Pharmacokinetics, Serum Stability and Immunogenicity of A Polypeptide Inhibitor of B2GPI/Antibody Complexes Tested in Mice.

Antiphospholipid syndrome (APS) is an autoimmune disease with clinical features of thrombosis and pregnancy loss. Beta2-glycoprotein I (B2GPI) is the major antigen for APS-related antibodies. We engineered a polypeptide consisting of two ligand-binding A1 modules from the ApoE receptor 2. Previously, we demonstrated that this polypeptide, A1-A1, preferentially binds B2GPI/antibody complexes compared to B2GPI alone and efficiently inhibits the binding of B2GPI/antibody complexes to negatively charged phospholipids. Therefore, A1-A1 effectively interferes with two pathological mechanisms of B2GPI/antibody complexes: the binding to anionic phospholipids and ApoER2. In order to use A1-A1 to study pathological mechanisms of B2GPI/antibody complexes in vivo, we tested its pharmacokinetic, serum stability and immunogenicity in mice. To visualize A1-A1, we labeled it with a fluorescent probe Atto-488 attached to the N-terminus.

We monitored clearance of A1-A1 from the circulation after intraperitoneal and intravenous administration. After intraperitoneal administration, the concentration of A1-A1 in the blood reached its maximum at 30 min after injection and cleared from the blood in 6–8 hours. When A1-A1 was injected intravenously, 14% of A1-A1 remained in the blood 1 hour after administration and decreased to 4% in 3 hours. We assessed the binding of A1-A1 to serum proteins in both mouse and human serum by gel-filtration chromatography. Chromatograms of A1-A1 in both mouse and human serum collected just after mixing of A1-A1 with serum were almost identical to those collected after 2 hours of incubation at 37° C. About 90% of A1-A1 stays free from serum proteins. Previously, we demonstrated that A1-A1 has a favorable stability in human serum. More than 35% of A1-A1 remained in human serum after 15 days of incubation at 37° C. Here, we determined whether A1-A1 is cleaved by proteases in mouse serum. Degradation of A1-A1 was monitored by the reversed-phase HPLC by comparing the peak corresponding to intact A1-A1 and A1-A1 incubated with serum for 2 hours at 37° C. After incubation with mouse serum, A1-A1 eluted at the same time as intact A1-A1 and the intensity of the elution peak did not decrease, indicating that A1-A1 remains intact in mouse serum. To evaluate immunogenecity of A1-A1, we immunized mice with A1-A1, A1-A1 in the presence of adjuvant and A1-A1 conjugated to a carrier protein. A1-A1 did not induce detectable anti-A1-A1 IgG production even in the presence of adjuvant or carrier protein.

A1-A1 has favorable properties for use in vivo. It stays in the circulation for more than one hour following intravenous injection. After intraperitoneal administration, A1-A1 is rapidly absorbed into blood and cleared in about 6 hours. A1-A1 is resistant to both human and mouse proteases, has low immunogenicity and its amount in the blood is not depleted by binding to serum proteins.

No relevant conflicts of interest to declare.