Christopher Laird

Transgenic expression of human leukocyte antigen-E attenuates GalKO.hCD46 porcine lung xenograft injury.

Lung xenografts remain susceptible to loss of vascular barrier function within hours in spite of significant incremental advances based on genetic engineering to remove the Gal 1,3‐αGal antigen (GalTKO) and express human membrane cofactor protein (hCD46). Natural killer cells rapidly disappear from the blood during perfusion of GalTKO.hCD46 porcine lungs with human blood and presumably are sequestered within the lung vasculature. Here we asked whether porcine expression of the human NK cell inhibitory ligand HLA‐E and β2 microglobulin inhibits GalTKO.hCD46 pig cell injury or prolongs lung function in two preclinical perfusion models.

N-glycolylneuraminic acid knockout reduces erythrocyte sequestration and thromboxane elaboration in an ex vivo pig-to-human xenoperfusion model

Wild‐type pigs express several carbohydrate moieties on their cell surfaces that differ from those expressed by humans. This difference in profile leads to pig tissue cell recognition of human blood cells causing sequestration, in addition to antibody‐mediated xenograft injury. One such carbohydrate is N‐glycolylneuraminic acid (Neu5Gc), a sialic acid molecule synthesized in pigs but not in humans. Here, we evaluate livers with and without Neu5Gc in an ex vivo liver xeno perfusion model.

Synthetic liver function is detectable in transgenic porcine livers perfused with human blood

In addition to immune barriers, molecular incompatibilities between species are predicted to limit pig liver survival in primate xenotransplantation models. Assessment and measurement of synthetic function of genetically modified porcine livers after ex vivo perfusion with human blood have not previously been described. Eight porcine livers from α1,3‐galactosyl transferase knockout and human membrane cofactor (GalTKO.hCD46), six livers from GalTKO.hCD46 and N‐glycolylneuraminic acid knockout (GalTKO.hCD46.Neu5GcKO), and six livers from GalTKO.hCD46 with humanized decay‐accelerating factor (hCD55), endothelial protein C receptor (hEPCR), tissue factor pathway inhibitor (hTFPI), and integrin‐associated protein (hCD47) (GalTKO.hCD46.hCD55.hEPCR.hTFPI.hCD47) pigs were perfused with human blood under physiologic conditions. Timed blood samples were tested for liver enzymes and for pig‐specific albumin production via Western blot. Porcine albumin levels increased with time in all experiments. By densitometry, GalTKO.hCD46.Neu5GcKO livers had the highest albumin levels, measured both as total produced, and when controlled for perfusion duration, compared to GalTKO.hCD46 (P = .068) and GalTKO.hCD46.hCD55.hEPCR.hTFPI.hCD47 livers (P = .04). Porcine livers perfused with human blood demonstrated the synthetic ability to produce albumin in all cases. GalTKO.hCD46.Neu5GcKO pig livers demonstrated the most robust albumin production. This suggests that the Neu5GcKO phenotype provides a protective effect on the graft due to decreased human antibody recognition and graft injury.

Interleukin-8 mediates neutrophil-endothelial interactions in pig-to-human xenogeneic models

Human neutrophils are sequestered by pig lung xenografts within minutes during ex vivo perfusion. This phenomenon is not prevented by pig genetic modifications that remove xeno‐antigens or added human regulatory molecules intended to down‐regulate activation of complement and coagulation pathways. This study investigated whether recipient and donor interleukin‐8 (IL‐8), a chemokine known to attract and activate neutrophils during inflammation, is elaborated in the context of xenogeneic injury, and whether human or pig IL‐8 promote the adhesion of human neutrophils in in vitro xenograft models.

Thromboxane and histamine mediate PVR elevation during xenogeneic pig lung perfusion with human blood

Elevated pulmonary vascular resistance (PVR), platelet adhesion, coagulation activation, and inflammation are prominent features of xenolung rejection. Here, we evaluate the role of thromboxane and histamine on PVR, and their contribution to other lung xenograft injury mechanisms.

Progress in Xenogeneic Lung Transplantation Using Multi-Transgenic Donor Pigs and Targeted Supportive Drug Treatments

Purpose Significant progress in genetic engineering has enabled the generation of knock-out pigs that additionally express multiple human “transgenes” chosen to address various known xenogeneic rejection pathways. We evaluated which transgene combinations are associated with prevention of lung xenograft injury in a rigorous life-supporting pig-to-baboon lung xenograft model. Methods GTKO.hCD46 porcine donor organs with up to 4 additional genetic modifications, including hCD55, hTBM, hEPCR, hTFPI, hCD47, hCD39, hHO-1, HLA-E, A20 and humanized vWF were used in 48 left single lung transplants into baboons. A rationally designed “platform drug regimen” consisted of steroids, sC1Inh, thromboxane synthase inhibitor, histamine receptor blockers, and anti-GPIb Fab. Desmopressin (DDAVP) was given to the donor animals prior to lung procurement, to deplete pig endothelial VWF. Immunosuppression consisted of aCD20, ATG, MMF, aCD40, coupled with anti-IL6R moAb and/or Alpha-1 Antitrypsin. Xenograft function was assessed intermittently by transplant blood flow measurements and radiographs. Results Xenogeneic lungs with 6 genetic modifications (6-GE) transplanted into baboons survived for up to 8d (hCD55.hEPCR.hTBM.hCD39) and 9d (hEPCR.hTBM.hCD47.HO-1), vs <12hrs for most 3- or 4-GE lungs. Several other donor transgene-combinations resulted in 47d survival. Lungs of GTKO.hCD46.hCD55.hEPCR.hCD47.hTFPI and a few 3- or 4-GE phenotypes (eg including HLA-E and hvWF) consistently exhibited life-supporting (LS) lung function for 24-30h, with relatively normal macro- and microscopic lung appearance until 2-3 d. Lung xenograft failure was usually associated with rebounding anti-pig antibody titer and loss of lung vascular barrier function leading to alveolar flooding and consolidation, consistent with “delayed xenograft rejection”. Accumulated evidence suggests that, in addition to anti-non-Gal antibody, recipient NK cells (HLA-E) and donor macrophage activation (thromboxane) each play important roles to drive the residual inflammation. Conclusion Combining multi-transgenic donor organs with mechanism-directed drug treatments significantly prolongs life-supportive lung xenograft function and recipient survival. Aggregation of existing targeted genetic modifications, along with antibody depletion and mechanism-based drug additions appear likely to successfully control known pathogenic pathways, and further advance lung xenotransplantation towards clinical application. United Therapeutics SRA. NIH U19A1090959.

Genetic Engineering in Xenotransplantation: What Human Transgenes Should the GTKO.hCD46 Pig Lung Donor Additionally Express?

Background Recent advances in genetic engineering have enabled generation of pigs expressing multiple transgenes as potential donors for xenotransplantation. What transgenes contribute to protect xenogeneic lungs from being injured by known xenorejection mechanisms, has not previously been systematically evaluated. Here we summarize results directly testing several individual transgenes on a platform GTKO.hCD46 in a xenogeneic lung perfusion model. Methods GTKO.hCD46 pig lungs, additionally expressing either hEPCR, HLAE, hvWF, hTBM, hCD55 or including Neu5GC knock-out were perfused with fresh heparinized human blood for up to 8 hours. Functional parameters, as well as blood and tissue samples were analyzed and compared to lungs that did not have the respective additional genetic modification. Results Lung “survival” was significantly increased in lungs with hEPCR and HLA-E. While HLA-E, hEPCR and hTBM expression led to reduced BTG elaboration, platelet sequestration was only markedly reduced by the expression of humanized vWF. Pulmonary vascular resistance as well as histamine elaboration showed significantly lower values in lungs with HLA-E or Neu5GcKO. The knock-out of the Neu5Gc epitope also significantly reduced thromboxane levels and activation of the coagulation cascade (F1+2). Conclusion Several mechanisms associated with GTKO.hCD46 xenolung injury are modulated by additional expression of individual pathway-targeting human transgenes. We conclude that each of the pathways targeted by the transgenes tested, including Neu5GcKO, provide protection from non-Gal-antibody-mediated lung rejection mechanisms. We hypothesize that combinations of the beneficial genetic modifications tested here will result in further improvement of xenogeneic lung function and extension of graft survival. United Therapeutics SRA. NIH U19A1090959.

Comparative Evaluation of αCD40 (2C10R4) and αCD154 (5C8H1 and IDEC-131) in a Nonhuman Primate Cardiac Allotransplant Model

Background Specific blockade of T cell costimulation pathway is a promising immunomodulatory approach being developed to replace our current clinical immunosuppression therapies. The goal of this study is to compare results associated with 3 monoclonal antibodies directed against the CD40/CD154 T cell costimulation pathway. Methods Cynomolgus monkey heterotopic cardiac allograft recipients were treated with either IDEC-131 (humanized &agr;CD154, n = 9), 5C8H1 (mouse-human chimeric &agr;CD154, n = 5), or 2C10R4 (mouse-rhesus chimeric &agr;CD40, n = 6) monotherapy using a consistent, comparable dosing regimen for 3 months after transplant. Results Relative to the previously reported IDEC-131-treated allografts, median survival time (35 ± 31 days) was significantly prolonged in both 5C8H1-treated (142 ± 26, P < 0.002) and 2C10R4-treated (124 ± 37, P < 0.020) allografts. IDEC-131-treated grafts had higher cardiac allograft vasculopathy severity scores during treatment relative to either 5C8H1 (P = 0.008) or 2C10R4 (P = 0.0002). Both 5C8H1 (5 of 5 animals, P = 0.02) and 2C10R4 (6/6, P = 0.007), but not IDEC-131 (2/9), completely attenuated IgM antidonor alloantibody (alloAb) production during treatment; 5C8H1 (5/5) more consistently attenuated IgG alloAb production compared to 2C10R4 (4/6) and IDEC-131 (0/9). All evaluable explanted grafts experienced antibody-mediated rejection. Only 2C10R4-treated animals exhibited a modest, transient drop in CD20+ lymphocytes from baseline at day 14 after transplant (−457 ± 152 cells/&mgr;L) compared with 5C8H1-treated animals (16 ± 25, P = 0.037), and the resurgent B cells were primarily of a naive phenotype. Conclusions In this model, CD154/CD40 axis blockade using IDEC-131 is an inferior immunomodulatory treatment than 5C8H1 or 2C10R4, which have similar efficacy to prolong graft survival and to delay cardiac allograft vasculopathy development and antidonor alloAb production during treatment.