Effect of intravenous immunoglobulin (IVIg) on primate complement-dependent cytotoxicity of genetically engineered pig cells: relevance to clinical xenotransplantation

Abstract

Triple-knockout (TKO) pigs may be ideal sources of organs for clinical xenotransplantation because many humans have no preformed antibody to TKO pig cells. Intravenous immunoglobulin (IVIg) is widely used for severe infection or the treatment/prevention of antibody-mediated rejection in allotransplantation. Anti-pig antibodies in IVIg could be harmful in clinical xenotransplantation. It is unknown whether anti-TKO pig antibodies are present in IVIg. The main aim of this study was to investigate in vitro whether IVIg contains anti-TKO pig antibodies with cytotoxic effect to pig cells. Undiluted pooled human serum (HS) and five different commercial preparations of IVIg were tested for IgM and IgG binding to red blood cells (RBCs) from wild-type (WT), α1,3-galactosyltransferase gene-knockout (GTKO), and TKO pigs by flow cytometry. Complement-dependent lysis of IVIg against these pig pRBCs was measured by hemolytic assay. Pooled HS and 4 of 5 IVIg commercial preparations contained anti-pig IgG that bound to WT and GTKO pRBCs, but not to TKO pRBCs. One preparation of IVIg contained antibodies that bound to TKO pRBCs, but there was no cytotoxicity of IVIg to TKO pRBCs. The results suggest that IVIg administration to human recipients of TKO pig grafts would be safe. However, the specific preparation of IVIg would need to be screened before its administration. Abbreviations Gal Galactose-α1,3-galactose GTKO α1,3-Galactosyltransfearse gene-knockout IVIg Intravenous immunoglobulin Neu5Gc N-Glycolylneuraminic acid NHP Nonhuman primate pAECs Pig aortic endothelial cells pRBCs Pig red blood cells TKO Triple gene-knockout WT Wild-type Genetically-modified pigs could be an alternative source of organs for clinical transplantation.

Initial experimental experience of triple‐knockout pig red blood cells as potential sources for transfusion in alloimmunized patients with sickle cell disease

“…At screening, IgM and IgG binding of C9 and C11 to TKO pRBCs was negative (Figures 3B and 5B). However, IgM binding was slightly positive immediately before xenotransfusion (rGM of C9:2.2 and C11:1.5 [cutoff level 1.2, as previously described 11,20 ]) (Figure 5B). IgG binding remained negative (Figure 5B).…”

“…RBCs from primates and pigs were separated from blood, as previously described. 20 RBCs from pigs or primates were stained for expression of Gal (by isolectin BSI-B4), Neu5Gc (chicken anti-Neu5Gc mAb), and Sda (Dolichos biflorus agglutinin, DBA), as previously described. 21 Details are described in supplementary Methods.…”

“…IgM/IgG binding of human plasma and capuchin monkey sera to RBCs was measured, as previously described. 18,20 Data acquisition was performed with a flow cytometer (BD LSRII, BD Biosciences), and binding was measured by the relative geometric mean (rGM) value, which was calculated as follows: rGM = each GM/GM in negative control.…”

“…CDC of human plasma and capuchin monkey sera to RBCs was measured, as previously described. 20 Details are described in supplementary Methods.…”

“…However, based on our previous study, TKO RBCs are associated with reduced antibody binding, and therefore complement-mediated lysis by human complement is reduced in vitro. 20 If one or more human complementregulatory proteins, for example, CD46, CD55, could be expressed in pRBCs (which is not yet possible), human serum cytotoxicity should be significantly reduced and would likely be associated with longer pRBC survival. 26 IgM binding to TKO pRBCs gradually increased 5 days after xenotransfusion in both capuchin monkeys (Figure 5B), as did CDC, especially in C11 in which the cells experienced shorter survival (Figure 5D).…”