Introduction: Dendritic cells (DC) are professional antigen-presenting cells. In patients, DC can induce an effective antitumor response by presenting a tumor-derived peptide in the groove of major histocompatibility complexes (MHC) to naΓ―ve T-cells. In our ongoing clinical trials, HLA-A2.1 + melanoma patients are vaccinated with mature DC loaded with tumor-derived gp100 peptides. However, little is known about the fate of the MHC-peptide complex after injection of these peptide-loaded DC. Therefore, we investigated the site-specific DTPA conjugation and subsequent 111 In labeling of tumorderived peptides and their binding to MHC class I.
Methods: To investigate the effect of the position of the chelator, the HLA-A2.1 binding peptide gp100:154-162mod (KTWGQYWAV) was conjugated with diethylene-pentaacetic acid (DTPA) either at the N-terminus ('alpha-DTPA') or at the epsilon amino group of the Lys 154 -residue ('epsilon-DTPA') and labeled with 111 InCl 3 . The specific activity was determined by RP-HPLC. The IC 50 of the radiolabeled peptides for the MHC molecule was determined both on a HLA-A2.1 + B lymphoblastoid cell line (JY) and on human DC in a competition-based assay with a FITC-labeled peptide and subsequent FACS-analysis. MHC binding studies with the 111 In-labeled peptides were performed on the same cell lines. Stability of the radiolabeled peptides in PBS and in the presence of DCs was analysed with RP-HPLC. To investigate whether the radiolabeled peptides could induce a cytotoxic Tlymphocyte response, a cytotoxicity assay was performed using 51 Cr-labeled JY cells as target cells.
Results: For both alpha-DTPA and epsilon-DTPA peptide, the maximum specific activity after 111 In-labeling was 13 GBq/Β΅mol and radiochemical purity exceeded 99%. The IC 50 of the 111 In-labeled alpha-DTPA peptide was >1000 Β΅M. The IC 50 of the 111 In-labeled epsilon-DTPA peptide was 3 Β΅M, which was similar to that of the unconjugated peptide. MHC binding studies showed specific binding of the 111 In-labeled epsilon-DTPA peptide to the JY cells at 4Β°C which could be blocked by an excess of unconjugated peptide. Interestingly, no specific binding was observed for the 111 In-alpha-DTPA peptide, most likely due to interference of the DTPA moiety. Incubation at 37Β°C resulted in less efficient binding to MHC, which may be due to instability of the MHC-peptide complex at 37Β°C. Moreover, RP-HPLC analysis showed partial degradation of the peptides, most likely due to DCassociated (amino)peptidase activity. The cytotoxicity assay revealed that in contrast to the 111 Inalpha-DTPA peptide, the 111 In-epsilon-DTPA peptide was able to elicit a specific cytotoxic response.
Conclusion:
We showed that DTPA conjugation can strongly affect the MHC binding and biological activity of the gp100-derived peptides. Conjugation of DTPA to the N-terminal NH 2 -group resulted in loss of affinity for the MHC. When DTPA was conjugated to the epsilon NH 2 -group of the Lys 154 -residue, MHC binding of the peptide was preserved. Moreover, cells loaded with the 111 Inepsilon-DTPA gp100:154-162mod peptide were still able to elicit a specific cytotoxic response. These studies allow the noninvasive determination of the behavior of MHC-peptide complexes on DC in vivo. This could allow the rational design of vaccination schemes for immunotherapy.