Pembrolizumab (anti-PD-1) Treatment Increases Anti-Tumor Activities of XBP1/CD138/CS1-Specific Cytotoxic T Lymphocytes Against Multiple Myeloma

In these studies, we investigated the role of Pembrolizumab (anti-PD-1)to enhance the function of myeloma-specific CD8⁺ cytotoxic T lymphocytes (CTL) and maintain their antigens-specific CTL activities against multiple myeloma cells. Bone marrow or peripheral blood mononuclear cells (BMMC, PBMC) from healthy donors or MGUS, smoldering multiple myeloma (SMM), newly diagnosed multiple myeloma (MM), relapsed MM, relapsed/refractory MM patients (N=5, respectively) were evaluated for expression of immune checkpoints that regulate antigen-specific CTL development and function. Active MM patients had higher levels of CD138⁺ tumor cells as compared to MGUS and SMM patients. PD-L1 and PD-L2 expression levels on myeloma cells were also higher in active MM patients than healthy donors. CD4 Treg (CD3⁺CD4⁺CD25⁺FoxP3⁺), but not CD8 Treg (CD3⁺CD8⁺CD25⁺FoxP3⁺), frequencies were highest in both BMMC and PBMC from MM patients. PD-L1, but not PD-L2 nor PD-1, was expressed on the CD4 Treg in BMMC and PBMC from MM patients. G-type MDSC (CD11b⁺CD33⁺HLA-DR^lowCD15⁺), but not M-type MDSC (CD11b⁺CD33⁺HLA-DR^lowCD14⁺), was increased in MM as compared to MGUS, SMM or healthy donors. The MDSC from active MM patients also had high levels of PD-L1 and PD-L2 expression. BMMC and PBMC from newly diagnosed MM patients (N=3) treated in vitro with Pembrolizumab enhanced the CD3⁺ T cell proliferation as compared to the untreated group. Interestingly, Pembrolizumab treatment induced proliferation of T cells expressing immune agonists (OX40, GITR) beneficial for cell function and immune checkpoint (LAG3) involved in down-regulating T cell activities. XBP1/CD138/CS1 antigens-specific CTL (XBP1/CD138/CS1-CTL) generated in vitro with a cocktail of immunogenic peptides [heteroclitic XBP1 US_184-192 (YISPWILAV), heteroclitic XBP1 SP_367-375 (YLFPQLISV), native CD138_260-268 (GLVGLIFAV), and native CS1_239-247 (SLFVLGLFL)] displayed increased proliferation of CD8⁺ CTL expressing the CD28 co-stimulatory and CD38 activation markers upon treatment with Pembrolizumab. Importantly, the XBP1/CD138/CS1-specific central memory and effector memory CTL subsets increased the cell proliferation, following treatment with the PD-1-specific antibody. In addition, XBP1/CD138/CS1-CTL treated with Pembrolizumab demonstrated increased cytotoxic activity and Th1-specific cytokines production against U266 myeloma cells. Thus, these studies suggest the potential benefit of Pembrolizumab (a-PD-1) therapy in MM patients to enhance XBP1/CD138/CS1 antigens-specific memory CTL proliferation and boost their anti-tumor activities through blockage of the PD-1 specific immune checkpoint specifically expressed on antigens-specific CD8⁺ CTL. These data provide the framework for an immunotherapeutic strategy encompassing a combination of multipeptide-based cancer vaccine with PD1-specific antibody to enhance the antigens-specific T cells efficacy and promote stronger and long-lasting CD8⁺ CTL immune responses in myeloma patients.