Domain Subclass Conversion Improved Activity of Anti-Mpl Agonist Antibodies in the Form of Whole IgG.

Since the molecular cloning of thrombopoietin (TPO), two types of recombinant human TPO, full-length recombinant human TPO (rhTPO) and pegylated recombinant human megakaryocyte growth and development factor (PEG-rhMGDF), have been tested in a variety of clinical trials. The agents showed clinical efficacy in some cases. However, neutralizing antibodies against endogenous TPO were generated in some healthy volunteers who received PEG-rhMGDF, resulting in thrombocytopenia. Recently, several TPO mimetics are being developed, such as peptide-based molecules, nonpeptidyl small compounds and agonist antibodies. Here we report generation of novel fully human anti-human Mpl agonist antibodies. Two lead monoclonal antibodies MA01 and MA02 were cloned from human antibody-producing mice (KM mice™) immunized with human Mpl-expressing cells. Both MA01 and MA02 were of immunoglobulin gamma 1 (IgG1). These IgG1 agonists promoted proliferation of UT7/TPO cells in vitro, but were >100-fold less effective than TPO. We, therefore, genetically modified MA01 and MA02 in order to enhance the activity and to minimize the effector function. First, constant heavy (CH) region of MA01 was converted to IgG4 (MA01G4) to reduce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). This conversion affected neither binding affinity nor agonistic activity of MA01. Next, hinge region of MA01G4 was changed to human IgG3 hinge (MA01G4344). MA01G4344 showed no change in binding affinity, but showed 10-fold higher activity than original MA01. In the case of MA02, the CH region was converted from G1 to G3344 to generate MA02G3344, in which CH1-hinge region was of IgG3 and CH2-CH3 region was of IgG4. The conversion drastically improved agonistic activity. Although MA02 (G1) activated Jak2 and Erk1/2 but not STAT5 and Akt, MA02G3344 activated all of them. These data suggest that conversion of CH1-hinge region to IgG3 type is essential for agonistic activity of anti-Mpl antibodies. The effective concentration 50 (EC50) values of MA01G4344 and MA02G3344 in UT7/TPO assay were in the range of 0.01–0.1nM. These antibodies stimulated human cord blood-derived CD34+ cells to give rise to CFU-MK colonies without any other supportive cytokines. A single injection of MA01G4344 increased platelet count of human Mpl transgenic mice for over one month. These domain subclass-converted antibodies are expected to provide a new medical option for treatment of chronic thrombocytopenia without losing good properties of natural antibody such as long serum half-life and low immunogenicity.

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