A monoclonal antibody with anti-D–like activity in murine immune thrombocytopenia requires Fc domain function for immune thrombocytopenia ameliorative effects (1)
The development of murine ITP models has enabled more in-depth studies of the molecular mechanism(s) of antibodies exhibiting anti-D–like activity in murine ITP. In a passive model of murine ITP, it was shown that ITP- ameliorative anti-RBC MoAbs also cause anemia and the level of RBC-binding activity seems to roughly relate with the level of therapeutic effect. In murine ITP models, one of the most widely used anti-D–like antibodies among independent research groups has been Ter119, a rat IgG2b antibody specific for the abundant surface glycoprotein glycophorin A complex on mouse RBCs. The ineffectiveness of Ter119 in ameliorating ITP when given via the intraperitoneal route, suggesting that routes of administration may play a significant role in therapeutic success.
The F(ab’)2 domain exhibits similar binding activity for the RBCs as intact Ter119. The absence of the Fc domain does not significantly modulate Ter119-binding activity for RBCs. Despite binding to RBCs, the Ter119 F(ab”)2 fragment was unable to significantly ameliorate ITP or induce significant anemia in vivo. The continued presence of F(ab’)2 fragments of Ter119 on RBCs in vivo was significantly reduced compared to the intact Ter119.
The Ter119 F(ab’)2 domain was also unable to mediate the phagocytosis of murine RBCs by RAW264.7 macrophages compared with intact Ter119.
Similar to the ineffectiveness of Ter119 F(ab’ )2 fragments, deglycosylation significantly inhibited Ter119’s ability to ameliorate ITP compared with the intact Ter119. Although deglycosylated Ter119 shows a moderate level of efficacy at 4 hours after ITP induction compared with the PBS control, this effect became undetectable at 24 hours. Similar to its inhibitory effect on ITP amelioration, deglycosylation also significantly reduced Ter119-induced anemia, an effect supported by the higher level of remaining deglycosylated Ter119 bound to RBCs.
Ter119 improves MWReg30-induced ITP, and that MWReg30 is known to induce ITP primarily through FcgRIII suggests a possible interaction between Ter119 and FcgRIII. The ability of Ter119 to ameliorate ITP induced by the IgG2a antibody 6A6, an antibody known to induce ITP predominantly through FccRIV was tested. Ter119 ameliorated 6A6-induced ITP, to a similar extent as IVIG.
The MoAb 2.4G2, a known antibody that blocks the phagocytic activity of FcgRIIB and FcgRIII was used. Ter119-mediated anemia was not reduced by pretreatment with 2.4G2 Fab.
FccRIV is the murine counterpart of human FccRIIIA, both of which are known to exhibit dramatically increased affinity for the afucosylated variant of IgG. An afucosylated variant of Ter119 was generated using the enzyme inhibitor kifunensine. Intact Ter119 exhibits a glycan profile typical of a recombinant IgG lacking F(ab’)2 glycosylation, comprising predominantly fucosylated, nonsialylated, glycan species. The afucosylated Ter119 exhibits a single uniform oligomannose glycoform devoid of the core fucose.
Unexpectedly, the afucosylated Ter119 induced slightly decreased anemia at the 24-hour time point compared with the intact Ter119. Similarly, afucosylated Ter119 mediates an equivalent level of ITP amelioration as the wild-type Ter119.
