Head: Prof. Balik Dzhambazov, PhD, CV
1) Assoc. Prof. Tsvetelina Batsalova, PhD CV 
2) Assoc. Prof. Elenka Georgieva, PhD, CV
3) Assoc. Prof. Ivanka Teneva, PhD, CV
4) Chief. Asst. Prof. Stela Stoyanova, PhD, CV

The planned activities in this WP are related to Component 3: “Industry for healthy lifestyle and biotechnologies”. The work package includes development of methods and products for personalized immunotherapy of autoimmune and tumor diseases. This is in line with the patient-oriented strategies of the healthcare system, namely better treatment with minimal side effects and transfer of the basic biological knowledge into the clinical practice and public health.

Results of the study will provide an assessment of the possibility to apply an antigen-based approach in the treatment of autoimmune and tumor diseases. It is expected to develop a set of useful molecular markers that can be used to characterize new participants in the autoimmune and antitumor response, and to use them for development of new therapeutic agents. It is envisaged development of new generation complexes between the key players in the immune response – antigens MHC, TCR, antibodies. The planned research is aimed at developing methods and products that can be used to implement an individual prevention / treatment of patients, which directly corresponds to the priorities of Component 3. “Industry for healthy lifestyle and biotechnologies” and contributes for the achievement of its objectives.

Most autoimmune and tumor diseases are complex with unclear etiology. It is assumed that it depends on both genetic and environmental factors.

Most prevalent autoimmune disorders are Sjögren’s syndrome, multiple sclerosis (MS), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), psoriasis, Crohn’s syndrome, celiac disease, type I diabetes (TID), Hashimoto’s disease, Myastenia gravis and others. These diseases are characterized by high frequency and chronicity, and significantly impair the quality of life of the patients and increased mortality rates among them (Marrack et al., 2001; Shoenfeld et al., 2008). This determines the priority and relevance of the research aimed at clarifying their pathogenesis and developing of adequate diagnostic, interventional and therapeutic methods.

On the other hand, tumors are the main cause of premature death among the population in Europe, as 90% of these cases are due to different types of carcinomas (Ferlay et al., 2013). Currently, the treatment of tumor diseases in humans includes surgery, radioactive therapy and chemotherapy. After surgical removal of advanced tumors, the metastatic tumor cells continue their development and penetrate in tissues other than the primary source. Radiation exposure affects not only the tumor, but also normal cells, causing serious side effects. Most chemotherapeutic agents that are used to treat primary and metastatic tumors do not distinguish malignant from normal cells and are equally toxic to both types of cells.

Recently, there is an increased interest to the immunotherapy by using induction of an autoimmune response against specific tumor markers (Munhoz and Postow, 2016). It relies on the induction of an immune response where the antibodies, T-cells or NK-cells play a major role (Zanetti, 2015). Best clinical results of treatment with monoclonal antibodies are demonstrated with anti-CTLA-4 and anti-PD-1/PD-L1 (Masucci et al., 2016; Menon and Shin, 2016). Experimental treatment with an adaptive T-cell transfer by the use of CD8+ and CD4+ cells did not show the expected results, which is due to increased expression of PD-1 and the relatively low expression of MHC molecules (Zanetti, 2015). Thus, the lack of co-stimulation leads to induction of tolerance rather than immune response.

Our research will be towards providing costimulatory signals by generating different protein complexes between the MHC and tumor antigens. At this stage, however, there are a small number of specific and permanent markers, which can be used to distinguish strictly the tumor from normal cells. Therefore, there is a need to find new specific biomarkers, which will enable the development and deployment of innovative methods for early diagnosis and effective treatment with new drugs that have improved pharmacological properties.

Accumulated experimental data support the involvement of specific receptors, cytokines and antibodies, the induction of which leads to activation of a cascade of sequential reactions in these diseases (Arbuckle et al., 2003; Rantapaa-Dahlqvist et al., 2003; Bizzaro et al., 2007). There are several unique properties of the immune responses of recognition, which could provide evidence for further understanding of the disease process, as well as providing opportunities for effective diagnosis and treatment.

The proposed package as a whole is innovative, since it is based on a novel, antigen-based approach oriented to the search of new biological and chemical agents or combined procedures against these diseases. The search is based on the information coming from sources and experimental models as well as based on the results from theoretical bioinformatics models supported by various calculations. Developed products will be based on the recent advances in cellular and molecular biology, immunology and post-genomic biology. The multidisciplinary approach allows developing of new drug candidates with high specificity and quality.

For effective treatment of the autoimmune and tumor disorders or for the development of immunological vaccines, the medicine and biotechnological industry need from additional funds. Applying the results of the basic biomedical research into the clinical practice is a key priority. The proposed research will lead to realization of some key objectives of the experimental medicine and applied research:

  • Development of specific markers for predicting disease, diagnosis, prognosis and prediction of the outcome from the therapeutic procedures;
  • Development and offering of new approaches and potential tools for an effective personal therapy.

The main goal of this package is to investigate the immunological and biochemical profile of patients with autoimmune and tumor diseases in order to identify antigens that are or could be immunodominant T- and B-cell epitopes and assessing the possibilities for their use in the development of new tools for treatment through immunotherapy.

To achieve this goal, it is necessary to perform several specific tasks:

  • Selection of patients diagnosed with an autoimmune or tumor disease
  • Collecting biological samples
  • Characterization of the biochemical profile (levels of various enzymes, reactive oxygen radicals /ROS/, nitric oxide /NO/, the presence of intermediate products from the metabolism of the antigens, etc.)
  • Phenotyping of the autoimmune T cells and characterization of T-cell responses to specific protein antigens and their modified forms
  • Characterization of the humoral immune response and autoreactivity
  • Establishment of new antigens and tools that can be used for treatment
  • Bioinformatics modeling of the interaction between the various antigens responsible for the development of autoimmune or tumor diseases with appropriate MHC molecules and T cell receptors (TCR)
  • Development of methods and products for personalized immunotherapy of autoimmune and tumor diseases.

The proposed research will help for development of personalized immunostimulatory vaccines based on known antigens and newly identified biomarkers combined in biological complexes with superantigens, MHC proteins or antibodies. Depending from the obtained results, such complexes can be generated with biocompatible polymers or nanoparticles used as carriers of drugs in the antitumor therapy.

The development of immunomodulatory complexes based on specific antigens and carriers will contribute to the development of new technologies for generation of biologically active molecules based on the individual characteristics of the expressed biomarkers in the patient to overcome the “Rejection” (tissue incompatibility) or general cytotoxicity, which occur frequently in the currently applied treatment of this type of disorders.

Although the basic studies are directed to the development of methods and products with practical applications, the results will be published in leading international scientific journals and presented at national and international scientific conferences and symposia. Our expectations are to be published at least three articles in journals with impact factor from which at least one in a journal among the top 10 in the certain field during the first five years of the project, and more additional materials after its completion (no less than 4).

We envisage to develop one useful model up to the fifth year of the project.

Also, we have planned specialization of 2 staff members in leading scientific organizations and / or biotechnology companies abroad to learn the main activities and stages in the implementation of developed method or useful model. The achieved scientific results will be presented at international congresses and conferences in the field. We plan up to 4 participations in international congresses and conferences for the presentation of scientific results.

For realization of the main tasks will be used:

  • Classical biochemical methods for conducting and optimizing the interaction between specific molecular markers (antigens) and cells, or functional groups on the surface of the carrier platform;
  • Immunological methods – ELISA for qualitative and quantitative assessment of specific molecular markers; Flow cytometry (FACS) analysis for phenotypic and functionality of molecular / cellular complexes;
  • HPLC methods, essentially gel-filtration, for purification and analysis of antigens;
  • Mechanical methods – density gradient centrifugation and membrane filtration for primary purification and concentration of the fractions;
  • Cultivation of human cell lines and conducting in vitro assays for detection of specific cellular markers, intercellular interaction or cytokines;
  • Performing in vitro assays for cytotoxicity, biological activity and selective antitumor activity.

By using these classic and recognized methods we will provide the necessary reliability and security in the interpretation of results and will avoid possible problems related to the validation of new methods or repetitions with alternative methods of analysis.

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