Convenient plasmid vectors for construction of chimeric mouse/human antibodies

Abstract

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 genes can be identified as rearranged bands by Southern hybridization of EcoRI‐ and HindIII‐digested DNAs with J_H and J_ϰ 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_ϰneo contains human C_ϰ and neo genes, and only one HindIII site upstream of the C_ϰ gene. An isolated EcoRI fragment containing a V_HD_HJ_H gene and a HindIII fragment containing a V_ϰJ_ϰ gene are inserted into pSV2‐HG1gpt and pSV2‐HC_ϰneo, 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.