Invest New Drugs DOI 10.1007/s10637-014-0102-9

PRECLINICAL STUDIES

Pharmacology, immunogenicity, and efficacy of a novel pegylated recombinant Erwinia chrysanthemi-derived L-asparaginase Wei-Wen Chien & Soraya Allas & Nicolas Rachinel & Pierre Sahakian & Michel Julien & Céline Le Beux & Claire-Emmanuelle Lacroix & Thierry Abribat & Gilles Salles

Received: 21 January 2014 / Accepted: 9 April 2014 # Springer Science+Business Media New York 2014

Summary Bacterial L-asparaginase (ASNase), hydrolyzing L-asparagine (Asn), is an indispensable component used in the treatment of acute lymphoblastic leukemia (ALL) and certain lymphoma entities. Native Erwinia chrysanthemi-derived ASNase (n-crisantaspase) has been approved as a second-line drug for treating patients exhibiting allergy syndromes to native and pegylated Escherichia coli-derived ASNase (EC-ASNase). However, it still induces hypersensitivity in at least 17 % of treated patients. In the present study, we investigated the pharmacological activity, immunogenicity and anti-leukemic activity of a new pegylated recombinant crisantaspase (PEG-r-crisantaspase). The results demonstrate that when compared to n-crisantaspase in vivo, PEG-rcrisantaspase maintains a complete depletion of plasma Asn for up to 72 h with a 50-fold lower dose. In mice receiving PEG-r-crisantaspase, specific antibodies against the enzyme were undetectable, indicating a lower immunogenicity of the pegylated enzyme. In vitro, PEG-r-crisantaspase exhibits similar cytotoxic effects (EC50 0.08 U/mL) were the T-ALL CEM and Jurkat cell lines. When comparing EC50 of PEG-r-crisantaspase with that of EC-ASNase, n-crisantaspase, and r-crisantaspase, the following observations were made: i) PEG-rcrisantaspase, n-crisantaspase, and r-crisantaspase were significantly more efficient than EC-ASNase on intermediate- and low-sensitive cell lines; ii) PEG-rcrisantaspase exhibited a similar efficacy profile to that of n-crisantaspase and r-crisantaspase in most cell lines; iii) PEG-r-crisantaspase demonstrated, however, a significantly greater activity than EC-ASNase, n-crisantaspase and r-crisantaspase, on highly-sensitive cell lines (Fig. 3, Supplementary Table 1). Asn and Gln hydrolysis by PEG-r-crisantaspase in vitro

Cytotoxic efficacy profile of PEG-r-crisantaspase in vitro Figure 3 illustrates the half-maximum effective concentration (EC50) of each of the tested ASNases for all studied cell lines, with detailed results summarized in Table 1. B-ALL RS(4,11) cell line and the MEC04, an NK/T-cell lymphoma derived cell line, were the highly sensitive (PEG-r-crisantaspase EC50 90 % degradation of Gln may be required for optimal Asn depletion by ASNase [47]. In our study, PEG-rcrisantaspase, as other tested ASNases, was shown to dramatically inhibit the viability of less sensitive cells, but only at the dose levels completely depleting Asn and depleting>88 % Gln in medium (depletion to around 310 μM with 0.05 U/mL of Erwinia chrysanthemi-ASNases or with 0.5 U/mL of ECASNase). Our results are in line with the previous published data indicating that glutamine restriction induces apoptosis of ASNase-less sensitive CEM, HL-60 cells when medium Gln concentration is lower than 300–400 μM for at least 24 h [49], and argue for the anti-tumor function of the glutaminase activity of ASNase. In our study, in vivo efficacy tests showed that three repeated PEG-r-crisantaspase injections at a 7-day interval were able to prevent leukemia development. However, the percentage of leukemic cells tended to re-increase prior to the 2nd and 3rd injections. In spite of the complete plasma Asn depletion observed, the 3rd PEG-r-crisantaspase injection was not as efficient in reducing leukemic cells in peripheral blood of mice as the 1st and 2nd injections. These results suggest that resistance mechanisms to ASNase, when used as a single agent, may occur during treatment, with leukemic cells escaping PEG-r-crisantaspase cytotoxic effects. In conclusion, based on the data presented here, PEG-rcrisantaspase exhibited promising PD/PK properties along with reduced immunogenicity, in comparison with the native protein. Moreover, data also showed that PEG-r-crisantaspase was able to inhibit leukemia and lymphoma cells proliferation in vitro, and to reduce leukemic cell expansion in leukemiabearing mice, thereby prolonging survival. Overall, these results support PEG-r-crisantaspase development as a promising ASNase for ALL patient treatment. The activity of PEGr-crisantaspase administered at appropriate intervals to ensure ASNase activity and Asn depletion would also have to be compared to repeat dosing of native Erwinia chrysanthemi ASNase, as commonly used in clinical practice. A Phase I dose-escalation clinical trial with PEG-r-crisantaspase involving adult patients presenting relapsed or refractory hematological malignancies has been recently initiated (NCT01551524).

Acknowledgments Special thanks to Dr. Philippe Gaulard (INSERM U955, France), Dr. Paul Coppo (Hôpital Saint-Antoine, Paris, France), and Dr. Hiroshi Kimura (Department of Virology, Japan) for providing MEC04 and KHYG1 cells lines, respectively. Sincere thanks to Dr. Christine Saban for amino acid measurement, Patrick Manas for his help in animal experiments, Martine Ffrench and Aline Billoud for patient samples, Lucile Baseggio for the immunocytochemistry technique, and to the ALL patients who donated blood samples for our studies. The project received partial financial support with a refundable grant from Oséo (Maisons Alfort, France) and an FUI (Fonds Unique Interministériel) grant contributed by both the European Regional Development Fund (ERDF), and the Grand Lyon and Rhône Alpes regions. Conflict of interest SA PS, MJ, and TA are employees, and TA is a shareholder of Alizé Pharma II. The remaining authors declare no competing financial interests.

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