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EFFECTS OF CHLORAMBUCIL/HYDROXYCHLOROQUINE-LOADED ANTI-CD20 NANOPARTICLES ON A HUMAN/MOUSE MODEL OF BURKITT LYMPHOMA Mezzaroba N,1 Mansilla E,2 Zorzet S,1 Larsen G,3 Tripodo C,4 Núñez L,5 Marín GH,2 Nabergoj M,6 Tedesco F,1 Pozzato G,6 Macor P1 1 Department of Life Science, University of Trieste, Italy; 2 CUCAIBA, Ministry of Health, La Plata, Buenos Aires, Argentina; 3 LNK Chemsolutions, Lincoln Nebraska, USA and Bio-Target, Chicago, USA; 4 Department of Human Pathology, University of Palermo, Italy; 5 University of Chicago, Chicago, USA and BioTarget, Chicago, USA; 6 Department of Medical and Surgical Sciences, University of Trieste, Italy Introduction. In Burkitt lymphoma (BL) chemotherapy shows variable responses since a fraction of younger patients are resistant or relapse and the patients over 40 have difficulties to follow the therapy. In nonresponders/relapsers BL cells often express low level of CD20 and/or mutant/deleted p53. To overcome these problems, we developed a new therapeutic approach in which the efficacy of chemotherapy is associated to the specificity and low side-effects of antibody-based therapy. This approach lies in biodegradable nanoparticles (NP) coated with an antibody to target the neoplastic cells and filled with Hydroxychloroquine (HCQ) (a p53-independent toxic substance) and chlorambucil (CLB) as modern “Trojan Horses” to destroy the cancer cells. Methods. NP were prepared by the copolymerization of polylactic acid and polycaprolactone (diameter:250nm). The NP with HCQ and CLB were prepared by encapsulation at concentrations of 165 µg per mg of polymer inside their core, and then modified by anti-CD20 adsorption. In vitro study: To investigate the ability of NP to kill Burkitt lymphoma cell lines, 5 x 105 BJAB cells were incubated for 48 hours and the number of residual viable cells was determined with MTT assay. Apoptosis was evaluated by analyzing PARP-1 cleavage and Annexin V detection. In vivo study: A xenograft model was developed in SCID mice inoculated i.p. with 2 x 106 labeled or unlabeled BJAB cells; a fraction of mice was treated with NP and all animals examined twice weekly up to 120 days. A small-animal time-domain eXplore Optix pre-clinical imager was used for the invivo and ex-vivo evaluation of labeled BJAB cell dissemination and Ritcy5.5 distribution. At 120 days the animals were sacrificed and peritoneal mass and organs, lymph nodes and bones were collected for immunohistochemical analysis. Results. In vitro study: The binding NP on cell surface and their internalization were documented with confocal microscopy. In BJAB cells, HCQ and CLM as free substances induced cell death in 82% and 38% respectively, while NP containing only 5,4 g of HCQ and CLB killed 100% of cells. This effect is due to the synergic proapoptotic effect as shown by Annexin V detection and by PARP activation. The effects of NP was tested on CD20-negative cell lines CHO, HUVEC, MEL-28, LoVo: No cell cytotoxicity was detectable using NP, whereas free HCQ+CLB induce cell killing between 58 and 71%. In vivo study: The intra-peritoneal injection of BJAB cells induced a fast (within 4 days) tumor cells spread in different organs and, without therapy, all animals died in 50-70 days as well as those receiving free HCQ and CLM. Conversely, survival at 120 days of the mice receiving 4 or 8 i.p. injections of NP was 50 and 90% respectively. Conclusions. These results indicate that NP are able to cure even the highly proliferating BL cell line BJAB in both in vitro and in vivo models. This suggests the introduction of this promising technology into human clinical trial.

DEVELOPMENT OF CHLORAMBUCIL/HYDROXYCHLOROQUINE-LOADED ANTI-CD20 NANOPARTICLES FOR THE TREATMENT OF CHRONIC LYMPHOCYTIC LEUKEMIA : IN VITRO MODEL Mezzaroba N,1 Mansilla E,2 Zorzet S,1 Calvaruso M,3 Tripodo C,3 Núñez L,4 Tedesco F,1 Nabergoj M,6 Sblattero D,5 Granzotto M,7 Pozzato G,6 Macor P1 1 Department of Life Science, University of Trieste, Italy; 2 CUCAIBA, Ministry of Health, La Plata, Buenos Aires, Argentina; 3 Department of Human Pathology, University of Palermo, Italy; 4 University of Chicago, Chicago, USA and BioTarget, Chicago, USA; 5 Department of Medical Sciences, University of Eastern Piedmont, Novara, Italy; 6 Department of Medical and Surgical Sciences, University of Trieste; 7 Department of Molecular and Laboratory Medicine, IRCCSS Burlo Garofolo, Trieste, Italy Introduction. The actual challenge of tumor therapy is to design new therapeutic agents able to maximize the efficacy minimizing the adverse effects. Nanoparticles (NP) have emerged as important tools to modify the release profile for a large number of drugs. The aim of this study was to determine whether anti-CD20-coated biocompatible FDA-approved NP loaded with Chlorambucil (CLB) and Hydroxychloroquine (HCQ) could induce apoptosis of the B-Chronic Lymphocytic Leukemia (CLL) cell line MEC-1, as well as of circulating neoplastic B-cells isolated from patients with B-CLL. Methods. Nanoparticles were prepared by the copolymerization of polylactic acid and polycaprolactone and had an diameter of 250 nm. The NP with HCQ and CLB were prepared by encapsulation of the two drugs at concentrations of 165 µg per mg of polymer inside their core, and then modified by anti-CD20 adsorption. The NP were resuspended in PBS with 10% Bovine Serum Albumin (BSA) and maintained at 4°C before use. To investigate the ability of NP to affect cell viability and induce cellular apoptosis, MEC-1 cells (2 x 105) were incubated with anti-CD20 coated and CLB-HCQ loaded NP for 48 hours and the residual viable cells was determined with MTT assay. Cell death program activation was also evaluated by analyzing PARP-1 cleavage and Annexin V detection. Monoclonal B-cells were obtained from 44 patients affected by CLL. In each case the IGHV gene mutational status was assessed using standard methods and CD38 and ZAP-70 expressions were determined by flow cytometry. Interphase FISH was performed on nuclei preparations of PBMC and each case was investigated for 13q deletion, 11q deletion, 17p deletion or presence of trisomy 12. Results. Anti-CD20 NP with HCQ and CLB were able to induce a strong reduction (95%) in cell viability of MEC-1 cells. The cytotoxicity was present in a dose-dependent manner and was apoptosis-dependent as confirmed by the PARP- 1 activation and by the binding of Annexin V on cell surface. The same high cytotoxicity rate (>90%) was found also in patient-derived B-cells. The killing rate was independent from IGVH mutational status or from ZAP70 and CD38 expression or from the presence of 17p deletion. An efficient cytotoxicity was present even in CLL B-lymphocytes expressing low CD20 antigen levels on cell surface. The effects of NP were also tested on CD20-negative human and animal cells (CHO, HUVEC, MEL- 28, LoVo cell lines) and no specific cytotoxicity was obtained. On the contrary, incubation of CD20-negative cells with free HCQ + CLB induced cell killing between 58 and 71%. Conclusions. These results indicate that anti-CD20 coated and CLB-HCQ loaded NP show a high toxicity rate in CLL cell line as well as in B-cell obtained from CLL patients. The toxic effect is present also in 17p deleted cases, usually resistant to chemo-immuno-therapy. These results should be confirmed in animal models in order to introduce this promising technology into human clinical trials

Monoclonal antibodies (mAbs), successfully adopted in the treatment of several haematological malignancies, have proved almost ineffective in multiple myeloma (MM), because of the lack of an appropriate antigen for targeting and killing MM cells. Here, we demonstrate that PSGL1, the major ligand of P-Selectin, a marker of plasmacytic differentiation expressed at high levels on normal and neoplastic plasma cells, may represent a novel target for mAb-mediated MM immunotherapy. The primary effectors of mAb-induced cell-death, complement-mediated lysis (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC), were investigated using U266B1 and LP1 cell-lines as models. Along with immunological mechanisms, the induction of apoptosis by PSGL1 cross-linking was assessed. The anti-PSGL1 murine mAb KPL1 induced death of MM cells in a dose- and time-dependent fashion and mediated a significant amount of ADCC. KPL1 alone mediated C1q deposition on target cells but proved unable to induce CDC due to inhibition of the lytic activity of complement by membrane complement regulators (mCRP) expressed on the cell surface. Consistently, CDC was induced by KPL1 upon mCRP blockage. Our results suggest a role for PSGL1 in MM humoral immunotherapy and support further in vivo studies assessing the effects of anti-PSGL1 mAbs on MM growth and interaction with the bone marrow microenvironment.

Complement activation plays a relevant role in the development of tissue damage under inflammatory conditions, and clinical and experimental observations emphasize its contribution to inflammatory vasculitides. Statins have recently been shown to reduce cardiovascular morbidity independently of plasma cholesterol lowering and in vitro studies support a direct anti-inflammatory action of these drugs. The aim of this study was to verify the in vivo effect of fluvastatin on complement-mediated acute peritoneal inflammation. The effect of oral treatment with fluvastatin was investigated in normo-cholesterolaemic rats that received intraperitoneal injection of either yeast-activated rat serum (Y-act RS) or lipopolysaccharide to induce peritoneal inflammation monitored by the number of PMN recruited in peritoneal fluid washes. In addition, vascular adherence and extravasation of leucocytes were evaluated by direct videomicroscopy examination on mesentery postcapillary venules topically exposed to Y-act RS. The number of PMN in the peritoneal washes of rats treated with fluvastatin was 38% lower than that of untreated animals (P < 0.05) 12 h after LPS injection, and was even lower (56%) in rats treated with Y-act RS already 8 h after injection (P < 0.02). Firm adhesion to endothelium and extravasation of leucocytes evaluated under direct videomicroscopy observation were significantly inhibited in fluvastatin treated rats (77% and 72%, respectively; P < 0.01), 120 min after treatment with Y-act RS. Our results demonstrate that fluvastatin inhibits in vivo complement-dependent acute peritoneal inflammation and suggest a role for statins in preventing the inflammatory flares usually associated with complement activation in chronic diseases, such as SLE or rheumatoid arthritis.