McCall A M, Adams G P, Amoroso A R, Nielsen U B, Zhang L, Horak E, Simmons H, Schier R, Marks J D, Weiner L M
Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
Mol Immunol. 1999 May;36(7):433-45. doi: 10.1016/s0161-5890(99)00057-7.
Bispecific antibody (bsAb)-based clinical trials of cancer have been conducted primarily using intact murine monoclonal antibody (mAb)-derived molecules. In some of these trials, toxicity resulting from the interactions of antibody Fc domains with cellular Fc receptors has limited the doses of antibody (Ab) that can be employed. Furthermore, human anti-mouse Ab responses prohibit multiple therapy courses. These factors have decreased the efficacy of the bsAb 2B1, which targets the extracellular domains (ECD) of the HER2/neu protooncogene product and the human FcgammaRIII (CD16). To address these obstacles, we have constructed and characterized a fully human gene-fused bsAb from single-chain Fv (scFv) molecules specific for HER2/neu and CD16. The human anti-CD16 scFv component, NM3E2, was isolated from a human scFv phage display library. As binding of NM3E2 to human neutrophil-associated CD16 decreased in the presence of plasma IgG, we have concluded that NM3E2 recognizes an epitope in the vicinity of the Fc binding pocket. Furthermore, the NM3E2 scFv was found by surface plasmon resonance-based epitope mapping to share an overlapping epitope with the Leu-11c mAb. The human anti-HER2/neu scFv component, C6.5, which was previously isolated from a human scFv phage display library, was employed as fusion partner for the creation of a bispecific scFv (bs-scFv). In the presence of the C6.5 x NM3E2 bs-scFv, peripheral blood lymphocytes promoted significant lysis of human SK-OV-3 ovarian cancer cells overexpressing HER2/neu. Biodistribution studies performed in SK-OV-3 tumor-bearing scid mice revealed that 1% ID/g of 125I-labeled C6.5 x NM3E2 bs-scFv was specifically retained in tumor at 23 h following injection. These results indicated that both scFv components of the bs-scFv retained their function in the fusion protein. This bsAb should overcome some of the problems associated with the 2B1 bsAb. C6.5 x NM3E2 bs-scFv offers promise as a platform for multifunctional binding proteins with potential clinical applications as a result of its human origin, lack of an Fc domain, ease of production, high level of in vitro tumor cell cytotoxicity and highly selective tumor targeting.
基于双特异性抗体（bsAb）的癌症临床试验主要使用完整的鼠源单克隆抗体（mAb）衍生分子进行。在其中一些试验中，抗体Fc结构域与细胞Fc受体相互作用产生的毒性限制了可使用的抗体剂量。此外，人抗鼠抗体反应阻碍了多疗程治疗。这些因素降低了靶向HER2/neu原癌基因产物的细胞外结构域（ECD）和人FcγRIII（CD16）的双特异性抗体2B1的疗效。为了解决这些障碍，我们从对HER2/neu和CD16具有特异性的单链Fv（scFv）分子构建并表征了一种完全人源的基因融合双特异性抗体。人抗CD16 scFv组分NM3E2是从人scFv噬菌体展示文库中分离出来的。由于在血浆IgG存在下NM3E2与人中性粒细胞相关CD16的结合减少，我们得出结论，NM3E2识别Fc结合口袋附近的一个表位。此外，通过基于表面等离子体共振的表位作图发现，NM3E2 scFv与Leu-11c单克隆抗体共享一个重叠表位。人抗HER2/neu scFv组分C6.5先前从人scFv噬菌体展示文库中分离出来，用作创建双特异性scFv（bs-scFv）的融合伙伴。在C6.5×NM3E2 bs-scFv存在下，外周血淋巴细胞促进了过表达HER2/neu的人SK-OV-3卵巢癌细胞的显著裂解。在荷SK-OV-3肿瘤的scid小鼠中进行的生物分布研究表明，注射后23小时，125I标记的C6.5×NM3E2 bs-scFv有1% ID/g特异性保留在肿瘤中。这些结果表明，bs-scFv的两个scFv组分在融合蛋白中都保留了其功能。这种双特异性抗体应该能克服与2B1双特异性抗体相关的一些问题。由于其源自人、缺乏Fc结构域、易于生产、具有高水平的体外肿瘤细胞细胞毒性和高度选择性的肿瘤靶向性，C6.5×NM3E2 bs-scFv有望作为多功能结合蛋白的平台，具有潜在的临床应用价值。
