Division of Hematology and Oncology, Department of Internal Medicine, University of Erlangen-Nuremberg, Erlangen, Germany.
J Immunol. 2010 Feb 1;184(3):1210-7. doi: 10.4049/jimmunol.0902033. Epub 2009 Dec 30.
Bispecific Abs offer new perspectives for cancer immunotherapy. In this study, we describe a recombinant bispecific single-chain fragment variable (bsscFv) directed against Fc alpha RI (CD89) on polymorphonuclear neutrophils (PMNs) or monocytes/macrophages and HLA class II on lymphoma target cells. Fc alpha RI and HLA class II-directed single-chain fragment variable (scFv) fragments were isolated from phage display libraries, established from the hybridomas A77 and F3.3, respectively. The two scFv molecules were connected with a 20 aa flexible linker sequence. After expression in SF21 insect cells and chromatographic purification, the bispecific molecule showed specific binding to both Ags at K(D) values of 148 +/- 42 nM and 113 +/- 25 nM for the anti-Fc alpha RI and anti-HLA class II scFv components in the bsscFv, respectively. In Ab-dependent cytotoxicity assays with PMNs as effectors and a series of lymphoma-derived cell lines (ARH-77, RAJI, REH, NALM-6, RS4;11), the bsscFv was significantly more cytotoxic than the parental murine IgG1 and its chimeric IgG1 derivative. When targeting primary tumor cell isolates from six patients with B cell malignancies, the killing capacity of the (Fc alphaRI x HLA class II) bsscFv compared favorably to conventional HLA class II mAb. Importantly, the cell lines NALM-6 and RS411, as well as two primary tumor cell isolates, were exclusively lysed by the bsscFv. To our knowledge, this is the first report of an Fc alpha RI-directed bsscFv effectively recruiting PMNs for redirected cytotoxicity against human B cell malignancies. Our data show that an (Fc alpha RI x HLA class II) bsscFv is an interesting candidate for further engineering of small, modular immunopharmaceuticals.
双特异性抗体为癌症免疫疗法提供了新的视角。在这项研究中，我们描述了一种针对多形核粒细胞（PMN）或单核细胞/巨噬细胞上的 FcαRI（CD89）和淋巴瘤靶细胞上的 HLA 类 II 的重组双特异性单链片段可变体（bsscFv）。FcαRI 和 HLA 类 II 导向的单链片段可变体（scFv）片段分别从杂交瘤 A77 和 F3.3 的噬菌体展示文库中分离出来。将两个 scFv 分子用 20 个 aa 柔性接头序列连接起来。在 SF21 昆虫细胞中表达并通过色谱纯化后，该双特异性分子显示出与两个抗原的特异性结合，在 bsscFv 中，抗 FcαRI 和抗 HLA 类 II scFv 成分的 K(D) 值分别为 148±42 nM 和 113±25 nM。在PMN 作为效应物和一系列淋巴瘤衍生细胞系（ARH-77、RAJI、REH、NALM-6、RS4；11）的 Ab 依赖性细胞毒性测定中，bsscFv 比亲本鼠 IgG1 及其嵌合 IgG1 衍生物更具细胞毒性。当靶向来自 6 名 B 细胞恶性肿瘤患者的原发性肿瘤细胞分离物时，（FcαRI x HLA 类 II）bsscFv 的杀伤能力与常规 HLA 类 II mAb 相比具有优势。重要的是，细胞系 NALM-6 和 RS411 以及两个原发性肿瘤细胞分离物仅被 bsscFv 溶解。据我们所知，这是第一个报道 FcαRI 定向 bsscFv 有效招募 PMN 以重新定向针对人类 B 细胞恶性肿瘤的细胞毒性的报告。我们的数据表明，（FcαRI x HLA 类 II）bsscFv 是进一步工程化小型模块化免疫药物的有前途的候选物。
