Project Summary: Enzyme by Design (EbD) is developing a safer asparaginase (ASNase) to maximize the potential clinical applications of this unique drug. ASNases are enzyme drugs that systemically deplete asparagine from the blood. In the US, the 1st-line ASNase is Oncaspar, a PEGylated version of the E. coli ASNase (EcA). Patients intolerant of Oncaspar switch to the naked Erwinia ASNase (ErA, Erwinaze). Despite being key drugs in pediatric acute lymphoblastic leukemia (ALL), the side effects of current FDA-approved ASNases are so pronounced in adults that their use is largely avoided. These side effects also prevent the use of ASNases in acute myeloid leukemia (AML) and in pancreatic, ovarian or triple-negative breast cancers, despite strong evidence that ASNases would be effective in treating those cancers. Side effects of EcA/ErA stem from i) their immunogenicity, due to their bacterial origin and ii) their glutaminase (GLNase) co-activity. To expand the use of this drug to adult ALL patients and to other indications, there is a strong need for an ASNase with (i) reduced immunogenicity, (ii) lack of GLNase co-activity, combined with (iii) long in vivo persistence. To mitigate the immunogenicity, EbD is developing the first mammalian ASNase from the guinea pig (GpA) that is much closer in identity to the human ASNase compared to the bacterial EcA/ErA. To reduce the immunological risk even more, we employed a structure-guided strategy to humanize GpA, generating GpAhum. An added advantage of GpAhum is that it is intrinsically GLNase-free, thereby reducing off-target drug toxicity. EbD successfully delivered the following STTR Phase 1 milestones: 1) 11 site-specific PEGylated versions of GpAhum were developed to increase its half-life and the best variant, PEG-GpAhum with ~3-fold increase in t1/2 was identified. 2) in T-ALL mouse xenograft models, PEG-GpAhum q.wk was more potent than GpAhum t.i.w, and 3) most importantly, benchmarking our molecule against the market leader, a strikingly undeniable superior toxicity profile of PEG-GpAhum vs. Oncaspar in both single and repeat dosing studies was observed, greatly de-risking PEG-GpAhum in the development pipeline. Our completed I-Corps programs with >130 interviews with leaders and stakeholders in the ALL field validated the expected adoptability of PEG-GpAhum, becoming best-in-class. This molecule would deliver similar therapeutic efficacy with reduced dosing frequency and total amount of drug injected, predicting less accumulated toxicity in patients, lowered drug-related therapy cost and increased ease of use. This SBIR Phase 2 proposal will 1) develop appropriate GMP scale-up manufacturing protocols for PEG-GpAhum, 2) advance it through key IND-enabling studies and 3) confirm a viable biomarker for patient stratification, using AML as the first example of expanded indication. Future SBIR Phase 2b will support the completion of the IND application package and GMP material for clinical use. Succes...