Initial Steps in the Generation of Transgenic Pigs Expressing a T Cell-Depleting Monoclonal Antibody

Abstract

Background: Developing an effective immunosuppressive regimen that can be used in the clinic is one of the great challenges in xenotransplantation. The use of genetically modified donors, specifically pigs that are α1,3-galactosyltransferase knockout (GTKO) and express human complement regulatory proteins, has not completely solved this problem. Local immunosuppression, whereby the graft itself secretes immunomodulatory proteins in the local environment, is a promising approach to reduce the level of systemic immunosuppression required to prevent rejection. We have generated and cloned a depleting, non-activating anti-CD2 monoclonal antibody that recognises human and primate (but not pig) T cells. We have shown that adenovirus-mediated expression and secretion of this antibody protects neonatal pig islets from infiltration by human T cells in a humanised mouse model, and that this effect is indeed local. In this study, we commenced the process of adding this genetic modification to our platform pig. Aim: To generate GTKO/hCD55-hCD59 pigs expressing a secreted anti-CD2 monoclonal antibody that will deplete human T cells. Methods: Early passage GTKO/hCD55-hCD59 pig fibroblasts were transfected with an expression construct for the humanised anti-CD2 monoclonal antibody. The construct consisted of heavy and light chain coding regions linked by 2A and driven by the mouse H-2Kb promoter. Stable transfectants were picked, expanded and screened for secretion of anti-CD2 by incubating culture supernatant with human T cells and analysing by flow cytometry. Expressing clones were used for somatic cell nuclear transfer. Results: Before commencing stable transfection of GTKO/hCD55-hCD59 cells, correct processing and secretion of anti-CD2 was confirmed by analysis of culture supernatant from COS-7 cells transiently transfected with the expression construct. 8 stable GTKO/hCD55-hCD59 clones expressing anti-CD2 at various levels were then isolated. One of the higher-expressing clones was used for 8 transfers, resulting in 2 pregnancies. After the first sow aborted at day 21, the second was sacrificed at 7 weeks and 3 embryos were collected. Fibroblasts were successfully cultured from 2 of the embryos and are being expanded for expression analysis and re-cloning. Conclusion: We have shown that GTKO/hCD55-hCD59 pig cells modified to secrete an anti-CD2 monoclonal antibody can be used for somatic cell nuclear transfer, without affecting gestational development to at least the 7 week stage. The next step will be to confirm anti-CD2 expression by the embryonic fibroblasts, followed by re-cloning to generate experimental animals.