Expression and characterization of recombinant soluble porcine CD3 ectodomain molecules: Mapping the epitope of an anti-porcine CD3 monoclonal antibody 898H2-6-15

详细信息    查看全文

• 作者：Jaclyn Stromp Peraino ; Christina E. Hermanrud ; Lauren Springett ; Huiping Zhang ; Guoying Li ; Srimathi Srinivasan ; Ashley Gusha ; David H. Sachs ; Christene A. Huang ; Zhirui Wang
• 关键词：Porcine CD3 ; 898H2-6-15 monoclonal antibody ; T cell ; Immunotoxin
• 刊名：Cellular Immunology
• 出版年：2012
• 出版时间：March-April, 2012
• 年：2012
• 卷：276
• 期：1-2
• 页码：162-167
• 全文大小：496 K

文摘

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 CD3¦Å, CD3¦Ã, CD3¦Ä, CD3¦Å¦Ã heterodimer, CD3¦Å¦Ä heterodimer, CD3¦Å¦Ã single-chain fusion protein and CD3¦Å¦Ä 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 CD3¦Å¦Ã ectodomain single-chain fusion protein can bind to the porcine CD3 specific monoclonal antibody 898H2-6-15. The availability of this porcine CD3¦Å¦Ã 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 CD3¦Å¦Ã ectodomain single-chain fusion protein will also be a very useful reagent to study the soluble phase interaction between porcine CD3¦Å¦Ã and porcine CD3 antibodies such as 898H2-6-15.