作者: Guo, Nancy ; Donnelly, Paul S ; Scott, Andrew M ; Gan, Hui ; Cao, Zhipeng ; Morgan, Katherine A ; Wichmann, Christian W ; Reilly, Edward ; Burvenich, Ingrid J G ; O'Keefe, Graeme J ; Osellame, Laura D

The epidermal growth factor receptor (EGFR) protein is highly expressed in a range of malignancies. Although therapeutic interventions directed toward EGFR have yielded therapeutic responses in cancer patients, side effects are common because of normal-tissue expression of wild-type EGFR. We developed a novel tumor-specific anti-EGFR chimeric antibody ch806 labeled with ²²⁵Ac and evaluated its in vitro properties and therapeutic efficacy in murine models of glioblastoma and colorectal cancer. Methods: ²²⁵Ac-ch806 was prepared using different chelators, yielding [²²⁵Ac]Ac-macropa-tzPEG₃Sq-ch806 and [²²⁵Ac]Ac-DOTA-dhPzPEG₄-ch806. Radiochemical yield, purity, apparent specific activity, and serum stability of ²²⁵Ac-ch806 were quantified. In vitro cell killing effect was examined. The biodistribution and therapeutic efficacy of ²²⁵Ac-ch806 were investigated in mice with U87MG.de2-7 and DiFi tumors. Pharmacodynamic analysis of tumors after therapy was performed, including DNA double-strand break immunofluorescence of γH2AX, as well as immunohistochemistry for proliferation, cell cycle arrest, and apoptosis. Results: [²²⁵Ac]Ac-macropa-tzPEG₃Sq-ch806 surpassed [²²⁵Ac]Ac-DOTA-dhPzPEG₄-ch806 in radiochemical yield, purity, apparent specific activity, and serum stability. [²²⁵Ac]Ac-macropa-tzPEG₃Sq-ch806 was therefore used for both in vitro and in vivo studies. It displayed a significant, specific, and dose-dependent in vitro cell-killing effect in U87MG.de2-7 cells. ²²⁵Ac-ch806 also displayed high tumor uptake and minimal uptake in normal tissues. ²²⁵Ac-ch806 significantly inhibited tumor growth and prolonged survival in both U87MG.de2-7 and DiFi models. Enhanced γH2AX staining was observed in ²²⁵Ac-ch806-treated tumors compared with controls. Reduced Ki-67 expression was evident in all ²²⁵Ac-ch806-treated tumors. Increased expression of p21 and cleaved caspase 3 was shown in U87MG.de2-7 and DiFi tumors treated with ²²⁵Ac-ch806. Conclusion: In glioblastoma and colorectal tumor models, ²²⁵Ac-ch806 significantly inhibited tumor growth via induction of double-strand breaks, thereby constraining cancer cell proliferation while inducing cell cycle arrest and apoptosis. These findings underscore the potential clinical applicability of ²²⁵Ac-ch806 as a potential therapy for EGFR-expressing solid tumors.

2012-01-01·The FEBS journal3区 · 生物学

Construction and humanization of a functional bispecific EGFR × CD16 diabody using a refolding system

3区 · 生物学

Article

作者: Makoto Nakayama ; Izumi Kumagai ; Hiroki Hayashi ; Yu Katayose ; Michiaki Unno ; Toshio Kudo ; Takeshi Nakanishi ; Tsuguo Kubota ; Ryutaro Asano ; Mitsuo Umetsu ; Hiroko Kawaguchi

We previously reported the construction and activity of a humanized, bispecific diabody (hEx3) that recruited T cells towards an epidermal growth factor receptor (EGFR) positive tumor. Herein, we describe the construction of a second functional, fully humanized, anti‐EGFR bispecific diabody that recruits another subset of lymphocyte effectors, the natural killer cells, to EGFR‐expressing tumor cells. After we confirmed that an anti‐EGFR × anti‐CD16 bispecific diabody (Ex16) consisting of a previously humanized anti‐EGFR variable fragment (Fv) and a mouse anti‐CD16 Fv had growth inhibitory activity, we designed a humanized anti‐CD16 Fv to construct the fully humanized Ex16 (hEx16). However, the humanized form had lower activity for inhibition of cancer growth. To restore its growth inhibitory activity, we introduced mutations into the Vernier zone, which is located near the complementarity‐determining regions and is involved in their binding activity. We efficiently prepared 15 different hEx16 mutants by expressing each chimeric single‐chain component for hEx16 separately. We then used our in vitro refolding system to select the most functional mutant, which had a growth inhibitory effect comparable with that of the commercially available chimeric anti‐EGFR antibody, cetuximab. Our refolding system could aid in the efficient optimization of other proteins with heterodimeric structure.

2001-09-01·Cancer research

Activated extracellular signal-regulated kinases: association with epidermal growth factor receptor/transforming growth factor alpha expression in head and neck squamous carcinoma and inhibition by anti-epidermal growth factor receptor treatments.

Article

作者: Anido, J ; Rojo, F ; Albanell, J ; Baselga, J ; Sauleda, S ; Roselló, J ; Nicholson, R I ; Raspall, G ; Codony-Servat, J ; Del Campo, J M ; Giralt, J ; Mendelsohn, J

The expression of the activated mitogen-activated kinases/extracellular signal-regulated kinases (ERKs) ERK1 and ERK2 was characterized in 101 humanhead and neck squamous carcinoma specimens. Activated ERK1/2were detected at different levels in the majority of these tumors, as assayed by immunostaining with an antibody specific for the dually phosphorylated and activated ERK1 and ERK2. ERK1/2 activation levels were higher in tumors with advanced regional lymph node metastasis (P = 0.048) and in relapsed tumors (P = 0.021). The expression of epidermal growth factor (EGF) receptor (P = 0.037), transforming growth factor alpha (TGF-alpha; P < 0.001), and HER2 (P = 0.066; positive trend) correlated with activation of ERK1/2. In a multivariate analysis, both TGF-alpha (P < 0.0001) and HER2 (P = 0.045) were independently correlated with ERK1/2 activation. In turn, activation of ERK1/2 was associated with a higher Ki-67 proliferative index (P = 0.002). In EGF receptor-dependent model cells (A431 and DiFi), a specific EGF receptor tyrosine kinase inhibitor ("Iressa"; ZD1839) and a chimeric anti-EGF receptor antibody ("Cetuximab"; C225) inhibited ERK 1/2 activation at concentrations that inhibited autocrine cell proliferation. In patients on treatment with C225, the activation of ERK1/2 in skin, an EGF receptor-dependent tissue, was lower compared with control skin. Parallel changes were seen in keratinocyte Ki67 proliferation indexes in skin from C225-treated patients. Taken together, these studies provide support for a role of activation of ERK1/2 in head and neck squamous carcinoma and a correlation with EGF receptor/TGF-alpha expression. The inhibition of ERK1/2 activation in vitro and in vivo by compounds targeting the EGF receptor points to the interest of ERK1/2 as potential surrogate markers of EGF-receptor signaling in clinical therapeutic studies.

100 项与重组抗EGFR人鼠嵌合单抗(上海张江生物技术有限公司) 相关的药物交易

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