title: 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.affiliation: Immunopharmacology and Targeted Therapy Laboratory, Department of Experimental Therapeutics, MD Anderson Cancer Center, Houston, TX 77054, USA.
authors.forename: Yu
authors.initials: Y
authors.lastname: Cao
authors.forename: Khalid A
authors.initials: KA
authors.lastname: Mohamedali
authors.forename: John W
authors.initials: JW
authors.lastname: Marks
authors.forename: Lawrence H
authors.initials: LH
authors.lastname: Cheung
authors.forename: Walter N
authors.initials: WN
authors.lastname: Hittelman
authors.forename: Michael G
authors.initials: MG
authors.lastname: Rosenblum
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D053804:Granzymes / Q000008:administration & dosage*; D006801:Humans; D051379:Mice; D058990:Molecular Targeted Therapy; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000008:administration & dosage* / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000276:immunology; D057057:Serine Proteases / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology; D015398:Signal Transduction / Q000187:drug effects; D057127:Single-Chain Antibodies / Q000008:administration & dosage* / Q000737:chemistry / Q000276:immunology
chemical_list: D053804:Granzymes; D019869:Phosphatidylinositol 3-Kinases; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D057057:Serine Proteases; D057127:Single-Chain Antibodies; C508053:ERBB2 protein, human