Clonidine inhibits anti-non-Gal IgM xenoantibody elicited in multiple pig-to-primate models

Background Survival of vascularized xenografts is dependent on pre-emptive inhibition of the xenoantibody response against galactosyltransferaseknockout (GTKO) porcine organs. Our analysis in multiple GTKO pig-to-primate models of xenotransplantationhas demonstrated that the anti-non-gal-α-1,3-gal (anti-non-Gal) xenoantibody response displays limited structural diversity. This allowed our group to identify an experimental compound which selectively inhibited induced anti-non-Gal IgM xenoantibodies. However, because this compound had an unknown safety profile, we extended this line of research to include screening small moleculeswith known safety profiles allowing rapid advancement to large animal models. Methods The NIH clinical collections of small moleculeswere screened by ELISA for their ability to inhibit xenoantibody binding to GTKO pig endothelial cells. Serum collected from non-immunosuppressed rhesus monkeys at day 14 post-injection with GTKO pig endothelial cells was utilized as a source of elicited xenoantibody for initial screening. Virtual small moleculescreening based on xenoantibody structure was used to assess the likelihood that the identified small moleculesbound xenoantibody directly. As a proxy for selectivity, ELISAs against tetanus toxoidand the natural antigens laminin, thyroglobulin, and single-stranded DNA (ssDNA) were utilized to assess the ability of the identified reagents to inhibit additional antibody responses. The identified inhibitory small moleculeswere further tested for their ability to inhibit xenoantibody elicited in multiple settings, including rhesus monkeys pre-treated with an anti-non-Gal selective anti- idiotypicantibody, non-immunosuppressed rhesus monkeys immunized with wild-type fetal pigisletlike cell clusters, and non-immunosuppressed baboons transplanted with GTKO multiple transgenic pig kidneys. Results Four clinically relevant small moleculesinhibited anti-non-Gal IgM binding to GTKO pig endothelial cells in vitro. Three of these drugs displayed a limited region of structural similarity suggesting they may inhibit xenoantibody by a similar mechanism. One of these, the anti-hypertensive agent clonidine, displayed only minimal inhibition of antibodies elicited by vaccination against tetanus toxoidor pre-existing natural antibodies against laminin, thyroglobulin, or ssDNA. Furthermore, clonidineinhibited elicited anti-non-Gal IgM from all animals that demonstrated a xenoantibody response in each experimental setting. Conclusions Clinically relevant small moleculedrugs with known safety profiles can inhibit xenoantibody elicited against non-Gal antigens in diverse experimental xenotransplantationsettings. These molecules are ready to be tested in large animal models. However, it will first be necessary to optimize the timing and dosing required to inhibit xenoantibodies in vivo.