Surface charge-modification prevents sequestration and enhances tumor-cell specificity of a recombinant granzyme...

Area of Science:

• Immunology and Cancer Therapeutics
• Protein Engineering and Drug Delivery

Background:

• Granzyme B (GrB) is a serine protease crucial for immune defense by cytotoxic lymphocytes.
• Recombinant GrB fused to tumor-targeting ligands show potential in cancer therapy.
• Unspecific binding of GrB fusion proteins to non-target tissues via electrostatic interactions limits their therapeutic application.

Purpose of the Study:

• To investigate cell binding and specific cytotoxicity of surface-charge-modified GrB fused to a tumor-targeting ligand.
• To develop optimized GrB fusion proteins with improved selectivity and functionality for cancer therapy.

Main Methods:

• Mutated cationic heparin-binding motifs in GrB to reduce electrostatic interactions, creating GrBcs.
• Genetically fused GrBcs to epidermal growth factor receptor (EGFR) ligand TGFα for expression in Pichia pastoris.
• Compared enzymatic activity, cell binding, and cytotoxicity of GrBcs-TGFα (GrBcs-T) with wild-type GrB-TGFα (GrB-T).

Main Results:

• GrBcs-T exhibited similar enzymatic activity and targeted cytotoxicity against EGFR-overexpressing cells as GrB-T.
• Unspecific binding of GrBcs-T to EGFR-negative cells was significantly reduced, enhancing tumor-cell specificity.
• Modification of GrB alleviated unspecific extracellular effects, such as cell detachment and extracellular matrix degradation.

Conclusions:

• Surface-charge modification of GrB fusion proteins dramatically improves tumor-cell specificity by reducing non-specific binding.
• This strategy offers a general approach for developing optimized GrB fusion proteins with enhanced selectivity and functionality for therapeutic applications.
• The modified GrBcs-T fusion protein represents a promising candidate for targeted cancer therapy.