Two types of oligonucleotides were synthesized with linker groups attached at the 5'-end. Both were repeating dimers of deoxyribocytidine and deoxyriboadenosine. A 20-mer was prepared with a thiol-containing linker, masked as a disulfide, and a 50-mer was prepared with a vicinal diol-containing linker. A tetraiodoacetylated poly(ethylene glycol) (PEG) derivative was synthesized and reacted with the thiol-containing 20-mer to provide an oligonucleotide PEG conjugate of precisely four oligonucleotides on each PEG carrier. The vicinal diol on the 50-mer was oxidized to an aldehyde and conjugated to keyhole limpet hemocyanin (KLH) to provide an oligonucleotide-KLH conjugate by reductive alkylation. The conjugates were annealed with complementary (TG)n strands. While the double-stranded oligonucleotide-KLH conjugate is an immunogen, eliciting the synthesis of antibodies against oligonucleotides, the PEG conjugate has the biological property of specifically suppressing (tolerizing) B cells which make antibodies against the immunizing oligonucleotide.

Alkylation↗

PubMedDate not supplied

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.

Amino Acid Sequence↗

PubMedDate not supplied

Multivalent thioether-peptide conjugates: B cell tolerance of an anti-peptide immune response.

Antibodies which bind beta2-glycoprotein I (beta2GPI) are associated with antiphospholipid syndrome. Synthetic peptide mimotopes have been discovered which compete with beta2GPI for binding to selected anti-beta2GPI. A thiol-containing linker was attached to the N-terminus of two cyclic thioether peptide mimotopes, peptides 1a and 1b. The resulting peptides, with linker attached, were reacted with two different haloacetylated platforms to prepare four tetravalent peptide-platform conjugates to be tested as B cell toleragens. The linker-containing peptides were reacted with maleimide-derivatized keyhole limpet hemocyanin (KLH) to provide peptide-KLH conjugates. Peptides 1a and 1b were also modified by acylation with 3-(4'-hydroxyphenyl)propionic acid N-hydroxysuccinimidyl ester. The resulting hydroxyphenyl peptides were radioiodinated and used to measure anti-peptide antibody levels. The KLH conjugates were used to immunize mice to generate an anti-peptide immune response. The immunized mice were treated with the conjugates or saline solution and boosted with the appropriate peptide-KLH conjugate. Three of the four conjugates suppressed the formation of anti-peptide antibody. The stabilities of the conjugates in mouse serum were measured, and the relative stabilities did not correlate with ability to suppress antibody formation.

Animals↗

PubMedDate not supplied

Body in the bog but no DNA.

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DNA↗