EFFECT OF aGAL VALENCY IN THE INHIBITION OF BINDING OF HUMAN ANTI-aGAL ANTIBODIES.

Richard Jack, Lin Yu, Fang Xie, Edcon Chang and Miriam Ramirez.

La Jolla Pharmaceutical Co., 6455 Nancy Ridge Drive, San Diego, CA USA

Natural human antibodies (Ab) to the galactose a1,3 galactose (aGal) epitope, which is expressed at high levels on porcine organs used in xenotransplantation, mediate both hyperacute rejection (HAR) and acute vascular rejection (AVR). Removal of these antibodies from non-human primates temporarily extends the lifespan of a xenograft. Removal and long-term suppression of the formation of anti-aGal antibodies (aGal B cell tolerance) would decrease dramatically these rejection events and thus significantly extend the functional lifespan of a xenograft. We have developed multivalent therapeutic drugs (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 increasing aGal valency on the ability of Toleragens to inhibit human anti-aGal AB from binding to aGal. Chemically synthesized aGal was coupled to non-immunogenic platforms at valencies of 2-8 to produce dimeric to octameric toleragens. Monomeric aGal, toleragens with valencies of 2-8, aGal glycoconjugates and control monosaccharides [200 nM-4 mM] or buffer were incubated with normal human serum. Doses which caused fifty percent inhibition (IC50) in the binding of IgM anti-aGal on ELISA plates bearing aGal-BSA were calculated for each molecule.

Results showed that increasing valency resulted in a linear dimunition in IC50. That is, while monomeric aGal molecules (aGal, tri- or pentasaccharide) displayed an IC50 of ~1 mM, increasing Toleragen valency to octameric 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 the increased ability of toleragens to bind circulating IgM through an increase in the avidity of anti-aGal IgM for the higher valency toleragen.

Preliminary in vivo results in primates showed that octameric aGal Toleragen binds both anti-aGal IgM and IgG by contrast to tetrameric anti-aGal Toleragen that only bound IgG in vivo. The ability of these multivalent Toleragens to clear anti-aGal Ab and to tolerize the anti-aGal IgM B cell response is being tested in both primates and galactosyl transferase knockout mice.

Presented at the 5th Congress of the International Xenotransplantation Association, Oct. 24-28, 1999, Nagoya, Japan.