Background: Pigs with deletion of 3 carbohydrate xenoantigens (triple knock-out, TKO) are expected to be optimal donors for human xenotransplantation. We have previously shown that long-term survival of the kidney xenograft from TKO pigs is possible even in old world monkeys (OWM) that exhibit higher binding to TKO cells compared to humans. We hypothesized that concomitantly inserted human transgenes (hTGs) are important to overcome rejection caused by higher anti-porcine antibodies.  In the current study, further addition of hTGs that encode coagulation regulatory proteins (CoagRPs), such as thrombomodulin (THBD) and endothelial cell protein C receptor (EPCR) to TKO was evaluated.  

Methods: Seventeen cynomolgus monkeys received kidneys from four different TKO pig lines (EGEN-2060 to 2734) with various expression of hTGs, encoding immune regulatory proteins (ImmuRPs; B2M/HLA-E, CD47, PDL1, and CD39), complement regulatory proteins (CompRPs; CD46, CD55 and CD59) and CoagRPs (THBD, tissue factor pathway inhibitor (TFPI), and EPCR). The recipients were treated with ATG and rituximab induction followed by weekly anti-CD154 antibody (20 mg/kg) and daily mycophenolate mofetil. Either rapamycin or tacrolimus and prednisone were also administered for the first two months.  

Results: EGEN-2528 expressed higher ImmuRP with lower CompRPs, while EGEN-2536 expressed high CompRPs with lower ImmuRPs. EGEN-2734 had moderately high THBD and EPCR expression, while EGEN-2060 expressed high CompRPs especially in the kidney, despite low CoagRP expression (Table 1). In EGEN-2528, the first recipient lost his xenograft on day 2 due to TMA, while the second recipient rejected on day 61 due to T cell mediated rejection and antibody mediated rejection (AMR). Among 6 recipients of EGEN-2536, 2 monkeys lost their xenografts due to ureteral complication on day 15, and acute graft thrombosis on day 20.  In the other four recipients, although one lost his graft on day 71 due to TMA, all other three recipients survived longer-term (135, 265 and 316 days) and only developed rejection after reduction of their immunosuppression due to infectious complications.  In 7 EGEN-2734 recipients, although 2 lost their xenografts early (day 8 and 9) due to TMA and another on day 45 due to AMR/TMA, the remaining 4 recipients have been currently doing well as long as 188 days without any rejection or infectious complications. Finally, both EGEN-2060 recipients have also been doing well for >90, >103 days (Table 2) with normal kidney function and without any evidence of rejection in their protocol biopsies.

Conclusion: More consistent long-term renal xenograft survival has been achieved by adding multiple hTGs, but the role of hTGs, including THBD and EPCR, in preventing xenograft rejection remains to be defined by longer-term observation in more recipients.