In vitro anti-tumour activities of a novel recombinant immunotoxin targeting differentially overexpressed Leucine-rich repeat-containing G-protein-coupled receptor 5 in cervical cancer.

Abstract

Cervical cancer is the fourth most common cancer among women worldwide and is associated with infection by high-risk human papillomaviruses (HPV). In 2022, an estimated 660,000 new cases and around 350,000 deaths were recorded. The burden of this disease remains disproportionately high in low- and middle-income countries, highlighting significant disparities in access to national HPV vaccination, screening, treatment, and social and economic determinants. Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a receptor that is differentially overexpressed in various cancers, including cervical cancer and is associated with tumour progression, metastasis, and poor prognosis. Targeting LGR5 with a novel recombinant immunotoxin offers a promising therapeutic approach.ObjectiveThe study aims to develop a novel recombinant anti-LGR5 immunotoxin candidate based on a truncated form of Pseudomonas exotoxin A (ETA).MethodsTo develop this LGR5-specific recombinant immunotoxin, a corresponding single chain antibody fragment (αLGR5(scFv)) fused to ETA, was expressed under osmotic stress in the presence of compatible solutes in Escherichia coli BL21 DE3 cells. Expression was monitored by Western blot analysis facilitated by an N-terminal 10x-His tag. Purification was done using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). The recombinant immunotoxin (rIT) was assessed for cell surface binding on cervical cancer cell lines using confocal microscopy and flow cytometry. The rIT was then used in an XTT cell viability assay to assess targeted cell killing.Results and discussionUpon confirmation of full-length protein by Western blot, purified protein was used to confirm binding on LGR5-positive cervical cancer cell lines via confocal microscopy and flow cytometry using anti-His PE antibody as a secondary antibody. Selective cell-killing of this novel recombinant immunotoxin was illustrated by the dose-dependent reduction in cell viability at IC50 values in nanomolar concentrations on antigen-positive but not antigen-negative cell lines.ConclusionsIn conclusion, the rIT described is a promising candidate to treat cervical cancer, which however, would finally need to be confirmed by preclinical in vivo studies.