Effects of complexation with in vivo enhancing monoclonal antibodies on activity of growth hormone in two responsive cell culture systems

We describe the properties of three monoclonal antibodies (MAbs) to ovine G H, two of which have previously been shown to enhance, in vivo, the biologi cal activity of bovine and ovine growth hormone. We have examined the effec ts of these MAbs on GH activity in two appropriate GH-responsive cell cultu re systems, investigating both acute signalling effects (Janus-activated ki nase (Jak)-2 tyrosine phosphorylation -5min) and longer-term (MTT-formazan production -24h) effects of hormone-antibody complexes. In the 3T3-F442A pr e-adipocyte cell line (which has been demonstrated to be GH responsive), we show that complexation of recombinant bovine (rb) GH with either of the tw o enhancing anti-ovine GH MAbs (OA11 and OA15) and the non-enhancing MAb, O A14, attenuates the ability of GH to stimulate tyrosine phosphorylation of Jak-2 at a 5-min time point. Using the mouse myeloid cell line, FDC-P1, sta bly transfected with the full-length ovine GH receptor (oGHR), we demonstra te that rbGH causes a dose-dependent increase in MTT-formazan production by these cells. Further, we demonstrate that OA11 and OA14, but not OA15, cau se a decrease in this stimulatory activity of rbGH over a hormone concentra tion range of 5-50 ng/ml at both 24 and 48 h. We conclude that the different in vitro activities of the two in vivo enhan cing MAbs are most probably related to the time-courses over which these tw o assays are performed, and also to the relative affinities between antibod y, hormone and receptor. In addition, the in vitro inhibitory activity of t he enhancing MAb OA11 in both short- and long-term bioassay lends further s upport to an exclusively in vivo model for MAb-mediated enhancement of GH a ction.