Thermoresponsive Polypeptide Fused L‐Asparaginase with Mitigated Immunogenicity and Enhanced Efficacy in Treating Hematologic Malignancies

Abstract

L‐Asparaginase (ASP) is well‐known for its excellent efficacy in treating hematological malignancies. Unfortunately, the intrinsic shortcomings of ASP, namely high immunogenicity, severe toxicity, short half‐life, and poor stability, restrict its clinical usage. Poly(ethylene glycol) conjugation (PEGylation) of ASP is an effective strategy to address these issues, but it is not ideal in clinical applications due to complex chemical synthesis procedures, reduced ASP activity after conjugation, and pre‐existing anti‐PEG antibodies in humans. Herein, the authors genetically engineered an elastin‐like polypeptide (ELP)‐fused ASP (ASP‐ELP), a core‐shell structured tetramer predicted by AlphaFold2, to overcome the limitations of ASP and PEG‐ASP. Notably, the unique thermosensitivity of ASP‐ELP enables the in situ formation of a sustained‐release depot post‐injection with zero‐order release kinetics over a long time. The in vitro and in vivo studies reveal that ASP‐ELP possesses increased activity retention, improved stability, extended half‐life, mitigated immunogenicity, reduced toxicity, and enhanced efficacy compared to ASP and PEG‐ASP. Indeed, ASP‐ELP treatment in leukemia or lymphoma mouse models of cell line‐derived xenograft (CDX) shows potent anti‐cancer effects with significantly prolonged survival. The findings also indicate that artificial intelligence (AI)‐assisted genetic engineering is instructive in designing protein‐polypeptide conjugates and may pave the way to develop next‐generation biologics to enhance cancer treatment.

Engineering and Expression Strategies for Optimization of L-Asparaginase Development and Production

“…This approach also resulted in L-ASNase with improved stability, half-life, reduced immunogenicity, in vitro and in vivo anti-cancer activity. In addition, this L-ASNase was more active than the native or PEG-conjugated enzyme [290].…”

“…Long after injection, the temperature sensitivity of the L-ASNase-ELP chimera resulted in the formation of an in situ depot with sustained release of the enzyme and zero-order release kinetics. In vitro and in vivo studies also showed that compared to L-ASNase and PEG-ASNase, L-ASNase-ELP had higher activity retention, superior stability, longer half-life, lower immunogenicity, lower toxicity and higher potency in vivo and in vitro [290].…”

Substrate Affinity Is Not Crucial for Therapeutic L-Asparaginases: Antileukemic Activity of Novel Bacterial Enzymes

“…The actions of any enzyme should also be tested in vivo to verify its stability, immunogenicity, and antineoplastic properties. Currently, reports from studies in animal models are available only for the commercial enzymes EcAII or ErAII [69][70][71], and there are no data for Nth-hydrolases (class 2) or Rhizobium etli-type (class 3) proteins.…”