Recombinant Immunomodulatory Anti-Cancer Preparations
Development of Recombinant Targeted Anti-Cancer Immunomodulatory Preparations
Tech Area / Field
- MED-DRG/Drug Discovery/Medicine
3 Approved without Funding
State Research Center for Applied Microbiology, Russia, Moscow reg., Obolensk
- Moffit Cancer Center & Research Institute, USA, FL, Tampa
Project summaryCancer diseases provide a great deal in human mortality yet, and chemotherapy is still one of 'three whales' in cancer therapy (surgery and irradiation are others). Unfortunately, low specificity of tumour chemotherapeutical remedies resulted in a plenty of complications and hard side effects due to injuries of such important organs as liver, spleen, bone marrow, kidney, gastro-intestinal tract, skin by chemical agents injected into the blood stream. Thus attempts are prolonged of employing every living organism natural protection system, its own immunity, capable of highly effective and specific supression of alien bioagent, for cancer cells elimination. There are two main obstacles for this: often very small differences (if any) in antigen structure of cancer cells relatively to normal ones making immune system tolerant to malignant cells, and failure of entire immune system in an ill organism. However, progress in molecular biology, immunology, genetic engineering, biotechnology give a chance for successful and ready development of the novel immunotherapeutic anti-cancer remedies effective, specific and safe as well. This project is dedicated to development of such a medicines.
The project author’s assume that solution of the two above-mentioned problems of cancer immunotherapy probably consists in simultaneous induction of cell pool capable for recognizing and elimination of cancer cells independently of their location in organism, within tumor site or in metastases, and general immunity stimulation in order to provide immune response of sufficient power. The authors propose providing these effects by administration of signal immunomodulatory protein molecules, designed in special way, into ill organism (so called immunotherapy), and as well making tumour cells produce such molecules by introduction of appropriate genes into the cells (gene therapy).
Modern genetic engineering methods will be applied for development of immunomodulating targeted or addressed molecules exploiting gene fusion technology, providing transcription of structure genes encoding different parts of complex molecule within the same reading frame. Expression of this composite gene produces a hybrid or fusion bifunction protein molecule consisting of recognizing domain represented by mini-antibody, mAb, capable of highly specific binding with tumour cell antigen, Ag, and of effector domain, represented by molecule of extremely powerful stimulator of cell immunity, superantigen, namely staphylococcal enterotoxin (serotype A or B), or by one of the immune system signal molecules, interleukin-2 (IL-2). The first construct is designed to re-direct the special immune cells, cytotoxic lymphocytes (CTL) function to tumor cells. The second construct is expected to increase efficiency of the first construct and to stimulate antitumor immunity in recipients. Soluble CD40 protein, capable for stimulating of a variety of immune responses, will be also cloned into E. coli and isolated. This protein was not isolated in its soluble form before and was never tested. Finally, especially designed DNA molecules, plasmid vectors, inducing production of the immune signal molecules in mammalian cells, will additively be used to stimulate anti-tumour immune response. Thus, the goal of the project is development of novel genetically engineered immunomodulatory preparations on the basis of recombinant proteins and plasmid DNA for combined immuno- and gene therapy of several human malignant tumors (adenocarcinomas). The antitumor effect of these preparations supposed to be executed by activating cellular immunity in the tumour site and in metastases.
One of main advantages of the proposed remedies believe to be their independance of so-called multi-drug resistance of tumor cells, MDR, arising during chemotherapy. MDR results in increasing doses of the drugs and subsequent enhancement of negative side effects of cytostatics very toxic by their nature. Targeted immunomodulators can be used in low doses and by virtue of highly specific interaction with tumour cells believed to give reduction of range and amplitude of side effects.
As a result of the project execution, genetic constructs will be developed, capable of inducing of expression of immunomodulatory proteins in the producer microorganism E. coli and in mammalian tumour cells: SEA-mAb(Ag4), IL2-mAb(Ag4), sCD40. Among other tasks are the following: isolation and purification of these recombinant proteins from the producer cultural media for subsequent trials; design, purification and accumulation of genetic constructs on a basis of plasmids, carrying structural genes encoding immunomodulators and regulatory sites controlling biosynthesis of these proteins into producer microorganism cell and into tumour cells; characterization of the proteins obtained in their physico-chemical and biological properties; testings of immune stimulating anti-cancer properties of the preparations based on the hybrid proteins and plasmid vectors using especially developed T-cell hybridoms and IL-2 dependent cell lines CTLL-2 (in vitro); development of in vivo model adequate for trials of immune modulating anti-tumour effect of the developed preparations in experimental animals.
This project is a prolongation of studies carrying out at the State Research Center for Applied Microbiology for recent years and aimed at development and characterizing the hybrid proteins such as tumor necrosis factor (TNF) fused with thymosin, interferon gamma (IFNg) fused with miniAb against mucin-like antigen Ag4 (derived from the hybridom ICO-25), and SEB fused with miniAb (Ag4) (the last two recombinant proteins and appropriate genetic constructs were developed within ISTC Project # 118B framework). Extremely restricted funding of the former Project 118B has not allowed to get development of the preparations finished. Techniques of genetic constructions suitable for producing fusion addressed protein immunomodulators, isolation technique for these recombinant proteins from bacterial E. coli cultures, and technology of plasmid introduction into tumour cells in vitro were developed by SRCAM team and can be used for development of so-called cell anti-cancer vaccines. Mini-antibodies or single-chain variable fragments of monoclonal antibodies to mucin-like Ag4 were cloned into E. coli producer strain, the recombinant mini-Ab were isolated and tested; plasmid vectors suitable for cloning and expression of fusion immunomodulatory proteins were obtained; recombinant hybrid targeted immunomodulators SEB-mAb(Ag4) and IFNg-mAb(Ag4) were cloned, isolated and purified.
The present project is a new development of novel generation of immunomodulatory preparations having controlled structure, properties and targets, in directions of preparation spectrum spread and improvement the preparations itselves by their specificity and efficacy in cell immunity induction, and also their technology. In fact, real preparations for immune and gene therapy will be obtained. Mini-Ab(Ag4) developed recently by the authors will be used in design novel genetic and protein constructs: SEA-miniAb(Ag4) and IL2-miniAb(Ag4). SEA will be modified in order to reduce its system toxicity down to a safe level. Novel wide range immunomodulatory recombinant protein, soluble sCD40 will be developed, isolated in purified form and tested in vitro and in vivo. Genetic engineering and preparative biochemistry works will be carried out by SRCAM, Obolensk, and Institute for Bioorganic Chemistry (Russian Academy of Sciences, Moscow) scientists highly skilled in this area. One of the most complicated stages in the works is testing of the immunomodulatory preparations in cell models and in animals due to the fact that human immune modulating proteins are inadequate in animals because of allospecificity of the immune reactions. Therefore a separate series of works by experienced cell and tumour immunologist (Russian Cancer Center of RAM, Moscow) within the project will be dedicated to development of an adequate model for immunological testings of such a kind. Development of the model would provide a basis for future development of human anti-cancer immunomodulating preparation. The results of the whole work will help obtaining new generation drugs for directed immunotherapy and can be applied for development of new methods for cancer gene therapy.
Effective and non-formal interaction and collaboration with our foreign colleagues is planned to be an integral part of the present project. Assistant Professor Hua Yu, Lee Moffitt Cancer Center and Research Institute, University of South Florida, USA, has agreed to be a Foreign Collaborator for the Project, and already provided with plasmid vectors bearing some immunomodulatory genes; purified recombinant sCD40 protein is planned to be obtained by the Project Executors and given for testing at Dr. Yu’s laboratory within the project framework. Dr. Howard Young and Professor John Ortaldo, National Cancer Institute at Frederick, USA, are ready to exchange results of experiments within the Project while testing developed preparations in their laboratories. Collaboration with the above mentioned scientist is planned also in a form of common seminars and joint publications; one of the most effective form of inclusion of ‘defense scientists’ from Obolensk and Moscow into solving peaceful problems and adaptation for the market economy. As the last problem is concerned, elaboration of recommendations for future pre-clinical and clinical trials of the preparations developed, as well as work results commercialization, for instance, in a form of laboratory technology patent sales, will be presented.