Cancer Research Institute of Scott & White Hospital and Department of Internal Medicine, Health Science Center, College of Medicine, Texas A&M University, 5701 South Airport Road, Temple, TX 76502, USA.
Bioconjug Chem. 2009 Oct 21;20(10):1975-82. doi: 10.1021/bc900333j. Epub 2009 Sep 28.
Immunotoxin potency is dependent on cell surface binding specificity as well as internalization efficiency. Current approaches for immunotoxin development are dependent on existing antibodies that were selected for high affinity and/or high production yield. However, these antibodies may demonstrate low internalization efficiency upon cell surface binding and thus are not necessarily the best candidates for immunotoxin design. Here, we have developed an assay with a novel protein, DTG3, to compare and evaluate the internalization efficiency of monoclonal antibodies in order to circumvent the possibility of low internalization. DTG3 is a fusion protein containing the N-terminus of diphtheria toxin (DT) and three copies of streptococci Protein G immunoglobulin binding domains. We show that antibody-DTG3 complexes formed in the test tube are able to bind their antigen on the target cell surface, resulting in cell internalization, DT-mediated protein synthesis inhibition, and host cell apoptosis. We tested this system with two well-studied antibodies, antihuman CD3ε, and anti-PSMA antibodies and were able to show efficiency of this assay. We further examined commercially available anti-CD123 antibodies for potential leukemia-targeting immunotoxin development. Finally, we applied this system in the early-stage screening of newly generated anti-CD123 hybridomas. Our data showed that this internalization assay system is sensitive, time efficient, and reproducible, and has provided a tool to compare monoclonal antibodies for the clinical development of effective immunotoxins for the treatment of a variety of neoplasms.
免疫毒素的效力取决于细胞表面结合的特异性和内化效率。目前的免疫毒素开发方法依赖于已选择的具有高亲和力和/或高生产产量的抗体。然而，这些抗体在细胞表面结合时可能表现出低内化效率，因此不一定是免疫毒素设计的最佳候选物。在这里，我们开发了一种使用新型蛋白 DTG3 的测定法，以比较和评估单克隆抗体的内化效率，从而避免内化效率低的可能性。DTG3 是一种融合蛋白，包含白喉毒素（DT）的 N 端和三个链球菌蛋白 G 免疫球蛋白结合结构域。我们表明，在试管中形成的抗体-DTG3 复合物能够结合靶细胞表面的抗原，导致细胞内化、DT 介导的蛋白质合成抑制和宿主细胞凋亡。我们用两种经过充分研究的抗体，抗人 CD3ε 和抗 PSMA 抗体测试了该系统，并能够证明该测定的效率。我们进一步研究了市售的抗 CD123 抗体，以开发潜在的白血病靶向免疫毒素。最后，我们将该系统应用于新生成的抗 CD123 杂交瘤的早期筛选。我们的数据表明，该内化测定系统灵敏、高效且可重复，为比较单克隆抗体用于开发治疗各种肿瘤的有效免疫毒素提供了一种工具。
