Leukemia (1998) 12 , 1527–1533  1998 Stockton Press All rights reserved 0887-6924/98 $12.00 http:/ / www.stockton-press.co.uk/ leu Anti-asparaginase antibodies following E. coli asparaginase therapy in pediatric acute lymphoblastic leukemia MH Woo 1 , LJ Hak 2 , MC Storm 2 , WE Evans 1,2,3 , JT Sandlund 3,4 , GK Rivera 3,4 , B Wang 2 , C-H Pui 3,4 and MV Relling 1,2 Departments of 1 Pharmaceutical Sciences and 4 Hematology-Oncology, St Jude Children’s Research Hospital, and Colleges of 2 Pharmacy and 3 Medicine, University of Tennessee, Memphis, TN, USA Asparaginase is an effective antileukemic agent and is included in most front-line protocols for pediatric acute lymphoblastic leukemia (ALL) worldwide; however, allergic reactions to aspar- aginase may be dose-limiting. We evaluated plasma anti-aspar- aginase antibody concentrations in a cohort of children with newly diagnosed ALL, who did and who did not exhibit clinical hypersensitivity, after Escherichia coli (E. coli) asparaginase therapy. Thirty-five children who received asparaginase 10 000 IU/m 2 i.m. three times weekly for nine doses as part of both multiagent induction and reinduction chemotherapy, and seven monthly doses during the first 7 months of continuation treatment, were studied. Twenty-two patients experienced initial allergic reactions to asparaginase during continuation (n = 20) or reinduction (n = 2) phases and 13 children did not exhibit any reaction. An enzyme-linked immunosorbent assay (ELISA) was used to measure anti-asparaginase antibodies in plasma samples, diluted 1:3200, using E. coli asparaginase as the antigen. The median anti-asparaginase antibody concen- tration (OD at 1:3200 dilution) increased from 0.039 at induction to 0.506 at reinduction in patients who exhibited clinical hyper- sensitivity (P = 0.0002). By comparison, median antibody level increased from 0.011 to 0.032 OD at identical time points in patients who did not react to asparaginase (P = 0.02). Both post-induction and post-reinduction anti-asparaginase anti- body levels were higher in reacting than in nonreacting patients (P = 0.004 and P = 0.01, respectively). Antibody levels were inversely related to the time elapsed between the reaction and sampling (P = 0.011). Although anti-asparaginase antibody lev- els increased from the post-induction plasma sample to the post-reinduction sample in 28 of 35 patients regardless of whether they exhibited clinical hypersensitivity, patients with hypersensitivity reactions had higher antibody levels than did identically treated control patients at comparable time points in therapy. Therefore, antibody analysis may be of clinical value in predicting future hypersensitivity. Keywords: asparaginase; antibodies; allergic reaction; acute lym- phoblastic leukemia Introduction Asparaginase has been used to treat leukemia for over 25 years. It exploits a metabolic difference between normal cells and malignant cells. Normal cells can synthesize asparagine as needed; however, asparagine synthetase activity in some malignant lymphoblasts is low, and thus endogenous pro- duction of this amino acid is diminished. 1,2 Asparaginase hydrolyzes asparagine to aspartic acid and ammonia extra- cellularly, depriving neoplastic cells of their normal source of asparagine. Asparagine-dependent protein synthesis is halted with subsequent inhibition of nucleic acid synthesis, which decreases leukemic cell proliferation. Because asparaginase is not expressed in humans, but is isolated from bacterial sources, one of the primary limitations Correspondence: MV Relling, Pharmaceutical Department, St Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA; Fax: 901 525 6869 Received 25 March 1998; accepted 5 June 1998 to its use is the development of anti-asparaginase antibodies. Available pharmaceutical preparations of asparaginase vary in their pharmacokinetic and immunogenic properties. 3–5 Aspar- aginase is isolated from two primary sources: Escherichia coli and Erwinia chrysanthemi. The former is the source of the most commonly used preparation in the United States and is also widely used throughout the world. PEG asparaginase is a conjugate of the native E. coli asparaginase covalently linked to polyethylene glycol, which may decrease the prob- ability of developing anti-asparaginase antibodies 6,7 and prolongs the elimination half-life of the drug. 3 Hypersensitivity reactions to E. coli preparations given intra- venously or intramuscularly are dose-limiting in 4–45% and 0–25% of patients, respectively. These reactions usually necessitate discontinuation of E. coli asparaginase and sub- sequent substitution with Erwinia asparaginase. This latter preparation is equally allergenic; 4,8 however, it exhibits lim- ited, but clinically relevant, immunologic cross-reactivity. 9 It has been suggested that development of antibodies may hamper the antileukemic effect of asparaginase by shortening its half-life, preventing or delaying absorption after intramus- cular injection, or interfering with enzyme activity. 3,10,11 The reported frequency of anti-asparaginase antibodies is quite variable, and so high in some subgroups of patients (ie over 70% in adults receiving E. coli asparaginase) 10,12 that it is not clear whether antibody levels are more frequently detected or higher in patients who exhibit overt clinical allergy than in identically treated patients without clinical evidence of hyper- sensitivity. Therefore, the objective of this study was to retro- spectively compare anti-asparaginase antibody concentrations at identical time points relative to therapy in ALL patients who did and who did not develop hypersensitivity reactions to asparaginase. These are the first data on anti-asparaginase antibodies at fixed time points relative to asparaginase dosing in patients with newly diagnosed ALL. Patients and methods Patient eligibility and treatment schedules Thirty-five children with newly diagnosed ALL enrolled on the St Jude Children’s Research Hospital front-line protocol (Total XIIIH) and with frozen plasma available at both the end of induction and reinduction phases were evaluated in this study. The criteria for eligibility, diagnosis, risk-group classi- fication, and treatment have been reported elsewhere. 13 Twenty-two patients who exhibited clinically overt hypersen- sitivity reactions to asparaginase were compared to 13 chil- dren without reactions. All allergic reactions were graded using the standard NCI common toxicity criteria. As per proto- col, E. coli asparaginase (Elspar, Merck, West Point, PA, USA) was administered at a dose of 10 000 IU/m 2 intramuscularly three times weekly for a total of nine doses during both induc-