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Alternative therapy for tumours

Development of the use of tumour necrosis factor related apoptosis inducing ligand or apo2 ligand (trail) as an efficient anti-tumour therapy as an alternative to chemo/radiotherapy.

A. Theoretical background. TRAIL (Tumour necrosis factor Related Apoptosis Inducing Ligand or Apo2 ligand) was described by Wiley et al. as another ligand from the TNF family capable of inducing apoptosis in tumour cell lines (1). Like most of the ligands of this family, TRAIL is a trimeric type II transmembrane protein expressed, similar to its receptors, in most tissues. There are several receptors for TRAIL, namely TRAIL-R1 (DR4), TRAIL-R2 (DR5), TRAIL-R3 (DcR1), TRAIL-R4 (DcR2) and osteoprotegerin - for recent reviews see (2-4). Only two of these, TRAIL-R1 and TRAIL-R2, contain the intracellular part with DD and transmit an apoptotic signal. The other two receptors (-R3 and -R4) have either truncated ICP or a GPI anchor and are believed to serve as anti-apoptotic, decoy receptors. Apoptotic signalling DISCs of TRAIL-R1 or -R2 are composed of the adaptor protein FADD and procaspase-8 or -10 (5-8). Another protein, DAP-3, was recently identified as an essential constituent of the TRAIL-R1 and -R2 DISC complexes (9,10). Similarly as for Fas, DISC-activated initiator caspases-8 (-10) amplify the apoptotic signal through processing of effector caspases and cleavage-induced activation of Bid. tBid and Bax-dependent release of Smac/DIABLO seems to constitute an essential step in TRAIL-induced apoptosis (11,12). Smac/DIABLO, through sequestering of cellular caspase inhibitors of the IAP family, allows for the effective amplification of TRAIL-induced apoptotic signalling - for review see (13). A unique feature of TRAIL-induced apoptotic signalling is its specificity towards damaged and malignant cells while sparing normal cells. Several recent communications as well as observations from TRAIL-deficient mice point to its specialised role in the elimination of cancer/damaged cells (14-16). Recently, recombinant TRAIL has received significant attention as a potential anti-tumour agent with high specificity and low side-toxicity. The first in vivo tests of recombinant TRAIL in animal models (from mice to monkeys) proved its high cytotoxicity against implanted tumours without any negative side effects (17,18). The apparent lack of TRAIL toxicity towards normal cells was later challenged by several communications describing the recombinant TRAIL-induced killing of ex-vivo human hepatocytes, neurons and prostate epithelial cells (19-21). However, it appears that some variants of recombinant TRAIL are less toxic (22) and that ex-vivo experiments do not necessary reflect the actual situation/processes in vivo. In the past 3 years a number of reports have documented the high potential of recombinant TRAIL in the killing of tumour cells of various origins. Especially remarkable appears to be the synergism of TRAIL with currently used anti-tumour drugs in inducing the apoptosis of malignant cells of epithelial, breast, prostate, mesotheliomal or hematopoeitic origin (23-29). At present, preclinical studies with recombinant TRAIL are in progress (30), and alternative approaches such as using an apoptosis-inducing anti-TRAIL-R2 monoclonal antibody with no hepatotoxicity are also being evaluated (31). B. Aim of the project. 1. Expression and purification of the recombinant soluble extracellular parts of TRAIL-R1 (DR4) and TRAIL-R2 (DR5). 2. Construction of hybridoma producing monoclonal antibodies against DR4 and DR5 receptors. 3. Characterisation of DR4- and DR5-specific monoclonal antibodies and selection of tumour apoptosis-inducing mAbs. 4. Preparation of apoptosis-inducing ScFv antibodies from anti-DR4 or anti-DR5 monoclonal antibodies. C. Experimental procedure. Extracellular parts (ECP) of DR4 or DR5 will be amplified by PCR and in frame subcloned into the eukaryotic expression plasmid (EF1? promoter) containing constant part of IgG1 heavy chain at the 3' end. Stable transfectants of human embryonic kidney cell line 293 or hamster cell line CHO with DR4(ECP)-IgG(hc) or DR5(ECP)-IgG(hc) expression constructs will be prepared and tested for expression of the recombinant fusion proteins. The fusion proteins will be purified from the cell culture media using protein G HiTrap column (AKTA FPLC, Amersham Bioscience) and their biological activity will be analysed (inhibition of TRAIL-induced apoptosis of permissive hematopoeitic cell lines). DR4(ECP) and DR5(ECP) fusion proteins will be used for immunisation of Balb/C mice. Reactivity of anti-DR4 or -DR5 antibodies in mouse serum and in selected hybridoma cells will be tested on the recombinant immunogens (by ELISA) and on DR4- or DR5-expressing hematopoietic cell line (by FACS). The immunisation procedure and screening for positive hybridomas will be carried out in EXBIO. Apoptosis-inducing abilities of soluble as well as plastic-immobilised monoclonal antibodies (mAbs) will be determined using TRAIL-sensitive tumour cell lines of various origins (colon, breast, leukaemia, etc.) both in Dr. Andera's and in Dr. Pintzs' laboratories. The best candidates (i.e. high affinity binding and apoptosis-inducing mAbs) will be extensively purified through combination of ion exchange and affinity columns (AKTA FPLC, Amersham Bioscience). Potential toxicity of the purified mAbs towards primary human hepatocytes and neural cells (both taken from biopsies) will be examined. The purified antibodies will also be in collaboration with laboratories in the INSTITUTE FOR HAEMATOLOGY AND BLOOD TRANSFUSION (IBHT, Prague), INSTITUTE OF MICROBIOLOGY and the 1st Medical Faculty of CHARLES UNIVERSITY (both in Prague) and in Dr. A. Pintzas laboratory tested both soluble and immobilised on the soluble polymers in their ability to suppress tumour growth in immuno-compromised NOD/scid or nude mice. The best performing mAb(s), selected on the basis of their in vitro and in vivo tumour apoptosis-inducing properties, will be considered for molecular cloning as single-chain fragment-variable (ScFv) antibodies with the help of commercially available Recombinant Phage Antibody System (RPAS, Amersham Bioscience). mRNA will be isolated from mAb producing hybridoma cells and variable regions of heavy and light chains will be after PCR amplification connected by a linker and subcloned into the phagemid display vector pCANTAB 5E. Phages expressing DR4- or DR5-specific ScFv antibodies will be selected using DR4(ECP) or DR5(ECP) fusion proteins with the help of the Recombinant Phage Selection Module. The recombinant ScFv antibodies will be analysed the same way as the "parental" anti-DR4 or anti-DR5 monoclonal antibodies. Alternatively, we will also prepare diabodies with a modified linker allowing dimerization of ScFv antibodies. D. Time schedule of the project 1st year: Preparation of DR4(ECP)-IgG(hc) or DR5(ECP)-IgG(hc) expression plasmids, expression and purification of the fusion proteins, mice immunisation, 2nd year: Hybridoma construction, selection of mAbs producing hybridomas and in vitro characterisation of the monoclonal antibodies. 3rd year: In vivo (NOD/scid mice) analysis of tumour-suppressing properties; cloning, expression and characterisation of anti-DR4 (DR5) ScFv antibodies. 4th year: Preparation of diabodies, in vitro and in vivo analysis of tumour-suppressing properties of ScFv antibodies and diabodies. References 1. Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C. P., Nicholl, J. K., Sutherland, G. R., Smith, T. D., Rauch, C., Smith, C. A., and et al. (1995) Immunity 3(6), 673-82. 2. Abe, K., Kurakin, A., Mohseni-Maybodi, M., Kay, B., and Khosravi-Far, R. (2000) Ann N Y Acad Sci 926, 52-63 3. Degli-Esposti, M. (1999) J Leukoc Biol 65(5), 535-42. 4. Griffith, T. S., and Lynch, D. H. (1998) Curr Opin Immunol 10(5), 559-63. 5. Kischkel, F. C., Lawrence, D. A., Chuntharapai, A., Schow, P., Kim, K. J., and Ashkenazi, A. (2000) Immunity 12(6), 611-20. 6. Kuang, A. A., Diehl, G. E., Zhang, J., and Winoto, A. (2000) J Biol Chem 275(33), 25065-8. 7. Sprick, M. R., Weigand, M. A., Rieser, E., Rauch, C. T., Juo, P., Blenis, J., Krammer, P. H., and Walczak, H. (2000) Immunity 12(6), 599-609. 8. Bodmer, J. L., Holler, N., Reynard, S., Vinciguerra, P., Schneider, P., Juo, P., Blenis, J., and Tschopp, J. (2000) Nat Cell Biol 2(4), 241-3. 9. Kissil, J. L., Cohen, O., Raveh, T., and Kimchi, A. (1999) Embo J 18(2), 353-62. 10. Miyazaki, T., and Reed, J. C. (2001) Nat Immunol 2(6), 493-500. 11. Deng, Y., Lin, Y., and Wu, X. (2002) Genes Dev 16(1), 33-45. 12. Zhang, X. D., Zhang, X. Y., Gray, C. P., Nguyen, T., and Hersey, P. (2001) Cancer Res 61(19), 7339-48. 13. Goyal, L. (2001) Cell 104(6), 805-8. 14. Takeda, K., Smyth, M. J., Cretney, E., Hayakawa, Y., Kayagaki, N., Yagita, H., and Okumura, K. (2002) J Exp Med 195(2), 161-169. 15. Dorr, J., Waiczies, S., Wendling, U., Seeger, B., and Zipp, F. (2002) J Neuroimmunol 122(1-2), 117-24. 16. Cretney, E., Takeda, K., Yagita, H., Glaccum, M., Peschon, J. J., and Smyth, M. J. (2002) J Immunol 168(3), 1356-61. 17. Walczak, H., Miller, R. E., Ariail, K., Gliniak, B., Griffith, T. S., Kubin, M., Chin, W., Jones, J., Woodward, A., Le, T., Smith, C., Smolak, P., Goodwin, R. G., Rauch, C. T., Schuh, J. C., and Lynch, D. H. (1999) Nat Med 5(2), 157-63. 18. Ashkenazi, A., Pai, R. C., Fong, S., Leung, S., Lawrence, D. A., Marsters, S. A., Blackie, C., Chang, L., McMurtrey, A. E., Hebert, A., DeForge, L., Koumenis, I. L., Lewis, D., Harris, L., Bussiere, J., Koeppen, H., Shahrokh, Z., and Schwall, R. H. (1999) J Clin Invest 104(2), 155-62. 19. Nesterov, A.,Ivashchenko, Y. and Kraft,A. S. (2002) Oncogene 21(7), 1135-40. 20. Nitsch, R., Bechmann, I., Deisz, R. A., Haas, D., Lehmann, T. N., Wendling, U., and Zipp, F. (2000) Lancet 356(9232), 827-8. 21. Jo, M., Kim, T. H., Seol, D. W., Esplen, J. E., Dorko, K., Billiar, T. R., and Strom, S. C. (2000) Nat Med 6(5), 564-7. 22. Lawrence, D., Shahrokh, Z., Marsters, S., Achilles, K., Shih, D., Mounho, B., Hillan, K., Totpal, K., DeForge, L., Schow, P., Hooley, J., Sherwood, S., Pai, R., Leung, S., Khan, L., Gliniak, B., Bussiere, J., Smith, C. A., Strom, S. S., Kelley, S., Fox, J. A., Thomas, D., and Ashkenazi, A. (2001) Nat Med 7(4), 383-5. 23. Plasilova, M., Zivny, J., Jelinek, J., Neuwirtova, R., Cermak, J., Necas, E., Andera, L., and Stopka, T. (2002) Leukemia 16(1), 67-73. 24. Gibson, S. B., Oyer, R., Spalding, A. C., Anderson, S. M., and Johnson, G. L. (2000) Mol Cell Biol 20(1), 205-12. 25. Chinnaiyan, A. M., Prasad, U., Shankar, S., Hamstra, D. A., Shanaiah, M., Chenevert, T. L., Ross, B. D., and Rehemtulla, A. (2000) Proc Natl Acad Sci U S A 97(4), 1754-9. 26. Nimmanapalli, R., Perkins, C. L., Orlando, M., O'Bryan, E., Nguyen, D., and Bhalla, K. N. (2001) Cancer Res 61(2), 759-63. 27. Di Pietro, R., Secchiero, P., Rana, R., Gibellini, D., Visani, G., Bemis, K., Zamai, L., Miscia, S., and Zauli, G. (2001) Blood 97(9), 2596-603. 28. Mitsiades, C. S., Treon, S. P., Mitsiades, N., Shima, Y., Richardson, P., Schlossman, R., Hideshima, T., and Anderson, K. C. (2001) Blood 98(3), 795-804. 29. Liu, W., Bodle, E., Chen, J. Y., Gao, M., Rosen, G. D., and Broaddus, V. C. (2001) Am J Respir Cell Mol Biol 25(1), 111-8. 30. Kelley, S. K., Harris, L. A., Xie, D., Deforge, L., Totpal, K., Bussiere, J., and Fox, J. A. (2001) J Pharmacol Exp Ther 299(1), 31-8. 31. Ichikawa, K., Liu, W., Zhao, L., Wang, Z., Liu, D., Ohtsuka, T., Zhang, H., Mountz, J. D., Koopman, W. J., Kimberly, R. P., and Zhou, T. (2001) Nat Med 7(8), 954-60. 32. Rihova, B. (1997) Crit Rev Biotechnol 17(2), 149-6933. Rihova, B. (1998) Adv Drug Deliv Rev 29(3), 273-289. 34. Rihova, B., Jelinkova, M., Strohalm, J., Subr, V., Plocova, D., Hovorka, O., Novak, M., Plundrova, D., Germano, Y., and Ulbrich, K. (2000) J Control Release 64(1-3), 241-61. 35. Ulbrich, K., Subr, V., Strohalm, J., Plocova, D., Jelinkova, M., and Rihova, B. (2000) J Control Release 64(1-3), 63-79. Keywords: apoptosis, recombinant protein, monoclonal antibodies.
Acronym: 
RETRAIL
Project ID: 
2 928
Start date: 
01-01-2003
Project Duration: 
48months
Project costs: 
1 000 000.00€
Technological Area: 
Gene Expression, Proteom Research
Market Area: 
Pharmaceuticals/fine chemicals

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