Knock‐out of N‐glycolylneuraminic acid attenuates antibody‐mediated rejection in xenogenically perfused porcine lungs

Abstract

Background Antibody‐mediated rejection has long been known to be one of the major organ failure mechanisms in xenotransplantation. In addition to the porcine α1,3‐galactose (α1,3Gal) epitope, N‐Glycolylneuraminic acid (Neu5Gc), a sialic acid, has been identified as an important porcine antigen against which most humans have pre‐formed antibodies. Here we evaluate GalTKO.hCD46 lungs with an additional cytidine monophospho‐N‐acetylneuraminic acid hydroxylase (CMAH) gene knock‐out (Neu5GcKO) in a xenogeneic ex vivo perfusion model Methods Eleven GalTKO.hCD46.Neu5GcKO pig lungs were perfused for up to 6 h with fresh heparinized human blood. Six of them were treated with histamine (H) blocker famotidine and 1‐thromboxane synthase inhibitor Benzylimidazole (BIA) and five were left untreated. GalTKO.hCD46 lungs without Neu5GcKO (n = 18: eight untreated and 10 BIA+H treated) served as a reference. Functional parameters, blood, and tissue samples were collected at pre‐defined time points throughout the perfusion Results All but one Neu5GcKO organs maintained adequate blood oxygenation and “survived” until elective termination at 6 h whereas two reference lungs failed before elective termination at 4 h. Human anti‐Neu5Gc antibody serum levels decreased during the perfusion of GalTKO.hCD46 lungs by flow cytometry (∼40% IgM, 60% IgG), whereas antibody levels in Neu5GcKO lung perfusions did not fall (IgM p = .007; IgG p < .001). Thromboxane elaboration, thrombin generation, and histamine levels were significantly reduced with Neu5GcKO lungs compared to reference in the untreated groups (p = .007, .005, and .037, respectively); treatment with BIA+H masked these changes. Activation of platelets, measured as CD62P expression on circulating platelets, was lower in Neu5GcKO experiments compared to reference lungs (p = .023), whereas complement activation (as C3a rise in plasma) was not altered. MCP‐1 and lactotransferin level elevations were blunted in Neu5GcKO lung perfusions (p = .007 and .032, respectively). Pulmonary vascular resistance (PVR) rise was significantly attenuated and delayed in untreated GalTKO.hCD46.Neu5GcKO lungs in comparison to the untreated GalTKO.hCD46 lungs (p = .003) Conclusion Additional Neu5GcKO in GalTKO.hCD46 lungs significantly reduces parameters associated with antibody‐mediated inflammation and activation of the coagulation cascade. Knock‐out of the Neu5Gc sialic acid should be beneficial to reduce innate immune antigenicity of porcine lungs in future human recipients.

A systematic review reveals conflicting evidence for the prevalence of antibodies against the sialic acid ‘xenoautoantigen’ Neu5Gc in humans and the need for a standardised approach to quantification

“…The anti-Neu5Gc antibody response is described to follow an inverse hormesis model, in which low antibody titres may stimulate chronic low-level inflammation, and high titres stimulate immune clearance mechanisms such as antibodydependent cellular cytotoxicity (Pearce et al, 2014). This dosedependency is especially relevant for attempts to exploit the increased presence of Neu5Gc on tumour tissues as a therapeutic target (Machado et al, 2011) or in efforts to develop new biotherapeutic products with reduced risk of rejection (Komoda et al, 2010;Chaban et al, 2022) as the outcome of this would depend heavily on the titres of circulating antibodies present in each individual. Therefore, an inability to agree on the extent of the anti-Neu5Gc antibody response both raises questions about the relevance of this unique xenoautoantigen to human health and presents an important roadblock for these areas of research.…”

“…Similarly, treatment of type 1 diabetes patients with anti-thymocyte globulin (ATG) produced in rabbits was associated with an increase in both titres and diversity of anti-Neu5Gc antibodies (Le Berre et al, 2019), and burn patients treated with porcine skin grafts showed a long-term increase in anti-Neu5Gc antibodies compared to patients treated with conventional allografts (Scobie et al, 2013). This potential for Neu5Gc-containing biotherapeutics to trigger an immune response in humans has also led to strong interest in Neu5Gc in the field of xenotransplantation, where graft failure and rejection have been attributed to the induction of anti-graft immune responses against carbohydrate xenoantigens such as αGal and Neu5Gc in multiple studies (Blixt et al, 2009;Zhang et al, 2018;Chaban et al, 2022;Senage et al, 2022).…”