Neutralization of biological activity and inhibition of receptor binding by antibodies against human thrombopoietin

Thrombopoietin (TPO) is a recently isolated cytokine that primarily regulates megakaryocytopoiesis and thrombopoiesis. We recently reported the development of a variety of antibodies (Abs) to synthetic peptides of human (h)TPO and to recombinant human TPO (rhTPO). In this study, we characterized the Abs and mapped immunologically distinct areas of the molecule. Among the five different antipeptide polyclonal Abs, only one, raised against synthetic peptide D⁸ to Q²⁸, neutralized the TPO-dependent growth of FDCP-2 cells expressing human Mpl (FDCP-hMpl5 cells). One out of seven anti-rhTPO monoclonal Abs, designated as TN1, also showed neutralizing activity. TN1 was found to be specifically reactive with two proteolytic fragments, residues S¹ to R¹¹⁷ and A⁶⁰ to K¹²² of hTPO, indicating that the epitope(s) of TN1 is localized in residues A⁶⁰ to R¹¹⁷ of the molecule. These two neutralizing Abs inhibited the binding of biotinylated rhTPO to FDCP-hMpl5 cells. On the other hand, the other Abs, which reacted with five polypeptides of S⁴⁷ to D⁶², L¹⁰⁸ to A¹²⁶, N¹⁷² to A¹⁹⁰, S²⁶² to T²⁸⁴, and P³⁰⁶ to G³³² of hTPO, did not show either the neutralizing activity or the ability to inhibit the binding of biotinylated rhTPO to the cell surface hMpl. These findings indicate that two regions, residues D⁸ to Q²⁸ and A⁶⁰ to R¹¹⁷ of hTPO, may contain the domains associated with its receptor, c-Mpl. These Abs characterized here are valuable for studying the structural analysis and the biological function of hTPO mediated by its receptor.