ABSTRACT
A humanized monoclonal antibody (mAb) against HM1.24 (AHM) caused antibody-dependent cellular cytotoxicity (ADCC) against multiple myeloma (MM) cells. Here, we constructed a conventional non-radioisotope method that quantifies the amount of HM1.24 using fluorescein-labeled AHM. More than 10(4) molecules/cell of HM1.24 were detected in 12 out of 14 patients' MM cells, and a linear correlation was found between ADCC by AHM and the amounts of HM1.24. Thus, AHM is likely to be more efficacious against MM cells with high levels of HM1.24. This conventional non-RI method to quantify HM1.24 will be useful to select patients most likely to respond to AHM.
Subject(s)
Antibodies, Monoclonal/immunology , Antibody-Dependent Cell Cytotoxicity/immunology , Antigens, CD/analysis , Membrane Glycoproteins/analysis , Multiple Myeloma/immunology , Antigen-Antibody Reactions , Antigens, CD/immunology , Cell Line, Tumor , Fluorescein/chemistry , GPI-Linked Proteins , Humans , Leukocytes, Mononuclear/immunology , Membrane Glycoproteins/immunology , Reference Values , Reproducibility of ResultsABSTRACT
A humanized monoclonal antibody against HM1.24 antigen (AHM), which is highly expressed on multiple myeloma (MM) cells, induced antibody-dependent cellular cytotoxicity (ADCC) in vitro. In this study, we further characterized AHM and evaluated its potency for clinical application. AHM bound to HM1.24 antigen with a dissociation constant of 0.35 nM, and its epitope resided between Leu116 and Leu127 of the HM1.24 antigen. Single intravenous injection of AHM significantly inhibited tumor growth in both orthotopic and ectopic human MM xenograft models. AHM reduced serum M protein levels and prolonged survival of mice intravenously inoculated with KPMM2 and ARH-77 cells. The number of KPMM2 cells in bone marrow or tumor volume of subcutaneously inoculated RPMI 8226 cells was also inhibited by AHM. The antitumor activity of AHM against tumor cells in bone marrow was diminished when the mice were pretreated with anti-Fcgamma receptor III/II antibody, demonstrating that antitumor activity by AHM requires effector cell functions in vivo. Experiments involving in vitro ADCC assays indicated that NK cells and monocytes/macrophages serve as effector cells for AHM-induced ADCC in mouse and human. Thus, AHM will provide an additional treatment option for MM.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibody Specificity/immunology , Membrane Glycoproteins/immunology , Multiple Myeloma/drug therapy , Animals , Antibody Affinity/immunology , Antibody-Dependent Cell Cytotoxicity/immunology , Antigens, CD , Epitope Mapping , Flow Cytometry , GPI-Linked Proteins , Humans , Killer Cells, Natural/immunology , Leukocytes, Mononuclear/immunology , Male , Mice , Mice, SCID , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: The purpose of this work was to test the suitability of the HM1.24 antigen as a CTL target for immunotherapy of patients with multiple myeloma. EXPERIMENTAL DESIGN: Antigen-specific T cells were generated from patients with multiple myeloma using stimulation with protein-pulsed dendritic cells and tested in ELISPOT and CTL assays. RESULTS: HM1.24-primed T cells responded selectively to HM1.24-loaded autologous peripheral blood mononuclear cells (PBMC) in an IFN-gamma ELISPOT assay (median, 342; range, 198-495 IFN-gamma-producing cells/10(5) cf. unloaded PBMC median, 98; range, 7-137; P < 0.05, n = 5) and also to autologous malignant plasma cells (MPC; median, 227; range, 153-335; P < 0.05 when compared with the response to allogeneic MPC median, 57; range, 22-158; n = 5). HM1.24-primed T cells lysed autologous MPC (at 20:1 E/T ratio: median, 48% specific killing; range, 23-88%; at 10:1 E/T ratio: median, 43%; range, 15-80%; n = 12) but not allogeneic MPC. Lysis of autologous MPC was inhibited by anti-MHC class I but not anti-MHC class II antibodies and was blocked by Concanamycin A. Lysis of autologous MPC was blocked by competition with autologous HM1.24-transfected dendritic cells (10:1 ratio with autologous MPC). Unmanipulated, or control plasmid-transfected dendritic cells had no effect on lysis of autologous MPC. CONCLUSION: Our results indicate that HM1.24 is a promising target for immunotherapy of multiple myeloma.
Subject(s)
Membrane Glycoproteins/immunology , Multiple Myeloma/immunology , T-Lymphocytes, Cytotoxic/immunology , Antigens, CD , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Line, Tumor , Cytotoxicity Tests, Immunologic , Cytotoxicity, Immunologic/immunology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Flow Cytometry/methods , GPI-Linked Proteins , Humans , Immunophenotyping , Interferon-gamma/metabolism , Leukocyte Common Antigens/immunology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/physiology , Multiple Myeloma/pathology , Perforin , Plasma Cells/immunology , Pore Forming Cytotoxic Proteins , Signal Transduction/immunology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , TransfectionABSTRACT
We investigated the effect of anti-IL-6 receptor monoclonal antibody (hPM1) on the in vitro proliferation of cloned and freshly isolated myeloma cells from 20 patients with advanced stage multiple myeloma (MM). Humanized PM1 significantly inhibited the growth of a myeloma cell line in a dose-dependent manner and inhibited more than 30% of the proliferation of fresh myeloma cells in 10 of the 19 cases. Flow cytometric analysis using annexin V and 7AAD showed that hPM1 induced apoptosis of myeloma cells. These observations suggest the possibility of using hPM1 for treating some patients with MM whose growth depends on IL-6.
