Surface Charge-Modification Prevents Sequestration and Enhances Tumor-Cell Specificity of a Recombinant Granzyme B–TGFα Fusion Protein

Abstract

The serine protease granzyme B (GrB) plays an important role in the immune defense mediated by cytotoxic lymphocytes. Recombinant derivatives of this pro-apoptotic protein fused to tumor-targeting ligands hold promise for cancer therapy, but their applicability may be limited by promiscuous binding to nontarget tissues via electrostatic interactions. Here, we investigated cell binding and specific cytotoxicity of chimeric molecules consisting of wild-type or surface-charge-modified human GrB and the natural EGFR ligand TGFα for tumor targeting. We mutated two cationic heparin-binding motifs responsible for electrostatic interactions of GrB with cell surface structures, and genetically fused the resulting GrBcs derivative to TGFα for expression in the yeast Pichia pastoris. Purified GrBcs-TGFα (GrBcs-T) and a corresponding fusion protein employing wild-type GrB (GrB-T) displayed similar enzymatic activity and targeted cytotoxicity against EGFR-overexpressing breast carcinoma cells in the presence of an endosomolytic reagent. However, unspecific binding of the modified GrBcs-T variant to EGFR-negative cells was dramatically reduced, preventing the sequestration by nontarget cells in mixed cell cultures and increasing tumor-cell specificity. Likewise, modification of the GrB domain alleviated unspecific extracellular effects such as cell detachment indicative of extracellular matrix degradation. Our data demonstrate improved selectivity and functionality of surface-charge-modified GrBcs, suggesting this strategy as a general approach for the development of optimized GrB fusion proteins for therapeutic applications.

Selective Induction of Cancer Cell Death by Targeted Granzyme B

“…This was confirmed by the inability of chloroquine to release unfused GrB from intracellular vesicles upon uptake via natural GrB internalization mechanisms [49]. In the presence of chloroquine concentrations of 50 to 100 µM, GrB-5 and GrB-T were able to specifically kill target cells with IC 50 values measured after 14 hours of treatment in the picomolar to nanomolar range, whereas non-target cells were not affected at considerably higher concentrations [23,38] (Table 1). Cytotoxic activity was accompanied by clear signs of apoptosis such as chromatin condensation, membrane blebbing, formation of apoptotic bodies and activation of endogenous initiator and effector caspases.…”

“…Receptor-mediated endocytosis then results in uptake into endosomes. Efficient endosome release and translocation to the cytosol can be achieved by addition of an endosomolytic activity such as chloroquine [23,38].…”

“…Cytotoxic activity was accompanied by clear signs of apoptosis such as chromatin condensation, membrane blebbing, formation of apoptotic bodies and activation of endogenous initiator and effector caspases. 50 values for granzyme B fusion proteins GrB-scFv(FRP5) (GrB-5) and GrB-TGFα (GrB-T) were determined in cell viability assays upon treatment of cells with recombinant proteins for 14 hours in the presence of chloroquine as described [23,38]. IC 50 values for Pseudomonas exotoxin A fusion proteins scFv(FRP5)-ETA (5-ETA) and TGFα-ETA (T-ETA) were determined in cell viability assays upon treatment of cells with recombinant proteins for 40 hours as described [61,62,64].…”

“…Interestingly, also GrB-T displayed cell killing in the absence of chloroquine. However, to achieve a measurable effect, high protein concentrations (≥12.5 nM) and incubation for 48 h were required [38]. These findings suggest that the type of target receptor determines uptake and intracellular routing of chimeric GrB molecules, and kinetics and efficiency of their access to the cytosol.…”

“…Yeast-expressed GrBcs retained the enzymatic activity of wildtype GrB, but displayed markedly reduced intrinsic cell binding [38]. When fused to TGFα for tumor targeting, the resulting GrBcs-T molecule showed enzymatic activity in cell-free assays that was indistinguishable from that of unmodified GrB-T.…”