Raben D, Buchsbaum D J, Khazaeli M B, Rosenfeld M E, Gillespie G Y, Grizzle W E, Liu T, Curiel D T
Department of Radiation Oncology, University of Alabama at Birmingham 35233, USA.
Gene Ther. 1996 Jul;3(7):567-80.
Conventional radiolabeled antibody targeting utilized in radioimmunotherapy has resulted in limited success clinically due in part to inadequate tumor localization resulting from low expression of human tumor-associated antigens on target cells. We hypothesized that one could improve upon these limitations by genetically inducing tumor cells to express high levels of a new membrane-associated receptor with high affinity for a radioligand. As a preliminary strategy, we induced a human glioma cell line (D54 MG) to express human carcinoembryonic antigen (CEA) in vitro. To accomplish this, we constructed a recombinant adenoviral vector encoding the CEA cDNA inserted downstream of a cytomegalovirus (CMV) promoter (AdCMVCEA). D54 MG cells were transfected with AdCMVCEA or an adenoviral vector encoding lacZ reporter gene as a control (AdCMVlacZ). LS174T human colon cancer cells, known to express CEA constitutively, served as positive controls. Immunofluorescence and immunohistochemistry assays employing unlabeled anti-CEA COL-1 monoclonal antibody demonstrated expression of CEA antigen on the cell surface of transduced D54 MG cells in culture. In addition, assays utilizing 125I-labeled COL-1 indicated high binding to transduced D54 MG cells expressing CEA (4.7 +/- 0.5 x 10(5) COL-1 molecules bound per cell) as compared with minimal binding to nontransduced D54 MG cells. LS174T cells demonstrated only 2.7 +/- 0.5 x 10(6) COL-1 molecules bound per cell. Thus, AdCMVCEA was able to induce levels of cell surface CEA in target cells at a higher level than CEA-overexpressing tumor cells (P < 0.01). The efficacy of transduction of recombinant AdCMVCEA by direct intratumoral injection into D54 MG xenografts was investigated by immunohistochemical analysis, immunofluorescence and by measuring 131I-labeled COL-1 uptake through external scintigraphic imaging and biodistribution studies. Expression of CEA in the tumor xenografts by, and radiolabeled antibody tumor targeting to, AdCMVCEA transduced D54 MG xenografts was comparable to that seen with LS174T xenografts. Results of these studies indicate the potential of adenovirus-mediated delivery of targets to improve radiopharmaceutical tumor localization.
放射免疫疗法中使用的传统放射性标记抗体靶向治疗在临床上取得的成功有限，部分原因是靶细胞上人类肿瘤相关抗原表达水平低导致肿瘤定位不足。我们假设可以通过基因诱导肿瘤细胞表达对放射性配体具有高亲和力的新型膜相关受体来改善这些局限性。作为初步策略，我们在体外诱导人胶质瘤细胞系（D54 MG）表达人癌胚抗原（CEA）。为实现这一目标，我们构建了一种重组腺病毒载体，其编码插入巨细胞病毒（CMV）启动子下游的CEA cDNA（AdCMVCEA）。用AdCMVCEA或编码lacZ报告基因的腺病毒载体作为对照（AdCMVlacZ）转染D54 MG细胞。已知组成性表达CEA的LS174T人结肠癌细胞用作阳性对照。采用未标记的抗CEA COL-1单克隆抗体的免疫荧光和免疫组织化学分析表明，在培养的转导D54 MG细胞的细胞表面上存在CEA抗原表达。此外，利用125I标记的COL-1的分析表明，与未转导的D54 MG细胞的最小结合相比，与表达CEA的转导D54 MG细胞有高结合（每个细胞结合4.7±0.5×10（5）个COL-1分子）。LS174T细胞每个细胞仅显示结合2.7±0.5×10（6）个COL-1分子。因此，AdCMVCEA能够在靶细胞中诱导出比CEA过表达肿瘤细胞更高水平的细胞表面CEA（P <0.01）。通过免疫组织化学分析、免疫荧光以及通过外部闪烁成像和生物分布研究测量131I标记的COL-1摄取，研究了通过直接瘤内注射重组AdCMVCEA到D54 MG异种移植物中的转导效率。AdCMVCEA转导的D54 MG异种移植物在肿瘤异种移植物中CEA的表达以及放射性标记抗体对肿瘤的靶向作用与LS174T异种移植物所见相当。这些研究结果表明腺病毒介导的靶点递送在改善放射性药物肿瘤定位方面的潜力。
