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Induction B-cell depletion, but not CD28/B7-directed costimulation blockade, prevents induced anti-non-Gal antibody responses after GalTKO.hCD46 cardiac xenotransplantation

Agnes Azimzadeh¹, Lars Burdorf¹, Avneesh Singh², Tianshu Zhang¹, Elana Rybak¹, Sean Kelishadi¹, Tiffany Stoddard¹, Emily Welty¹, Chris Avon¹, Amal Laaris¹, Keith Horvath², Bernard Vanhove³, David Ayares⁴, Muhammad Mohiuddin², Richard Pierson III¹

¹Surgery, University of Maryland School of Medicine, Baltimore, MD, United States; ²NHLBI/CSRP, NIH, Bethesda, MD, United States; ³U643, INSERM, Nantes, France; ⁴Revivicor, Blacksburg, VA, United States

Purpose: The aim of this study was to determine whether targeting CD28/B7 costimulation or transient B-cell depletion prevents elaboration of anti-non-Gal antibody that is often seen in conjunction with a well-studied but incompletely effective a-CD154 monoclonal antibody−based regimen.

Methods: 20 baboons received an heterotopic heart xenograft from GalTKO pigs, most of which (19) also expressed human CD46 (hCD46). All baboons received the standard a-CD154-based immunosuppressive regimen utilized by Kuwaki et al (Nature Med 2005). Three recipients were additionally given human CTLA4-Ig (10-20mg/kg d0,5,14), and one received a non-activating anti-human CD28 antibody fragment (sc28AT, 4mg/kg, qd d0-14). Ten instead received anti-CD20 (rituximab 19 mg/kg D-2,0,7,14). If graft failure occurred before day 14, immunosuppression was continued until day 14. Anti-non-gal antibody (aNGAb) levels were measured by flow cytometry using GalTKO endothelial cells.

Results: With a-CD154 treatment cardiac xenografts survived for a median of 9 days (range 7-28d) and 7d (2-10d) with additional CD28/B7 blockade, but >60d with additional aCD20 treatment, which efficiently depleted recipient B-cells. All recipients treated with the aCD154 regimen exhibited strong IgM and intermediate-to-high IgG induced aNGAb responses by two weeks post-transplant. Addition of CTLA4-Ig or sc28AT did not significantly modulate the aNGAb response (Table). In contrast, no induced aNGAb response was detected with additional rituximab-induced B cell depletion.

Conclusion: CD28/B7 inhibition with CTLA4-Ig or selective CD28 blockade as dosed here did not prevent elicited aNGAb production in baboons treated with an a-CD154-based immunosuppressive regimen. The prevalence of pretransplant aNGAb levels above those typically found in humans suggests that pre-existing anti-non-Gal immunity may have limited the efficacy of CD28/B7 inhibition and anti-CD154 treatment in this experience, although other factors (receptor coverage, innate immune activation) may also have contributed. In contrast B cell depletion prevents early elicited a-non-Gal responses, perhaps contributing to the prolonged survival previously reported with this regimen.