Project Summary: The goal of this proposal is to extensively broaden the clinical applications of the unique anti-cancer biologic drug L-asparaginase (ASNase) by developing a longer-acting next generation version of our novel variant, EBD- 300, currently in IND-enabling studies. ASNases are enzyme drugs that exploit a metabolic vulnerability of specific tumor cells via systemic depletion of L-asparagine (Asn) from the blood, and they are critical in the treatment of pediatric acute lymphoblastic leukemia (ALL). Cure rates greatly improved upon incorporation of ASNase into pediatric treatment protocols, however, adult ALL patients are largely precluded from ASNase treatment due to the toxicity from current FDA-approved versions. The result is significantly lower overall cure rates for adults with ALL. The side effects of current ASNases stem from two causes – one is their immunogenicity due to their bacterial origin and the second is their off-target L-glutaminase (GLNase) co- activity. To expand the use of this drug to adult ALL patients and other indications, there is a need for a superior ASNase with (1) reduced immunogenicity, (2) devoid of GLNase activity, and with (3) long in vivo persistence. To begin addressing this need, EbD developed EBD-300, the 1st mammalian ASNase (derived from guinea pig and further humanized to reduce immunological risk) and the 1st super specific ASNase (zero GLNase). In vivo studies demonstrated high efficacy of EBD-300 in ALL, with vastly superior safety compared to the current 1st-line (Oncaspar) and 2nd-line (Rylaze) FDA-approved ASNases. The in vivo persistence (t1/2) of EBD-300 is much longer than Rylaze, making it a promising candidate to become the preferred 2nd-line ASNase. However, EBD-300 is not competitive with 1st-line Oncaspar/Asparlas because of its need for a higher and more frequent dosing schedule. Notably, over 130 customer discovery interviews with KOLs in ALL revealed the strong need for a long-acting EBD-300 variant to be able to break into 1st line. The work proposed here will engineer and test next generation EBD-300 variants with long in vivo persistence, and such a long-acting variant will both impact ALL patients and provide the ultimate ASNase for additional indications (where, until now, current ASNases are simply too toxic for these adult populations). This includes blood cancers such as acute myeloid leukemia and solid tumors, like liver and colorectal cancers, all of which have been shown preclinically to be sensitive to ASNase. To achieve this goal, we will engineer EBD-300 with a novel half-life extension (HLE) technology in a 3-step process. Step 1 will generate HLE EBD-300 variants and confirm that the modifications maintain the required enzymatic properties for clinical efficacy. In Step 2, the top 3 variants passing stringent QC will have their in vivo persistence evaluated. The top candidate will proceed to Step 3 to confirm its anti-ALL power in mouse models. The feasibility ...