Hybrid nanoparticles loaded with ALK-degrader and functionalized with anti-CD30 antibody for the treatment of anaplastic large cell lymphoma

Anaplastic large cell lymphoma (ALCL), specifically anaplastic lymphoma kinase (ALK)-positive ALCL, is a rare and aggressive type of non-Hodgkin’s lymphoma (NHL), that accounts for 10-20% of pediatric NHL and 1-3% of adult NHL with high risk of relapse. ALK-positive ALCL is characterized by high CD30 and abnormal ALK protein expression. Currently available therapies for ALK-positive ALCL are mainly based on chemotherapy, that suffers from variable efficacy, significant side effects, and poor selectivity. Several ALK inhibitors have been proposed to treat this disease, but the emergence of drug resistance has limited their clinical application. Therefore, the development of novel therapeutic strategies is essential to improve the overall clinical outcomes for ALK-positive ALCL. Nano-dimensioned drug delivery systems are valuable tools in cancer treatment, able to reduce drug toxicity, control drug release, and selectively target cancer cells thereby maximizing tumor accumulation of treatments. This project aims to develop and characterize a novel treatment approach involving hybrid lipid/polymer nanoparticles (NPs) designed to deliver an ALK-degrader (ALK-proteolysis targeting chimera, ALK-PROTAC) (AD), to induce ALK degradation. NPs were further functionalized with anti-CD30 antibody (Ab) for targeted delivery to CD30-expressing ALCL cells. NPs were prepared through nanoprecipitation and characterized in terms of size and surface charge by dynamic light scattering, confirming small size distribution (180 ± 24 nm) and a negative surface charge (–33 ± 3 mV). AD-loaded NPs presented a high encapsulation efficacy (69 ± 4%), and an optimal release kinetic sustaining the release for up to 7 days. To confirm NPs biocompatibility, three ALCL cell lines (TS, COST, and K299) were treated with empty NPs and cell viability was measured by CellTiter-Glo: no signs of cytotoxicity were observed in all tested conditions. NPs internalization rate was analyzed by FACS after different incubation timepoints, achieving a 46-78% internalization after 30 min, an 82-96% internalization after 3 h, and a 78-97% internalization after 24 h, depending on the cell line. Subsequently, the same cell lines were treated with AD-loaded NPs and western blot confirmed successful degradation of ALK in all cases, with comparable results to those obtained with free ALK-degrader at the same concentration. The functionalization of NPs was optimized using a control monoclonal Ab, which was coupled to the activated carboxyl groups on the pegylated lipid on NPs, through carbodiimide chemistry. Different antibody/carboxyl groups molar ratios were tested (1:32 and 1:16), resulting in high Ab functionalization efficiency (63 ± 21% and 67 ± 8% respectively), evaluated by BCA assay. The selectivity of NPs functionalized with anti-CD30 Ab was evaluated by FACS analysis against TS cells (high CD30 expression), AMO-1, and U937 cells, with respectively low and intermediate expression of the receptor. Good NPs internalization rate was observed in TS for both functionalized and unfunctionalized NPs. In future studies, Ab-functionalized NPs will be tested on additional ALCL cell lines and their targeting ability evaluated in vivo using mouse models of ALCL.