Division of Radiobiology & Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Germany.
Strahlenther Onkol. 2012 Sep;188(9):823-32. doi: 10.1007/s00066-012-0121-4. Epub 2012 Aug 10.
Anti-EGFR antibody cetuximab (C225) is used in combination with radiotherapy of head and neck squamous cell carcinoma (HNSCC) patients. We investigated whether conjugation of cetuximab with trans-cyclohexyl-diethylene-triamine-pentaacetic acid (CHX-A″-DTPA) and radiolabeling with (90)Yttrium affect the molecular and cellular function of cetuximab and improve its combined effect with external-beam irradiation (EBI).
The following cell lines were used: HNSCC UT5, SAS, FaDu, as well as A43, Chinese hamster ovary cells (CHO), and human skin fibroblast HSF7. Binding affinity and kinetics, specificity, retention, and the combination of (90)Y-cetuximab with EBI were evaluated.
Control cetuximab and CHX-A″-DTPA-cetuximab blocked the proliferation activity of UT5 cells. In combination with EBI, CHX-A″-DTPA-cetuximab increased the radiosensitivity of UT5 to a similar degree as control cetuximab did. In contrast, in SAS and HSF7 cells neither proliferation nor radiosensitivity was affected by either of the antibodies. Binding [(90)Y]Y-CHX-A″-DTPA-cetuximab ((90)Y-cetuximab) to EGFR in HNSCC cells occurred time dependently with a maximum binding at 24 h. Retention of (90)Y-cetuximab was similar in both HNSCC cell lines; 24 h after treatment, approximately 90% of bound activity remained in the cell layer. Competition assays, using cell membranes in the absence of an internalized fraction of cetuximab, showed that the cetuximab affinity is not lost as a result of conjugation with CHX-A″-DTPA. Cetuximab and CHX-A″-DTPA-cetuximab blocked EGF-induced Y1068 phosphorylation of EGFR. The lack of an effect of cetuximab on EGF-induced Akt and ERK1/2 phosphorylation and the inhibition of irradiation (IR)-induced Akt and ERK1/2 phosphorylation by cetuximab were not affected by DTPA conjugation. (90)Y-cetuximab in combination with EBI resulted in a pronounced inhibition of colony formation of HNSCC cells.
Conjugation of CHX-A″-DTPA to cetuximab does not alter the cellular and biological function of cetuximab. (90)Y-labeling of cetuximab in combination with EBI may improve radiotherapy outcome.
抗 EGFR 抗体西妥昔单抗（C225）与头颈鳞癌（HNSCC）患者的放射治疗联合使用。我们研究了将西妥昔单抗与反式环己基二乙烯三胺五乙酸（CHX-A″-DTPA）缀合以及用（90）Yttrium 放射性标记是否会影响西妥昔单抗的分子和细胞功能，并改善其与外照射（EBI）的联合作用。
使用以下细胞系：HNSCC UT5、SAS、FaDu 以及 A43、中国仓鼠卵巢细胞（CHO）和人皮肤成纤维细胞 HSF7。评估了结合亲和力和动力学、特异性、保留以及（90）Y-西妥昔单抗与 EBI 的结合。
对照西妥昔单抗和 CHX-A″-DTPA-西妥昔单抗均阻断了 UT5 细胞的增殖活性。与 EBI 联合使用时，CHX-A″-DTPA-西妥昔单抗增加了 UT5 对放射的敏感性，与对照西妥昔单抗相似。相比之下，在 SAS 和 HSF7 细胞中，两种抗体均未影响增殖或放射敏感性。EGFR 在 HNSCC 细胞中与[（90）Y]Y-CHX-A″-DTPA-西妥昔单抗（（90）Y-西妥昔单抗）的结合随时间呈依赖性发生，最大结合发生在 24 小时。两种 HNSCC 细胞系中（90）Y-西妥昔单抗的保留率相似；治疗后 24 小时，约 90%的结合活性仍保留在细胞层中。使用没有内化西妥昔单抗部分的细胞膜进行竞争测定表明，西妥昔单抗与 CHX-A″-DTPA 的缀合不会导致其亲和力丧失。西妥昔单抗和 CHX-A″-DTPA-西妥昔单抗阻断了 EGF 诱导的 EGFR 的 Y1068 磷酸化。西妥昔单抗对 EGF 诱导的 Akt 和 ERK1/2 磷酸化的缺乏作用以及西妥昔单抗对辐射（IR）诱导的 Akt 和 ERK1/2 磷酸化的抑制作用不受 DTPA 缀合的影响。（90）Y-西妥昔单抗与 EBI 联合使用可显著抑制 HNSCC 细胞集落的形成。
CHX-A″-DTPA 与西妥昔单抗缀合不会改变西妥昔单抗的细胞和生物学功能。（90）Y 标记的西妥昔单抗与 EBI 联合使用可能改善放射治疗效果。
