Anti‐non‐Gal‐specific combination treatment with an anti‐idiotypic Ab and an inhibitory small molecule mitigates the xenoantibody response

Abstract

Background B cell depletion significantly extends survival of α-1,3-galactosyltranferase knockout (GTKO) porcine organs in pig-to-primate models. Our previous work demonstrated that the anti-nonGal xenoantibody response is structurally restricted. Selective inhibition of xenoantigen-xenoantibody interactions could prolong xenograft survival while preserving B cell-mediated immune surveillance. Methods The anti-idiotypic antibody, B4N190, was selected from a synthetic human phage display library after enrichment against a recombinant anti-nonGal xenoantibody followed by functional testing in vitro. The inhibitory small molecule, JMS022, was selected from the NCI diversity set III using virtual screening based on predicted xenoantibody structure. Three rhesus monkeys were pretreated with anti-nonGal specific single chain anti-idiotypic antibody, B4N190. A total of five monkeys, including two untreated controls, were then immunized with GTKO porcine endothelial cells to initiate an anti-non α-1,3-gal (nonGal) xenoantibody response. The efficacy of the inhibitory small molecule specific for anti-nonGal xenoantibody, JMS022, was tested in vitro. Results After the combination of in vivo anti-id and in vitro small molecule treatments, IgM xenoantibody binding to GTKO cells was reduced to pre-immunization levels in 2/3 animals, however, some xenoantibodies remained in the third animal. Furthermore, when treated with anti-id alone all three experimental animals displayed a lower anti-nonGal IgG xenoantibody response compared to controls. Treatment with anti-idiotypic antibody alone reduced IgM xenoantibody response intensity in only one of three monkeys injected with GTKO pig endothelial cells. In the one experimental animal which displayed reduced IgM and IgG responses select B cell subsets were also reduced by anti-id therapy alone. Furthermore, natural antibody responses, including anti-laminin, anti-ssDNA, and anti-throglobulin antibodies were intact despite targeted depletion of anti-nonGal xenoantibodies in vivo indicating that selective reduction of xenoantibodies can be accomplished without total B cell depletion. Conclusions This preliminary study demonstrates the strength of approaches designed to selectively inhibit anti-nonGal xenoantibody. Both anti-nonGal specific xenoantibody and small molecules can be used to selectively limit xenoantibody responses.

Clonidine inhibits anti‐non‐Gal IgM xenoantibody elicited in multiple pig‐to‐primate models

“…This is consistent with our previous analysis suggesting that the majority of the anti-non-Gal IgM antibody response is structurally restricted in multiple settings [12,13]. However, the previously identified experimental compound JMS022 was only capable of inhibiting the IgM xenoantibody response of three of five rhesus monkeys tested (NIS.A, AIA.A, and AIA.C) [14]. This suggested some degree of structural variation at the site to which JMS022 binds.…”

“…Fifteen juvenile rhesus monkeys were screened for low levels of non‐Gal‐reactive xenoantibody by ELISA as previously reported . Five animals with the lowest levels of pre‐existing anti‐non‐Gal IgM xenoantibody were selected for further study.…”

“…Our group has previously demonstrated that the elicited anti‐non‐Gal xenoantibody response displays limited structural diversity in multiple galactosyltransferase knockout (GTKO) pig‐to‐primate models of xenotransplantation . This enabled us to identify an anti‐non‐Gal selective anti‐idiotypic single‐chain antibody and an experimental small molecule capable of selectively inhibiting induced anti‐non‐Gal IgM xenoantibodies . Although in vitro this small molecule could inhibit the binding of residual IgM xenoantibody in animals pre‐treated with anti‐idiotypic antibody, it had an unknown safety profile.…”

Xenotransplantation literature update, May–June 2014

“…Stewart et al. used anti‐idiotypic antibodies (think Fab fragments with mirror image binding domains) and a small inhibitory molecule to reduce the xenoantibody response in rhesus monkeys previously immunized with GTKO endothelial cells . The antibody therapy, B4N190, had little if any effect on natural antibodies.…”

“…In lieu of genetically modifying pigs, it may be possible to identify the target of xeno-directed antibodies and block the interaction with small molecules that mimic the immune-dominant epitope. Stewart et al used anti-idiotypic antibodies (think Fab fragments with mirror image binding domains) and a small inhibitory molecule to reduce the xenoantibody response in rhesus monkeys previously immunized with GTKO endothelial cells [6]. The antibody therapy, B4N190, had little if any effect on natural antibodies.…”