Purpose: Monotherapy using αCD28 or αCD40 antibodies significantly prolongs graft survival but does not induce tolerance in non-human primates (NHP). Here, we evaluate the relative efficacy of combined αCD28 and αCD40 treatment to attenuate pathogenic alloimmunity in a preclinical cardiac NHP model.

Methods: Cynomolgus cardiac transplant recipients were treated with either αB7 (belatacept, n=4), αCD28 (FR104, n=7), αCD40 (2C10R4, n=6), or αCD28+αCD40 (n=5) for 90 days. Acute rejection (ISHLT score) and chronic allograft vasculopathy (CAV) severity were quantified in protocol biopsies and explanted grafts using published scoring systems. Antidonor alloantibody production was detected by flow cytometry. Peripheral CD4⁺ and CD8⁺ T memory phenotypes were characterized as naïve (N: CD45RA⁺ or CD95^–CD62L⁺), central memory (CM: CD45RA^– or CD95⁺CD62L⁺), or effector memory (EM: CD45RA^+/- or CD95^+/- CD62L^–) using flow cytometry.

Results: αCD28+αCD40 prolonged allograft median survival time (MST 173 days, range 100-210) compared to αB7 (122, 7-124, p=0.006) but not αCD28 (163, 51-180) or αCD40 (124, 89-178). One of 4 αB7-treated, 3/7 αCD28-treated, and 1/6 αCD40-treated allografts, but none in the αCD28+αCD40 group, rejected during treatment. αCD28+αCD40 attenuated acute rejection (0.2±0.1) and CAV scores (0.02±0.07) relative to αCD40 (1.1±0.3, p=0.03; 1.13±0.3, p=0.03, respectively) or αCD28 (1.2±0.3, p=0.06; 0.92±0.3, p=0.08, respectively). Suppression of alloantibody production was associated with graft protection in all treatment groups, and rejection coincided with a rise in both IgM and IgG. CD8⁺ T_EM cells increased in all groups after transplant, but significantly fewer CD8⁺CD28^– T cells were detected in the αCD28+αCD40 group (295±62) compared to the αB7 (740±54, p<0.0001) and αCD40 groups (517±79, p=0.02). Similarly, fewer CD4⁺ T_EM cells (308±26) were observed with combined treatment compared to each monotherapy group (αB7:423±30, p=0.0047, αCD28: 521±44, p=0.0002, αCD40:582±51, p<0.0001).

Conclusions: αCD28+αCD40 significantly attenuated acute rejection and CAV during treatment, in association with reduced circulating T_EM, compared to aB7, αCD28, or αCD40 monotherapy. These apparently synergistic effects suggest that combined αCD28+αCD40 costimulation blockade is an effective immunosuppressive strategy, one that appears promising for clinical translation.

CITATION INFORMATION: O'Neill N, Zhang T, Sun W, Braileanu G, Cheng X, Tatarov I, Hassanein W, Azimzadeh A, Pierson R. Combination αCD28+αCD40 Costimulation Blockade Attenuates Acute and Chronic Rejection in Non-Human Primates. Am J Transplant. 2017;17 (suppl 3).

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