To explore the potential for monoclonal antibodies as a treatment for immune thrombocytopenia (ITP) and to further explore their mechanisms of action, we tested 8 monoclonal CD44 antibodies in murine ITP and found 4 antibodies that could successfully ameliorate ITP; 2 of these antibodies function at a full 3-log fold lower dosage compared with IVIg. Further characterization of the 2 most successful antibodies (5035-41.1D and KM114) demonstrated that, similar to IVIg: (1) the presence of the inhibitory IgG receptor FcγRIIB was required for their ameliorative function, (2) complement-deficient mice responded to anti-CD44 treatment, and (3) human transgenic FcγRIIA-expressing mice also responded to the CD44 therapeutic modality. Dissimilar to IVIg, the Fc portion of the CD44 antibody was not required. These data demonstrate that CD44 antibodies can function therapeutically in murine ITP and that they could potentially provide a very-low-dose recombinant therapy for the amelioration of human ITP.
2528 Intravenous immunoglobulin (IVIg) is used to treat autoimmune diseases such as ITP. IVIg is a limited resource and its dosage and cost are both high. Although considered safe, it will always carry a theoretical risk of transferring infectious disease. Thus it would thus be highly desirable to improve the efficacy of IVIg or develop monoclonal antibodies, capable of mimicking the clinical effects of IVIg. Work by others has successfully improved the efficaciousness of IVIg in murine models of autoimmunity: It has been shown that enriched sialylated IVIg has a therapeutic effect at a 1 log fold lower dosage than IVIg; and a sialylated recombinant Fc fragment of IgG functions successfully at a 2 log fold lower dosage than IVIg. CD44 is a widely expressed cell surface polymorphic glycoprotein. It is involved in many processes including tumour metastasis and inflammation. CD44 antibodies have been successfully used to treat several murine autoimmune disease models, including inflammatory arthritis and experimental autoimmune encephalomyelitis, although their mechanism of action remains unclear. To explore the potential for monoclonal antibodies as a treatment for ITP as well as further explore their mechanisms of action, we tested 8 monoclonal CD44 antibodies in murine ITP, and found 4 antibodies which could successfully ameliorate ITP; 2 of these antibodies function at a full 3 log fold lower dosage as compared to IVIg. Further characterization of the 2 most successful antibodies (5035-41.1D and KM114) demonstrated that, similar to IVIg, i) the expression of the inhibitory IgG receptor FcγRIIB was required for their ameliorative function and ii) complement-depleted mice also responded to anti-CD44 treatment. Dissimilar to IVIg, the Fc portion of the CD44 antibody was not required: an F(ab')2 fragment of antibody KM114 also significantly ameliorated thrombocytopenia at an equivalent molar concentration as intact KM114. Thus while KM114 functions by an FcγRIIB sensitive mechanism, FcγRIIB is unlikely a direct target of the Fc region of this antibody and likely plays a downstream role in the amelioration of immune thrombocytopenia, similar to IVIg. These data demonstrate that CD44 antibodies can function therapeutically in murine ITP and we speculate that they could potentially provide a very low dose recombinant therapy for the treatment of ITP. Disclosures: No relevant conflicts of interest to declare.
2529 There are several theories as to the mechanism of intravenous immunoglobulin (IVIg) in the treatment of autoimmune diseases such as immune thrombocytopenia (ITP). One prominent theory involves accelerated pathogenic autoantibody clearance by saturation of the neonatal Fc Receptor (FcRn). FcRn is an IgG receptor, and FcRn within endosomes binds endocytosed IgG and diverts IgG from degradation. In the treatment of ITP, it has been theorized that high concentrations of IVIg saturate FcRn, reducing the ability of the pathogenic anti-platelet antibodies to bind FcRn, increasing their catabolism, and thus rapidly decreasing their serum concentration which results in a decrease in their ability to induce platelet clearance. Mice lacking FcRn have only 20–30% of the level of endogenous IgG as compared with wild-type mice and have an accelerated clearance rate of both endogenous and injected IgG. We have utilized these mice in a murine model of ITP to help understand the role, if any, that FcRn plays in IVIg's therapeutic activity. Both IVIg (2 g/kg body weight) and monoclonal antibodies to CD44 (2 mg/kg body weight; please see submitted abstract ID# 29504, “Amelioration of murine immune thrombocytopenia by CD44 antibodies: a potential therapy for ITP?”) successfully ameliorate murine ITP. To definitively determine if FcRn is required for the acute amelioration of ITP by these two therapeutics, we employed FcRn deficient mice in the murine ITP model. Here, we demonstrate that FcRn deficient mice (B6.129X1-Fcgrttm1Dcr/DcrJ) injected with an anti-platelet antibody exhibit a slightly more profound degree of thrombocytopenia than wild-type mice. FcRn deficient mice treated with IVIg or the anti-CD44 antibody KM114 (at a 3 log fold lower dosage than IVIg) were protected from ITP to the same extent as wild-type mice. FcRn has an absolute requirement for the protein β2 microglobulin (β2M) to be functionally expressed. Specifically, β2M deficient mice do not possess functional FcRn and also show low endogenous IgG levels and increased clearance of IgG. To verify and substantiate the results found with FcRn deficient mice, we next employed β2M deficient mice in the murine ITP model and found that β2M deficient mice (B6.129P2-B2mtm1Unc/J) treated with IVIg or KM114 were also protected from ITP to the same extent as wild-type mice. These data suggest that for both high dose IVIg as well as low dose monoclonal CD44 antibody treatment in an acute ITP model, FcRn expression is dispensable. Disclosures: No relevant conflicts of interest to declare.
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