An Assay to Predict L-Asparaginase Neutralization and Enzyme Loaded Red Blood Cells to Maintain in Vivo Efficacy.

E. coli. L-Asparaginase repeated injections induce immunization. Anti-Asparaginase antibodies can provoke clinical hypersensitivity reactions and/or silently inactivate enzyme activity. Consequently, L-Asparaginase clearance is increased, implying a lack of L-asparagine deamination. Firstly, we developed an assay able to detect the presence of neutralizing factors including anti-Asparaginase antibodies. Next we investigated in a mouse model if loading L-Asparaginase into red blood cells (RBC) may be a way to protect its activity against neutralizing factors. A rabbit was immunized injecting 0.5 mg of L-Asparaginase (167 IU) mixed with Freund’s adjuvant every 3 weeks for 4-fold. The animal was euthanized and the final serum collected. Part of this final serum was immuno-adsorbed onto protein A for IgG antibodies purification. L-Asparaginase activity was measured by monitoring the kinetics of ammonia generation from the hydrolysis of asparagine. This assay was adapted to a biochemistry automated analyzer. When mixed with undiluted serum from the immunized rabbit, L-Asparaginase activity (0.8 to 100 IU/ml) was totally inhibited for all the concentration range within 15 min at 37°C. In the other hand, up to 1/128 serial dilutions of serum totally inhibited 2 IU/ml L-Asparaginase. As a control, undiluted pre-immunization serum from the same animal did not significantly affect L-Asparaginase activity. To identify the neutralizing factors, IgG from serum were purified by protein-A. As performed with serum, successive dilutions of IgG were mixed with 1.25 IU/ml L-Asparaginase. The IgG inhibited enzyme activity at the 1/128 dilution by 97%, thus proving their neutralizing effect on L-Asparaginase. To simulate the presence of neutralizing antibodies in the patient, we injected 7.5 μg of rabbit IgG into OF1 mice. Control mice were injected with phosphate buffered saline (PBS). Twenty minutes later mice either received 80 IU/kg of native E. coli L-Asparaginase or the same dose entrapped into OF1 mouse RBC. L-Asparaginase was loaded into murine RBC by reversible hypotonic dialysis, followed by a resealing step. The RBC thus acts as a bioreactor where plasmatic asparagine enters and is cleaved by the entrapped L-Asparaginase inside the erythrocyte. L-Asparaginase activity inside the erythrocyte was quantified at 68 IU per ml of erythrocytes, and the extracellular enzyme activity was less to 9% of total enzyme activity. Mice were sacrificed 6 hours after the administration of native or encapsulated L-Asparaginase. Free L-Asparaginase was totally inactivated in plasma of anti-Asparaginase IgG pre-treated mice: 0.002 ±0.002 IU/ml vs 0.417 ±0.103 IU/ml in PBS pre-treated mice. In addition, when L-Asparaginase is loaded inside RBC the activity is maintained irrespective of the presence of antibodies (0.798 ±0.126 IU/ml with IgG vs 0.879 ±0.146 IU/ml without). Moreover asparagine was not deaminated in IgG pre-treated mice who received free L-Asparaginase (27 ±1.6 μmol/L), while below 2 μmol/L in all the other groups. In conclusion, this newly developed assay can predict in vivo L-Asparaginase inefficacy. In addition, L-Asparaginase loaded into RBC is protected against neutralizing antibodies and its efficacy is maintained.