Miescher S, Zahn-Zabal M, De Jesus M, Moudry R, Fisch I, Vogel M, Kobr M, Imboden M A, Kragten E, Bichler J, Mermod N, Stadler B M, Amstutz H, Wurm F
ZLB Central Laboratory, Swiss Red Cross, Inselspital, Bern, Switzerland.
Br J Haematol. 2000 Oct;111(1):157-66. doi: 10.1046/j.1365-2141.2000.02322.x.
Replacement of the hyperimmune anti-Rhesus (Rh) D immunoglobulin, currently used to prevent haemolytic disease of the newborn, by fully recombinant human anti-RhD antibodies would solve the current logistic problems associated with supply and demand. The combination of phage display repertoire cloning with precise selection procedures enables isolation of specific genes that can then be inserted into mammalian expression systems allowing production of large quantities of recombinant human proteins. With the aim of selecting high-affinity anti-RhD antibodies, two human Fab libraries were constructed from a hyperimmune donor. Use of a new phage panning procedure involving bromelin-treated red blood cells enabled the isolation of two high-affinity Fab-expressing phage clones. LD-6-3 and LD-6-33, specific for RhD. These showed a novel reaction pattern by recognizing the D variants D(III), D(IVa), D(IVb), D(Va), D(VI) types I and II. D(VII), Rh33 and DFR. Full-length immunoglobulin molecules were constructed by cloning the variable regions into expression vectors containing genomic DNA encoding the immunoglobulin constant regions. We describe the first, stable, suspension growth-adapted Chinese hamster ovary (CHO) cell line producing a high affinity recombinant human IgG1 anti-RhD antibody adapted to pilot-scale production. Evaluation of the Fc region of this recombinant antibody by either chemiluminescence or antibody-dependent cell cytotoxicity (ADCC) assays demonstrated macrophage activation and lysis of red blood cells by human lymphocytes. A consistent source of recombinant human anti-RhD immunoglobulin produced by CHO cells is expected to meet the stringent safety and regulatory requirements for prophylactic application.
用完全重组的人抗RhD抗体替代目前用于预防新生儿溶血病的超免疫抗RhD免疫球蛋白，将解决当前与供需相关的物流问题。噬菌体展示文库克隆与精确筛选程序相结合，能够分离出特定基因，然后将其插入哺乳动物表达系统，从而大量生产重组人蛋白。为了筛选高亲和力的抗RhD抗体，从一名超免疫供体构建了两个人Fab文库。使用一种涉及菠萝蛋白酶处理红细胞的新噬菌体淘选程序，能够分离出两个表达高亲和力Fab的噬菌体克隆，即对RhD具有特异性的LD-6-3和LD-6-33。这些克隆通过识别D变体D(III)、D(IVa)、D(IVb)、D(Va)、I型和II型D(VI)、D(VII)、Rh33和DFR，显示出一种新的反应模式。通过将可变区克隆到含有编码免疫球蛋白恒定区的基因组DNA的表达载体中，构建全长免疫球蛋白分子。我们描述了第一个适应悬浮生长的稳定中国仓鼠卵巢（CHO）细胞系，该细胞系可生产适合中试规模生产的高亲和力重组人IgG1抗RhD抗体。通过化学发光或抗体依赖性细胞毒性（ADCC）试验对该重组抗体的Fc区进行评估，结果表明人淋巴细胞可激活巨噬细胞并裂解红细胞。预计CHO细胞产生的重组人抗RhD免疫球蛋白的稳定来源将满足预防性应用的严格安全和监管要求。
