Multi-database transcriptomic screening to identify subtype-selective cell surface targets for development of SNAP-tag based immunotherapeutics

Abstract

Aims: Cervical cancer is the second most common cancer globally in women of reproductive age. Current immunotherapies offer only moderate improvement in overall survival for metastatic or refractory disease, highlighting the need for more effective, patient-directed immunotherapies. Methods: A unified pipelinereprocessed all The Cancer Genome Atlas (TCGA) and genotype tissue expression (GTEx) samples from raw reads using identical alignment and quantification parameters to screen a curated set of 259 high-confidence surface protein genes. This analysis provided the foundation for selecting differentially overexpressed cell surface receptors as potential antibody-drug conjugate (ADCs) targets . Recombinant single chain variable fragment (scFv)-O6-alkylguanine-DNA alkyltransferase (SNAP) fusion proteins were expressed, purified and characterised using immunoblotting. The dose-dependent and selective cytotoxicity of an auristatin F-conjugated (scFv)-SNAP fusion protein was verified in vitro. Results: Bioinformatic screening revealed 30 potential targets overexpressed in cervical carcinoma (Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (CESC)) vs. healthy tissues. Tissue-of-origin analysis (endocervix vs. ectocervix) further refined priorities. These results, together with supporting evidence from the literature, justified drug production.. The (scFv)-SNAP fusion proteins exhibited strong surface binding across cervical cancer cell lines and, when conjugated to auristatin F, showed dose-dependent cytotoxicity at nanomolar concentrations, confirming the differences observed in transcriptomic analyses of endo- vs. ectocervix profiling. Conclusion: Bioinformatics and in vitro validation synergize powerfully from initial screening through subtype refinement to iterative target confirmation, enabling a precision medicine approach for cervical cancer-specific immunotherapies. The (scFv)-SNAP-auristatin F (ADC) conjugates reproduced this refinement, showing highly promising potential.