Engenharia de nanossistemas biespecíficos para mediar uma estratégia multialvo de imuno-quimioterapia para cancro do pulmão

The main goal of this proposal is to develop novel bispecific T-cell engagers-based nanosystems (nanoBiTEs), consisting in core-shell polymer-lipid nanocarriers with three targeting ligands/engagers, with the ability to simultaneously bind T-cells and NSCLC cells, including cancer stem cells (CSCs), and to co-deliver immunomodulatory and anticancer drugs in the tumor, in order to overcome and revert the immunosuppressive tumor microenvironment (TME), including the phenotype repolarization of tumor-associated macrophages (TAMs), and induce T-cells activation for a strong antitumor activity with reduced side effects. The specific aims of the present project are: - Develop targeted polymer-lipid-based nanoBiTEs, loaded with therapeutic agents and prepared in different molar ratios of PLGA, lipids, ligands and drugs, and perform their extensive physicochemical characterization (composition, functionalization, size, polydispersity, surface charge, stability, structure, morphology, drug loading and release, and interaction with serum), since the physicochemical properties are determinant for the therapeutic efficacy of nanosystems. We are expecting to produce nanoBiTEs with suitable properties for in vivo application. - Evaluate and understand the mechanisms involved in the cells-nanoBITES interactions (including cytocompatibility, binding, affinity of engagers, co-localization), with target and non-target cells, in particular, the advantage of incorporating anti-CD3, anti-EGFR and anti-CD44 antibodies in nanoBiTEs formulation, as they should result in high levels of both specificity and binding between T-cells and NSCLC cells, including CSCs. Moreover, we expect to obtain highly biocompatible nanosystems. - Assess the antitumor activity of the individual and combined strategies (nanoBiTEs loaded or not with CA-170 and/or AMD3100) and clarify the underlying cell death mechanisms. In particular, we will evaluate the effect of the drugs and the effector function of T-cells, in 2D cell cultures of NSCLC and in 3D multicellular systems, by measuring cell proliferation; cytotoxic activity; mechanisms of cell death; T-cell activation, through analysis of their surface markers and the secretion of pro-inflammatory cytokines; proportion of T-cell sub-populations; mRNA and protein levels of molecular targets involved in immunosuppression and tumor progression; T-cells infiltration and morphology; and levels of M1 and M2 TAMs. The combined antitumor strategies are expected to result in a high and synergistic cancer cell death effect. - Assess the therapeutic potential of the selected combined strategy in an animal model of NSCLC, by evaluating tumor volume and progression, body weight, toxicity profile, antitumor activity of the immune system, including immune cells infiltration analysis, polarization of macrophages and memory response. It is expected that the developed nanoBiTEs will have the ability to specifically and efficiently mediate this innovative multitarget therapeutic strategy, inducing a much higher antitumor activity and much less side effects, due to the specificity of the nanosystems, than that obtained with the conventional approaches. The multifunctionality of this nanoplatform may constitute a novel T-cell-based therapy for NSCLC, solving the main challenges of standard treatment and, therefore, contributing to the generation of new antitumor strategies with high socioeconomic impact.