GENERATION OF A HIGH-PRODUCING CLONE OF A HUMANIZED ANTI-B-CELL LYMPHOMA MONOCLONAL-ANTIBODY (HLL2)

BACKGROUND, LL2 is a murine immunoglobulin (Ig)G2a-kappa anti-B-cell m onoclonal antibody with proven targeting and therapeutic efficacy in t he management of non-Hodgkin's lymphoma (NHL). The authors had previou sly generated a humanized LL2 (hLL2) that demonstrated binding propert ies identical to those of LL2. Nevertheless, the productivity of the c ell line was insufficient for large-scale production of the antibody f or clinical studies. Therefore, the authors chose an amplifiable syste m for the generation of hLL2. METHODS. The hLL2 sequences were ligated into the expression vector pdHL2, which has a dhfr amplifiable gene, and were incorporated into the SP2/0 cells by electroporation. A metho trexate (MTX) resistant clone producing hLL2 was identified. Stepwise increases in MTX concentrations, from 0.1 to 5 mu M, and subcloning of the cells by limiting dilution were performed. RESULTS, By amplifying the dhfr and hLL2 genes with stepwise increases in the MTX concentrat ion, the antibody production was enhanced from its original 1.4 to 70 +/- 5 mg per liter of culture media. Subsequent subcloning further imp roved the productivity. Immunoreactivity of the antibody was conserved , as proven by enzyme-linked immunosorbent assay and cell-binding assa ys. By isoelectrofocusing, the isoelectric point (pi) of the antibody was measured at approximately 9.6. The productivity of the clone was n ot affected by culture conditions or storage of the cells in liquid ni trogen. CONCLUSIONS, By means of gene amplification, the authors have generated a high-producing hLL2-IgG clone suitable for production of t he quantity of antibody necessary for clinical diagnostic and therapeu tic trials of NHL patients. (C) 1997 American Cancer Society.