Immunonanoparticelle biodegradabili: sviluppo preclinico di un nuovo strumento terapeutico per il trattamento di patologie oncoematologiche

2010/2011Il linfoma di Burkitt (LB) e la leucemia linfatica cronica a cellule B (B-LLC) sono due malattie oncoematologiche accomunate dalla mancanza di un trattamento che sia in grado di eradicare completamente la malattia in tutti i pazienti. Il lavoro di questi tre anni si è inserito in questo contesto, con lo scopo di trovare uno strumento terapeutico alternativo che possa superare i limiti dei trattamenti attuali. La nostra attenzione si è spostata sulle nanotecnologie, in particolar modo una delle applicazioni dei nanomateriali in campo tumorale è il “drug delivery”, cioè l’utilizzo di veicoli di dimensioni nanometriche per il trasporto di farmaci o altre molecole all’interno delle cellule. Sono in corso di studio numerosi nanovettori di questo tipo, e questo progetto si è focalizzato sullo studio di un tipo di immunonanoparticelle biodegradabili (BNP: “Biodegradable NanoParticles”) costituite da un involucro polimerico ricoperto dall’anticorpo anti-CD20 Rituximab e contenenti al loro interno due farmaci: il clorambucile e l’idrossiclorochina. Lo scopo di questo progetto è stato quello di effettuare studi preclinici in vitro ed in vivo per valutare il potenziale terapeutico delle BNP nel trattamento di pazienti affetti da LNH e LLC. La raccolta di questi dati preclinici permette una prima analisi del potenziale terapeutico di questo nuovo approccio e permette di giustificare i successivi studi preclinici e clinici necessari a completare la valutazione dell’uso clinico delle BNP.XXIV Ciclo198

New Potential Therapeutic Approach for the Treatment of B-Cell Malignancies Using Chlorambucil/ Hydroxychloroquine-Loaded Anti-CD20 Nanoparticles

Current B-cell disorder treatments take advantage of dose-intensive chemotherapy regimens and immunotherapy via use of monoclonal antibodies. Unfortunately, they may lead to insufficient tumor distribution of therapeutic agents, and often cause adverse effects on patients. In this contribution, we propose a novel therapeutic approach in which relatively high doses of Hydroxychloroquine and Chlorambucil were loaded into biodegradable nanoparticles coated with an anti-CD20 antibody. We demonstrate their ability to effectively target and internalize in tumor B-cells. Moreover, these nanoparticles were able to kill not only p53 mutated/deleted lymphoma cell lines expressing a low amount of CD20, but also circulating primary cells purified from chronic lymphocitic leukemia patients. Their safety was demonstrated in healthy mice, and their therapeutic effects in a new model of Burkitt’s lymphoma. The latter serves as a prototype of an aggressive lymphoproliferative disease. In vitro and in vivo data showed the ability of anti-CD20 nanoparticles loaded with Hydroxychloroquine and Chlorambucil to increase tumor cell killing in comparison to free cytotoxic agents or Rituximab. These results shed light on the potential of anti-CD20 nanoparticles carrying Hydroxychloroquine and Chlorambucil for controlling a disseminated model of aggressive lymphoma, and lend credence to the idea of adopting this therapeutic approach for the treatment of B-cell disorders

A new approach for the treatment of CLL using chlorambucil/hydroxychloroquine-loaded anti-CD20 nanoparticles

16siCurrent approaches for the treatment of chronic lymphocytic leukemia (CLL) have greatly improved the prognosis for survival, but some patients remain refractive to these therapeutic regimens. Hence, in addition to reducing the long-term sideeffects of therapeutics for all leukemia patients, there is an urgent need for novel therapeutic strategies for difficult-to-treat leukemia cases. Due to the cytotoxicity of drugs, the major challenge currently is to deliver the therapeutic agents to neoplastic cells while preserving the viability of non-malignant cells. In this study, we propose a therapeutic approach in which high doses of hydroxychloroquine and chlorambucil were loaded into biodegradable polymeric nanoparticles coated with an anti-CD20 antibody.We first demonstrated the ability of the nanoparticles to target and internalize in tumor B-cells. Moreover, these nanoparticles could kill not only p53-mutated/deleted leukemia cells expressing a low amount of CD20, but also circulating primary cells isolated from chronic lymphocytic leukemia patients. The safety of these nanoparticles was also demonstrated in healthy mice, and their therapeutic effects were shown in a new model of aggressive leukemia. These results showed that anti-CD20 nanoparticles containing hydroxychloroquine and chlorambucil can be effective in controlling aggressive leukemia and provided a rationale for adopting this approach for the treatment of other B-cell disorders.reservedmixedCapolla, Sara; Mezzaroba, Nelly; Zorzet, Sonia; Tripodo, Claudio; Mendoza-Maldonado, Ramiro; Granzotto, Marilena; Vita, Francesca; Spretz, Ruben; Larsen, Gustavo; Noriega, Sandra; Mansilla, Eduardo; Dal Bo, Michele; Gattei, Valter; Pozzato, Gabriele; Núñez, Luis; Macor, PaoloCapolla, Sara; Mezzaroba, Nelly; Zorzet, Sonia; Tripodo, Claudio; Mendoza Maldonado, Ramiro; Granzotto, Marilena; Vita, Francesca; Spretz, Ruben; Larsen, Gustavo; Noriega, Sandra; Mansilla, Eduardo; Dal Bo, Michele; Gattei, Valter; Pozzato, Gabriele; Núñez, Luis; Macor, Paol

Comparison between BNP2 and Rituximab effects.

<p>BJAB cells were sorted to obtain High-CD20 and Low-CD20 cells. Mononuclear cells were purified from untreated CLL patients. Cells were analyzed for CD20 expression (MFI-mean fluorescence intensity) and then incubated with BNP2 for 48 hours to induce apoptosis. Cells were also incubated with Rituximab + NHS as a source of Complement for 1 h to induce CDC. Residual viable cells were measure using MTT assay.</p

<i>In vitro</i> characterization of the cytotoxic effect of BNP2.

<p>BJAB (A) and Raji (B) cells were incubated with 0.5, 1 and 2 μL of BNPs or HCQ+CLB for 48 hours at 37°C and residual viable cells were measured. Data are expressed as mean ± SD. *: p<0.01 vs BNP1. C) BJAB cells wer incubated with 1 μL of BNPs for only 16 hours at 37°C and apoptotic cells were analyzed using AnnexinV/PI test. D) Western blot analysis of activated PARP-1, LC3 and p62 from cell lysates obtained from BJAB cells incubated with 0, 0.5, 1 and 2 μL of BNP2.</p

Toxicological studies.

<p>C57/BL mice received BNP1, BNP2, BNP3 and HCQ+CLB at different doses. A) Animal survival and B) total body weight was measured for 28 days to evaluate toxicity of the treatments. *: p<0.001 vs PBS.</p

Therapeutic effect of BNPs, HCQ+CLB and Rituximab.

<p>A) SCID mice (n = 5 per group) received 2×10⁶ BJAB cells i.p.; Cy5.5 labeled-BNP1 or BNP2 (40 μL for 3 times in 5 days) were injected in tumor-bearing mice with a visible peritoneal tumor mass at day 25; the animals were sacrificed 7 days after the end of the treatment and the tumor masses were visualized by confocal microscopy and analyzed by H&E. Original magnification 200×. B) Survival curve. SCID mice (n = 7–10 per group) received 2×10⁶ BJAB cells i.p. and BNP1, BNP2, BNP3, HCQ+CLB or Rituximab as described in the results. P values. Untreated vs. BNP1: Not significant; Untreated vs. BNP2×4: p<0.0001; Untreated vs. BNP2×8: p<0.0001; Untreated vs. (HCQ+CLB)×4: p<0.0003; Untreated vs. Rituximab: p<0.0005; BNP2×4 vs. (HCQ+CLB) ×4: Not significant; BNP2×4 vs. Rituximab: Not significant; BNP2×8 vs. (HCQ+CLB) ×8: p<0.0001; BNP2×8 vs. Rituximab: p<0.0003; BNP3×8 vs BNP1: Not significant; BNP3×8 vs BNP2×4: p<0.0005; BNP3×8 vs BNP2×8: p<0.0001.</p

Characterization of Burkitt model in SCID mice.

<p>Labeled BJAB (2×10⁶ cells) were injected i.p. in SCID mice and fluorescence intensity emissions were acquired <i>in vivo</i> for 25 days. (A) Whole body scans at indicated post-injection time are reported. (B) To evaluate the dissemination to multiple organs by <i>ex vivo</i> analysis, mice were injected with labeled cells and they were sacrificed 7 days after the injection. (C) The same <i>ex vivo</i> analysis was performed on mice 25 days after the BJAB injection, when the tumor mass was already developed. NC =  Normalized Count. D) Peritoneal tumor mass displays a solid cohesive pattern of growth with round small/medium sized elements and a high number of mitotic and apoptotic figures (Hematoxilin and Eosin, original magnification 200×). Neoplastic cells show strong immunoreactivity to CD20 (anti-CD20 immunostaining, Strept-ABC method, original magnification 200×) and Bcl-6 (anti-Bcl-6 immunostaining, Strept-ABC, original magnification 200×). The high proliferation rate of neoplastic cells (nearly 100% of cells) is highlighted by Ki-67 immunostaining (anti-Ki-67, Strep-ABC method, original magnification 100×).</p