Identification of CS1 Peptides for Induction of Antigen-Specific CTLs in Multiple Myeloma.
Authors: Song Weihua , Tai Yu-Tzu , Sasada Tetsuro , Burger Peter , Fulciniti Mariateresa , Li Xianfeng , Anderdon Kenneth C. , Munshi Nikhil C. 
Published: April 2020

Abstract
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.