Human antibody reactivity against xenogeneic <i>N</i>‐glycolylneuraminic acid and galactose‐α‐1,3‐galactose antigen

<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>Despite the development of α1,3‐galactosyl transferase‐knockout (<jats:styled-content style="fixed-case">GTKO</jats:styled-content>) pigs, acute humoral xenograft rejection caused by antibodies against non‐Gal antigens, along with complement activation, are hurdles that need to be overcome. Among non‐Gal antigens, <jats:italic>N</jats:italic>‐glycolylneuraminic acid (Neu5Gc) is considered to play an important role in xenograft rejection in human.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>We generated human embryonic kidney 293 (<jats:styled-content style="fixed-case">HEK</jats:styled-content>293) cells that expressed xenogeneic Neu5Gc (<jats:styled-content style="fixed-case">HEK</jats:styled-content>293‐<jats:styled-content style="fixed-case">pCMAH</jats:styled-content>) or α1,3Gal (<jats:styled-content style="fixed-case">HEK</jats:styled-content>293‐<jats:styled-content style="fixed-case">pGT</jats:styled-content>) antigen and investigated the degree of human antibody binding and complement‐dependent cytotoxicity (<jats:styled-content style="fixed-case">CDC</jats:styled-content>) against these antigens using 100 individual human sera.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Both IgM and IgG bound to α1,3Gal, while only IgG bound to Neu5Gc. Of the <jats:styled-content style="fixed-case">ABO</jats:styled-content> blood groups, the degree of IgG binding to α1,3Gal was highest for blood group A. The degree of <jats:styled-content style="fixed-case">CDC</jats:styled-content> against <jats:styled-content style="fixed-case">HEK</jats:styled-content>293‐<jats:styled-content style="fixed-case">pCMAH</jats:styled-content> cells was significantly lower than that against <jats:styled-content style="fixed-case">HEK</jats:styled-content>293‐<jats:styled-content style="fixed-case">pGT</jats:styled-content> cells. However, <jats:styled-content style="fixed-case">CDC</jats:styled-content> against <jats:styled-content style="fixed-case">HEK</jats:styled-content>293‐<jats:styled-content style="fixed-case">pCMAH</jats:styled-content> cells was significantly higher than that against control <jats:styled-content style="fixed-case">HEK</jats:styled-content>293 cells. In addition, the severity of <jats:styled-content style="fixed-case">CDC</jats:styled-content> against <jats:styled-content style="fixed-case">HEK</jats:styled-content>293‐<jats:styled-content style="fixed-case">pCMAH</jats:styled-content> cells positively correlated with that against <jats:styled-content style="fixed-case">GTKO</jats:styled-content> pig aortic endothelial cells (<jats:styled-content style="fixed-case">PAEC</jats:styled-content>s), suggesting that Neu5Gc is the main antigen in <jats:styled-content style="fixed-case">GTKO PAEC</jats:styled-content>s. Similar to antibody‐binding activity, only IgG binding correlated with <jats:styled-content style="fixed-case">CDC</jats:styled-content> against <jats:styled-content style="fixed-case">HEK</jats:styled-content>293‐<jats:styled-content style="fixed-case">pCMAH</jats:styled-content> cells. The most common subclass of IgGs against Neu5Gc was IgG1, which typically induces strong complement activation.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>We showed that IgG‐mediated <jats:styled-content style="fixed-case">CDC</jats:styled-content> was detected in Neu5Gc‐overexpressed <jats:styled-content style="fixed-case">HEK</jats:styled-content>293 cells incubated with human sera; however, this antibody reactivity to Neu5Gc was highly variable among individuals. Our results suggest that additional modifications to the <jats:italic><jats:styled-content style="fixed-case">CMAH</jats:styled-content></jats:italic> gene should be considered for widespread use of pig organs for human transplants.</jats:p></jats:sec>