Itoh K, Inoue K, Hirooka K, Maruyama K, Ohkawa M, Matsui K, Tada H, Enomoto T, Hashimoto Y, Suzuki T, Masuko T
Department of Pharmaceutical Science, Akita University Hospital, Akita 010-8543, Japan.
Jpn J Cancer Res. 2001 Dec;92(12):1313-21. doi: 10.1111/j.1349-7006.2001.tb02155.x.
The Fab gene of anti-CD98 heavy chain (h.c.) monoclonal antibody (mAb) HBJ127 was cloned and expressed as a recombinant Fab (rFab) fragment by means of a phage display system. The variable heavy and light chain genes of HBJ127 were found to be derived from VOx-1 and IgVk8-30 germline, respectively. Extensive somatic mutation was found in the heavy chain complementarity determining region 2. rFab fragment was purified homogeneously from crude bacterial lysates by Ni-chelate chromatography in a yield of 71.4 mg from 100 ml of culture. rFab fragment was reactive with the cell surface of CD98-positive cells irrespective of tissues of origin, but not with CD98-negative cells. The recognition site of the rFab fragment was identical to that of mAb since the binding of rFab fragment to HeLaS(3) cells was completely inhibited by pretreatment with an excess of mAb. The relative affinity values of rFab fragment and mAb were found to be 0.11 x 10(8) and 0.35 x 10(8) M(-1), respectively. Three-fold lower affinity of rFab fragment may be due to the difference of valency of the antibody preparation. Cell growth inhibition in vitro by rFab fragment preincubated with anti-Fab suggests that the rFab fragment produced by cloned gene-bearing Escherichia coli was identical to the Fab part of HBJ127 mAb. These results show that a small fragment with antigen binding activity similar to that of the parent mAb can easily be prepared by using a phage display system. To our knowledge, this is a first report of the production of anti-CD98 h.c. rFab fragment.
利用噬菌体展示系统克隆了抗CD98重链单克隆抗体（mAb）HBJ127的Fab基因，并将其表达为重组Fab（rFab）片段。发现HBJ127的重链可变区基因和轻链可变区基因分别来源于VOx-1和IgVk8-30种系。在重链互补决定区2中发现了广泛的体细胞突变。通过镍螯合层析从粗细菌裂解物中纯化得到均一的rFab片段，从100 ml培养物中获得的产量为71.4 mg。rFab片段与CD98阳性细胞的细胞表面反应，与细胞来源组织无关，但与CD98阴性细胞不反应。rFab片段的识别位点与mAb相同，因为用过量mAb预处理可完全抑制rFab片段与HeLaS(3)细胞的结合。发现rFab片段和mAb的相对亲和力值分别为0.11×10(8)和0.35×10(8) M(-1)。rFab片段亲和力低三倍可能是由于抗体制剂的价态不同。与抗Fab预孵育的rFab片段在体外对细胞生长的抑制作用表明，携带克隆基因的大肠杆菌产生的rFab片段与HBJ127 mAb的Fab部分相同。这些结果表明，利用噬菌体展示系统可以很容易地制备出具有与亲本mAb相似抗原结合活性的小片段。据我们所知，这是关于抗CD98重链rFab片段生产的首次报道。
