title: 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.
authors: Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Paul-Ehrlich-Str. 42-44, 60596 Frankfurt am Main, Germany.
authors: Robert A
authors: RA
authors: Jabulowsky
authors: Pranav
authors: P
authors: Oberoi
authors: Hayat
authors: H
authors: Bähr-Mahmud
authors: Benjamin
authors: B
authors: Dälken
authors: Winfried S
authors: WS
authors: Wels
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000378:metabolism; D053804:Granzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D006801:Humans; D008024:Ligands; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D013379:Substrate Specificity; D013499:Surface Properties; D016211:Transforming Growth Factor alpha / Q000235:genetics*
chemical_list: D000970:Antineoplastic Agents; D008024:Ligands; D011993:Recombinant Fusion Proteins; D016211:Transforming Growth Factor alpha; C512478:EGFR protein, human; D066246:ErbB Receptors; D053804:Granzymes