Evaluation of expression of a human T-cell depleting monoclonal antibody in islets and other tissues of transgenic pigs
Evelyn Salvaris1, Nella Fisicaro1, Stephen McIlfatrick2, Adwin Thomas3, Erin Fuller3, Andrew M Lew4, Mark B Nottle2, Wayne J Hawthorne3,5, Peter J Cowan1,6.
1Immunology Reseach Centre, St Vincent's Hospital , Melbourne, Australia; 2Reproductive Biotechnology Group, Robinson Research Institute and School of Biomedicine, University of Adelaide, Adelaide, Australia; 3The Centre for Transplant and Renal Research, , Westmead Institute for Medical Research, Westmead, Australia; 4Walter and Eliza Hall Insititute, Department of Medical Biology and Department of Microbiology and Immunology, University of Melbourne, Melbourne, Australia; 5Department of Surgery, Westmead Hospital, School of Medical Sciences, University of Sydney, Westmead, Australia; 6Department of Medicine, University of Melbourne, Melbourne, Australia
Background: Our preclinical model demonstrates long-term restoration of blood glucose control in diabetic immunosuppressed baboons transplanted with GTKO-hCD55-hCD59 porcine neonatal islet cell clusters (NICCs). However, T cell-mediated rejection inevitably occurs following withdrawal of maintenance immunosuppression. We hypothesise local expression of an anti-T-cell monoclonal antibody by the xenografts may prevent rejection. We previously reported generation of two transgenic (Tg) pig lines, using CRISPR to integrate a transgene for diliximab, a chimeric anti-human CD2 monoclonal antibody, into GGTA1. Transgene expression was directed by either a mouse MHC class I promoter (MHCIP) for ubiquitous tissue expression, or the pig insulin promoter (PIP) for islet-specific expression. Aim: To compare diliximab expression in the Tg pig lines and to examine local expression in a pig-to-mouse islet xenograft model.
Methods: Diliximab expression was tested by ELISA (serum), RT-qPCR and immunohistochemistry (tissues). MHCIP-diliximab and control GTKO NICCs were transplanted under the kidney capsule of streptozotocin-diabetic SCID mice. NICC xenograft maturation and function were assessed by blood sugar levels (BSL) and intraperitoneal glucose tolerance tests.
Results: Diliximab mRNA and protein were detected in spleen, kidney, heart, liver, lung, and pancreas of MHCIP-diliximab pigs, and in serum at a mean concentration of 1.8 μg/ml. Tissue expression ranged from strong in spleen to patchy in pancreas, with weak expression in islets. MHCIP-diliximab NICC xenografts restored normoglycemia in diabetic immunodeficient mice, indicating no overt effect of the transgene on islet function, and demonstrated weak expression of diliximab in situ on day 85. Diliximab was not detected in serum or non-pancreatic tissues of PIP-diliximab pigs, but unexpectedly was also absent in islets.
Discussion/Conclusion: Diliximab was expressed in all tested organs of MHCIP-diliximab pigs including the pancreas. Islet cell function appeared to be unaffected by the transgene, and expression by islet xenografts in mice was detected at the transplant site. Despite weak basal expression in MHCIP-diliximab islets, we predict that in the transplant setting, pro-inflammatory cytokines produced by activated T cells recruited to the site will induce the MHCIP to upregulate diliximab expression; whether this will be sufficient to prevent T-cell mediated islet xenograft rejection awaits further investigation. Surprisingly, the PIP-diliximab pigs failed to express diliximab in islets, despite in vitro validation of the transgene construct and confirmation of correct integration into GGTA1. Sequencing analysis is underway to assess the integrity of the transgene in the genome of the PIP-diliximab pigs.
National Health & Medical Research Council of Australia (Project Grants #1061868 and #1156889) and the Juvenile Diabetes Research Foundation (Project 3-SRA-2017-366-S-B).
