Engineered calcium-regulated affinity protein for efficient internalization and lysosomal toxin delivery

Area of Science:

• Biochemistry
• Molecular Biology
• Cancer Therapy

Background:

• Protein-drug conjugates (PDCs) offer targeted cancer therapy by delivering cytotoxic payloads to tumor cells.
• Effective PDC therapy relies on payload internalization and retention within target cells.
• Current strategies face challenges in controlling payload release and receptor recycling.

Purpose of the Study:

• To engineer a protein domain with calcium-regulated affinity for controlled target binding and lysosomal trafficking.
• To develop a novel drug delivery system for targeted cancer treatment.
• To assess the efficacy of the calcium-regulated binder in delivering toxins to cancer cells.

Main Methods:

• Engineering of an Epidermal Growth Factor Receptor (EGFR) binder with calcium-regulated affinity (CaRA).
• Affinity measurements and structural modeling to understand calcium-modulated binding.
• Live cell imaging to track internalization, lysosomal trafficking, and receptor fate.
• Cytotoxicity assays to determine the efficacy of the toxin delivery system.

Main Results:

• The CaRA_EGFR binder demonstrated calcium-dependent affinity, facilitating endosomal dissociation.
• Live cell imaging confirmed efficient internalization and lysosomal trafficking of the binder, with EGFR recycling.
• CaRA_EGFR effectively delivered toxins to lysosomes, achieving potent cytotoxicity (IC50 = 0.8 nM) in EGFR-expressing cancer cells.

Conclusions:

• Engineered calcium-regulated protein domains can achieve targeted payload delivery and lysosomal trafficking independent of receptor fate.
• This approach offers a novel strategy for enhancing the efficacy of protein-drug conjugates in cancer therapy.
• CaRA_EGFR represents a promising tool for developing next-generation targeted cancer therapeutics.