Development of Cell Based Therapeutic Vaccines for the Treatment of Multiple Myeloma.

Abstract

Immunotherapy may provide alternative or supplementary treatment of multiple myeloma (MM). We proposed that semi-autologous myeloma hybrid cells, formed by fusing malignant plasma cells with the human lymphoblastoid cell line, HMy2, would generate immortalised cell lines with enhanced immunogenicity for T cell responses, which could be used as potential therapeutic vaccines. The specific goals of the project were: to generate stable hybrid cell lines in vitro, by fusion of HMy2 with ex vivo tumour cells from patients with MM; to characterise the hybrid cells with respect to growth in culture, stability, and surface marker phenotype; and to investigate the ability of the hybrid cells to stimulate T cell immune responses in vitro as compared with parental tumour cells. The parent HMy2 cell line was fused with ex vivo myeloma plasma cells (purified from the marrow using CD138+ magnetic beads) from 5 patients, and chemically selected in HAT medium (to which HMy2 is sensitive), to form stable cell lines that grew continuously in culture. Efforts of growing myeloma cell lines from all of the patients were unsuccessful. The hybrid status of 4 of the 5 fused cell lines was confirmed by microsatellite testing using polymorphic loci on chromosomes X, 16q, 7q, 13q, 5q, 2p, 11p, 12p. Furthermore, the microsatellite analysis was checked after 3 months of continuous culture (approximately 40 passages), and all of the hybrids remained genetically stable over this period. Hybrid cells were analysed for expression of immunologically relevant accessory molecules using flow cytometry, in comparison with parent tumour cells and HMy2. The hybrids retained CD138 expression, although expression of CD56 was reduced in hybrids from two of three CD56+ myeloma cases. CD54 (ICAM-1) and CD50 (ICAM-3) were uniformly expressed in all hybrids. CD58 (LFA-3) was expressed more strongly in the hybrids than in original tumour cells, but CD11a (LFA-1) was weakly expressed or absent, in line with expression on HMy2 cells. All of the hybrids expressed CD40, CD80 (B7-1) and CD86 (B7-2), although CD80 was weakly or not expressed by myeloma plasma cells. Both MHC class I and II were expressed in all the hybrids, suggesting the potential for antigen presentation to both CD8+ and CD4+ T cells, in contrast to the majority of myeloma plasma cells, which failed to express MHC class II. The hybrid cells therefore expressed important markers to facilitate effective antitumour responses, which were absent on the parent tumour cells. The hybrid cells stimulated allogeneic T cell proliferative (3H thymidine uptake) responses in vitro to a considerably greater degree than their respective parent myeloma plasma cells, which failed to induce T cell proliferation above background levels. In addition, T cells from patients from whom hybrid cell lines had been derived responded to stimulation with both autologous and allogeneic hybrid cell lines, whilst failing to proliferate in response to autologous or allogeneic myeloma plasma cells. These data indicate that myeloma patients are immunocompetent, suggesting that active immunisation with semi-autologous hybrid cell lines represents a valid approach to immunotherapy in these patients. We have shown the generation of myeloma hybrid cell lines that grow continuously in tissue culture and have increased immunogenicity for T cell responses in vitro. This approach represents a novel strategy for the development of therapeutic vaccines for the treatment of multiple myeloma.