Extending the in vivo half-life of a novel engineered mammalian asparaginase via its binding to human serum albumin

PROJECT SUMMARY: Enzyme by Design (EbD) is developing a safer L-asparaginase (ASNase) to maximize the potential clinical applications of this unique drug. ASNases are enzyme drugs that systematically deplete L- 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 other hematological malignancies (e.g. acute myeloid leukemia) and in solid tumors (e.g. 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 L-glutaminase co-activity. To expand the use of this drug to adult ALL patients and to other indications, there is a need for an ASNase with (i) reduced immunogenicity, (ii) lack of L-glutaminase co- activity, combined with (iii) long in vivo persistence. To mitigate the immunogenicity, EbD is developing the first mammalian ASNase. In sharp contrast to the bacterial EcA/ErA, which are very distant from the human homolog, EbD is developing the guinea pig ASNase (GpA), which is much closer in identity to the human ASNase. Moreover, by following a humanization process, EbD increased GpA’s % sequence identity to the human homolog. Together, this humanized enzyme, referred to as GpAhum, is predicted to be much less immunogenic compared to the bacterial ASNases. An added advantage of this enzyme is that it is intrinsically GLNase-free, thereby reducing off-target drug toxicity. We have extensive data that shows strong in vivo anti-ALL potency of GpAhum with little toxicity. However, the half-life (t1/2) of GpAhum is not optimal for clinical use. Whereas PEGylation offers one solution for increasing the t1/2, as seen in Oncaspar, recent clinical data reporting anti-PEG antibodies in patients previously treated with Oncaspar made it clear that PEGylation is not a viable path to the clinic for any new ASNase. Therefore, to achieve an increased t1/2, we pivoted to human serum albumin (HSA)-binding technology via fusing our ASNase to an immunologically neutral human serum albumin binding domain (ABD). The product, ABD-GpAhum, more than doubled the parental GpAhum’s blood circulation time with excellent HSA binding affinity. In vivo efficacy in mouse xenograft models confirms its superior potency: 1 dose of ABD-GpAhum is equivalent to 3 doses of GpAhum, allowing similar therapeutic efficacy with reduced dosing frequency and total amount of drug injected. This predicts less accumulated toxicity in patients, lowered drug-related therapy cost and ease of use for hospital-related staffs. This proposal will further evaluate the anti-ALL efficacy of ABD-GpAhum and supply proof-of-concept toxicity data to confirm that ABD-GpAhum is indeed a viable, safe and competitive therapeutic candidate, thus providing the rationale for advancing it through IND-enabling studies.PROJECT NARRATIVE: Bacterial asparaginases are critical drugs used to treat acute lymphoblastic leukemia patients, but they are plagued by side effects caused by (i) the immunogenicity of these bacterial enzyme drugs and (ii) their glutaminase coactivity, which often forces halting of treatment in pediatric patients and mostly precludes their use in adult patients, whose cure rate is therefore significantly lower. We developed ABD-GpAhum, a novel L- asparaginase with three virtues: MAMMALIAN hence less immunogenic, GLUTAMINASE-FREE thus less off- target side effects, and LONG-ACTING via binding to serum albumin which allows similar therapeutic efficacy with less dosing frequency and total amount of drug injected. This proposal will confirm that ABD-GpAhum is indeed a viable and competitive therapeutic candidate by showing its in vivo efficacy and superior toxicity compared to current standard of care.