*Purpose: Triple-knockout (TKO) pigs are likely to be an optimal source of organs for transplantation into human recipients, many of whom do not have natural antibodies against TKO pig cells. However, Old World monkeys (OWMs), e.g., baboons, have natural antibodies directed to TKO cells, i.e., to a ‘fourth’ xenoantigen that appears when N-glycolylneuraminic acid (Neu5Gc) is deleted. The aim of this study was to evaluate in vitro (i) anti-pig IgM/IgG binding, and (ii) complement-dependent cytotoxicity (CDC) to several kinds of pig peripheral blood mononuclear cells (PBMCs) using primate sera. In addition, (iii) in vivo we compared the survival in baboons of renal grafts from pigs that either (a) expressed Neu5Gc (GTKO pigs) or (b) did not express Neu5Gc (GTKO/CMAHKO or TKO pigs) and therefore expressed the ‘fourth’ xenoantigen.

*Methods: In vitro, we measured (i) anti-pig IgM/IgG binding, and (ii) CDC to wild-type (WT), Gal-knockout (GTKO), and TKO pig PBMCs (none of which expressed any human ‘protective’ transgenes, e.g., CD46, CD55) using sera from humans (n=9), several OWMs (n=24; baboon [n=6], rhesus [n=6], cynomolgus [n=6], African green [n=6]), and two NWMs; (n=15; capuchin [n=9], squirrel [n=6]). In vivo, life-supporting renal transplants were carried out in baboons using GTKO (n=6) or CMAHKO (TKO or GTKO/CMAHKO) (n=6) pig kidneys under an anti-CD40mAb-based regimen.

*Results: In vitro studies: Mean anti-TKO IgM was significantly higher in OWMs (8.1) than in humans (3.4) (p<0.01), but was lower in NWMs (2.1) (p<0.05). CDC in OWMs to TKO PBMCs (74%) was significantly higher than in humans (3%) (p<0.01), but there was no significant difference between humans and NWMs (19%). In vivo studies: One baboon in each group required euthanasia within the first post-transplant week for acute gastric dilatation, and were excluded from further analysis. GTKO pig kidneys functioned for 90-260 days (median 237 days, mean 196 days), with histopathological features of antibody-mediated rejection (AMR) being seen in only 2 of 5. The rejection-free survival of pig kidneys not expressing Neu5Gc (TKO or GTKO/CMAHKO) was significantly shorter (p<0.05), with 4 of 5 grafts failing from AMR within 1-61 days (median 21 days; mean 26 days), though one baboon is ongoing at 5 months without features of rejection.

*Conclusions: Even though considered ideal for clinical xenotransplantation, the presence of natural antibodies to CMAHKO(TKO or GTKO/CMAHKO) pig cells in OWMs complicates the survival of CMAHKO pig kidneys in OWMs. This will make it difficult to provide the regulatory authorities with positive data to support the concept that TKO pig organs will provide long-term graft survival in humans. NWMs more closely mimic the human immune response to CMAHKO pig cells, but transplantation of pig organs into NWMs would be difficult because of the small size of these monkeys available in the USA.

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