Study of the influence of selected kinase inhibitors involved in signaling in neoplastic cells on the cytotoxicity of NK cells

The aim of the project is to investigate the effect of selected kinase inhibitors (such as Syk, Btk, Akt, PI3K, mTOR) on the anti-tumor efficacy of monoclonal antibodies related to the mechanism of antibody dependent cellular cytotoxicity (ADCC). There are currently many clinical trials in which inhibitors of the cell signaling pathway are tested in combination with monoclonal antibodies used in cancer therapy. The results of our preliminary studies indicate that some of the tested inhibitors of kinases involved in signal transduction from the B lymphocyte receptor almost completely inhibit the cytotoxic functions of NK cells (which are the main effector cells in the ADCC process), and thus significantly weaken the anti-cancer effect of monoclonal antibodies in ADCC mechanism. As part of the project, the scope of research will be extended. Experiments will be carried out to test a series of various inhibitors of the aforementioned kinases on the activity of NK cells in the ADCC process induced by many different monoclonal antibodies (such as trastuzumab, obinutuzumab, alemtuzumab, cetuximab or daratumumab). The project will develop and optimize methods of studying the mechanisms of NK cell cytotoxicity, in particular the ability to kill target cells by the mechanism of cell cytotoxicity dependent on monoclonal antibodies commonly used in cancer therapy: anti-CD20 antibodies (rituximab, ofatumumab, obinutuzumab), anti-HER2 (trastuzumab), anti-EGFR (cetuximab) as well as anti-CD38 (daratuumab) used in clinical trials. In the ADCC assay models, the target cells will be cells of various human cell lines expressing the antigens recognized by the respective monoclonal antibodies: CD20 antigen-expressing lymphomas (Raji, Daudi, DoHH2, Ly-4), HER2/Neu expressing breast cancer lines (SKBR3 , T47D, ZR75-30) and ovarian cancer (SK-OV-3), showing EGFR expression: lung cancer (SW-900), bowel cancer (HCT116, LoVo), prostate cancer (Du-145). In addition, a method for studying NK cell degranulation and cytokine secretion using flow cytometry will be developed and optimized. Screening will then be performed using established methods to identify inhibitors of the cell signaling pathway that may affect the anti-tumor activity of NK cells. Inhibition of selected signaling pathways in NK cells will be confirmed by Western blotting. The project also plans to introduce a sequence encoding the FcγRIIIa (CD16) receptor, which is a key receptor involved in ADCC, into an NK-derived cell line by viral transduction. All commercial NK cell lines lack the surface of the FcγRIIIa receptor. The line created will serve as a model for ADCC research. Monoclonal antibodies are widely used in the treatment of cancer. There are currently 15 monoclonal antibodies registered in oncology. Moreover, new monoclonal antibodies modified in such a way as to maximize their antitumor effectiveness are still being tested. In the light of ongoing clinical trials combining monoclonal antibodies with many kinase inhibitors, it is extremely important to investigate how the tested chemical compounds affect the mechanisms of action of monoclonal antibodies already used in therapy. Understanding the interactions between the tested drugs will allow the selection of those therapeutic combinations that should be contraindicated, as well as those that should be used in combination therapy. The project's research will also contribute to a better understanding of NK cell biology and a more detailed understanding of what role different signal transduction pathways play in NK cell activation.