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Heart xenotransplantation using genetically-engineered pigs: can CTLA4-Ig replace anti-CD154mAb in the immunosuppressive regimen?

Burcin Ekser^1,2, Cassandra Long¹, Goutham Kumar¹, Hidetaka Hara¹, Eefje M. Dons¹, Jnanesh Thacker³, Yoshiya Toyoda³, Massimiliano Veroux², David Ayares⁴, David K.C. Cooper¹, Mohamed Ezzelarab¹

¹Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, PA, United States; ²Department of Surgery, Transplantation and Advanced Technologies, Vascular Surgery and Organ Transplant Unit, University Hospital of Catania, Catania, Italy; ³Department of Cardiothoracic Surgery, Division of Cardiac Surgery, University of Pittsburgh, Pittsburgh, PA, United States; ⁴Revivicor, Inc., Blacksburg, VA, United States

Background: Heterotopic heart xenotransplantation (HTx) using genetically-engineered pigs can be achieved without sensitization to pig antigens with an anti-CD154mAb-based immunosuppressive regimen (IS). We investigated whether CTLA4-Ig could replace anti-CD154mAb.

Methods: Study 1: GTKO pig-to-baboon artery patch Tx was carried out (n=6). IS was anti-CD154mAb-based (n=4) or CTLA4-Ig-based (n=2) with ATG, MMF, and steroids in both regimens. Study 2: HTx was carried out in baboons using GTKO.hCD46.hCD55 pigs (n=4). IS was anti-CD154mAb-based (n=2) or CTLA4-Ig-based (n=2), but HTx recipients received additional anti-CD20mAb. CD3+, CD4+, CD8+ T cell and CD19+, CD20+, CD21+ B cell counts and IgG and IgM antibody (Ab) binding against WT and GTKO PBMCs were determined by flow cytometry. Follow-up in both studies was for one month.

Results: Study 1: Baboons with GTKO patch grafts did not develop anti-pig Abs if IS was anti-CD154mAb-based, but did develop sensitization (5.5-fold increase in anti-pig IgG, no increase in anti-pig IgM) if IS was CTLA4-Ig-based. Study 2: The addition of anti-CD20mAb to the regimen (compared to historical controls) depleted all B cells in the blood for >1m (mean pre-Tx CD19#, CD20# and CD21# were 142, 650, 395, while post-Tx were 2, 2, 4, respectively). Despite this additional IS, baboons receiving a CTLA4-Ig-based IS regimen developed anti-pig IgG (with a 2.3-fold increase against WT and 1.6-fold increase against GTKO PBMCs), whereas those receiving anti-CD154mAb did not. After ATG, repopulation of CD3+T cells occurred more rapidly in CTLA4-Ig-treated baboons (73% recovery) than in anti-CD154mAb-treated baboons (9% recovery).

Conclusions: When either GTKO artery patch grafts or GTKO.CD46.CD55 heart grafts were transplanted into baboons, an anti-CD154mAb-based IS regimen prevented sensitization to pig antigens, whereas a CTLA4-Ig-based regimen did not, even with additional B cell depletion, though less sensitization occurred despite increased antigen load. Potent clinically-applicable IS or additional pig genetic modifications will be required to prevent T cell-dependent Ab development. Alternative approaches to block CD40-CD154 signaling pathway might be necessary.