Abstract

Abstract 2205

Background:

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.

Results:

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.

Conclusions:

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.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.