A recombinant bispecific single-chain Fv antibody against HLA class II and FcγRIII (CD16) triggers effective lysis of lymphoma cells
A recombinant bispecific single-chain Fv antibody against HLA class II and FcγRIII (CD16) triggers effective lysis of lymphoma cells
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DOI：
10.1111/j.1365-2141.2004.04893.x
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发表时间：
2004-04-01
影响因子：
6.5
通讯作者：
Fey, GH
中科院分区：
文献类型：
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作者：
Bruenke, J;Fischer, B;Fey, GH
双特异性抗体为抗体治疗提供了改善效应细胞募集的可能性。为此，构建了针对 FcgammaRIII (CD16) 和 II 类人类白细胞抗原 (HLA) 的重组双特异性单链 Fv 抗体 (bsscFv)，并在功能测定中进行了测试。来自杂交瘤3G8和F3.3的RNA分别与CD16和HLA II类反应，用于生成噬菌体展示文库。从这些文库中分离出反应性噬菌体，并通过 20 个氨基酸的柔性接头连接两个单链 Fv 组件来构建 bsscFv。在 SF21 昆虫细胞中表达并层析纯化后，bsscFv 特异性且同时结合两种抗原。 bsscFv的抗CD16和抗HLA II类scFv组分的亲和力分别为8.6 x 10(-8) mol/l和13.7 x 10(-8) mol/l，比亲本抗体的F(ab)片段低约七倍。在以人单核细胞作为效应器的抗体依赖性细胞毒性实验中，bsscFv 介导的 HLA II 类阳性恶性人 B 淋巴细胞系和来自慢性 B 细胞淋巴细胞白血病患者的原代细胞的特异性裂解。在大约 400 ng/ml 的 bsscFv 浓度下获得最佳裂解，类似于相应化学连接的双特异性抗体最大裂解所需的浓度。因此，这种重组 bsscFv 抗体是效应细胞介导的恶性人 B 淋巴细胞裂解的有效分子。
Bispecific antibodies offer the possibility of improving effector-cell recruitment for antibody therapy. For this purpose, a recombinant bispecific single-chain Fv antibody (bsscFv), directed against FcgammaRIII (CD16) and human leucocyte antigen (HLA) class II, was constructed and tested in functional assays. RNA from the hybridomas 3G8 and F3.3, reacting with CD16 and HLA class II, respectively, was used to generate phage display libraries. From these libraries, reactive phages were isolated and the bsscFv was constructed by connecting both single-chain Fv components through a 20 amino acid flexible linker. After expression in SF21 insect cells and chromatographic purification, the bsscFv bound specifically and simultaneously to both antigens. The affinities of the anti-CD16 and the anti-HLA class II scFv components of the bsscFv were 8.6 x 10(-8) mol/l and 13.7 x 10(-8) mol/l, respectively, which was approximately sevenfold lower than the F(ab) fragments of the parental antibodies. In antibody-dependent cellular cytotoxicity experiments with human mononuclear cells as effectors, the bsscFv-mediated specific lysis of both HLA class II-positive, malignant human B-lymphoid cell lines and primary cells from patients with chronic B-cell lymphocytic leukaemia. Optimal lysis was obtained at bsscFv concentrations of approximately 400 ng/ml, similar to the concentration required for maximum lysis by the corresponding chemically linked bispecific antibody. Thus, this recombinant bsscFv-antibody is an efficient molecule for effector-cell mediated lysis of malignant human B-lymphoid cells.