L-Asparaginase Loaded into Erythrocytes (GRASPA): Principle and Interests in Acute Lymphoblastic Leukemia.
Authors: Godfrin Yann , Thomas Xavier , Bertrand Yves , Duget Corinne 
Published: October 2019

Abstract
L-asparaginase is an essential drug in the treatment of acute lymphoblastic leukemia (ALL) in cleaving plasmatic asparagine, an aminoacid involved in lymphoblastic cell proliferation. However, the short half-life (24h) of the native L-asparaginase requests massive and repeated injections (5000 to 10000 IU/m2) leading to potential adverse events. Reactions of hypersensitivity and/or anti-asparaginase neutralizing antibodies are often observed. The encapsulation of L-asparaginase into erythrocytes is an interestic way to improve the therapeutic index and decreasing side effects. The technique of encapsulation is carried out by reversible osmotic lysis: qualified red blood cells (RBCs) are subjected to hypo-osmotic conditions by dialysis followed by one return to the isotonicity. During dialysis, the RBCs inflate and pores are formed on the membrane. L-asparaginase penetrates by these pores into the erythrocytes. Back to isotonicity, RBCs recover their original shape and size, the pores are then definitively closed, and L-asparaginase is definitively encapsulated. An automated device allows achievement of an accurate reproducibility of the technique of entrapment which is assessed by the quality of the red cells and quantity of drug entrapped. In addition, the technology employed allows a 2-hour preparation of the product which can be shipped to physician the same day of the prescription. An intra corpuscular activity of L-asparaginase of 112±11,3 IU/ml of pure RBCs is obtained with an output of L-asparaginase encapsulation of 29,8± 2,1%. 250 ml-packed RBCs (hematocrit 50%), contains about 14000 IU. The encapsulation of L-asparaginase in the erythrocytes is a technology which strongly increases the half-life of the enzyme (29,2±9,7 days versus 24 hours) and reduces the immunogenicity of the enzyme. Here the red cell works as a bioreactor: L-asparaginase is maintained active inside the erythrocyte as long as this one is circulating. The plasmatic asparagine, a small size protein, penetrates passively and continuously towards the intra-erythrocyte compartment, where it is cleaved by the enzyme. In addition, the membrane of red blood cells avoids bindings between anti-asparaginase antibodies and the enzyme leading to decreased reactions of hypersensibility. A randomized multicenter phase II clinical trial including ALL patients in first relapse (GRASPALL) is currently ongoing in France and Belgium. The main objective is to establish a dose/effect relationship between 3 doses of entrapped L-asparaginase (50, 100, 150 IU/kg) or the free form of the enzyme and the duration of the plasmatic asparagine depletion (<2 μmol/L). All patients receive a 6-drugs 4-weeks induction chemotherapy combining L-asparaginase (entrapped or non-) with dexamethasone, prednisone, vincristine, methotrexate and cytarabine as recommended by French and Belgium pediatric groups (COOPRALL). Twenty-one patients on the twenty-four patients planned, aged from 1 to 55 years (2 strates children and adults (more than 18yr) are already enrolled. GRASPA is given as a single injection once a month. The mean dosage (100 IU/kg) should be able to reach approximately 30 days of continuous plasmatic asparagine depletion. Thanks to the improvement of the pharmacokinetic and pharmacodynamic and especially to the side effects reduction, this new form of L-asparaginase is very promising especially in patients with a poor tolerance to the free form such as intolerant, hypersensitive or elderly patients.