Generation of HM1.24-Specific Cytotoxic T Lymphocytes from Peripheral Blood Stem Cell Harvests of Patients with Multiple Myeloma. (2004)

Abstract HM1.24 is a type II transmembrane glycoprotein that was originally identified as a myeloma cell-specific antigen and expected to be an attractive target molecule for immunotherapy of multiple myeloma (MM). Our previous studies have demonstrated that HM1.24-specific cytotoxic T lymphocytes (CTL) can be induced by the stimulation with dendritic cells (DC) pulsed with HM1.24-derived peptides such as HM1.24-126 (KLQDASAEV) and HM1.24-165 (APQLLIVLL) in normal HLA-A2 and/or A24 individuals. Recent clinical studies have shown that autologous peripheral blood stem cell transplantation (PBSCT) significantly improves the remission rate and overall survival of patients with MM. However, additional therapeutic strategies are necessary to eradicate residual disease. To explore the possibility of HM1.24-targeting cellular immunotherapy after PBSCT, we evaluated the ability of PBSC harvests as a source of DC and investigated the efficacy of these DC to induce HM1.24 peptide-specific CTL. Eight MM patients with HLA-A2+ and/or A24+ type undergoing autologous PBSCT were studied. PBSC were collected after high-dose cyclophosphamide (100 mg/kg) and granulocyte colony-stimulating factor (400 μg/m2/day) mobilization using a COBE Spectra. The median percentage of CD34+ cells in the harvest product was 1.5% (range 0.61–13.0). PBSC were cryopreserved in 5% DMSO and 6% hydroxyethyl starch until use. DC were derived by culture of plastic-adherent mononuclear cells from thawed PBSC in medium containing granulocyte-macrophage colony-stimulating factor and interleukin (IL) -4 for 6 days, and then in the presence of tumor necrosis factor-alpha for 1 day. To generate HM1.24-specific CTL, 1x106 cells of non-adherent fraction from thawed PBSC were stimulated with 1x105 DC pulsed with 10 μg/mL of HM1.24-126 or HM1.24-165 peptides. Cells were stimulated weekly with the peptide-pulsed DC in the presence of IL-2. After two rounds of stimulation, CD8+ cells were enriched using MACS column. EBV-transformed B cells (HLA-A2+, A24+) were used as target cells. Specific recognition of peptide-pulsed target cells by CTL was determined by ELISPOT assay for interferon-gamma. These CTL were also tested for cytotoxic activity using Granzyme B ELISPOT assay. Phenotypically mature DC (CD1a+, CD40+, CD80+, CD83+, CD86+) were generated from all PBSC harvests of MM patients. CD8+ cell expansion was observed in 6 of 8 products. HM1.24-126 and HM1.24-165 peptides successfully induced peptide-specific CTL in 4 of 4 HLA-A2 patients and 2 of 4 HLA-A24 patients, respectively. More importantly, these HM1.24-specific CTL showed cytotoxic activity against myeloma cell lines such as ARH-77 (HLA-A2+) and TSPC-1 (HLA-A24+) in an HLA-restricted and antigen-specific manner. No response was observed against K562 cells, indicating that this cytotoxicity was not mediated by non-specific NK activity. These results suggest the existence of functional DC and HM1.24-specific CTL precursors in PBSC harvests of patients with MM and provide the basis for cellular immunotherapy with these HM1.24-derived peptides using PBSC harvests in combination with PBSCT.