Matthew D. Linnik, David S. Jones, Mary-Ann Campbell, Michael J. Branks, G. Michael Iverson, Edward J. Victoria, Patricia A. McNeeley, Keith A. Cockerill

La Jolla Pharmaceutical Company, San Diego, CA.

Autoantibodies directed against ß2-glycoprotein I (ß2-GPI) have been implicated in the thrombotic events associated with antiphospholipid syndrome (APS). The presence of these antibodies is an independent risk factor for arterial and venous thrombosis and greatly increases the risk of a recurrent thrombotic event. Our labs have recently demonstrated that anti- ß2-GPI antibodies from APS patients specifically target the amino terminal domain (domain 1) of ß2-GPI (PNAS 95:15542, 1998). This discovery led to the development of a B cell toleragen-based approach to specifically deplete anti- ß2-GPI antibodies and the B cells that produce these antibodies. Four copies of recombinant human ß2-GPI (h ß2-GPI) domain 1 were covalently attached to a polyethylene glycol-containing platform to produce a tetravalent bioconjugate of domain 1, LJP 1082. LJP 1082 bound to immobilized affinity purified APS antibodies with a higher affinity than domain 1 alone (Kd'= 42-65 nM for LJP 1082 and 326-376 nM for domain 1 binding to antibodies from 3 APS patients). An immunized rat model was developed to evaluate the ability of LJP 1082 to tolerize ß2-GPI B cells in vivo. Rats were immunized with h ß2-GPI domain 1 coupled to keyhole limpet hemocyanin (D1-KLH) and treated approximately 3 weeks later with LJP 1082. Five days after exposure to LJP 1082 rats were boosted with D1-KLH and splenic B cells and serum antibody levels were evaluated at 7 days. Vehicle-treated control rats developed h ß2-GPI specific B cells (35 positive cells/106 spleen cells) and anti-h ß2-GPI antibodies (7000 U/ml serum). Treatment with a single dose of LJP 1082 (4.5 mg/kg) prior to boosting with D1-KLH caused almost complete tolerance as evidenced by the reduction in hß2-GPI B cells (< 3 positive cells/106 spleen cells) and anti-hß2-GPI antibodies (< 300 U/ml serum). We conclude that a multivalent conjugate of hß2-GPI domain 1 is capable of inducing significant B cell tolerance in an immunized rat model. Treatment of APS patients with a B cell toleragen would remove the pro-thrombotic risk factor, anti- ß2-GPI antibodies, without the risks associated with current anticoagulation therapy.

Presented at the 26th International Stroke Conference
Fort Lauderdale, FL.
February 14-16, 2001.