Anti-Leukemic Activity of a Novel Pegylated Recombinant Erwinia Chrysanthemi-Derived L-Asparaginase On Lymphoid Cell Lines and Leukemia-Bearing Mouse Models.

Abstract

AbstractAbstract 2571 Background:Bacterial-derived L-asparaginase (ASNase) is an indispensable component of the therapy of acute lymphoblastic leukemia (ALL). Despite the high rate of successful treatment, hypersensitivity reactions occur in 20–40 % of patients receiving this non-human protein, which limits the use of ASNase. Native Erwinia chrysanthemi-derived ASNase (n-crisantaspase) has been used for treating patients exhibiting allergic symptoms to native and PEGylated Escherichia coli-derived ASNases (EC-ASNase). However, hypersensitivity reactions are still observed in at least 17 % of patients receiving n-crisantaspase. A PEGylated recombinant Erwinia chrysanthemi-derived ASNase (PEG-r-crisantaspase) with improved pharmacokinetic and pharmacodynamics properties and reduced immunogenicity has been developed recently (Allas et al., abstracts #2003, #2034, ASH 2009). We present here the in vitro and in vivo evaluation of PEG-r-crisantaspase on leukemia and lymphoma cell lines and on a leukemia-bearing mouse model, respectively. Material and methods:Different cell lines (ALL, B, T and NK/T lymphoma) and bone marrow aspiration samples obtained from patients with B-ALL or T-ALL were exposed in vitro to increasing doses (0.00005 to 5 U/ml) of PEG-r-crisantaspase, n-crisantaspase, or native EC-ASNase for 3 days. The cytotoxicity of each molecule was evaluated using 3- (4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. For the in vivo study, 5 millions of ASNase-sensitive cells, RS(4,11) were xenografted by intravenous injection (iv) into immunodeficient NOD/SCID mice. When 5% of leukemic blasts were observed in the blood of leukemia-bearing mice, 2, 5, 10 or 20 U/kg of PEG-r-crisantaspase or vehicle was injected intravenously in those mice. Two additional injections were performed with an interval of 7 days. Mice having significant weight loss (>20%) and/or extensive expansion of leukemic cells in blood (> 25%) and/or impaired general condition were sacrificed. Results:In vitro, PEG-r-crisantaspase and n-crisantaspase exhibited similar half maximal effective concentration (EC50) values for inhibiting the proliferation of leukemia and lymphoma cells. PEG-r-crisantaspase had a greater cytotoxicity effect on two high-sensitive cell lines than n-crisantaspase did, as shown by 4.5 and 8.1-fold-weaker EC50 values of PEG-r-crisantaspase relative to n-crisantaspase. PEG-r-crisantaspase, r-crisantaspase and n-crisantaspase were more efficient than EC-ASNase on less sensitive cell lines, which might be related to the 10-fold-greater glutaminase activity of crisantaspases than EC-ASNase. In vivo, after the first administration of PEG-r-crisantaspase in animals with ≥ 5% of leukemic cells, the leukemic cells were reduced to almost 0 % in mice within 4 days with all doses of PEG-r-crisantaspase tested, whereas the leukemic cells kept multiplying in the mice receiving vehicle. The 3 repeated injections of PEG-r-crisantaspase with an interval of 7 days delayed leukemia development for 17 days. All control mice receiving the vehicle had to be sacrificed at day 44 after leukemia inoculation, whereas of 28 mice receiving PEG-r-crisantaspase, 5 reached this endpoint at day 51 and the others at day 56. PEG-r-crisantaspase significantly improved the survival of leukemia-bearing mice for 7 to 12 days, regardless of the dose tested. Conclusions:These data provide evidence that PEG-r-crisantaspase has similar in vitro cytotoxic effect to n-crisantaspase on leukemia and lymphoma cell lines and significantly reduces the expansion of leukemic cells in leukemia-bearing mice, prolonging the survival of the animals. These results, together with preclinical PK/PD and immunogenicity data, support the clinical development of PEG-r-crisantaspase. A phase I dose escalation study in adult patients with relapsed or refractory hematological malignancies has been recently initiated.Research support from Alizé Pharma, the European Regional Development Fund (ERDF) and Grand Lyon. Disclosures:Allas:Alizé pharma: Employment. Sahakian:Alizé pharma: Employment. Julien:Alizé pharma: Employment. Abribat:Alizé pharma: Employment.