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
8 Project completed
Senior Project Manager
Melnikov V G
Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany
Project summarySearch for the novel therapeutic approaches in cancer therapy actually results in identification of the 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 tumor 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 tumor-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 -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 tumor 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 characterized 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 tumor 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.
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 rAFP for its potential commercial use for medical application. 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-tumor drug on the base of yeast-derived human recAFP selectively suppressing tumor 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 recAFP. The recAFP-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 of the carbohydrate chain of the various types of human AFP: serum embryonic AFP, cancer-derived AFP and recombinant yeast recAFP and determination of its role in functional activity of the protein.
- Comparative experimental data about the functional pro-apoptotic activity of the various samples of serum AFP and recAFP.
Results of applied and commercial value.
- AFP-producing secreting recombinant yeast strain, production technology of a substance of yeast-derived recAFP could be used to initiate industrial process of AFP 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 AFP secretion in laboratory conditions and in a pilot scale.
- Technological protocol of the purification process of recombinant human AFP isolation from the concentrated cultural liquid of the recombinant yeast strain.
- Design of the novel type of anticancer drug on the base of human recombinant AFP for the targeting tumor 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, confocal 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.