Neelakshi Mungra

Conference 2022 Live Talk


Talk title

Development of SNAP-tag based fusion proteins as novel photoimmunotheranostics in the detection and treatment of triple-negative breast cancer


Authors and Affiliations

Neelakshi Mungra1, Stefan Barth1,2

1. Medical Biotechnology and Immunotherapy Research Unit, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
2. South African Research Chair in Cancer Biotechnology, Department of Integrative Biomedical Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa




Targeted therapy for breast cancer represents the most promising state-of-the-art technology in the field of oncology. Such therapy directly addresses the lack of specificity encountered with conventional treatment regimes. This is achievable through receptor-specific monoclonal antibodies eventually conjugated to potent cytotoxic agents. However, despite showing great promise as anti-cancer compounds, these agents, also known as antibody-drug conjugates (ADCs) display several limitations: they feature stoichiometrically undefined chemical linkages, poor tumor penetration, unpredictable pharmacokinetic profiles and their generation results in a heterogeneous mixture of products. In this project, SNAP-tag based fusion proteins were generated, targeting chondroitin-sulphate proteoglycan 4 (CSPG4), CD44 and aspartate β-hydroxylase (ASPH) – biomarkers of aggressive and treatment-resistant cancers – for the treatment of triple-negative breast cancer (TNBC). By conjugating these fusion proteins to the potent near-infrared photosensitizer IRDye700DX (IR700), this study proposes to alleviate the challenges of early ADC development, while ensuing target-specific tumor cell death.


Following molecular cloning, protein expression was carried out using a transient secretory mammalian expression system and purified from the cell culture supernatant by ion metal affinity chromatography. Integration of the fluorescent dye Alexa Fluor 488 into the fusion proteins was carried out to investigate the self-labeling activity of the SNAP-tag moiety, as well as to provide qualitative and quantitative insights into the binding potential of the antibody fragments towards their cognate antigens. Subsequently, the IR700-based immunoconjugates were generated by conjugating scFv-SNAP with their respective benzylguanine (BG)-modified substrates, in a defined 1:1 stoichiometric reaction. The specific and dose-dependent biological activities of the resulting bifunctional therapeutic proteins were then assessed on TNBC cells.


In this study, all 3 fusion proteins were effectively expressed, although with moderate yields and purity, yet adequate for downstream in vitro characterization. After showcasing the self-labeling potential of the SNAP-tag component, surface binding of the fluorescently labeled products were demonstrated on TNBC cell lines through confocal microscopy and flow cytometry. The cell killing ability of the novel IR700 photoimmunoconjugates, was illustrated by the induction of a 50% reduction in cell viability (IC50 value) at nano to micromolar concentrations on target-positive cell lines. This observable selective cytotoxicity revealed that conjugation of BG derivatives to SNAP-tag, did not affect the binding potential of the antibody fragment, nor abrogated the cytocidal activity of the payload.


While various architectural modifications could further improve cytotoxic activities of future combination products, this research underscores the duality of SNAP-tag in the development of immunodiagnostics and therapeutics, that could potentially be instrumental in instilling a shift towards a personalized medicine stratagem. In conclusion, the combination of such immunoconjugates with a robust companion diagnostic panel provided by SNAP-tag, represents a first step towards the effective management of TNBC, with potential impact on the economic, social and clinical settings.