New Therapeutic Approach for the Treatment of B-Cell Disorders Using Chlorambucil/Hydroxychloroquine-Loaded AntiCD20 Nanoparticles
Authors: Pozzato Gabriele , Mezzaroba Nelly , Zorzet Sonia , Secco Erika , Biffi Stefania , Tripodo Claudio , Calvaruso Marco , Nuñez Luis , Spretz Rubens , Larsen Gustavo , Noriega Sandra , Garrovo Chiara , Marin Gustavo , Mansilla Edoardo , Macor Paolo 
Published: October 2019

Abstract
Abstract 158
B-cell disorders show highly variable clinical courses, ranging between indolent diseases like the chronic lymphocytic leukemia (CLL) and highly aggressive lymphoproliferative disorders like Burkitt Lymphoma. The treatments of these disorders have been characterized by the development of new approaches, including dose-intensive chemotherapy regimens and immunotherapy via monoclonal antibodies (Ab). Despite the promising survival rates, these multi-agent treatments are flawed by a high degree of toxicity and a significant fraction of patients do not respond. The use of core shell nanoparticles design with specific Ab-coating represents a new strategy to target only tumor cells with high-dose chemotherapy. We characterized, both in vitro and in vivo, the effects of a new kind of biodegradable nanoparticles (BNP) coated with an anti-CD20 antibody and loaded with Chlorambucil (CLB) and Hydroxychloroquine (HCQ).
BJAB and MEC1 are two well characterized Burkitt lymphoma and CLL cell line, respectively; both are mutated in P53, express different amount of CD20 and result quite resistant to CLB, but more susceptible to HCQ. HCQ/CLB-loaded antiCD20 BNP can release enough amounts of drugs inside the cancer cells and overcome multi-drug resistance mechanisms, which are over-expressed in many B-cell disorders. The presence of antiCD20 antibody targets BNP on cells expressing this antigen, inducing a selective penetration in these cells, as demonstrated by electron microscopy studies; the cytotoxic effect was evident analyzing apoptosis and after 24–48 hours all the cancer B-cells resulted destroyed. Data indicate that BNP were able to kill also cells expressing very low amount of CD20, where Rituximab was almost ineffective.
These results were confirmed using primary cells isolated from more than 40 patients with untreated chronic lymphocytic leukemia. These data were obtained incubating fresh isolated blood with HCQ/CLB-loaded antiCD20 BNP and analyzing residual viable or apoptotic cells.
Endovenous or intraperitoneal injection of HCQ/CLB-loaded antiCD20 BNP in mice did not induced tissue damages, weight loss or death, while the same amount of free cytotoxic agent cause death of all the animals.
Biodistribution analysis demonstrated a selective targeting of the antiCD20 BNP to tumor cells, with a peak 48 hours after injection; BNP elimination was mainly documented in liver and intestine but not by spleen, kidney, lungs or other organs.
The intraperitoneal injection of BJAB cells in SCID mice determined the formation of a tumor mass visible at the site of injection followed by early multi-organ involvement and death of all animals. Eight injections of antiCD20 BNP containing CLB/HCQ (400 micrograms each) in 17 days were able to increase survival in 100% of mice and 90% of animals were cured. The same amount of CLB+HCQ cannot be used as free drugs because of their toxicity able to kill all the animals in about a week.
The intravenous injection of MEC1 cells in SCID mice allowed the development a new human/mouse model of leukemia. The treatment with antiCD20 BNP containing CLB/HCQ showed comparable therapeutic efficacy, while free drugs were almost uneffective.
In conclusion, the results of the present study demonstrate that antiCD20 BNP containing HCQ/CLB can be effective as a single agent in controlling both aggressive and indolent models of disseminated B-cell disorders and provide a rationale for adopting this new therapeutic approach in clinical trials. Furthermore, the results demonstrate that nanoparticles are effective via different administration methods.

Disclosures:
No relevant conflicts of interest to declare.