Ames R S, Tornetta M A, Deen K, Jones C S, Swift A M, Ganguly S
Department of Molecular Immunology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406-0939, USA.
J Immunol Methods. 1995 Aug 18;184(2):177-86. doi: 10.1016/0022-1759(95)00086-p.
The use of combinatorial Ig libraries displayed on the surface of bacteriophage has advantages over traditional hybridoma techniques for the generation of mAbs but in many instances full length Igs may be more desirable than Fab fragments. Two murine Fabs reactive with the human complement component C5a, recovered from a combinatorial library, were converted to full length IgG2a mAbs. The VH and VL domains of these antibodies were removed from the bacterial expression vector used for the combinatorial library construction, and subcloned into individual mammalian expression vectors containing the corresponding Ig heavy and light chain constant regions. The subcloning relied on 5' restriction endonuclease sites encoded by the oligonucleotide primers originally used to amplify the Ig cDNAs and 3' sites conserved in CH1 and C kappa. These vectors were co-transfected into COS cells yielding full length IgG2a versions of the anti-C5a antibodies. The mAbs, purified from the culture supernatant, retained the full activity of the Fabs, binding specifically to and neutralizing human recombinant C5a. Refined versions of the mammalian expression vectors have been constructed for single step conversion of murine recombinant Fabs, recovered from combinatorial libraries, to IgG2a mAbs.
与传统杂交瘤技术相比，利用展示在噬菌体表面的组合免疫球蛋白文库来产生单克隆抗体具有诸多优势，但在许多情况下，全长免疫球蛋白可能比Fab片段更具优势。从一个组合文库中筛选出的两种与人类补体成分C5a反应的鼠源Fab片段，被转化为全长IgG2a单克隆抗体。这些抗体的VH和VL结构域从用于构建组合文库的细菌表达载体中移除，并亚克隆到含有相应免疫球蛋白重链和轻链恒定区的单个哺乳动物表达载体中。亚克隆依赖于最初用于扩增免疫球蛋白cDNA的寡核苷酸引物编码的5'限制性内切酶位点以及CH1和Cκ中保守的3'位点。这些载体共转染到COS细胞中，产生了抗C5a抗体的全长IgG2a版本。从培养上清液中纯化得到的单克隆抗体保留了Fab片段的全部活性，能够特异性结合并中和人类重组C5a。已构建了优化版的哺乳动物表达载体，用于将从组合文库中筛选出的鼠源重组Fab片段一步转化为IgG2a单克隆抗体。
