LJP 993, a tetravalent conjugate of the a
mino-ter
minal do
main (do
main 1) of
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2GPI, was synthesized,and studies were carried out to explore the ability of LJP 993 to bind anti-
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2GPI antibodies and tofunction as a B cell toleragen. Do
main 1 was expressed in
Pichia pastoris, and the N-ter
minus wassite-specifically
modified by a transa
mination reaction converting the N-ter
minal glycine to a glyoxylgroup. A tetravalent platfor
m was synthesized with linkers that ter
minate in a
minooxy groups. Thiswas acco
mplished by preparing an ethylene glycol-based heterobifunctional linker that contains botha Boc-protected a
minooxy group and a free pri
mary a
mine. The linker was used to
modify a tetravalentplatfor
m molecule by reacting the a
mino groups on the linker with 4-nitrophenyl carbonate esters onthe platfor
m to provide a linker-
modified platfor
m, and the Boc protecting groups were re
moved toprovide a tetravalent a
minooxy platfor
m. Glyoxylated do
main 1 was attached to the platfor
m to provideLJP 993 by for
mation of oxi
me bonds. The protein do
mains of LJP 993 retain activity as evidenced bythe ability of LJP 993 to bind to anti-
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2GPI antibodies. Dissociation constants (
Kd) for do
main 1 andLJP 993 bound to i
mmobilized affinity-purified anti-
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2GPI antibodies fro
m autoi
mmune thro
mbosispatients were deter
mined using surface plas
mon resonance. An i
mmunized
mouse
model was developedto test the ability of LJP 993 to act as a toleragen. A thiol containing do
main 1 analogue was expressedin insect cells using the baculovirus expression syste
m, and it was used to prepare an i
mmunogenicconjugate of do
main 1 and
malei
mide-derivatized keyhole li
mpet he
mocyanin (KLH). Mice werei
mmunized with the KLH conjugate, and spleen cells were harvested fro
m the i
mmunized
mice. Thecells were incubated with various concentrations of LJP 993 and transferred to
mice whose i
mmunesyste
ms had been co
mpro
mised by irradiation. The hosts were then boosted with the KLH-do
main 1conjugate, and after 7 days their antibody levels were
measured. Host
mice receiving cells that weretreated with LJP 993 produced significantly lower a
mounts of anti-do
main 1 antibodies than controlswhich received untreated cells, indicative of B cell tolerance.
