Effects of an agonist interleukin-2/Fc fusion protein, a mutant antagonist interleukin-15/Fc fusion protein, and sirolimus on cardiac allograft survival in non-human primates

Abstract

Background: To tilt the immunologic balance toward tolerance and away from rejection, non-human primate recipients of cardiac allografts were treated with interleukin (IL)-2/Fc, mutant (m) antagonist type mIL-15/Fc, and sirolimus.

Methods: Heterotopic heart transplants were performed on 8 fully mismatched cynomolgus macaques. An untreated control recipient rejected its graft by post-operative Day 6. The remaining 7 animals received oral or intramuscular immunosuppression with sirolimus. A recipient treated with sirolimus alone rejected at the end of 28 days of immunosuppression. The remaining 6 monkeys also received IL-2/Fc and mIL-15/Fc intramuscularly until 28 days after transplant. One animal received a second 28-day course of fusion protein starting at Day 50. In these 6 animals, sirolimus was continued for 28 days (n = 4) or until protein levels were low (n = 2).

Results: In the 4 monkeys treated with a 28-day course of sirolimus and fusion proteins, mean graft survival was 51.5 days (range, 28-76 days). The animal receiving a second course of fusion protein rejected its graft on Day 177, despite detectable levels of the fusion proteins and sirolimus. The central memory, effector memory, and naïve CD4(+) and CD8(+) T-cell populations in the peripheral blood did not change significantly during fusion protein administration. A 2.5-fold expansion in CD4(+)CD25(+) lymphocytes occurred in recipients treated with fusion proteins and sirolimus that was not observed in the recipient treated with sirolimus alone.

Conclusions: Although IL-2/Fc, mIL-15/Fc, and sirolimus administered in this manner permitted modest prolongation of graft survival and expansion of CD4(+)CD25(+) T cells, tolerance was not achieved.

Figure 1

Representative sirolimus and fusion protein levels from a recipient treated with oral (M3605) and intramuscular sirolimus (M206). A Oral administration of sirolimus (1.0-2.0 mg/kg) was associated with low serum drug levels (dark line, mean level = 8.6±3.5) whereas intramuscular administration at a lower dose (0.05-0.3mg/kg) led to higher, prolonged serum levels (light line, mean level = 21.4±2.3). B Fusion proteins were administered by intramuscular injection in both cases. The target level of 0.5 O.D, was maintained throughout the life of the graft. A pre-transplant loading period was used in all animals other than M4205.

Figure 2

Gross and muscopic appearance of cardiac allografts at time of explantation after treatment with fusion proteins and either oral (A-C; M3605) or IM (D-F; M206) sirolimus. Acute cellular rejection was present in both sets grafts at time of explantation (B, E) and cardiac allograft vasculopathy was present in the longer-surviving grafts (C, F).

Figure 3

Expansion of CD4⁺CD25⁺ lymphocytes occurred during the first two weeks of combined IL2/Fc and mIL15/FC and sirolimus administration.

Figure 4

Changes in the proportion of naïve (CD28⁺CD95-), effector (CD95⁺CD28-) and central (CD95⁺CD28⁺) memory cells during sirolimus and fusion protein administration. There was no consistent reduction in CD8+ memory T-cells.

Figure 5

Anti-donor sensitization assessed using the ELISPOT assay. A, B Animals treated with 28 days of fusion proteins and oral sirolimus (M3605, A; M4205 B), increasing anti-donor sensitization was present on day 50 and 100. C Animal treated with 28 days of fusion proteins and intramuscular sirolimus (M206). D, E Animals treated with a prolonged course of fusion