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Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a ligand of EGF receptor, is involved in the growth and malignant progression of cancers. Cross-reacting material 197, CRM197, a non-toxic mutant of diphtheria toxin (DT), specifically binds to the EGF-like domain of HB-EGF and inhibits its mitogenic activity, thus CRM197 is currently under evaluation in clinical trials for cancer therapy. To develop more potent DT mutants than CRM197, we screened various mutant proteins of R domain of DT, the binding site for HB-EGF. A variety of R-domain mutant proteins fused with maltose-binding protein were produced and their inhibitory activity was evaluated in vitro. We found four R domain mutants that showed much higher inhibitory activity against HB-EGF than wild-type (WT) R domain. These R domain mutants suppressed HB-EGF-dependent cell proliferation more effectively than WT R domain. Surface plasmon resonance revealed their higher affinity to HB-EGF than WT R domain. CRM197(R460H) carrying the newly identified mutation showed increased cell proliferation inhibitory activity and affinity to HB-EGF. These results suggest that CRM197(R460H) or other recombinant proteins carrying newly identified mutation(s) in the R domain are potential therapeutics targeting HB-EGF., (© The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

HB-EGF is a member of the EGF family of growth factors that bind and activate the EGF receptor. HB-EGF is synthesized as a membrane-anchored protein (proHB-EGF), and then proteolytically cleaved, resulting in the mitogenically active soluble form. ProHB-EGF functions as the receptor for the diphtheria toxin (DT). HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Monoclonal antibodies (mAbs) specific for HB-EGF could be an important tool in HB-EGF research. However, few such mAbs have been established to date. In this study, we newly generated seven clones of hybridoma-derived mAbs by immunizing HB-EGF null mice with recombinant human HB-EGF protein. All mAbs specifically bound to human HB-EGF but not to mouse HB-EGF. Epitope mapping analysis showed that most of the mAbs recognized the EGF-like domain. Although none of the newly isolated mAbs directly inhibited the mitogenic activity of HB-EGF for EGFR-expressing cells, some strongly inhibited DT-binding. Interestingly, some of the mAbs efficiently inhibited ectodomain shedding of proHB-EGF, and consequently prevented the cell growth of the EGFR-expressing cells in a co-culture system with proHB-EGF-expressing cells. Hence, these new anti-HB-EGF mAbs may advance clinical as well as basic research on HB-EGF.

Heparin-binding EGF-like growth factor (HB-EGF) plays a pivotal role in tumor growth and clinical outcomes in patients with ovarian cancer, leading to the validation of HB-EGF as a target for ovarian cancer therapy. In this study, we investigated the anti-tumor effects of paclitaxel, as an anti-cancer agent, and CRM197, as a specific inhibitor off HB-EGF, in ovarian cancer. Paclitaxel induced transient ERK activation and sustained activation of JNK and p38 MAPK through the ectodomain shedding of HB-EGF in SKOV3 cells. In addition, the overexpression of HB-EGF in paclitaxel-treated SKOV3 cells resulted in modulation of paclitaxel-evoked MAPK signaling, including marked activation of ERK and Akt, and minimized activation of JNK and p38 MAPK, indicating that HB-EGF is involved in drug sensitivity through the balance of anti-apoptotic and pro-apoptotic signals induced by paclitaxel. The combination of paclitaxel with CRM197 had an inhibitory effect on cell proliferation and enhanced apoptosis via the inhibition of ERK and Akt activation and the stimulation of p38 and JNK activation. More prominently, the administration of paclitaxel with CRM197 resulted in synergistic anti-tumor effects in SKOV3 cells and in SKOV3 cells overexpressing HB-EGF in xenografted mice. Accordingly, inhibitory agents against HB-EGF, such as CRM197, represent possible chemotherapeutic and chemosensitizing agents for ovarian cancer.

CRM197, a mutated diphtheria toxin (DT), has long been recognized to be a non-toxic protein. Based on its non-toxic feature, this protein has been utilized for various purposes, including as an inhibitor of heparin-binding EGF-like growth factor (HB-EGF) and as an immunological adjuvant for vaccination. Here we show evidence that CRM197 has a weak toxicity. This toxicity was observed in cells over-expressing the DT receptor/proHB-EGF, but not in parental cells, indicating that the toxicity was mediated through DT receptor. CRM197 did not show any toxicity toward DT-resistant cells, which have a mutation in elongation factor 2, and a cell-free assay revealed the existence of weak EF-2-ADP ribosylation activity in fragment A of CRM197. Thus, the present study indicates a requirement for specific care in the use of CRM197 at a high dosage, although the toxicity of CRM197 is about 106 times less than that of wild-type DT. We found that a monoclonal antibody to DT inhibited CRM197 toxicity, but did not affect the inhibitory activity of CRM197 toward HB-EGF-induced mitogenic activity. CRM197 strongly inhibits tumour growth in nude mice. The anti-DT monoclonal antibody administered with CRM197 reduced the anti- tumourigenic effect of CRM197, indicating that the toxicity of CRM197 potentiates its anti- tumourigenic effect. [ABSTRACT FROM PUBLISHER]

Specific cell ablation is a useful method for analyzing the in vivo function of cells. We have developed a simple and sensitive method for conditional cell ablation in transgenic mice, called "toxin receptor -- mediated cell knockout." We expressed the diphtheria toxin (DT) receptor in transgenic mice using a hepatocyte-specific promoter and found that injection of DT caused fulminant hepatitis. Three independently established transgenic lines demonstrated a good correlation between the sensitivity of hepatocytes to DT and the expression level of the DT receptors. Moreover, the degree of hepatocyte damage was easily controlled over a wide range of doses of injected DT without any obvious abnormalities in other cells or tissues. This system is useful for generating mouse models of disease and for studying the recovery or regeneration of tissues from cell damage or loss. As DT is a potent inhibitor of protein synthesis in both growing and non-growing cells, the method is applicable to a wide range of cells and tissues in mice or in other DT-insensitive animals. [ABSTRACT FROM AUTHOR]