid-siRNAs. The concentration of lipofectamine 2000 corresponded to the Protocol of the manufacturer. After incubation of 48 h the cells were collected for mRNA analysis or incubated with recombinant Apo2L/TRAIL man, etoposide or staurosporine for 24 to 72 hours to research the viability or for 4, 8 or 24 h for Western blotting.
Inhibition of O-glycosylation under the action of benzyl-a-GalNAc.
The Colo205 cells were grown in the presence of benzyl-a-GalNAc (2 mm or 4 mm) for 72 hours. At this point they were transferred in 96-well plates, leaving for adhesion for 24 hours, still in the presence of inhibitor. Then, stimulated by increasing concentrations of Apo2L/TRAIL, as indicated, and subjected to examination for viability.
If the natural enemy PCR
The levels of expression of transcripts GalNacT-14 and GalNacT-3 was evaluated using quantitative RT-PCR using standard Taqman methodology. The levels of transcripts was normalized to obligate gene, GAPDH, and the results were expressed as normalized values of the expression (=2-DCt). Sets of primer/probe for GalNacT-14 (cat#: Hs00226180_m1_GT14), GalNacT-3 (cat#:Hs00237084_m1_GT3) and GAPDH (cat#: 402869) were purchased from Applied Biosystems (Foster City, CA).
Immunoprecipitation, Western blotting and antibodies
Immunoprecipitate: anti-Apo2L (2E11; ATCC registration number HB-12256), anti-DR4 (3G1 and 4G7, ATCC registration number PTA-99) and anti-DR5 (3H3, ATCC registration number and 12534 5C7) monoclonal antibodies were obtained in Genentech, Inc., using as antigens fused proteins receptor-Fc. Anti-DR4 (4G7) and anti-DR5 (5C7) monoclonal antibodies used for thus Apo2L/TRAIL DISC, conjugatively with agarose using a set of ImmunoPure Protein G IgG Plus orientation kit (catalog number 44990) from Pierce. Anti-DR4 (3G1) and anti-DR5 (3H3) monoclonal antibodies used for immunodetection DR4/5 immunoprecipitated DISC, biotinilated using the kit biotinidase EZ-link Sulfo-NHS-LC (catalogue number 21217) from Pierce. Received Apo2L/TRAIL marked FLAG, and a cross was made with anti-FLAG antibody M2 (Sigma)as described (Kischkel, Immunity, 12:611 (2000)). These experiments were performed as described previously for Apo2L/TRAIL-FLAG + anti-FLAG DISC analysis (Kischkel, supra). Experiments on DR4/5 DISC IMM is coprecipitate conducted, as described, except that the anti-DR4 (4G7) and anti-DR5 (5C7) monoclonal antibodies directly conjugatively with agarose for thus (Sharp et al., J. Biol. Chem., 280:19401 (2005)).
Western blotting: 5×105cells per well were sown in 6-well plates. For the experiments on RNC the knockdown cells were treated with different siRNAs for 48 hours, then Apo2L/TRAIL for 4, 8 or 24 h After the indicated time periods, cells were washed in ice-cold PBS and literally in 1% Triton X-100 containing hypotonic lyse 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 separated in reducing conditions on 10% or 10-20% gradient SDS-polyacrylamide gels. After transfer to nitrocellulose membranes (Schleicher and Schuell) membranes were incubated for 1 h in 10% non-fatty milk powder, followed by 1 h incubation with the following primary antibodies: goat antibody to caspase-3 (1:1000, R&D), rabbit antibody to caspase-8 (1:1000, Pharmingen), mouse antibody to caspase-9 5B4 (1:1000, MBL), rabbit antibody to Bid (1:1000, Pharmingen), rabbit antibody to DR5 (1:500, Cayman) or goat antibody to actin (1:200, Santa Cruz Biotechnology). Membranes were washed five times with TBS/0.05% Tween, and then incubated with the appropriate conjugated with peroxidase affinity purified secondary antibody (1:5000, Biorad) for 30 min, the Membrane was again washed the five times and showed, using the enhanced chemiluminescence (ECL, Amersham) and film Kodak Biomax.
Flow cytometry/FACS analysis.
Surface expression of receptors of the TNF family DR4 and DR5 were determined using fluorescence activated cell sorting (FACS)using flow cytometer FACS Calibur (Becton Dickinson Immunocytometry System, San Jose, CA). Cells C170 and PSN-1, transfection indicated siRNAs for 48 h, were stained with 10 μg/ml primary antibody 4G7 (anti-DR4) or 3H3 (anti-DR5), or control antibody mouse IgG for 1 h at 4°C. Then cells were washed in PBS, and then incubated with secondary goat artemisinin antibody conjugated with fluorescein (FITC) (Jackson Laboratories) for 30 min at 4°C. Then cells were analyzed by flow cytometry using flow cytometer FACS Calibur.
The activity of caspase-3/-7 investigated at 37°C in 40 μl Casanova buffer (50 mm HEPES pH of 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 during the specified time on the release of the AFC DEVD-AFC, using molecular devices fluorimetry in dynamic mode with a pair of filters 405-510. To assess caspase activity 20 μg total cellular protein extracts Triton X-100) was used in 40 µl Casanova buffer (containing 100 μm DEVD-AFC).
Analysis of carbohydrates DR5 originating from CHO
The composition of monosaccharides DR5 from CHO cells was obtained after hydrolysis with 4 N. TFA. Analysis of the extracted monosaccharides was performed on the system HPLC Dionex BioLC using anion-exchange chromatography high-resolution connected with pulse ammeter.
Animals and study of subcutaneous xenograft
Female Nude mice "nude" (The Jackson Laboratory, Bar Harbor, ME, USA) were accustomed to the conditions of animals Genentech at least 1 week prior to the registration of a pilot study. All experimental procedures were approved by the bioethics Committee Genentech (IACAUC). The mice were infected subcutaneously with 5 × 106cells/mouse Colo205, DLD-1 and RKO or 20 × 106cells/mouse cells carcinoma human colon Colo320HSR (American type culture collection). Tumor sizes were obtained using a digital caliper, and tumor volumes were calculated using the formula □~□ (A= length B=width)2. When the tumor volume of ~ 150-200 mm3mice were randomly grouped and used intraperitoneal treatment (I.P.) with the carrier or Apo2L/TRAIL (60 mg/kg/day) on days 0-4.
1. A method for predicting the sensitivity of a tissue sample or cells of a malignant tumor of a mammal the polypeptide Apo2L/TRAIL, including the stage of the research sample of tissue or cells in immunohistochemical analysis to identify the former is ressie GalNac-T14, where specified immunohistochemical analysis performed using antibodies against GalNac-T14, 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 polypeptide Apo2L/TRAIL.
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 studies of a sample of tissue or cells in immunohistochemical analysis to detect the expression of polypeptide GalNac-T14, where specified immunohistochemical analysis performed on OSU antibodies against GalNac-T14, linking 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 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 the polypeptide Apo2L/TRAIL;
and, after detection of the expression of the specified polypeptide GalNac-T14, the stage of exposure to the specified sample of tissue or cells an effective amount of the polypeptide Apo2L/TRAIL.
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 the polypeptide Apo2L/TRAIL containing amino acids 114-281 indicated in figure 1 (SEQ ID NO:1).
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, in which these cells are also treated with a cytokine, a cytotoxic agent or a growth inhibitor.
11. The way is about to claim 5, wherein said polypeptide Apo2L/TRAIL associated with a molecule of polyethylene glycol.
12. The method according to claim 5, wherein said polypeptide Apo2L/TRAIL contains amino acids 41-281 shown on figure 1 (SEQ ID NO:1)or a biologically active fragment.
13. The method according to claim 5, in which cells of malignant tumor cells are non-small cell lung cancer.
14. The method according to item 12, wherein said polypeptide Apo2L/TRAIL consists of amino acids 114-281 indicated in figure 1 (SEQ ID NO:1).
15. The use of antibodies or polynucleotide to detect the expression of protein GalNac-T14 or mRNA in a tissue sample or cells of a malignant tumor of a mammal to obtain a set or articles for predicting sensitivity of a sample of tissue or mammalian cells to Apo2L/TRAIL, 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 Apo2L/TRAIL.
16. The application of clause 15, wherein the specified expression of GalNac-T14 evaluate the mRNA expression GalNac-T14.
17. The application of clause 15, wherein the specified expression of GalNac-T14 examined in immunohistochemical analysis.
18. The application of clause 15, which additionally carry out stage study the expression of receptors DR4, DR5, DcR1 or DcR2 in a specified sample of tissue or cells.
19. The application of clause 15, when the cat the rum 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 cells of the mammal the polypeptide Apo2L/TRAIL, 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 by polypeptide Apo2L/TRAIL, where the kit includes a container with a label on the specified container with the specified container is 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 the neck is 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.
SUBSTANCE: five-percent placental tissue extract is analysed for the concentration of arginine and histidine to be related, and if the relation is lower than 0.86, development of cerebral affection in a newborn is predicted.
EFFECT: more precise and specific prediction of perinatal CNS affection in the newborn and enabled well-timed pathogenetic therapy.
3 dwg, 1 tbl
SUBSTANCE: patient's blood serum is treated with 7% solution of polyethylene glycol-6000, incubated and with dye Sudan B at 40°C for 1 hour, separated electrophoretically in agarose gel. After that, additionally, before treatment of blood serum with 7% PEG-6000 to 0.6 ml of sample 0.2 ml of 0.1% tritone X-100 solution is added, incubated for 15 minutes at 20°C, after which mixture is mixed by shaking 120 times per 1 minute. Application of method makes it possible to detect additional intensive minor fraction of modified LP(a).
EFFECT: increase of diagnostics accuracy.
3 dwg, 1 tbl
SUBSTANCE: method of determination of triterpene saponins in vegetable raw material and medications includes dissolution of saponin-containing fraction in mixture water-ammonium buffer, determination of its optic density and calculation of saponin content in terms of oleanolic acid, under specified conditions.
EFFECT: claimed method represents express method, facilitates analysis and increases degree of reliability of obtained results.
SUBSTANCE: in order to estimate efficiency of treating ischemic nephropathy in newborn babies in early neonatal period activity of gamma-glutamyltransferase and cholinesterase in child's urine is determined in dynamics of treatment. If activity of said enzymes decreases with respect to initial level, treatment is estimated as efficient, if activity increases or does not change - as inefficient.
EFFECT: application of method makes it possible to increase accuracy of estimation of ischemic nephropathy treatment in newborns, carry out correction of therapeutic measures in due time and improve disease outcome.
1 tbl, 3 ex
SUBSTANCE: in a first trimester of pregnancy, the microalbuminuria level is determined. If the value is 45 mg\l and more, placental insufficiency is predicted.
EFFECT: method enables early prediction of placental insufficiency by a simple quantitative estimation and thereby ensures early adequate preventive treatment.
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
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
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
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.
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, 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.
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.
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.
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.
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.
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.
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.
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
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.