Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Building 149-9019, 13th St., Boston, MA 02129, USA.
Cell Immunol. 2012 Mar-Apr;276(1-2):162-7. doi: 10.1016/j.cellimm.2012.05.004. Epub 2012 May 18.
The porcine CD3 specific monoclonal antibody 898H2-6-15 has been used in allo- and xeno-transplantation studies as a porcine CD3 marker and as an effective T cell depletion reagent when conjugated to the diphtheria toxin mutant, CRM9. A recombinant anti-porcine CD3 immuntoxin was recently developed using single-chain variable fragments (scFv) derived from 898H2-6-15. In this study, using published sequence data, we have expressed the porcine CD3 ectodomain molecules in E. coli through inclusion body isolation and in vitro refolding approach. The expressed and refolded porcine CD3 ectodomain molecules include CD3ε, CD3γ, CD3δ, CD3εγ heterodimer, CD3εδ heterodimer, CD3εγ single-chain fusion protein and CD3εδ single-chain fusion protein. These refolded porcine CD3 ectodomain molecules were purified with a strong anion exchange resin Poros 50HQ. ELISA analysis demonstrated that only the porcine CD3εγ ectodomain single-chain fusion protein can bind to the porcine CD3 specific monoclonal antibody 898H2-6-15. The availability of this porcine CD3εγ ectodomain single-chain fusion protein will allow screening for affinity matured variants of scFv derived from 898H2-6-15 to improve the recombinant anti-porcine CD3 immunotoxin. Porcine CD3εγ ectodomain single-chain fusion protein will also be a very useful reagent to study the soluble phase interaction between porcine CD3εγ and porcine CD3 antibodies such as 898H2-6-15.
猪 CD3 特异性单克隆抗体 898H2-6-15 已在同种异体和异种移植研究中用作猪 CD3 标记物，并在与白喉毒素突变体 CRM9 缀合时用作有效的 T 细胞耗竭试剂。最近使用源自 898H2-6-15 的单链可变片段 (scFv) 开发了重组抗猪 CD3 免疫毒素。在这项研究中，我们使用已发表的序列数据通过包涵体分离和体外重折叠方法在大肠杆菌中表达猪 CD3 细胞外结构域分子。表达和重折叠的猪 CD3 细胞外结构域分子包括 CD3ε、CD3γ、CD3δ、CD3εγ 异二聚体、CD3εδ 异二聚体、CD3εγ 单链融合蛋白和 CD3εδ 单链融合蛋白。这些重折叠的猪 CD3 细胞外结构域分子用强阴离子交换树脂 Poros 50HQ 进行纯化。ELISA 分析表明，只有猪 CD3εγ 细胞外结构域单链融合蛋白可以与猪 CD3 特异性单克隆抗体 898H2-6-15 结合。这种猪 CD3εγ 细胞外结构域单链融合蛋白的可用性将允许筛选源自 898H2-6-15 的 scFv 的亲和力成熟变体，以改善重组抗猪 CD3 免疫毒素。猪 CD3εγ 细胞外结构域单链融合蛋白也将是一种非常有用的试剂，可用于研究猪 CD3εγ 与猪 CD3 抗体（如 898H2-6-15）之间的可溶性相相互作用。
