Synthesis of LJP 993, a multivalent conjugate of the N-terminal domain of beta(2)GPI and suppression of an anti-beta(9)2GPI immune response

LJP 993, a tetravalent conjugate of the amino-terminal domain (domain 1) of beta (2)GPI, was synthesized, and studies were carried out to explore the ability of LJP 993 to bind anti-beta (2)GPI antibodies and to function as a B cell toleragen. Domain 1 was expressed in Pichia pastoris, and the N-ter minus was site-specifically modified by a transamination reaction convertin g the N-terminal glycine to a glyoxyl group. A tetravalent platform was syn thesized with linkers that terminate in aminooxy groups. This was accomplis hed by preparing an ethylene glycol-based heterobifunctional linker that co ntains both a Boc-protected aminooxy group and a free primary amine. The li nker was used to modify a tetravalent platform molecule by reacting the ami no groups on the linker with 4-nitrophenyl carbonate esters on the platform to provide a linker-modified platform, and the Boc protecting groups were removed to provide a tetravalent amino,oxy platform. Glyoxylated domain 1 w as attached to the platform to provide LJP 993 by formation of oxime bonds. The protein domains of LJP 993 retain activity as evidenced by the ability of LJP 993 to bind to anti-beta (2)GPI antibodies. Dissociation constants (K-d) for domain I and LJP 993 bound to immobilized affinity-purified anti- beta (2)GPI antibodies from autoimmune thrombosis patients were determined using surface plasmon resonance. An immunized mouse model was developed to test the ability of LJP 993 to act as a toleragen. A thiol containing domai n 1 analogue was expressed in insect cells using the baculovirus expression system, and it was used to prepare an immunogenic conjugate of domain 1 an d maleimide-derivatized keyhole limpet hemocyanin (KLH). Mice were immunize d with the KLH conjugate, and spleen cells were harvested from the immunize d mice. The cells were incubated with various concentrations of LJP 993 and transferred to mice whose immune systems had been compromised by irradiati on. The hosts were then boosted with the KLH-domain 1 conjugate, and after 7 days their antibody levels were measured. Host mice receiving cells that were treated with LJP 993 produced significantly lower amounts of anti-doma in I antibodies than controls which received untreated cells, indicative of B cell tolerance.