Construction and characterization of novel, completely human serine protease therapeutics targeting Her2/neu.

Abstract

Immunotoxins containing bacterial or plant toxins have shown promise in cancer-targeted therapy, but their long-term clinical use may be hampered by vascular leak syndrome and immunogenicity of the toxin. We incorporated human granzyme B (GrB) as an effector and generated completely human chimeric fusion proteins containing the humanized anti-Her2/neu single-chain antibody 4D5 (designated GrB/4D5). Introduction of a pH-sensitive fusogenic peptide (designated GrB/4D5/26) resulted in comparatively greater specific cytotoxicity although both constructs showed similar affinity to Her2/neu-positive tumor cells. Compared with GrB/4D5, GrB/4D5/26 showed enhanced and long-lasting cellular uptake and improved delivery of GrB to the cytosol of target cells. Treatment with nanomolar concentrations of GrB/4D5/26 resulted in specific cytotoxicity, induction of apoptosis, and efficient downregulation of PI3K/Akt and Ras/ERK pathways. The endogenous presence of the GrB proteinase inhibitor 9 did not impact the response of cells to the fusion construct. Surprisingly, tumor cells resistant to lapatinib or Herceptin, and cells expressing MDR-1 resistant to chemotherapeutic agents showed no cross-resistance to the GrB-based fusion proteins. Administration (intravenous, tail vein) of GrB/4D5/26 to mice bearing BT474 M1 breast tumors resulted in significant tumor suppression. In addition, tumor tissue excised from GrB/4D5/26-treated mice showed excellent delivery of GrB to tumors and a dramatic induction of apoptosis compared with saline treatment. This study clearly showed that the completely human, functionalized GrB construct can effectively target Her2/neu-expressing cells and displays impressive in vitro and in vivo activity. This construct should be evaluated further for clinical use.

• Authors: Yu Cao, Khalid A Mohamedali, John W Marks, Lawrence H Cheung, Walter N Hittelman, Michael G Rosenblum
• Journal: Molecular cancer therapeutics (Mol Cancer Ther) Vol. 12 Issue 6 Pg. 979-91 (Jun 2013) ISSN: 1538-8514 [Electronic] United States
• PMID: 23493312 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: ©2013 AACR

Chemical References

• Recombinant Fusion Proteins
• Single-Chain Antibodies
• Phosphatidylinositol 3-Kinases
• ERBB2 protein, human
• Receptor, ErbB-2
• Serine Proteases
• Granzymes

Topics

• Animals
• Apoptosis (drug effects)
• Breast Neoplasms (drug therapy, genetics, pathology)
• Cell Survival (drug effects)
• Female
• Granzymes (administration & dosage)
• Humans
• Mice
• Molecular Targeted Therapy
• Phosphatidylinositol 3-Kinases (metabolism)
• Receptor, ErbB-2 (administration & dosage, genetics, immunology)
• Recombinant Fusion Proteins (administration & dosage, immunology)
• Serine Proteases (administration & dosage, genetics, immunology)
• Signal Transduction (drug effects)
• Single-Chain Antibodies (administration & dosage, chemistry, immunology)