111 Binding and signalling properties of a GH-enhancing monoclonal antibody

Abstract

We have recently described in detail the structure of an epitope defined by a monoclonal antibody which enhances in vivo the biological activity of bovine GH @GH) [ 11. Although several theories have been advanced in relation to the mechanism associated with this enhancement phenomenon, there is little conclusive data supporting any hypothesis [2]. To examine the molecular and cellular aspects of this phenomenon more closely, we have undertaken studies using the well characterised GH-responsive 3T3-F442A preadipocyte cell line and the FDCP-I cell line transfected with full length mouse growth hormone receptor (GHR). As a prelude to this, we examined the binding properties of OA15 in relation to the subsequent interaction between bGH and its receptor. Initial examination of the epitope defined on bGH by OA15 suggested that it was removed from binding sites 1 and 2 on GH required for interaction with the GH receptor [3]. Using the recombinant bGH binding protein (bGHBP), which represents the extracellular domain of the receptor, we have demonstrated, using optical evanescent wave biosensor technology, that engagement of bGH by OA15 still allows for the interaction between bGH and bGHBP. Although further experiments are underway to investigate the effects on the affinity of bGH-bGHBP interaction following bGH binding to OA15, at this stage we can tentatively conclude that these real time binding studies agree with our initial epitope mapping work ie that the epitope defined by OA15 is removed from receptor interaction sites on the hormone. The results of these experiments suggested that in cell culture studies tnpartite complexes of antibody-hormone and receptor would exist. In the context of mechanistic studies, this in turn would suggest that OA15 would not inhibit GH signalling events through disruption of GH-GHR interactions. To examine this hypothesis in more detail we used two GH-responsive cell culture systems the 3T3-F442A preadipocyte cell line and IL-3 dependant FDCP-I cell line which has been transfected with the full length mouse GHR [4]. In F442A cells, treatment with GH leads to the rapid tyrosine phosphorylation of proteins of the JAK-STAT-MAP pathway(s). Figure 1 is an antiphosphotyrosine (py) blot of lysates from F442A cells treated with bGH alone or hormone pre-complexed with OA15. As can be clearly seen, GH treatment stimulates the level of a 9lkDa tyrosine phosphorylated protein. This is most likely to be a member of the Stat (signal transducers and activators of transcription) family of transcription factors. The level of tyrosine phoshporylated 9 lkDa