CSPG4 as a target for the specific killing of triple-negative breast cancer cells by a recombinant SNAP-tag-based antibody-auristatin F drug conjugate.

PURPOSE: Triple-negative breast cancer (TNBC) is phenotypic of breast tumors lacking expression of the estrogen receptor (ER), the progesterone receptor (PgR), and the human epidermal growth factor receptor 2 (HER2). The paucity of well-defined molecular targets in TNBC, coupled with the increasing burden of breast cancer-related mortality, emphasizes the need to develop targeted diagnostics and therapeutics. While antibody-drug conjugates (ADCs) have emerged as revolutionary tools in the selective delivery of drugs to malignant cells, their widespread clinical use has been hampered by traditional strategies which often give rise to heterogeneous mixtures of ADC products. METHODS: Utilizing SNAP-tag technology as a cutting-edge site-specific conjugation method, a chondroitin sulfate proteoglycan 4 (CSPG4)-targeting ADC was engineered, encompassing a single-chain antibody fragment (scFv) conjugated to auristatin F (AURIF) via a click chemistry strategy. RESULTS: After showcasing the self-labeling potential of the SNAP-tag component, surface binding and internalization of the fluorescently labeled product were demonstrated on CSPG4-positive TNBC cell lines through confocal microscopy and flow cytometry. The cell-killing ability of the novel AURIF-based recombinant ADC was illustrated by the induction of a 50% reduction in cell viability at nanomolar to micromolar concentrations on target cell lines. CONCLUSION: This research underscores the applicability of SNAP-tag in the unambiguous generation of homogeneous and pharmaceutically relevant immunoconjugates that could potentially be instrumental in the management of a daunting disease like TNBC.

Click Chemistry-Generated Auristatin F-Linker-Benzylguanine for a SNAP-Tag-Based Recombinant Antibody-Drug Conjugate Demonstrating Selective Cytotoxicity toward EGFR-Overexpressing Tumor Cells.

Antibody-drug conjugates (ADCs) are bifunctional molecules combining the targeting potential of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs. This simple yet intelligently designed system directly addresses the lack of specificity encountered with conventional anti-cancer treatment regimes. However, despite their initial success, the generation of clinically sustainable and effective ADCs has been plagued by poor tumor penetration, undefined chemical linkages, unpredictable pharmacokinetic profiles, and heterogeneous mixtures of products. To this end, we generated a SNAP-tag-based fusion protein targeting the epidermal growth factor receptor (EGFR)-a biomarker of aggressive and drug-resistant cancers. Here, we demonstrate the use of a novel click coupling strategy to engineer a benzylguanine (BG)-linker-auristatin F (AuriF) piece that can be covalently tethered to the EGFR-targeting SNAP-tag-based fusion protein in an irreversible 1:1 stoichiometric reaction to form a homogeneous product. Furthermore, using these recombinant ADCs to target EGFR-overexpressing tumor cells, we provide a proof-of-principle for generating biologically active antimitotic therapeutic proteins capable of inducing cell death in a dose-dependent manner, thus alleviating some of the challenges of early ADC development.