Efficient Lysis of Myeloma Cells by CD16 Positive Effector Cells with Recombinant Single Chain Bispecific Antibodies Targeting HM1.24.

Different therapeutic options are available for the treatment of multiple myeloma patients, but conventional chemotherapy often is not able to completely eradicate the tumor. After high dose chemotherapy, complete remission with only minimal residual disease could be achieved in many patients, but additional targeted strategies may help to eradicate residual cells and improve prognosis. Therapy with monoclonal antibodies is well established in CD20 positive B-cell lymphomas, but in contrast not many suitable target antigens are defined / expressed by multiple myeloma cells. HM1.24 a surface molecule expressed on terminally differentiated B-lineage cells represents a promising candidate antigen that is overexpressed on multiple myeloma cells. Here the development of a recombinant bispecific single chain Fv HM1.24 × CD16 antibody (tandem format) with novel features is presented. The HM1.24×CD16 bispecific antibody was expressed in 293T cells and purified to homogeneity by two-step affinity chromatography. Binding to HM1.24 and CD16 was demonstrated by immunofluorescence staining and flow cytometry with antigen positive and negative cells. The lytic activity of the bispecific HM1.24×CD16 scFv was evaluated in an antibody-dependent cellular cytotoxicity (ADCC) assay with different myeloma cell lines (RPMI 8226, INA-6, U266, JK6L) and primary patient derived cells as targets. Mononuclear cells (MNC), isolated from healthy donors served as effector cells. The bispecific HM1.24×CD16 scFv mediated efficient lysis of all tested cell lines at concentrations as low as 1 nM. In direct comparison to an HM1.24-IgG1 control molecule, the recombinant bispecific antibody demonstrated superior lytic activity at saturating concentrations and showed significant enhanced killing capacity. In conclusion, the recombinant bispecific HM1.24×CD16 retained its antigen specificity and demonstrated efficient lytic activity against patient-derived tumor cell lines and primary material. These results indicate that the bispecific antibody may be promising as a new therapeutic strategy in multiple myeloma.