title: Construction and expression of a human/mouse chimeric CD19 monoclonal antibody: Successful modification of a murine IgM to a chimeric IgG.
abstract: CD19 is a specific surface marker of B cells. A murine IgM-subtype antibody, 2E8, was generated previously and assigned to the CD19 category by the 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens in 1996. In the present study, the 2E8 Fv gene was inserted into a baculovirus shuttle vector and novel protein was expressed in an IgG1 form in the Sf9 insect cell line. VH2E8and VL2E8genes were cloned and inserted into the baculovirus shuttle vector pAc-κ-CH3 to form pAc-κ-CH3-VH2E8-VL2E8. Sf9 cells were then transfected with the reconstructed baculovirus shuttle vector. Novel protein expressed by the Sf9 cells was identified by immunofluorescence and western blot analysis, while activity levels were analyzed by flow cytometry (FCM). Sequencing demonstrated that the VH2E8and VL2E8fragments were inserted into pAc-κ-CH3 correctly. The immunofluorescence, western blot analysis and FCM results indicated that active recombinant antibody was expressed in the cytoplasm of Sf9 cells, but not in the culture supernatant. Thus, functional recombinant antibody was expressed successfully in the cytoplasm of Sf9 cells, but was not secreted into the culture supernatant. Therefore, the present study demonstrates that it is possible to modify mouse IgM to mouse-human chimeric IgG1 while retaining reasonable biological activity.
authors.affiliation: Department of Hematology and Oncology, School of Medicine, Children's Hospital of Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China.
authors.forename: Weiqun
authors.initials: W
authors.lastname: Xu
authors.affiliation: Department of Hematology and Oncology, School of Medicine, Children's Hospital of Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China.
authors.forename: Lingyan
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Key Laboratory, The First Hospital of Ningbo City, Ningbo, Zhejiang 315700, P.R. China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Hematology and Oncology, School of Medicine, Children's Hospital of Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China.
authors.forename: Yongmin
authors.initials: Y
authors.lastname: Tang
keywords: CD19; antibody engineering; baculovirus shuttle vector; chimeric antibody; leukemia