Both L- and D-isomers of allotrap 2702 prolong cardiac allograft survival in mice

Background: Peptides derived from a conserved region of the human leukocyte antigen class I heavy chain (a.a. 75-84) have been shown to have immunomodulatory activity. The peptide 07.75-84, derived from HLA-B7, prolonged Lewis 1.W cardiac allograft survival in Lewis 1.A recipients. In combination with cyclosporine A, permanent heart allograft acceptance was induced in the Lewis to ACI donor/recipient combination. In mice, 2702.75-84, derived from HLA-B2702, prolonged the survival of C57B1/6 skin allograft in CBA recipients. In vitro, 2702.75-84 inhibited NK and cytotoxic T cells. To further evaluate the immunomodulatory activity of both L- and D-isoforms of 2702.75-84, we studied their effects in a mouse cardiac allograft model.

Methods: Peptides consisting of L- and D-amino acids were synthesized and administered to CBA (H-2k) recipients of a C57B1/6 (H-2b) cardiac allografts. In addition to peptide monotherapy, combinations of peptide with cyclosporine A and azathioprine were evaluated. Graft survival was monitored by palpation. Breakdown products of 2702.75-84 were identified by a combination of high-performance liquid chromatography and mass spectrometry.

Results: Daily administration of 2702.75-84 to CBA (H-2k) recipients of a C57B1/6 (H-2b) cardiac allograft prolonged graft survival to 11.4 +/- 2.6 days compared with 7.5 +/- 1.2 days in untreated control Animals (n = 8; p = 0.0003, Mann-Whitney U Test). Other major histocompatibility complex class I-derived peptides including HLA-G.75-84, HLA-07.75-84, HLA-2705.75-84, H-2Kk.75-84, H-2Dk.75-84, H-Kb.75-84, and H-2Db.75-84 had no effect. In combination with a subtherapeutic dose of cyclosporine A (days 0 to 4), 2702.75-84 prolonged graft survival to at least 70 days (five of eight grafts still beating, p = 0.0002) compared with a median graft survival of 14 days in Animals that received cyclosporine A monotherapy. Like Other peptides consisting of L-amino acids, 2702.75-84 is subject to degradation by serum proteases. Breakdown products of 2702.75-84 were identified by a combination of high-performance liquid chromatography and mass spectrometry. These compounds were found to be inactive when tested in vivo. In contrast to peptides consisting of L-amino acids, peptides consisting of D-amino acids are resistant to proteolytic degradation. When D2702.75-84 was administered to graft recipients at 5 to 10 mg/kg/day (days 0 to 10), graft survival was prolonged to more than 50 days (5 of 13 grafts still beating, p = 0.0001). In combination with azathioprine, administration of D2702.75-84 induced graft acceptance (> 100 days) in 100% of Animals (all grafts still beating).

Conclusions: This study shows that administration of 2702.75-84 consisting of L- or D-amino acids will prolong graft survival in mice. In addition, the data suggest that the D2702 isomer is more potent than the L-isomer in vivo and may allow reduced dosage or frequency of administration.