Synthesis of LJP 993, a multivalent conjugate of the N-terminal domain of beta2GPI and suppression of an anti-beta2GPI immune response.
LJP 993, a tetravalent conjugate of the amino-terminal domain (domain 1) of beta2GPI, was synthesized, and studies were carried out to explore the ability of LJP 993 to bind anti-beta2GPI antibodies and to function as a B cell toleragen. Domain 1 was expressed in Pichia pastoris, and the N-terminus was site-specifically modified by a transamination reaction converting the N-terminal glycine to a glyoxyl group. A tetravalent platform was synthesized with linkers that terminate in aminooxy groups. This was accomplished by preparing an ethylene glycol-based heterobifunctional linker that contains both a Boc-protected aminooxy group and a free primary amine. The linker was used to modify a tetravalent platform molecule by reacting the amino 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 aminooxy platform. Glyoxylated domain 1 was 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-beta2GPI antibodies. Dissociation constants (Kd) for domain 1 and LJP 993 bound to immobilized affinity-purified anti-beta2GPI 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 domain 1 analogue was expressed in insect cells using the baculovirus expression system, and it was used to prepare an immunogenic conjugate of domain 1 and maleimide-derivatized keyhole limpet hemocyanin (KLH). Mice were immunized with the KLH conjugate, and spleen cells were harvested from the immunized mice. The cells were incubated with various concentrations of LJP 993 and transferred to mice whose immune systems had been compromised by irradiation. 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-domain 1 antibodies than controls which received untreated cells, indicative of B cell tolerance.
Jones
D S
DS
La Jolla Pharmaceutical Company, 6455 Nancy Ridge Drive, San Diego, California 92121, USA. dave.jones@ljpc.com
Cockerill
K A
KA
Gamino
C A
CA
Hammaker
J R
JR
Hayag
M S
MS
Iverson
G M
GM
Linnik
M D
MD
McNeeley
P A
PA
Tedder
M E
ME
Ton-Nu
H T
HT
Victoria
E J
EJ
D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation / Q000187:drug effects; D000925:Anticoagulants / Q000008:administration & dosage / Q000737:chemistry / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001323:Autoantibodies / Q000276:immunology; D001562:Benzimidazoles / Q000138:chemical synthesis* / Q000378:metabolism; D017690:Cell Transplantation; D003432:Cross-Linking Reagents / Q000737:chemistry; D004987:Ethers / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D005260:Female; D006023:Glycoproteins / Q000008:administration & dosage / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007108:Immune Tolerance / Q000187:drug effects*; D007659:Ketones / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000008:administration & dosage / Q000276:immunology*; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D013154:Spleen / Q000166:cytology; D013927:Thrombosis / Q000276:immunology; D053482:beta 2-Glycoprotein I
D000925:Anticoagulants; D001323:Autoantibodies; D001562:Benzimidazoles; D003432:Cross-Linking Reagents; D004987:Ethers; D006023:Glycoproteins; D007659:Ketones; C447679:LJP 993; D010446:Peptide Fragments; D053482:beta 2-Glycoprotein I; C447680:hoechst 32258