Tese de Doutoramento Biomedicina 2023 Faculdade de Ciências Médicas, Universidade NOVA de Lisboa

CLL is the most common form of leukemia worldwide, affects manly males above 60 years old and still remains incurable, due to prolonged disease progression and development of drug resistance. Combination of chemotherapy and immunotherapy are effective in controlling the disease, however in the lymph node proliferation centers, signals from the microenvironment particularly through the interaction with follicular T helper cells shield CLL cells from apoptosis. Previous observations have shown that the activation of co-stimulatory receptor CD40 can activate downstream signalling and induce changes in the regulation of apoptotic-mediator proteins, resulting in CLL cell survival. For other hand, CD40 activation can also induce sensitivity to treatment with anti-CD20 monoclonal antibodies, as rituximab and obinutuzumab. To understand whether these observations could be clinically further exploited, the proposed study aimed to investigate the distinct and potentially opposing effects of CD40 activation by a novel agonistic CD40 monoclonal antibody (selicrelumab). We described for the first time in primary CLL cells that selicrelumab can promote cell activation, a moderate pro-survival profile and significantly sensitize anti-CD20-mediated cell death of CLL cells, when enhanced by the presence of a Fc antibody crosslinker. With respect to pro-survival profile and drug resistance to venetoclax, crosslinked selicrelumab showed a moderated effect, predominantly mediated by MCL-1 upregulation, rather than BCL-XL. Since CD40 signalling activates distinct downstream pathways and contributes to pathogenesis of many cancer types, we investigated if the specific inhibition of PI3K/AKT, NF-kB, and MEK/ERK signalling pathways could rescue cell sensitivity to venetoclax. We demonstrated that AKT inhibitors were able to partially rescue venetoclax resistance induced by CD40 stimulation. Regulation of the PI3K/AKT kinases was important as a direct interference with other survival signalling as NK-kB and these observations were demonstrated in CLL cells. To add another perspective to the investigation of potential immunotherapy options against CLL, we investigated the activity of anti-HLA-DR X CD5 bispecific monoclonal antibodies (bA0024, bA0025 e bA0180) for the first time in hematological cancers cell lines. Complement-mediated killing, antibody-dependent cytotoxicity and direct-cell death features from the bispecific antibodies were tested. Results showed an efficient effect regarding the HLA-DR biding side in depleting CD5+ /HLA-DR+ target cells. New developments in Immunotherapy for CLL are showing great results in improving overall response rate. Novel strategies including monoclonal and bispecific antibody therapy for the treatment of CLL are under investigation, to potentiate cell death and to optimize the activation of the immune system. We here concluded that agonistic CD40 antibodies should be considered in combination with obinutuzumab, and HLA-DR is an effective target to promote tumor depletion. Additionally, multiple small-molecule inhibitors targeting different components of the PI3K/AKT pathway are currently at various stages of clinical development, and new combination strategies for CLL should consider the use of AKT inhibitors in clinical studies.