Preliminary evaluation of local expression of a human T-cell depleting monoclonal antibody by diliximab-hCD55-hCD59-GalTKO pig islet xenografts in diabetic mice and baboons
Evelyn J. Salvaris1, Erin Fuller2, Thanh Le2, Nella Fisicaro1, Stephen McIlfatrick3, Mark B. Nottle3, Wayne J. Hawthorne2,4, Peter J. Cowan1,5.
1Immunology Research Centre, St Vincent's Hospital, Melbourne, VIC, Australia; 2The Centre for Transplant and Renal Research, Westmead Institute for Medical Research, Westmead, NSW, Australia; 3Reproductive Biotechnology Group, Robinson Research Institute and School of Biomedicine, University of Adelaide, Adelaide, SA, Australia; 4Department of Surgery, Westmead Hospital, School of Medical Sciences, University of Sydney, Westmead, NSW, Australia; 5Department of Medicine, University of Melbourne, Melbourne, VIC, Australia
Background: Our preclinical model clearly demonstrates restoration of blood glucose control and long-term islet cell function after transplantation of GTKO-hCD55-hCD59 porcine neonatal islet cell clusters (NICCs) into diabetic immunosuppressed baboons. However, T cell-mediated rejection inevitably occurs following withdrawal of maintenance immunosuppression. We hypothesised local expression of an anti-T-cell monoclonal antibody by the xenografts may prevent rejection after withdrawal of all exogenous immunosuppression. We previously generated a transgenic (Tg) pig line, using CRISPR to integrate a transgene for diliximab, a chimeric anti-human CD2 monoclonal antibody, into GGTA1. Transgene expression was driven by a mouse MHC class I promoter for ubiquitous tissue expression. Diliximab expression was detected in all pig organs tested including pancreas.
Aim: To examine local expression of diliximab produced by Tg pig NICC in pig-to-mouse and pig-to-NHP NICC xenograft models.
Methods: Diliximab-GalTKO and hCD55-hCD59-GalTKOpigs were crossbred to produce diliximab-hCD55-hCD59-GalTKO pigs. Genotyping was performed by PCR. Tg pigs were tested for diliximab expression by ELISA (serum), RT-qPCR and immunohistochemistry (tissues). Diliximab-GalTKO and control GTKO NICCs were transplanted under the kidney capsule of streptozotocin-diabetic SCID mice and diliximab-hCD55-hCD59-GalTKO NICCs were transplanted intraportally into two diabetic baboons. NICC xenograft maturation and function and diliximab expression were assessed histologically following biopsy and NICC function by daily blood sugar levels (BSL) and glucose tolerance tests.
Results: Diliximab NICC xenografts restored normoglycemia in diabetic immunodeficient mice, and in our diabetic baboons. In the mouse, detectable diliximab expression was seen in situ on day 85 and from transplanted baboon biopsies at day 112, 114 and endpoint. Systemic porcine C-peptide was detected from day 30 onwards. Baboon liver biopsies showed well preserved morphology with no cellular infiltrate (H&E) and staining for insulin, somatostatin and glucagon. Moderate to strong expression of diliximab was detected on the islets and in the surrounding biopsy tissues.
Discussion/Conclusion: Islet cell function remains unaffected by the addition of the diliximab transgene in both mouse and baboon models. Preliminary immunohistochemistry data in baboon model demonstrates islet xenografts express diliximab on the NICCs and in the surrounding recipient liver tissue confirming local secretion of anti-CD2 by the Tg pig islets. Whether this level of diliximab expression will be sufficient to prevent T-cell mediated islet xenograft rejection is pending ongoing investigation.