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 Vn and V, genes can be identified as rearranged bands by Southern hybridization of EcoRI-and HindHI-digested DNAs with Jn and J, probes, respectively, and such fragments can be isolated in I-EcoRI and I-Hind111 vectors, respectively. We constructed two plasmids: pSV2-HGlgpt contains human C;., and Ecogpt genes, and only one EcoRI site upstream of the C,, gene; pSV2-HC,neo contains human C, and neo genes, and only one Hind111 site upstream of the C, gene. An isolated EcoRI fragment containing a VuDnJu gene and a Hind111 fragment containing a V,J, gene are inserted into pSV2-HGlgpt and pSV2-HC,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.

A mouse/human chimeric anti-(ganglioside GD3) antibody with enhanced antitumor activities

“…A murine anti-GD3 mAb KM641 (IgG3, ~:) established in our laboratories was used for the isolation of KM641 H-and L-chain cDNA. A cell line producing chimeric mouse/human anti-(phosphorylchofine) antibody, SP2-PC Chimera-1 [ 16], kindly provided by Dr. Kurosawa (Fujita-gakuen Health University, Aichi, Japan), was used for the isolation of human C~;-and Cyl-chain cDNA.…”

“…A cDNA library was generated from SP2-PC Chimera-1 cells and screened for human C~c-and Cyl-chain clones with probes from the V~: and VH genomic genes respectively, which code for the anti-PC antibody [16]. The human C~c-and Cyl-chain genes were subcloned in pBhiescript and sequenced.…”

A new system for the expression of recombinant antibody in mammalian cells

“…The B~mHI-Hindlll fragment (0.17 kbp) of the resulting plasmid was inserted in pECEdhfr (Yasukawa et al, 1990) digested by B__g]ll and Hi._n.ndlll to give the plasmid pECEdhfrVHL. The Ec,,,QRI-Pstl fragment (0.89 kbp) of pSV2-HGlgpt (Kameyama et al, 1989) was inserted in M13mp19. A Sail site was introduced at the 5' portion of the gene for the CH1 region by site-directed mutagenesis using the primer CHISAL1, followed by an insertion of the PstI-E09521 fragment of pSV2-HGlgpt, in which the Eco521 cohesive end had been converted to be blunt, at the Pstl and Hi._.Endlll sites, in which the Hindlll cohesive end had been converted to be blund, to give mp19HCG1.…”

“…Several vector systems have been devised for mammalian expression of the complete immunoglobulin heavy and light chain genes (Kameyama et al, 1989, Orlandi, 1989. Such systems are typically composed of two expression vectors, one for the heavy (H) chain and the other for the light (L) chain.…”

“…Such systems are typically composed of two expression vectors, one for the heavy (H) chain and the other for the light (L) chain. The insert DNA for variable regions are cloned from the genomic DNA library (Kameyama et al, 1989) or amplified from cDNA or genomic DNA by the polymerase chain reaction (PCR) with primers designed to be specific for regions at each end of the gene and incorporate restriction enzyme sites for subsequent cloning (Orlandi, 1989).…”