ALBERT

Description: Despite progress in the treatment of multiple myeloma (MM), the disease remains incurable. Therefore novel therapeutic approaches are required to achieve better outcome in patients. Development of peptide-based immunotherapy against tumor specific or associated antigens offers an attractive approach that may lead to tumor-targeted cellular therapy in MM patients. Recently, CS1 (CD2 subset 1, CRACC, SLAMF7), a cell surface glycoprotein of the CD2 family, was found to be highly expressed by the tumor cells of the majority of MM patients but not other normal tissues. Importantly, a novel anti-CS1 HuLuc63 mAb induced significant anti-myeloma killing in vitro and in vivo (Abstract #950059). In addition, CS1 may play a role in MM cell survival (Abstract #953659). In this study, we asked whether CS1 is a suitable myeloma-associated antigen for cellular therapy in MM. We wanted to identify potential immunogenic peptides derived from CS1 and tested whether these peptides evoke MM-specific cytotoxic T lymphocytes (CTLs). First, we predicted the potential peptide sequences of CS1 that could bind to HLA-A*0201 molecule using three databases (RANKPEP, BIMAS and NetMHC). A total of four peptides were selected and synthesized according to the high binding scores among all the databases. Next, peptide-T2 cells binding assay confirmed these peptides with high binding affinity to HLA-A*0201 molecule (Fluorescence index: 2.19, 3.28, 3.44 and 3.49). To generate the peptide-specific CTLs, HLA-A*0201-positive normal human CD8+ T lymphocytes were stimulated with autologuous dendritic cells (DCs) or T2 cells pulsed with candidate CS1-peptides. The CTL cell lines were established after several rounds restimulation. We have evaluated the immunogenicity of the expanded CTLs by the stimulation with peptide-pulsed or unpulsed T2 cells. Our data demonstrated that one peptide-induced CTLs (CS1-CTLs) possessed high antigen-specific immune responses with the HLA-A*0201-restriction relative to a certain level of immune responses displayed by another three peptide-induced CTLs. We further evaluated the proliferative activity of CS1-CTLs by 3H-TdR incorporation assay. Upon stimulation by peptide-pulsed T2 cells, the cell proliferation of CS1-specific CTLs increased approximately 55-fold, compared with unstimulated cells (pulsed vs. unpulsed: 22894 vs. 423 cpm). The activation of CS1-CTLs was monitored by the expression of CD25 (IL-2Rα chain) on peptide-stimulated CS1-CTLs (%CD25+ cells: pulsed vs. unpulsed: 98% vs. 13%). CS1-CTLs significantly secreted 14-fold higher levels of IFN- γ in response to peptide-pulsed T2 cells (pulsed vs. unpulsed: 615 vs. 46 pg/ml). Importantly, the expanded CS1-CTLs generated high cytotoxicity (93%) in response to peptide pulsed T2 cells. Furthermore, more than 30% of killing activity by CS1-CTLs against HLA-A*0201+CS1+ MCCAR cells was observed at the effector: target ratio of 20:1. In contrast, these CS1-CLTs did not kill HLA-A*0201+/CS1− U266 and HLA-A*0201−/CS1+ MM1S target cells. These results support CS1 as an antigenic target for the induction of peptide-specific CTLs against MM cells. Further functional studies are underway to confirm potential CS1-specific cellular therapy to treat MM.