DEVELOPMENT OF MULTIVALENT TOLERAGENS TO INHIBIT BINDING OF HUMAN ANTI-GALACTOSE a1,3 GALACTOSE ANTIBODIES.

Lin Yu, Ph.D., Fang Xie, Ph.D., David S. Jones, Ph.D., Miriam Ramirez, B.S. and Richard M. Jack, Ph.D.

Departments of Chemistry and Immunology, La Jolla Pharmaceutical, San Diego, CA 92121

Natural human antibodies to the galactose a1,3 galactose (aGal) epitope expressed on porcine organs for xenografting mediate both hyperacute rejection (HAR) and delayed xenograft rejection (DXR). Suppression of the formation of anti-aGal antibodies would decrease dramatically these rejection events. We developed Toleragens designed to both clear circulating aGal antibodies and stop their production by tolerizing the B cell population producing the pathogenic antibody. We sought to test the effect of aGal valency on the ability of Toleragens to inhibit anti-aGal Ab from binding to aGal-expressing proteins and cells. Chemically synthesized aGal was coupled to non-immunogenic platforms at valencies of 2-8 aGal molecules/platform to produce dimeric to octameric toleragens. Monomeric aGal, toleragens, aGal neoglycoconjugates and irrelevant monosaccharides at final concentrations of 200 nM to 4 mM or buffer alone were incubated with normal human serum. Doses which caused fifty percent inhibition (IC50) in the binding of IgM anti-aGal IgM on ELISA plates bearing aGal-BSA or on PK-15 cells by flow cytometry were calculated for each molecule. Results showed that there was a valency-dependent linear diminution in IC50. That is, while monomeric aGal molecules (aGal, trisaccharide or pentasaccharide) displayed an IC50 of 1 mM, increasing Toleragen valency to octamer aGal resulted in an IC50 of 50 uM, a twenty-fold increase in efficacy. We conclude that increased aGal valency on Toleragen platforms results in much increased ability of toleragens to bind circulating IgM likely through an increase in the avidity of anti-aGal IgM for the higher valency toleragen. Preliminary in vivo results show that octameric aGal Toleragen is also able to bind both anti-aGal IgM and IgG by contrast to tetrameric anti-aGal Toleragen which only bound IgG in vivo. The ability of these Toleragens to tolerize the anti-aGal IgM B cell response is being tested.

Presented at the 25th Annual Scientific Meeting of the American Society of Transplant Surgeons, May 19-21, 1999, Chicago, IL.