Immunogenicity Profile in Mice of a Pegylated Recombinant Erwinia Chrysanthemi-Derived L-Asparaginase.

Hypersensitivity is the most common dose-limiting toxicity observed with any of the currently available L-asparaginases, vital components of multi-agent chemotherapy for childhood acute lymphoblastic leukemia (ALL). In a significant proportion of patients, administration of L-asparaginase preparations also results in the development of high titers of serum IgG antibodies which in the majority of cases interfere with the therapeutic effect of the enzyme. L-asparaginases are extracted either from Escherichia coli or Erwinia chrysanthemi and when signs of hypersensitivity occur with the Escherichia coliproducts, used as front line therapy, patients are switched to the Erwinia chrysanthemi preparations. Despite the availability of second line products, hypersensitivity reactions and neutralizing antibodies remained frequently observed, providing the rationale for developing new L-asparaginases. Using recombinant and state-of-the-art PEGylation technologies, Asparec®, a new Erwinia chrysanthemi-derived L-asparaginase (crisantaspase), has been designed with the objective of developing a compound with improved pharmacokinetic (PK) and pharmacodynamic (PD) properties and reduced immunogenicity as compared to the native enzyme.

A recombinant crisantaspase has been engineered and further modified by covalent conjugation to methoxy-polyethylene glycol (PEG) molecules of 2, 5 or 10 kDa using a stable PEG-protein linkage, yielding PEG conjugates of high specific activity (range: 483 to 543 U/mg). Studies performed in mice have shown that the 5 kDa PEG r-crisantaspase conjugate showed improved PK/PD properties as compared to other tested PEG-r-crisantaspase conjugates and Erwinase®, the commercially available crisantaspase. In order to characterize the immunogenicity profile of PEG-r-crisantaspase conjugates and select a candidate for further development, groups of 8 female mice received intravenous administration of PEG-r-crisantaspase conjugates (5 U/kg) versus Erwinase® (250 U/kg) twice a week at Week 1, 2, 3, 4 and 8. Levels of specific antibodies against r-crisantaspase or PEG-r-crisantaspase conjugates were measured using indirect Elisa method with absorbance read at 450 nm.

High absorbance values reflective of high titers of anti-crisantaspase antibodies were observed for Erwinase® starting at Week 2 of treatment (mean±SD: 1.816±0.57) and were maintained for the whole study period (2.946±0.283 at Week 8). In contrast, no significant anti-crisantaspase antibody levels were noted following treatment with the 2, 5 or 10 kDa PEG-r-crisantaspase conjugates (at Week 8: 0.026±0.008, 0.019±0.010, and 0.021±0.004, respectively). Treatment with the 2 kDa PEG-r-crisantaspase conjugate did not induce significant levels of specific anti-conjugate antibodies within the first 4 weeks of treatment. Mean absorbance values slightly increased with time resulting in weak positive signals at the end of the treatment period (0.183±0.422). Mean values were slightly greater following treatment with the 5 kDa and 10 kDa PEG- r-crisantaspase conjugates starting week 1 and increased with time. At the end of the treatment period, values were markedly higher with the 10 kDa (1.094±0.578) as compared to the 5 kDa PEG-r-crisantaspase conjugate (0.371±0.392). Similarly, the number of animals with significant mean absorbance values (>0.5) was higher with 10 kDa PEG-r-crisantaspase conjugate as compared to the 2 and 5 kDa PEG-r-crisantaspase conjugates.

These data clearly indicate that PEGylation significantly reduced immunogenicity of r-crisantaspase. Data are in agreement with PK/PD results, providing additional support for the development of the 5 kDa PEG-r-crisantaspase conjugate as a promising second line treatment for ALL.

Allas:Alize Pharma: Employment. Sahakian:Alize Pharma: Employment. Abribat:Alize Pharma: Employment.