Immunology Resarch Centre
Generation of transgenic pigs expressing a T cell-depleting anti-CD2 monoclonal antibody
Evelyn Salvaris1, Ivan Vassiliev2, Stephen McIlfatrick2, Nella Fisicaro1, Jamie Brady3, Wayne Hawthrone4,5, Andrew Lew3, Mark Nottle2, Peter Cowan1,6.
1Immunology Research Centre, St Vincent's Hospital, Melbourne, Australia; 2Obstetrics and Gynaecology, University of Adelaide, Adelaide, Australia; 3Immunology Unit, Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia; 4Transplant and Renal Research, Millennium Institute for Medical Research, Westmead, Australia; 5Department of Surgery, University of Sydney, Sydney, Australia; 6Department of Medicine, University of Melbourne, Melbourne, Australia
Background: Achieving a clinically acceptable level of systemic immunosuppression remains a major challenge in xenotransplantation. One transgenic approach to this problem is local immunosuppression, in which the graft protects itself by secreting immunomodulatory molecules in the local environment. We have chosen to target CD2, which is expressed exclusively on T cells and NK cells. We previously generated CD2hb11, an anti-CD2 monoclonal antibody (mAb) that binds human and primate (but not pig) CD2. We made a partly humanised (chimeric) form of CD2hb11 and showed that it: (i) depletes human T cells in vitro and baboon T cells in vivo; (ii) blocks costimulation of human T cells via the CD2-CD58 axis; and (iii) protects adenovirus-transfected pig islets from infiltration by human T cells in a humanised mouse model (Brady et al, Xenotransplantation 2013; 20:100). In the current study, we pursued the next step in this strategy, which was to move the technology into the pig.
Aim: To generate transgenic pigs expressing the chimeric anti-CD2 mAb CD2hb11.
Methods: Pig kidney cells (PKC) were isolated. Minimum passage number PKC were transfected by nucleofection with a transgenic construct consisting of the coding regions of the heavy and light chains of CD2hb11 (linked by an F2A signal for equimolar synthesis) under the control of an MHC class I promoter to ensure widespread expression. Stable clones were selected using G418 and screened for secretion of CD2hb11 by FACS using culture supernatant as described below. Expressing clones were used for somatic cell nuclear transfer (SCNT).
Results: Following transfection, 90 stable clones were isolated and expanded. 14 clones secreted CD2hb11 of which three were used for SCNT. 12 transfers were performed with reconstructed embryos generated from one clone producing three pregnancies, two litters and a total of 5 healthy live born piglets. All piglets were confirmed transgenic by PCR. To detect CD2hb11 secretion, human T cells or CHO cells expressing baboon CD2 were analysed by FACS after sequential incubation with piglet serum and a labeled anti-human IgG mAb. CD2hb11 was present in sera from all 5 piglets. Dilutions of purified CD2hb11 were included in the FACS assay to estimate the concentration of CD2hb11 in the sera, indicating a range between 230 and 600 ng/ml.
Conclusion: We have generated transgenic pigs that secrete the chimeric anti-CD2 mAb CD2hb11, without obvious side effects. The CD2hb11 transgene will be bred onto our existing GM pig background (GTKO/hCD55/hCD59) to determine whether ‘local’ immunosuppression protects xenografts in pig-to-primate kidney and islet xenotransplantation models. The ultimate goal is to reduce or even eliminate systemic immunosuppression and its associated risks and side effects.
15:30 - 17:00
|Genetic Engineering; Methodology||Generation of transgenic pigs expressing a T cell-depleting anti-CD2 monoclonal antibody||Room 109|