Convenient plasmid vectors for construction of chimeric mouse/human antibodies

Chimeric antibodies composed of mouse-derived variable regions and human-derived constant regions have been developed for clinical use. However, construction of chimeric mouse/human genes in expression vectors is time-consuming work. In this study, we developed convenient vectors for construction of chimeric mouse/human antibodies. The protocols are as follows: In mouse hybridomas and B cells, most active V_H and V_κ{script} genes can be identified as rearranged bands by Southern hybridization of EcoRI- and HindIII-digested DNAs with J_H and J_κ{script} probes, respectively, and such fragments can be isolated in λ-EcoRI and λ-HindIII vectors, respectively. We constructed two plasmids: pSV2-HG1gpt contains human C_γ1 and Ecogpt genes, and only one EcoRI site upstream of the C_γ1 gene; pSV2-HC_κ{script}neo contains human C_κ{script} and neo genes, and only one HindIII site upstream of the C_κ{script} gene. An isolated EcoRI fragment containing a V_HD_HJ_H gene and a HindIII fragment containing a V_κ{script}J_κ{script} gene are inserted into pSV2-HG1gpt and pSV2-HC_κ{script}neo, respectively. Both resulting plasmid DNAs are co-transfected into SP2/0 cell, a non-Ig-secreting mouse myeloma. Transformants are selected by both mycophenolic acid and G418. With this procedure, it takes only 2 months to obtain chimeric antibodies.