A NOVEL THERAPEUTIC APPROACH FOR TREATING ANTIPHOSPHOLIPID SYNDROME BASED ON TOLERIZING ANTI-BETA2 GLYCOPROTEIN I B CELLS

Linnik MD, Jones DS, Campbell MA, Iverson GM, Cockerill KA

Introduction: Autoantibodies directed against the phospholipid binding protein _2-glycoprotein I (_2-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 recurrent thrombotic events. We demonstrated that anti-_2-GPI antibodies from APS patients specifically bind the amino terminal domain (D1) of _2-GPI (PNAS 95:15542, 1998). Approximately 90% of APS patients have antibodies specific for D1 (Thromb. Haemost. 86:590, 2001). This discovery led to the development of a B-cell Toleragen® designed to specifically deplete anti-_2-GPI antibodies and the B cells that produce them. B-cell toleragens act by cross-linking antigen-specific receptors. In the absence of T cell help, targeted B cells become unresponsive to further antigen stimulation.

Methods: Four copies of recombinant human D1 were covalently attached to a polyethylene glycol-containing platform to produce LJP 1082, a tetravalent bioconjugate. LJP 1082 was tested for T cell epitopes by culturing for six days with peripheral blood mononuclear cells (PBMCs) from healthy donors or patients with APS. On day six the cultures were pulsed with 3H-thymidine, and a stimulation index was calculated from the amount of 3H incorporated relative to PBMCs cultured with medium alone.

A rat model was developed to evaluate the ability of LJP 1082 to tolerize specific B cells in vivo. Rats were immunized with D1 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. Splenic B cells were evaluated 5 days after boost and serum antibody levels were evaluated 7 days after boost. Antibodies to KLH or D1 were detected by direct binding to KLH or recombinant human _2-GPI coated on microplate wells. D1-specific antibody forming cells were detected using an ELISPOT assay in which spleen cells were incubated in wells coated with _2-GPI. Both assays were developed using alkaline phosphatase conjugated goat anti-rat IgG.

Results: LJP 1082 did not stimulate proliferation of T cells in cultured PBMCs from healthy donors or APS patients. Rats immunized with D1-KLH developed antibodies to both D1 and KLH. Treatment with a single dose of LJP 1082 (2.5 mg/kg) prior to boosting with D1-KLH conferred almost complete tolerance as evidenced by a reduction in specific antibody forming B cells and anti-_2-GPI antibodies. The suppressive effect of LJP 1082 was dose-dependent, and antibodies to KLH were not affected.

Conclusions: LJP 1082 is a multivalent conjugate of domain 1 that suppresses specific antibodies and B cells in immunized rats.LJP 1082 does not stimulate in vitro proliferation of T cells from APS patients. A phase I-II clinical trial is evaluating the safety and tolerability of LJP 1082 in APS patients.

Presented at
FOCIS 2nd Annual Meeting
San Francisco, CA
June 28-July 1, 2002