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.

Figure 1

Construction and preparation of GrB-based fusion constructs. A. Schematic diagram of GrB fusion constructs containing scFv 4D5 and GrB without or with fusogenic peptide 26. B. Purified GrB-based chimeric proteins were analyzed by SDS-PAGE under non-reducing conditions.

Figure 2

Characterization and comparison of GrB-based fusion proteins. A. K_d of the fusion constructs to Her2/neu ECD, Her2/neu-positive BT474 M1 cells, and Her2/neu-negative Me180 cells by ELISA. B. Enzymatic activity of GrB moiety of fusion proteins compared with native GrB. C. Internalization analysis of BT474 M1 cells and Me180 cells after 4 h of treatment with 25 nM functionalized GrB fusions. Cells were subjected to immunofluorescent staining with anti-GrB antibody (FITC-conjugated secondary), with PI nuclear counterstaining. D. Western blot analysis of intracellular behavior of 25 nM GrB fusion constructs in BT474 M1 cells.

Figure 3

Effects of GrB-based fusions on apoptotic pathways of BT474 M1 parental, HR and LR cells. A. Detection of apoptosis of GrB/4D5/26 by Annexin V/PI staining assay. Me180 cells served as a Her2/neu-negative control group. B. Western blot analysis of cleavage and activation of caspases-3 and -9 as well as PARP by GrB-based fusion constructs. C. Western blot investigation of apoptosis kinetics and specificity of GrB/4D5/26. Cells were treated with GrB/4D5/26 for up to 24 h with or without 100 μM zVAD-fmk for 24 h in parental or HR cells and for up to 48 h in LR cells.

Figure 4

Effects of GrB fusion constructs on mitochondrial pathway in BT474 M1 parental, HR and LR cells. A. Effects of GrB-based fusion proteins on the upstream components Bcl-2 and BID in mitochondrial pathway. B. Effects of the fusion constructs on cytochrome c release and Bax translocation.

Figure 5

Western blot analyses of the effects of GrB/4D5 and GrB/4D5/26 in BT474 M1 parental, HR and LR cells on Her2/neu and ER signaling pathways. Cells were treated with 100 nM functionalized GrB constructs for 24 or 48 h, and total cell lysates were quantified and further measured by western blot analysis of pHer2/neu, pAkt, pmTOR, pERK, ER, PR and PI-9 level.

Figure 6

Tumor apoptotic activity of GrB/4D5/26 in BT474 M1 tumor xenografts. A. Mice with BT474 M1 flank tumors were intravenously injected with saline or 44 mg/kg GrB/4D5/26 at the indicated times (arrows). Mean tumor volume was calculated as W×L×H as measured with digital calipers. B. Immunofluorescence staining of tumor samples after i.v. injection of saline and GrB/4D5/26. 24h after injection, animal was sacrificed and frozen tumor sections were prepared and detected by anti-GrB antibody (green) and anti-mouse CD31 antibody (red). Hoechst 33342 (blue) was used for DNA staining. C. Apoptosis detection in tumor tissue by TUNEL assay.