Old World Monkeys are less than ideal transplantation models for testing pig organs lacking three carbohydrate antigens (Triple-Knockout)

Abstract

Triple-knockout (TKO) pigs (with added protective human transgenes) are likely to be optimal sources of organs for clinical organ xenotransplantation because many humans have minimal or no natural antibody to TKO pig cells. However, Old World monkeys (OWMs) have naturally-existing antibodies directed to TKO cells. We measured anti-pig IgM/IgG binding, and complement-dependent cytotoxicity to wild-type (WT), α1,3-galactosyltransferase gene-knockout (GTKO), and TKO pig peripheral blood mononuclear cells (PBMCs) using sera from humans, several OWMs, and two New World monkeys (NWMs). Furthermore, we compared survival of GTKO (n = 5) and TKO (n = 3) pig kidneys in baboons. OWMs had significantly greater IgM binding and cytotoxicity to TKO PBMCs than humans or NWMs. Mean anti-TKO IgM was significantly higher in OWMs and significantly lower in NWMs than in humans. Cytotoxicity of OWM sera to TKO PBMCs was significantly greater than of human serum, but there was no significant difference between human and NWM sera. The median survival of TKO pig kidneys (4 days) in baboons was significantly shorter than that of GTKO kidneys (136 days) (p < 0.05). Even though considered ideal for clinical xenotransplantation, the presence of naturally-existing antibodies to TKO pig cells in OWMs complicates the transplantation of TKO pig kidneys in OWMs.

Evidence for GTKO/β4GalNT2KO Pigs as the Preferred Organ-source for Old World Nonhuman Primates as a Preclinical Model of Xenotransplantation

“…PBMCs from pigs were stained for expression of Gal (by isolectin BSI-B4 ), Neu5Gc(chicken anti-Neu5Gc mAb), and Sd a ( Dolichos biflorus agglutinin, DBA) as previously described. 10 …”

“…IgM and IgG binding to pig PBMCs was measured after exposure to baboon or human serum, as previously described. 10 , 13 Data acquisition was performed with a flow cytometer (Becton Dickinson, San Jose, CA). Binding of IgM and IgG was assessed as the relative geometric mean (rGM), calculated by dividing the geometric mean fluorescence for each sample by that of the negative control, as previously described.…”

“… 8 In contrast, if CMAH is not knocked out (eg, in α1,3-galactosyltransferase gene-knockout [GTKO] and GTKO/β-1,4 N-acetylgalactosaminyltransferase gene-knockout [β4GalNT2KO] pigs), the pig appears not to express the “fourth antigen” (or expresses it at a lower level). 9 , 10 …”

“…We have studied serum antibody binding in several different Old World NHPs (including 6 baboons) to various genetically engineered pig cells (eg, GTKO and TKO). 10 TKO pigs are not an ideal source of organs for Old World NHPs. 10 Therefore, a pig of a different genotype is required that more closely mimics the TKO pig-to-human model.…”

“… 10 TKO pigs are not an ideal source of organs for Old World NHPs. 10 Therefore, a pig of a different genotype is required that more closely mimics the TKO pig-to-human model.…”