GGTA1/CMAH/β4GalNT2 knockout porcine cells have reduced human antibody binding and achieved near background thresholds for most transplant patients and remaining antibody binding is partially attributable to swine leukocyte antigen
Gregory R. Martens1, Ping Li1, Luz M. Reyes1, Jose L. Estrada1, James R. Butler1, Zheng-Yu Wang1, Joseph Ladowski1, Richard A Sidner1, Matt Tector1, A. Joseph Tector1,2.
1Department of Surgery, Indiana University School School of Medicine, Indianapolis, IN, United States; 2IU Health Transplant Institute, Indiana University School School of Medicine , Indianapolis, IN, United States
Background: Organ shortage limits the clinical application of transplantation, particularly for highly sensitized patients. An increased level of preexisting antibodies to human leukocyte antigen (HLA) makes it difficult to find matching organs and increases likelihood for rejection of human grafts. Xenotransplantation represents a possible solution, but its application has been precluded by human anti-pig xenoantibodies. A significant decrease in antibody binding has been shown following disruption of GGTA1/CMAH genes in healthy human sera. β4GalNT2 has been identified by cDNA screening to produce a xenoantigen in primates, but its importance for human transplantation requires further study. We evaluated serum from transplant patients for binding to cells from a pig lacking expression of GGTA1/CMAH/β4GalNT2 genes and investigated highly xenoreactive samples for swine leukocyte antigen (SLA) reactivity with SLA Class I deficient pig cells.
Methods: We created pigs lacking expression of GGTA1/CMAH/β4GalNT2, GGTA1/CMAH, GGTA1/SLA Class I, and GGTA1 genes using Cas9 genetic modification and somatic cell nuclear transfer. Sera from transplant patients were assessed for antibody binding to porcine peripheral blood mononuclear cells (PBMC) on knockout (KO) cells. Flow cytometric crossmatch analysis was used to assess for IgG and IgM antibody binding, and results were expressed as median fluorescence intensities (MFI). Transplant recipient sera (n=1017) were tested for IgG and IgM reactivity on cells from GGTA1/CMAH/β4GalNT2 KO pig. A subset of serum samples with high xenoreactivity were absorbed on wild type red cells and tested on PBMCs from GGTA1/SLA Class I, GGTA1/CMAH and GGTA1 KO pigs. Paired t-test and ANOVA statistical analysis was completed.
Results: A human sera screening assay (n=39) confirmed statistically significant decreases in IgG and IgM antibody binding to PBMCs from GGTA1/CMAH/β4GalNT2 compared to both GGTA1/CMAH and GGTA1 KO pig cells. 1,017 transplant recipients sera were tested on GGTA1/CMAH/β4GalNT2 KO PMBC, and IgG and IgM MFI were 3,446 and 3,101, respectively. Eighty percent of screened samples had IgG and IgM MFI of less than 5,000. A subset of highly xenoreactive samples were selected, absorbed, and tested on SLA Class I deficient cells. IgG antibody binding on GGTA1/CMAH/β4GalNT2 KO cells was statistically unchanged following absorption on porcine red cells (mean MFI intact 7,490 and depleted 6,373 p=0.062). IgG binding decreased when absorbed serum was tested on GGTA1/SLA Class I KO cells when compared to absorbed serum binding to triple KO cells (mean MFI 2,039 p=0.033).
Conclusion: Eighty percent of transplant patients screened on GGTA1/CMAH/β4GalNT2 PBMCs achieved near background levels. Remaining IgG reactivity is not changed following absorption on wild type cells and this suggests minimal remaining red cell surface glycan reactivity. The statistically significant decrease in binding on GGTA1/SLA Class I KO cells indicates likely cross-reactive HLA/SLA antibodies in some individuals. A SLA class I deficient pig with inactive GGTA1/CMAH/β4GalNT2 genes may decrease antibody binding for reactive individuals and represent a broadly acceptable xenotransplant donor background. The humoral barrier to clinical trials of human xenotransplantation has been significantly reduced and further studies will investigate this knockout model, MHC Class II, and cell mediated rejection.
 Reyes, L. M."Creating class I MHC-null pigs using guide RNA and the Cas9 endonuclease."
15:30 - 16:30
|Non-Gal Antibodies||GGTA1/CMAH/β4GalNT2 knockout porcine cells have reduced human antibody binding and achieved near background thresholds for most transplant patients and remaining antibody binding is partially attributable to swine leukocyte antigen||Room 109|