Inactivation of Cancer Cell
Construction of Directed Action Molecules on Chosen Classes of Eucariotic Cells
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
- MED-DRG/Drug Discovery/Medicine
8 Project completed
Senior Project Manager
Evstyukhin K N
State Research Center for Applied Microbiology, Russia, Moscow reg., Obolensk
- University of Uppsala / Department of Medical Genetics, Sweden, Uppsala\nBrown University / Roger Williams Hospital, USA, RI, Providence\nUniversity of Texas at El Paso / Department of Biological Sciences, USA, TX, El Paso
Project summaryWorking plan presented is the result of project proposal #402 feasibility stage investigation. The project aims and tasks have been specified and discussed with foreign collaborators approved one. Work carried out brought us to more strong belief in importance and need of creating of a set of immunotoxins of various structures and mechanisms action for treatment prostate tumors being now one of the most common cancers.
The main objective of the project is to develop recombinant immunotoxins of various structures and mechanisms action. Possibility of overgrowing cancer cells resistant to toxic action of some toxins lead us to use for this purpose toxin interfering with protein synthesis (diphtheria toxin, DT) and surface active toxic Pyrularia pubera thionin (PT), one of useful property of which was shown is inability to elicit immune response in mice. Prostate surface membrane antigen (PSM) has been chosen as target on unneeded cells because of its almost entire specificity for prostate tissue and outer membrane localization. Necessity of using immunotoxins with small sizes for penetrating in solid tumors was one of causes of recombinant molecules construction based on minimal antigen-binding unit - so called single chain anti-PSM antibody (scFv). Metastase process was shown as early event in prostate cancer. Clinical trials showed more usefulness of conventional conjugated immunotoxins for eliminating of disseminated target cells due to their large sizes and as a consequence more stability and half-life in patients serum with comparing to small ones. So we propose to create fusion proteins toxin-streptavidin (SA) for obtaining large size immunotoxins with anti-PSM antibodies or their variable fragments. Besides hybrid protein diphtheria toxin-streptavidin will be useful for determining of an antigen ability to mediate endocytosis.
The investigation will be performed as described behind. Hybridoma cells producing anti-PSM monoclonal antibodies (Mabs) will be obtained and their dissociation constants themselves and ones combined with toxins will be determined. Diphtheria toxin lacked receptor-binding region CDTaeg), streptavidin gene, synthesized PT gene and anti-PSM scFv genes will be cloned. Recombinant plasmids coding hybrid proteins DT389 scFv, PTscFv, DT389SA, PTSA and toxins DT389 and PT under the control of late promoter of phage T7 will be constructed. Expressed in Escherichia coli and purified fusion proteins based on scFv and toxins-SA bound to biotinilated Mabs or their variable regions will be investigated on cytotoxic activity on tumor cells bearing or no PSM in vitro.
As a result of the research a set of immunotoxins for treatment prostate tumors will be constructed with assumption into account of data of clinical trials of readdressed toxins. A panel of monoclonal antibodies to extracellular part of PSM which as was shown is an attractive target for killing prostate cancer cells will be obtained and dissociation constants will be determined. Immunotoxins of various sizes and mechanisms action based on conventionally used toxin interfered with protein synthesis and on surface active Pyrularia pubera toxic thionin and on anti PSM scFv and Mabs variable regions will be compared on their selective cytotoxic effect on prostate cancer cells in vitro.
Thus a set of immunotoxins of various mechanisms of action and structures for treatment one of the most common prostate tumors will be created and investigated as a result of the research.