Human Alpha-Fetoprotein for Anticancer Therapy
Development of the Biotechnology of the Yeast Recombinant Alpha-Fetoprotein: Design of the Highly Specific Anticancer Drug for the Tumor-Selective Triggering of Apoptosis
Tech Area / Field
- MED-DRG/Drug Discovery/Medicine
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
3 Approved without Funding
Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany
- State Scientific Center of Genetics and Selection of Industrial Microorganisms (GosNIIGenetica), Russia, Moscow
- National Research Council Canada / Biotechnology Research Institute, Canada, QC, Montreal\nBoston University / School of Medicine, USA, MA, Boston\nMassachusetts General Hospital, USA, MA, Boston
Project summaryThe main objective of the project is to design a new anticancer drug on the base of human recombinant α-fetoprotein (rhAFP) expressed in yeast, which will selectively induce apoptosis in tumour cells without any toxic effects against normal tissues. This goal determine a set of biotechnological tasks leading to creation of recombinant yeast strain - high-yield producer of natively folded active protein suitable for medical use.
Search for the novel therapeutic approaches in cancer therapy actually results in identification of the novel factors capable of targeting triggering apoptosis in cancer cells with minimal side effects against normal cells. The another extremely actual problem is to overcome resistance of tumour cells to the cytotoxic effects of chemotherapeutic drugs and radiation therapy, which rises from their insensitivity to apoptotic stimuli. Thus, the elaboration of the new type of drug, which will be capable of targeting triggering or positive regulation of tumour-selective apoptosis with minimal unspecific toxicity, could be considered to be the most perspective direction in the cancer biotechnology and pharmacology.
Recently, it had been established that oncoembryonal marker α-fetoprotein (AFP) is capable to induce apoptosis selectively in cancer cells being simultaneously completely untoxic for normal cells. This allowed proposing possible use of AFP as a component of the targeting anticancer drugs. The tumoricidal effect of AFP was shown to be due to the rapid induction of tumor cell apoptosis, and was observed for the various types of tumour cells lines in vitro and in vivo. Our data also demonstrated the ability of AFP to modulate apoptotic signals, generated by other factors by enhancing of the total anticancer effect. Basing on these studies, the new types of targeting anti-cancer drugs on the base of pure human AFP or this one in combination with other cytotoxic anticancer drugs (TNF, antibiotics, retinol or steroids), which characterised by high degree of tumor-selective cytotoxicity, could be designed. Additionally, AFP could be used as a highly specific carrier protein to deliver anticancer drugs selectively into tumour cells bypassing normal cells. This application of AFP is based on its ability to bind various biologically active ligands, such as arachidonic acid, hormones, drugs, retinoic acid and others to deliver them to the developing cells, which express specific AFP receptors. In the scope of the ISTC project #1878 bacterial recombinant strain E. Coli expressing rhAFP was obtained. Structural and functional studies of bacterial rhAFP demonstrated close similarity with natural serum AFP of embryonic origin. In vivo studies demonstrated that rhAFP expressed in bacterial cells and natural serum AFP induced tumour suppression by significant inhibition of transplanted of human hepatoma in nude mice.
So, there exist good arguments for high effectiveness of AFP for targeting therapy of cancer. However, these good perspectives are restricted because of the deficit of pure human serum AFP that could be used in medical investigations. The another problem is connected with the risk of transmission of pathogenic viruses or prions by using plasma-derived materials. Genetic engineering is the best approach toward solving these problems.
The aims of the project: We propose here to develop a technology of obtaining considerable amounts of AFP from an acceptable and unlimited source - a proprietary strain of commercial brewing yeast. The main goal of the project is to design a recombinant yeast strain producing secreted human rhAFP for its potential commercial use for medical application. Yeast microorganisms are classified to belong to a group of GRAS organisms (generally recognised as safe) and could be considered as the most potentially safe candidates to use them for heterologous expression of recombinant human AFP for medical use. It is proposed to develop the technology of production of a novel biologically active substance on the base of human recombinant AFP for potential medical use with the activity of the native serum embryonic AFP, but obtained from the material, which have no relation to human sources. We propose to design a novel highly specific anti-tumour drug on the base of yeast-derived human rhAFP selectively suppressing tumour survival in humans without significant unspecific toxicity. If positive results would be received (documentation of the tumor-suppressive activity in laboratory investigations), it is planned to recommend medical trials of these multidrug compositions on the base of human yeast-derived rhAFP. The rhAFP-based anticancer drugs are proposed to have high tumor-directed selectivity and pro-apoptotic activity with minimized unwanted side effects in the course of the medical treatment.
Results of theoretical importance.
- Experimental data about the most optimal structure of the gene construction carrying AFP gene and corresponding leader sequence to provide high-yield gene expression in heterologous yeast system.
- Comparative experimental data about the secondary and tertiary structure of the serum and recombinant human AFP.
- Comparative data about the structure and functional significance of the carbohydrate chain of the various types of hyman AFP: serum embrionic AFP, cancer-derived AFP and recombinant yeast rhAFP.
- Comparative experimental data about the functional pro-apoptotic activity of the various samples of serum AFP and rhAFP.
Results of applied and commercial value.
- AFP-producing secreting recombinant yeast strain of commercial value, production technology of a substance of yeast-derived rhAFP could be used to initiate industrial process of rhAFP production for medical use and could be a matter of patenting and potential technological commercialization.
- Technological protocol of recombinant yeast strain cultivation for high-yield rhAFP secretion in laboratory conditions and in a pilot scale.
- Technological protocol of the purification process of rhAFP isolation from the concentrated cultural liquid of the recombinant yeast strain.
- Design of the novel type of anticancer drug on the base of rhAFP for the targeting tumour therapy.
- Elaboration of the new approach for anti-cancer therapy basing on the experimental data of the laboratory and pre-clinical studies of human recombinant AFP.
Technical approach and methodology: To perform the tasks set up in the project standard techniques of molecular biology, including gene synthesis and cloning, site-directed mutagenesis, chromatography, PAGE-electrophoresis, DNA-electrophoresis, Southern, Northern, and Western blot analysis will be employed. Cultivation of the recombinant yeast strains will be performed using the fermentation apparatus with the automatic monitoring of the temperature and pH under conditions of the automatic feeding regimen. Various techniques: flow cytometry, fluorescent, conofocal and phase contrast microscopy, assays for proliferation, cytotoxicity and DNA-fragmentation, will be employed for documentation of the recAFP-mediated programmed cell death. Study of the conformational and structural properties of recAFP will be done by techniques of fluorescence and adsorption spectroscopy, circular dichroism, and adiabatic differential microcalorimetry.
Patents and recent publications on the subject of the project:
- Dudich EI, Benevolensky SV, Marchenko AN, Zatcepin SS, Dudich DI, Koslov DI, Shingarova LN, Dudich IV, Semenkova LN, Tatoulov EB (2004) “Method of production and application of the recombinant alpha-fetoprotein and compositions” Eurasian Patent No 2420-300467EA.
- Dudich EI, Benevolensky SV, Marchenko AN, Zatcepin SS, Dudich DI, Koslov DI, Shingarova LN, Dudich IV, Semenkova LN, Tatoulov EB (2005) “Recombinant alpha- fetoprotein, method and means for preparation thereof, compositions on the base of thereof, and use of thereof” International PCT 655/3 – 300467EA.253.
- Dudich E., Semenkova L., Dudich I., Tatoulov E., Zubov D., Korpela T. (2004) “Peptides modulating caspase activity”, International PCT No WO2004033500.
- Dudich E., Semenkova L., Dudich I., Tatoulov E., Zubov D., Korpela T. (2005) “Peptides modulating caspase activity”, USA patent application 10/530,779.
- Dudich E.I., Semenkova L.N., Dudich I.V., Gorbatova E.A., Nikolaeva M.A., Tokhtamysheva, N., Tatulov E.B., Sukhikh G.T. (1999) -Fetoprotein causeses apoptosis in tumor cells via Fas-, TNFR1, TNFR2-independent pathway through activation of caspase-3-like proteases. Eur. J. Biochem. 266: 750-761.
- Semenkova, L., Dudich, E., Dudich, I., Tokhtamisheva, N., Tatulov, E., Okruzhnov, Y., Garcia-Foncillas, J., Palop-Cubillo J.-A., Korpela T. (2003) Alpha-fetoprotein positively regulates cytochrome c-mediated caspase activation and apoptosome complex formation, Eur. J. Biochem. 270: 4388-4399.