Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/β4GalNT2 genes

Abstract

Background: Simultaneous inactivation of pig GGTA1 and CMAH genes eliminates carbohydrate xenoantigens recognized by human antibodies. The β4GalNT2 glycosyltransferase may also synthesize xenoantigens. To further characterize glycan-based species incompatibilities, we examined human and non-human primate antibody binding to cells derived from genetically modified pigs lacking these carbohydrate-modifying genes.

Methods: The Cas9 endonuclease and gRNA were used to create pigs lacking GGTA1, GGTA1/CMAH, or GGTA1/CMAH/β4GalNT2 genes. Peripheral blood mononuclear cells were isolated from these animals and examined for binding to IgM and IgG from humans, rhesus macaques, and baboons.

Results: Cells from GGTA1/CMAH/β4GalNT2 deficient pigs exhibited reduced human IgM and IgG binding compared to cells lacking both GGTA1 and CMAH. Non-human primate antibody reactivity with cells from the various pigs exhibited a slightly different pattern of reactivity than that seen in humans. Simultaneous inactivation of the GGTA1 and CMAH genes increased non-human primate antibody binding compared to cells lacking either GGTA1 only or to those deficient in GGTA1/CMAH/β4GalNT2.

Conclusions: Inactivation of the β4GalNT2 gene reduces human and non-human primate antibody binding resulting in diminished porcine xenoantigenicity. The increased humoral immunity of non-human primates toward GGTA1-/CMAH-deficient cells compared to pigs lacking either GGTA1 or GGTA1/CMAH/β4GalNT2 highlights the complexities of carbohydrate xenoantigens and suggests potential limitations of the non-human primate model for examining some genetic modifications. The progressive reduction of swine xenoantigens recognized by human immunoglobulin through inactivation of pig GGTA1/CMAH/β4GalNT2 genes demonstrates that the antibody barrier to xenotransplantation can be minimized by genetic engineering.

Keywords: CRISPR; Cas9; antibody; genetic engineering; primate; swine; xenoantigen; β4GalNT2.

Figure 1. Genetic modification and cell selection

Step 1: Wild type liver-derived cells were treated with the Cas9 endonuclease and gRNA targeting the GGTA1, CMAH, and β4GalNT2 genes. Step 2: This heterogenous population was subjected to an IB4 lectin column to enrich for GGTA1 mutant cell types. Step 3: GGTA1 mutant cells were incubated with DBA lectin and sorted to isolate DBA-negative cells, which do not express β4GalNT2-derived carbohydrates. Step 4: SCNT was performed with cells determined to lack GGTA1 and β4GalNT2 gene function by the phenotypic selection. Phenotypic analysis of CMAH gene activity is difficult to perform on cells in culture and was not attempted prior to SCNT.

Figure 2. Phenotype and genotype of GGTA1/CMAH/β4GalNT2 knockout pig

A) PBMCs from wild type (WT) or KO pigs were incubated with fluorescent probes: IB4 lectin to detect αGal carbohydrates (green histograms); chicken antibodies specific for the Neu5Gc carbohydrate (red histograms); DBA lectin specific for carbohydrates produced by the β4GalNT2 gene (blue histograms). Gray histograms outlined in black represent negative controls that were unstained cells for lectin experiments and an irrelevant isotype matched antibody for Neu5Gc staining. The appearance of a single histogram in KO samples indicates overlap with negative control. B) DNA sequence analysis was performed on isolated alleles of GGTA1, CMAH and β4GalNT2. Electropherograms revealed mutations in all alleles of each gene. WT sequence (WT) is shown for comparison to the modified alleles.

Figure 3. Normal Karyotype of the GGTA1/CMAH//β4GalNT2 knockout pig

Chromosome 12 contains the β4GalNT2 gene.

Figure 4. Plots of NHP antibody binding to single, double, and triple KO PBMCs

NHP sera (34 rhesus macaques and 10 baboons) were incubated with PBMC from swine containing inactivated GGTA1 (KO-1), GGTA1/CMAH (KO-2), or GGTA1/CMAH/β4GALNT2 (KO-3). Secondary fluorescent antibodies were used to detect IgM and IgG binding to the cells with median fluorescence intensity being evaluated (MFI). A) Summary data are shown in box and whisker plots comparing binding of antibodies to swine PBMC of each genotype. Oneway ANOVA, and Tukey post hoc analyses were used to analyze the differences between each group. All comparisons of rhesus IgM and IgG binding yielded p values less than 0.006. GGTA1 and CMAH deficient pig cells bound the most baboon IgM (KO-1 vs. KO-2, p=0.041, and KO-2 vs. KO-3, p=0.010). Comparisons of baboon IgM binding to KO-1 versus KO-3 cells (NS, p=0.626), and baboon IgG binding comparisons did not achieve statistical significance (IgG not shown). B) MFI from baboon IgG binding to KO-2 and KO-1 PBMC were plotted individually. The diagonal line indicates equivalent binding to both KO-1 and KO-2 cells. Dots falling below the line represent samples where KO-1 cells bound fewer antibodies than KO-2 PBMC. C) Baboon IgG binding to KO-2 relative to KO-3 where data points below the line represent samples where KO-3 cells bound fewer antibodies than KO-2 PBMC.

Figure 5. Plot of human IgG and IgM antibody binding to PBMCs from GGTA1/CMAH KO and GGTA1/CMAH/β4GALNT2 KO pigs

Human sera (n=82) were incubated with PBMC from swine having inactivated GGTA1/CMAH (KO-2), or GGTA1/CMAH/β4GALNT2 (KO-3). Secondary fluorescent antibodies were used to detect IgM and IgG binding and fluorescence reported as median fluorescence intensity (MFI). A) Grouped analysis of human IgM and IgG binding to KO-2 and KO-3 swine PBMC. One-way ANOVA and Tukey post hoc analyses indicate significantly different antibody binding in ever comparison (p<0.01). B) Each data point represents a matched IgG and IgM MFI from individual human sera samples to either KO-2 or KO-3 PBMC. Dotted lines were placed at 6,000 MFI to highlight transitions from minimal to elevated antibody binding as determined by visual inspection of the data. More than 90% of samples are in left lower quadrant for KO-3. C) Individual data is plotted to allow simultaneous comparison of human IgG binding to KO-2 and KO-3 cells. The diagonal line represents equivalent binding to both sources of PBMC. Values falling below the line indicate less antibody binding to the KO-3 PBMCs relative to KO-2 cells. D) Human IgM binding to KO-2 and KO-3 was analyzed exactly as described in panel C. Dots falling below the line represent samples where KO-3 cells bound less IgM than KO-2 PBMC.