Identification of RHAMM-Derived CD8⁺ Restricted, Heteroclitical, Cryptic Epitope R9Y as a Promising Target for Immunotherapy of Chronic Lymphocytic Leukemia.

Abstract 3034

Poster Board II-1010

The receptor for hyaluronic acid mediated motility (RHAMM) is leukemia associated antigen (LAA) strongly correlated with proliferation and bearing negative prognosis for chronic lymphocytic leukemia (CLL). Recently, we completed a peptide vaccination clinical trial with dominant RHAMM-derived epitope (R3) for patients with CLL. To our surprise, R3 vaccination induced regulatory T cells (Treg) in 66% vaccinated patients which might limit the clinical efficacy of peptide immunotherapy. Obtained results also pointed to a pre-existing tolerance to LAA-derived peptides. While the tolerance is limited to dominant epitopes we aimed to identify a crypitic epitopes that might circumvent tolerance mechanisms and in order to increase affinity modified them heteroclitically.

We screened in silico 50 peptides with different heteroclitical modifications using SYFPEITHI algorithm and calculate affinity to MHC-I complex. Further, we choose 10 peptide pairs (cryptic – modified) which affinity increased to the highest extent and screened them in vitro assay for the affinity as well as the peptide-MHC-I complex stability in T2 binding test. In functional studies we screened 5 peptide pairs by ELISPOT analysis using CD8+ T cells isolated from peripheral blood (PB) of CLL patients and healthy donors. We found that CD8+ cells from CLL patients presensitized with peptides: R9Y (YLQLDAFEV); R13YL (YLVQSLEDV); R15Y (YLKQTLDEL); R17YL (YLQEQLNKI) as well as R19YL (YLIKHVVKL) specifically recognized T2 cells pulsed with respective peptides with the highest frequency for R9Y (90%). In chromium-51 release assays, R9Y-primed CD8⁺ T cells from CLL patients were able to effectively lyse R9Y-peptide pulsed T2 cells as well as CLL leukemic cells presenting RHAMM in a HLA-A2 restricted manner; in contrast, K562 cells and T2 cells lacking either RHAMM or HLA-A2 expression were not lysed above background levels. In addition, mAb-based MHC I blockade demonstrated that the observed peptide-specific lysis was MHC I-restricted.

In conclusion, we defined a novel RHAMM-derived CD8+ restricted, heteroclitical, cryptic epitope R9Y which might represent an interesting target for immunotherapy of CLL. In future clinical administration the R9Y peptide might circumvent tolerance to dominant epitopes and thereby increase efficacy of peptide vaccination.