Non-Gal antigens Neu5Gc and SDa still trigger rejection

The first generation of xenotransplant pigs knocked out the alpha-1,3-galactosyltransferase (GGTA1) gene to eliminate the Gal antigen, the primary target of human natural antibodies against pig tissue. This was a breakthrough, but it revealed two additional carbohydrate antigens that human antibodies also attack: N-glycolylneuraminic acid (Neu5Gc, produced by the CMAH gene) and the SDa blood group antigen (produced by B4GALNT2). Humans are one of the few mammals that lost the ability to produce Neu5Gc, and because we are exposed to it through diet (red meat), we develop anti-Neu5Gc antibodies. Even with all three genes knocked out (triple knockout pigs), recipients still develop antibody-mediated rejection, suggesting there are additional unknown pig antigens that the human immune system targets. Researchers do not have a complete map of all immunogenic pig carbohydrate and protein antigens recognized by the human immune system. Each new antigen discovered requires another gene knockout, but each additional knockout increases the risk of unintended consequences on organ development, function, and pig health, and the combinatorial complexity of testing multi-gene edits in preclinical models is enormous.