Expression of functional mouse antibodies directed against the tumour marker human placental alkaline phosphatase in non-lymphoid cells

Abstract

A mouse hybridoma cell line was isolated which produces monoclonal antibodies (MAbs) of the IgGZb, K subtype directed against the tumour-associated marker human placental alkaline phosphatase (hPLAP). The mRNAs coding for the heavy (H) and light (L) chains were cloned as cDNA copies. These genes were then separately inserted into the eukaryotic expression vector pSV23p, under control of the SV40 early promoter. Both genes were introduced with the DEAE-dextrane technique in COSI cells, and 72 hr after transfection, 10 ng/ml functional antibodies could be detected in the supernatant of the cells. Permanent CHO cell lines secreting 100 n g h l functional antibodies were established upon Previously, we constructed a mouse hybridoma cell line (E6j) secreting highly specific antibodies against human placental alkaline phosphatase (hPLAP), an onco-developmental protein present on the cell surface of certain tumours and in sera of cancer patients (Van de Voorde et al., 1985).Ouchterlony immunodiffusion demonstrated that the E6 antibodies were of the IgG2b, K subclass. This isotype was further confirmed by polyacrylamide gel electrophoresis of immunoprecipitated, in vivo-labelled antibodies.Total RNA from E6j hybridoma cells was isolated by oligo-dT cellulose chromatography followed by sucrosegradient centrifugation. Fractions containing mRNAs coding for H and L chains were identified by in vitro translation and immuno-precipitation and used for cDNA synthesis. To this end, mRNA is transcribed by AMV-reverse transcriptase (RTase) using an oligo-dT12-18 primer resulting in an RNA-DNA heteroduplex. After dC-tailing, the mRNA template is removed by alkaline hydrolysis and secondstrand synthesis with RTase is primed by oligo-dG6. Double-stranded cDNA copies are tailed with dC and inserted into a dG-tailed PstI-site of pBR322, restoring the PstI restriction site. By this method, the use of S1-nuclease to remove hairpin loop structures is circumvented, resulting in more efficient cloning of 5'-ends of mRNAs. E. coli MC1061X transformants were screened by colony hybridization using IgG2b and K-specific probes. From the total number of positive clones obtained, we could calculate that both mRNAs represent only 0.13% of the total mRNA population.Detailed restriction analysis of clone pBRE6-H and clone pBRE6-L indicated that we had full-length copies of the mRNAs coding for the H and L chains of our E6j MAb. The nucleotide sequence confirmed the IgGZb, K isotype.The coding information of both the light and the heavy chain was recloned separately into pSP64 plasmids under control of the Salmonella typhimurium bacteriophage SP6 promoter. In the presence of SP6 polymerase and in a transcription mix that contained additional CAP-structures [7mG(5')ppp(5 ')GI, transcripts were made that could be efficiently translated in an in vitro rabbit reticulocyte translation system. SDS-polyacrylamide gel electrophoresis of such in vitro translation products showed a 56-kDa immunoprecipitable heavy chain directed by the H-chain clone and a 28-kDa ...

scFv Antibody: Principles and Clinical Application

“…Since then, monoclonal antibodies (mAbs) have been favored as they can be produced in unlimited quantities to practically bind to any antigen and are more easily standardized [ 7 , 8 ]. Besides monoclonal antibodies, hybridoma cells that are successfully produced could then serve as a starting material in the generation of Fab, or Fv fragments in lymphoid or nonlymphoid cells [ 9 ]. Monoclonal antibodies face several difficulties, as they are almost exclusively murine in origin thus could create human anti-mouse antibody (HAMA) when introduced to human therefore limits their clinical applications [ 7 , 8 ].…”