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 galactosyltransferase knockout (GTKO) porcine organs. Our analysis in multiple GTKO pig-to-primate models of xenotransplantation has 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 molecules with known safety profiles allowing rapid advancement to large animal models.


METHODS
The NIH clinical collections of small molecules were 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 molecule screening based on xenoantibody structure was used to assess the likelihood that the identified small molecules bound xenoantibody directly. As a proxy for selectivity, ELISAs against tetanus toxoid and 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 molecules were further tested for their ability to inhibit xenoantibody elicited in multiple settings, including rhesus monkeys pre-treated with an anti-non-Gal selective anti-idiotypic antibody, non-immunosuppressed rhesus monkeys immunized with wild-type fetal pig isletlike cell clusters, and non-immunosuppressed baboons transplanted with GTKO multiple transgenic pig kidneys.


RESULTS
Four clinically relevant small molecules inhibited 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 toxoid or pre-existing natural antibodies against laminin, thyroglobulin, or ssDNA. Furthermore, clonidine inhibited elicited anti-non-Gal IgM from all animals that demonstrated a xenoantibody response in each experimental setting.


CONCLUSIONS
Clinically relevant small molecule drugs with known safety profiles can inhibit xenoantibody elicited against non-Gal antigens in diverse experimental xenotransplantation settings. 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.
Department of Human Anatomy, Loma Linda University School of Medicine, Loma Linda, CA, USA.
John M
http://orcid.org/0000-0002-6400-4188
JM
Stewart
Departments of Pediatrics and Cell Biology and Human Anatomy, and California National Primate Research Center, University of California, Davis, CA, USA.
Alice F
AF
Tarantal
Centre for Transplant and Renal Research, Westmead Millennium Institute, Westmead, NSW, Australia.|National Pancreas Transplant Unit, University of Sydney at Westmead Hospital, Westmead, NSW, Australia.
Wayne J
WJ
Hawthorne
Immunology Research Centre, St. Vincent's Hospital Melbourne, Melbourne, Vic., Australia.
Evelyn J
EJ
Salvaris
Centre for Transplant and Renal Research, Westmead Millennium Institute, Westmead, NSW, Australia.|National Pancreas Transplant Unit, University of Sydney at Westmead Hospital, Westmead, NSW, Australia.
Philip J
PJ
O'Connell
Discipline of Obstetrics and Gynaecology, University of Adelaide, Adelaide, SA, Australia.
Mark B
MB
Nottle
Immunology Research Centre, St. Vincent's Hospital Melbourne, Melbourne, Vic., Australia.|Department of Medicine, University of Melbourne, Melbourne, Vic., Australia.
Anthony J F
AJ
d'Apice
Immunology Research Centre, St. Vincent's Hospital Melbourne, Melbourne, Vic., Australia.|Department of Medicine, University of Melbourne, Melbourne, Vic., Australia.
Peter J
http://orcid.org/0000-0001-9016-4954
PJ
Cowan
Department of Human Anatomy, Loma Linda University School of Medicine, Loma Linda, CA, USA.
Mary
M
Kearns-Jonker
D000818:Animals; D000910:Antibodies, Heterophile / Q000097:blood*; D003000:Clonidine / Q000494:pharmacology*; D055786:Gene Knockout Techniques; D064593:Heterografts / Q000276:immunology*; D007075:Immunoglobulin M / Q000276:immunology; D008253:Macaca mulatta; D023421:Models, Animal; D010215:Papio / Q000276:immunology*; D034421:Sus scrofa; D013552:Swine; D014183:Transplantation, Heterologous / Q000379:methods
D000910:Antibodies, Heterophile; D007075:Immunoglobulin M; D003000:Clonidine
baboon; clonidine; endothelial cell; islet; kidney; pig; rhesus monkey; small molecule; xenotransplantation