The goal of this proposal is to develop a significantly deimmunized variant of the anti-cancer biologic drug L- asparaginase (ASNase). ASNases are enzyme drugs that systematically deplete L-asparagine from the blood and are a mainstay of treatment for acute lymphoblastic leukemia (ALL), a cancer of the white blood cells and the most prevalent pediatric cancer. Despite being highly effective in the treatment of pediatric ALL, ASNases are associated with a multitude of toxic side effects, some so severe as to be fatal. Their high toxicity precludes routine use to treat adult ALL, contributes to the much lower cure rate of <40% for adult ALL, and prevents their use in other hematological malignancies (e.g. acute myeloid leukemia) and solid tumors (e.g. pancreatic cancer), despite strong preclinical evidence for efficacy. There are two main sources for the toxicity: L- glutaminase (GLNase) coactivity and immunogenicity. All current FDA-approved ASNases have intrinsic GLNase coactivity AND are immunogenic due to their bacterial origins from E. coli (Elspar) or Erwinia chrysanthemi (Erwinaze). A PEGylated version of Elspar called Oncaspar was developed and is now the 1st- line treatment for pediatric ALL in the US. However, there is now evidence that antibodies against the PEG moiety are being generated. Moreover, some patients even have pre-existing PEG antibodies prior to treatment due to the increasing prevalence of PEG in everyday products. Hypersensitivity can abolish the drug's efficacy and halt ASNase treatment, which correlates with poorer outcomes. With these toxic side effects that can prevent or terminate treatment of this life-saving drug, there is a clear unmet clinical need for an ASNase that lacks GLNase coactivity and has reduced immunogenicity. We are currently developing an ASNase variant that addresses both problems as it is devoid of GLNase coactivity and predicted to be less immunogenic since it is mammalian in origin, specifically guinea pig (GpA), sharing 70% sequence identity to the human homolog. This is in sharp contrast to Elspar and Erwinaze, sharing only ~25% identity to the human enzyme. To further reduce the risk of immunogenicity, we used a structure-guided approach to truncate and further humanize GpA, resulting in our lead called GpA369hum, which is >80% identical to the human homolog. However, since even fully human proteins can be immunogenic, in the proposed work we will identify those regions in GpA369hum that are efficiently presented by MHCII molecules (Aim 1). We will then incorporate in silico predictors and our recently obtained crystal structure of GpA369hum to design deimmunized variants - GpA369hum-DI (Aim 2). Finally, we will confirm the reduced immunogenicity of GpA369hum-DI using an ex vivo dendritic cell/T (DC-T) cell proliferation assay (Aim 3). Whereas clinical immunogenicity is extremely complex and is impacted by many intrinsic and extrinsic factors, the proposed work will allow us to mitigate this risk by assessing potential immunogenicity issues at this early preclinical stage to avoid possible failure in the clinic due to immunogenicity, where costs are much higher, and more importantly, patients' lives are at risk.
Public Health Relevance Statement: Project narrative: All current FDA-approved L-asparaginases (ASNases) are plagued by a wide variety of toxic side effects, foremost being immunogenicity due to these enzymes' bacterial origins as well as the PEG present in Oncaspar, which is the first-line treatment for acute lymphoblastic leukemia (ALL) in the US. To supply an alternative ASNase to relapsed/refractory ALL patients and a safer, tolerable option for adult ALL patients, we are developing an ASNase that, because of its mammalian origin, is expected to be less immunogenic. In this proposal we will further de-risk the enzyme's immunogenicity by revealing its most immunogenic regions and exchanging them with non-immunogenic amino acids that maintain the anti-ALL potency of the biologic.
Project Terms: 3-Dimensional; Acute Lymphocytic Leukemia; Acute Myelocytic Leukemia; Address; Adult Acute Lymphocytic Leukemia; Affinity; Amino Acids; anti-cancer; anti-cancer therapeutic; Antibodies; asparaginase; Asparagine; Binding; Biological; Biological Assay; Blood; Cavia; CD8-Positive T-Lymphocytes; Childhood; Childhood Acute Lymphocytic Leukemia; Clinic; Clinical; Clinical Trials; Complex; Computer Simulation; cost; Crystallization; cytokine release syndrome; Dendritic Cells; design; drug efficacy; Endotoxins; Engineering; Enzymes; Erwinia; Escherichia coli; Failure; FDA approved; Glutaminase; Goals; Hematologic Neoplasms; Homologous Gene; Human; Hypersensitivity; Immunization; immunogenic; immunogenicity; Immunologics; improved; In Vitro; indexing; Lead; lead candidate; Leukocytes; Life; lymph nodes; Malignant Childhood Neoplasm; Malignant neoplasm of pancreas; Malignant Neoplasms; novel; Outcome; outcome forecast; Patients; Pectobacterium chrysanthemi; Pegaspargase; Peptides; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Phase; Polyethylene Glycols; Polymers; pre-clinical; Prevalence; prevent; Property; Proteins; Refractory; Relapse; Reporting; response; Risk; Safety; Savings; side effect; Solid Neoplasm; Source; Structure; System; T-Cell Proliferation; T-Lymphocyte; T-Lymphocyte Epitopes; Target Populations; Therapeutic; TimeLine; Toxic effect; trait; Transgenic Mice; Treatment Efficacy; Variant; Work
