Convenient plasmid vectors for construction of chimeric mouse/human antibodies

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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 VH and Vϰ genes can be identified as rearranged bands by Southern hybridization of EcoRI‐ and HindIII‐digested DNAs with JH and Jϰ probes, respectively, and such fragments can be isolated in λ‐EcoRI and λ‐HindIII vectors, respectively. We constructed two plasmids: pSV2‐HG1gpt contains human Cγ1 and Ecogpt genes, and only one EcoRI site upstream of the Cγ1 gene; pSV2‐HCϰneo contains human Cϰ and neo genes, and only one HindIII site upstream of the Cϰ gene. An isolated EcoRI fragment containing a VHDHJH gene and a HindIII fragment containing a VϰJϰ gene are inserted into pSV2‐HG1gpt and pSV2‐HCϰneo, 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.