SBIR-STTR Award

The goal of this proposal is to develop a significantly safer variant of the anti-cancer biologic drug L- asparaginase (ASNase). ASNases are enzyme drugs that systematically deplete L-asparagine from the blood. First generation ASNases are approved by the FDA for the treatment of acute lymphoblastic leukemia (ALL), a cancer of white blood cells. This generation, including enzymes from E. coli (Elspar®) and Erwinia chrysanthemi (Erwinaze®), is severely hampered by toxicity which not only reduces the quality of life for patients, but whose effects can be so severe as to be fatal. Side effects stem from two causes ? one coming from the L-glutaminase coactivity of these enzyme drugs and the second from the immunogenicity due to their bacterial origin. To address the immunogenic side effects, second generation ASNases are PEGylated. They include the PEGylated E. coli enzyme (Oncaspar®), which is currently the standard of care in the USA, and the PEGylated Erwinia enzyme (Asparec®), which is currently in clinical trials. Whereas PEGylation reduces but does not eliminate immunogenicity problems, the side effects caused by their high L- glutaminase coactivity remains a major clinical challenge. Despite ASNases being key drugs in pediatric ALL treatment, these side effects are so pronounced in adults that their use is largely avoided. This factors into the much lower cure rate (~40%) of the adult ALL population. Additionally, ASNase-associated side effects prevent the use of this unique cancer drug in other hematological malignancies (e.g. acute myeloid leukemia) and in solid tumors (e.g. pancreatic cancer), despite strong evidence that ASNases would be effective in treating those cancers. Hence, there is a clear unmet need for an ASNase with reduced immunogenicity and that lacks L-glutaminase coactivity. Recently, we characterized a guinea pig ASNase (GpA) that possess the required low KM property for clinical efficacy and that has exhibited in vivo tumor cell-killing. Notably, we also discovered that GpA is devoid of the toxicity-causing L-glutaminase coactivity. With ~70% sequence identity to human ASNase, GpA should be less immunogenic compared to the bacterial enzymes that share only ~25% sequence identity with the human enzyme. We recently used a genetic screen to identify highly humanized (~86% identity) GpA variants and identified a stable and active C-terminal truncation of GpA comprising the catalytic domain. We call this variant 63N. We also employed structural information to identify surface residues on 63N that can be mutated into cysteines and are appropriately spaced to permit site-specific PEGylation that results in masking the drug from the immune system as well as proteases. This technology is superior to that used in second generation enzymes where lysine PEGylation results in a non-homogenous product, variations between batches, and reduced enzymatic (i.e. drug) activity. An additional advantage of PEGylation is increased in vivo stability that will allow lower dosing at more extended intervals. The goal of this Phase I STTR application is to develop a proof of concept for such a third generation ASNase drug.

Project Terms:
Acute; Acute Lymphocytic Leukemia; Acute Myelocytic Leukemia; Address; Adult; Adult Acute Lymphocytic Leukemia; Adverse effects; Amides; anti-cancer; Antineoplastic Agents; asparaginase; Asparagine; Autopsy; base; Biological Assay; bioluminescence imaging; Blood; Blood Circulation; Body Weight; Body Weight Changes; C-terminal; cancer therapy; Catalytic Domain; Cavia; cell killing; Clinical; clinical efficacy; Clinical Trials; Cysteine; Data; design; DNA Shuffling; Dose; Drug usage; efficacy study; Engineering; Enzymes; Erwinia; Escherichia coli; Exhibits; Eye; FDA approved; Fibrinogen; Formulation; Gel; Generations; Genetic Screening; Glutaminase; Glutamine; Goals; Half-Life; Health Care Costs; Hematologic Neoplasms; Homeostasis; Human; Immune system; immunogenic; immunogenicity; immunoreaction; improved; In Vitro; in vivo; in vivo bioluminescence imaging; Kinetics; Length; leukemia treatment; Leukemia, Lymphocytic, Acute, L1; Leukocytes; Libraries; Liver; Lysine; Malignant neoplasm of pancreas; Malignant Neoplasms; Masks; Mass Spectrum Analysis; Measurable; Measures; Monitor; Mus; Mutate; neoplastic cell; novel; older patient; Patients; Pectobacterium chrysanthemi; pediatric patients; Pegaspargase; Peptide Hydrolases; Pharmaceutical Preparations; Phase; Population; Posture; pre-clinical; prevent; Property; Quality of life; Safety; Serum; Signal Transduction; Site; Small Business Technology Transfer Research; Solid; Solid Neoplasm; Spleen; standard of care; stem; Structure; Surface; Technology; thioether; Time; TimeLine; Toxic effect; Toxicology; Translating; Treatment outcome; Variant; Weight; Work; Xenograft Model; Xenograft procedure;

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. Successful development of PEG-GpAhum will supply a much safer ASNase drug with immediate clinical implications for ALL and likely future expansion to additional indications of unmet need. Public Health Relevance Statement

Project narrative:
L-asparaginases (ASNases) are critical drugs for acute lymphoblastic leukemia (ALL) with great potential use in other cancers, but (i) the immunogenicity and (ii) the L-glutaminase (GLNase)-related toxic side effects of current bacterial ASNases often forces halting of treatment in pediatric ALL patients; mostly precludes their use in adult ALL patients and prevents their application in other cancers including acute myeloid leukemia (AML). We developed a long-acting version of a mammalian GLNase-free ASNase, allowing persistent asparagine depletion at lower doses and less frequent drug administrations, but most importantly its low immunogenicity and free of GLNase-related complications make it a safer ASNase eligible for expanded indications in cancer therapy. This proposal will 1. develop an appropriate manufacturing protocols for the lead biologic, 2. advance it through key IND-enabling studies and 3. identify a patient stratification strategy for its use in AML.