Recent human decedent model studies^1,2 and compassionate xenograft use³ have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts.

Porcine cells devoid of three major carbohydrate xenoantigens, αGal, Neu5GC, and SDa (TKO) exhibit markedly reduced binding of human natural antibodies. Therefore, it is anticipated that TKO pigs will be better donors for human xenotransplantation. However, previous studies on TKO pigs using Old World Monkeys (OWMs) have been disappointing because of higher anti-TKO pig antibodies in OWMs than humans. Here we show that long-term survival of renal xenografts from TKO pigs that express additional human transgenes (hTGs) can be achieved in cynomolgus monkeys. Kidney xenografts from TKO-hTG pigs were transplanted into eight cynomolgus recipients without pre-screening for low anti-pig antibody titers. Two recipients of TKO-hTG xenografts with low expression of human complement regulatory proteins (CRPs)(TKO-A) survived for 2 and 61 days, whereas six recipients of TKO-hTG xenografts with high CRP expression (TKO-B) survived for 15, 20, 71, 135, 265 and 316 days. Prolonged CD4⁺T cell depletion and low anti-pig antibody titers, which were previously reported important for long-term survival of αGal knock-out (GTKO) xenografts, were not always required for long-term survival of TKO-hTG renal xenografts. This study indicates that OWMs such as cynomolgus monkeys can be used as a relevant model for clinical application of xenotransplantation using TKO pigs.

Porcine kidney xenotransplantation is accelerating towards clinical translation. However, despite the demonstrated ability of porcine kidneys to remove metabolic waste products, questions remain about their ability to faithfully recapitulate renal endocrine functions after transplantation. Here we analyze xenograft growth and function of two kidney dependent endocrine pathways in seventeen cynomolgus macaques after kidney xenotransplantation from gene edited Yucatan minipigs. Xenograft growth, the renin-angiotensinogen aldosterone-system, and the calcium-vitamin D-parathyroid hormone axis are assessed using clinical chemistries data, renin activity and beta-C-terminal-telopeptide assays, kidney graft RNA-sequencing and serial ultrasonography. We demonstrate that xenografts transplanted from minipigs show only modest growth and do not substantially contribute to recipient RAAS pathway activity. However, parathyroid hormone-independent hypercalcemia and hypophosphatemia are observed, suggesting a need for close monitoring and timely intervention during human testing. Further study of these phenotypes is warranted in designing prospective clinical trials.

The blockade of the CD154-CD40 pathway with anti-CD154 monoclonal antibody has been a promising immunomodulatory approach to prevent allograft rejection. However, clinical trials of immunoglobulin G1 antibodies targeting this pathway revealed thrombogenic properties, which were subsequently shown to be mediated by crystallizable fragment (Fc)-gamma receptor IIa-dependent platelet activation. To prevent thromboembolic complications, an immunoglobulin G4 anti-CD154 monoclonal antibody, TNX-1500, which retains the fragment antigen binding region of ruplizumab (humanized 5c8, BG9588), was modified by protein engineering to decrease Fc binding to Fc-gamma receptor IIa while retaining certain other effector functions and pharmacokinetics comparable with natural antibodies. Here, we report that TNX-1500 treatment is not associated with platelet activation in vitro and consistently inhibits kidney allograft rejection in vivo without clinical or histologic evidence of prothrombotic phenomena. We conclude that TNX-1500 retains efficacy similar to that of 5c8 to prevent kidney allograft rejection while avoiding previously identified pathway-associated thromboembolic complications.

B cells constitute a complex system of antigen-presenting cells (APCs) and exist as distinct subsets that differ in their lineage affiliation, surface molecule expression, and biological function, thus potentially regulating the immune response. In this study, we investigated the immune-regulatory roles of murine B cell subsets as regulatory APCs targeting alloreactive T cells. Either splenic B cells, peritoneal cavity (PerC) B cells, or non-B cells from Balb/c mice were intravenously injected into B6 mice. Serum levels of anti-Balb/c antibodies in the recipients of PerC B cells were significantly lower than those in the recipients of splenic B cells and PerC non-B cells, as determined over a 4-week period after the injection. Mixed-lymphocyte reaction (MLR) assays using splenocytes from the B6 mice at 2 weeks after the injection revealed the significantly reduced anti-Balb/c T cell-responses in the recipients of PerC B cells, as compared to those in the recipients of splenic B cells or untreated control mice. Since PerC B cells contained MHC class II⁺ CD80⁺ CD86⁺ PD-L1⁺ PD-L2⁺ cells among the CD5⁺ B-1a cell subset, PerC B cells from Balb/c mice were pre-incubated with anti-PD-L1/PD-L2 mAbs prior to injection. This treatment abrogated their immune-regulatory effects on anti-Balb/c T cells in the MLR assays. In addition, the inoculation with Balb/c PerC B cells significantly prolonged the survival of subsequently grafted Balb/c hearts in B6 mouse recipients, whereas that with SPL B cells did not. These findings indicate that the PerC B cells, including PD-L1/PD-L2 B-1a cells, may suppress T cells responding to allostimulation, and thus may be optimal for donor lymphocyte injection.

Hematopoietic stem cell transplantation (HSCT) has many potential applications beyond current standard indications, including treatment of autoimmune disease, gene therapy, and transplant tolerance induction. However, severe myelosuppression and other toxicities after myeloablative conditioning regimens have hampered wider clinical use. To achieve donor hematopoietic stem cell (HSC) engraftment, it appears essential to establish niches for the donor HSCs by depleting the host HSCs. To date, this has been achievable only by nonselective treatments such as irradiation or chemotherapeutic drugs. An approach that is capable of more selectively depleting host HSCs is needed to widen the clinical application of HSCT. Here, we show in a clinically relevant nonhuman primate model that selective inhibition of B cell lymphoma 2 (Bcl-2) promoted hematopoietic chimerism and renal allograft tolerance after partial deletion of HSCs and effective peripheral lymphocyte deletion while preserving myeloid cells and regulatory T cells. Although Bcl-2 inhibition alone was insufficient to induce hematopoietic chimerism, the addition of a Bcl-2 inhibitor resulted in promotion of hematopoietic chimerism and renal allograft tolerance despite using only half of the dose of total body irradiation previously required. Selective inhibition of Bcl-2 is therefore a promising approach to induce hematopoietic chimerism without myelosuppression and has the potential to render HSCT more feasible for a variety of clinical indications.