Experimental Study On Therapeutic Effect Of BB-102-modified Myeloma Cell Vaccine

Multiple myeloma (MM) is a clonal B cell neoplasia affecting both the immune system and the skeletal system. Little progress in the treatment of this disease has been made despite decades of treatment with different chemotherapy regimens, all of which have only resulted in a 5-year survival rate of <10%. Although high-dose chemotherapy and stem cell transplantation have improved the rate of complete remission, some myeloma cells escape the treatment and all MM patients experienced relapse. Therefore, new therapeutic modalities are needed for this disease. Immunotherapy is expected to offer a cure or long-term disease control. In this study, we attempted to provide an individualized immunotherapic strategy of gene-modified MM vaccination. We have constructed a recombinant adenovirus termed BB-102 carrying human wild-type p53, granulocyte-macrophage colony-stimulating factor (GM-CSF), and B7-1 genes previously. Co-transfer of immunomodulatory genes GM-CSF, B7-1 and apoptosis-inducing gene p53 into malignant plasma cells for making a tumor vaccine might invoke an autologous immune response to tumor cells and be of potential therapeutic value in MM. In this study, we aimed to develop a novel therapeutic strategy for MM by using a BB-102-modified tumor vaccine.As indicated in previous studies, rather than the absence of tumor-specific antigens, myeloma cells escape from immune surveillance mainly by down-regulating the expression of costimulatory molecules (especially B7-1 molecule) and inhibiting the induction and maturation of dendritic cells (DCs). Our hypothesis was that co-transfer of B7-1, GM-CSF and p53 genes should result in a synergistic effect on augmenting the immunogenicity of myeloma cells. It is known B7-1 (i.e. CD80) molecule plays a critical costimulatory role in the activation of TCR-stimulated CTLs by binding to CD28. GM-CSF has the ability to promote maturation of precursor cells into DCs, and P53 protein induces the apoptosis of MM cells. Recent studies haveshown that DCs cultured with apoptotic bodies stimulated significantly greater T cell proliferation than MM lysate-plused DCs or MM cells alone did. Mature DCs generate tumor antigen epitopes for cross-presentation on HLA-I class to stimulate CD8+ cytotoxic T cells, or for conventional presentation on HLA-II class to stimulate CD4+ helper T cells.In ex vivo experiments, we evaluated the effect of a BB-102-modified myeloma cell vaccine strategy aiming to induce the apoptosis and augment the immunogenicity of MM cells. Three myeloma cell lines Sko-007, U266 and RPMI8226 were used in this study. Human primary MM cells were separated from bone marrow aspirates of MM patients. BB-102 and other 4 control Ads, Ad-GFP, Ad-p53, Ad-p53/GM-CSF and Ad-p53/B7-l were obtained by large-scale amplification in 293 cells and ultra-centrifugating in CsCl step gradient solutions. Infectious efficiency was assayed by flow cytometry using MM cells transfected with Ag-GFP. Both MM cell line and purified primary myeloma cells were infected with BB-102. The high-level expressions of these three genes were confirmed separately by Western blot, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. By introduction of wild-type p53, GM-CSF and B7-1 genes, the growth of MM cells was inhibited via enhanced apoptosis and the immunogenicity of tumor cells was augmented. The combinative effect of these 3 genes on inducing cytotoxic T lymphocytes (CTLs) was more evident than that of p53 individually or in combination with one of the other two genes (p53 plus GM-CSF or p53 plus B7-1). Furthermore, significant proliferation of autologous peripheral blood lymphocytes (PBLs) and specific cytotoxicity against autologous primary MM cells were induced in vitro.We also evaluated the in vivo antitumor effect of genetically modified myeloma cell vaccine on human myeloma xenografts implanted into non-obese diabetic / severe combined immunodeficiency (NOD/SCID) mice. Human immune system was reconstructed in NOD/SCID mouse by intraperitoneal injection of human peripheral blood lymphocytes (PBLs). After inoculated subcutaneously with irradiated myeloma cell line sko-007, adenoviral transduced to express either the GFP or p53, GM-CSF and 57-7 genes, huPBL-NOD/SCID mice were challenged by subcutaneous injection of sko-007 cells. As a result, Ad-p53/GM-CSF/B7-l -infected sko-007 cell vaccination significantly reduced local tumor growth compared with controls. Histological analysis showed that tumor tissues displayed diffuse necrosis, mainly caused by apoptosis,...