Group Leader, Senior Scientist University Medical Center Schleswig-Holstein Kiel, Schleswig-Holstein, Germany
Introduction: In recent years, a variety of antibody-based immunotherapies were approved for multiple myeloma (MM) treatment and substantially improved patients’ outcome. Nonetheless, the vast majority of MM immunotherapeutics including bispecific and monoclonal antibodies, antibody-drug conjugates as well as chimeric antigen-receptor T cells are directed against a very limited number of antigens on myeloma cells, e.g., CD38, SLAMF-7 and B cell maturation antigen (BCMA). This enables the development of resistances and relapsed/refractory patients still represent a major challenge. Novel target structures on MM cells may open additional avenues to develop effective antibody-based immunotherapies.
Methods: Therefore, we aimed to identify new antibodies targeting alternative antigens on myeloma cells by immunizing mice with patient-derived malignant plasma cells, enriching myeloma cell binders by phage display and analyzing the antibody repertoire by next-generation sequencing (NGS). Binding properties of monoclonal single-chain fragment variable (scFv) antibodies were initially tested by cellular ELISA, including Chinese hamster ovary (CHO) cells transfected with known myeloma antigens. For testing the modes of action of the most promising candidates, scFv-Fc fusion proteins were produced by transient transfection and purified by affinity chromatography. Direct anti-tumor effects were investigated by flow cytometry using plasma cell lines as well as freshly isolated tumor cells.
Results: Immunization of mice with primary myeloma cells enabled the generation of an immune scFv antibody library from 1.9 million different B cell clones for MM antibody selection through phage display. Accompanied NGS analyses revealed successful enrichment of more than 218 promising candidates. Among 41 initially tested scFv antibodies evolving from different B cell clones, 9 showed strong binding to myeloma cells and only weak binding to healthy donor peripheral blood mononuclear cells. Analyses with transfected CHO cells expressing individual myeloma antigens revealed that the most promising candidates neither bound CD38, SLAMF-7, CD138, CD56, IL-6R, FGFR3 nor BCMA. Beside a novel CD38 and CD54 antibody, respectively, 7 antibodies targeting yet unknown antigens on MM cells were isolated. Two of these antibodies, #9 and #16, were capable to directly induce rapid homotypic aggregation of myeloma cells. Incubation with and without cross-linking of the antibodies on MM cell surface resulted in up to 70 % 7-AAD/Annexin V+ MM cells.
Conclusions: Our results show that phage display and NGS provide an innovative way to develop new antibodies with therapeutic potential for MM immunotherapy. Further characterization of the antibodies and investigations to identify the respective antigens are on-going.
