Identification of diphtheria toxin R domain mutants with enhanced inhibitory activity against HB-EGF
Identification of diphtheria toxin R domain mutants with enhanced inhibitory activity against HB-EGF
复制标题
鉴定对 HB-EGF 具有增强抑制活性的白喉毒素 R 结构域突变体
作者：
Suzuki K;Mizushima H;Abe H;Iwamoto R;Nakamura H;Mekada E
肝素结合表皮生长因子样生长因子（HB-EGF）是EGF受体的配体，参与肿瘤的生长和恶性进展。交叉反应物质197，CRM 197，白喉毒素（DT）的无毒突变体，特异性结合HB-EGF的EGF样结构域并抑制其促有丝分裂活性，因此CRM 197目前正在癌症治疗的临床试验中进行评估。为了开发比CRM 197更有效的DT突变体，我们筛选了DT的R结构域（HB-EGF结合位点）的各种突变蛋白。制备了多种与麦芽糖结合蛋白融合的R结构域突变体蛋白，并在体外对其抑制活性进行了评价。我们发现四个R结构域突变体对HB-EGF的抑制活性比野生型（WT）R结构域高得多。这些R结构域突变体比WT R结构域更有效地抑制HB-EGF依赖的细胞增殖。表面等离子体共振显示它们对HB-EGF的亲和力高于WT R结构域。携带新鉴定的突变的CRM 197（R460 H）显示增加的细胞增殖抑制活性和对HB-EGF的亲和力。这些结果表明，CRM 197（R460 H）或其他重组蛋白携带新鉴定的突变的R域是潜在的治疗靶向HB-EGF。
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 evaluatedin 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.