Abstract
Abstract 3916
Introduction:
Treatment of B-cell malignancies with rituximab, an antibody specific for the B-cell antigen CD20, in combination with chemotherapy has been established as standard clinical practice during recent years. Despite the impressive clinical benefit of rituximab-chemotherapy combination treatment, there remains a substantial need for improved treatment modalities. Here we describe two novel Fc-engineered antibodies to CD37, mAb 37.1 and mAb 37.2, which mediate potent pro-apoptotic and ADCC activity against malignant B-cells. The tetraspanin CD37 is predominantly expressed on the cell surface of normal and malignant B-cells and therefore qualifies as target for antibody mediated tumor therapy.
Methods:
MAb 37.1 is an Fc-engineered, mouse-human chimeric IgG1-type of antibody which binds CD37 with low nanomolar affinity as determined by FACS scatchard analysis. MAb 37.2 is a humanized version derived from mAb 37.1. Affinity to Fc-receptors was determined by BIAcore analysis. ADCC and pro-apoptotic activity was determined using Ramos Burkitt lymphoma cells and primary CLL cells. ADCC assays were performed using PBMCs from healthy donors as effector cells. Apoptosis induction was determined by AnnexinV/PI staining. The effect in blood from healthy individuals was assessed in whole blood assays ex vivo. In vivo anti-tumor efficacy was assessed in a Ramos xenograft model in immuno-compromised mice.
Results:
Both CD37 antibodies are able to induce apoptosis of Ramos Burkitt lymphoma cells and primary CLL cells in vitro. Direct comparison of mAb 37.1 to Rituximab demonstrated superior apoptosis induction both on Ramos and primary CLL cells. The pro-apoptotic activity of mAb 37.1 is not dependent on cross-linking of the antibody, representing a novel mode of CD37-specific apoptosis induction. Phenotypic analysis of mAb 37.1 treated Ramos cells revealed early aggregation of tumor cells resembling homotypic aggregation. In order to further improve the cytotoxic potential of the antibodies an Fc-engineering approach was chosen to enhance binding to FcgRIIIa. By applying a two amino acid substitution in the Fc-region of the antibody the affinities of mAb 37.1 and mAb 37.2 to human FcgRIIIa was increased by 40 to 80-fold, depending on the FcgRIIIa allotype, as compared to the parental antibody. This increase directly translates into a strongly enhanced ADCC activity compared to the parental antibody as detected in an in vitro ADCC assay with Ramos and primary CLL cells, resulting in superior ADCC activity compared to Rituximab. The potent B-cell specific depleting activity of mAb 37.1 and mAb 37.2 was confirmed with naïve B-cells and spiked Ramos Burkitt lymphoma cells in vitro in whole blood conditions, whereas no significant effects on T-cells and monocytes were observed. Rituximab, which was used as comparator antibody in parallel, showed no significant reduction of viable B-cells in this assay format. Finally, the in vivo anti-tumor effect of CD37 mAbs was assessed using a Ramos xenograft. Treatment of established subcutaneous tumors with doses ranging from 2.5 to 25 mg/kg in a twice weekly treatment schedule resulted in tumor growth inhibition up to 100%.
Conclusion:
MAb 37.1 and mAb 37.2 represent novel, Fc-engineered antibodies specific for CD37 with potent pro-apoptotic and enhanced ADCC activity against lymphoma cell lines and primary CLL cells. Based on these data mAb 37.1 and mAb 37.2 are considered as promising candidates for treatment of patients with CD37-positive B-cell malignancies.
Disclosures:
Heider: Boehringer Ingelheim: Employment. Ostermann:Boehringer Ingelheim: Employment. Lamche:Boehringer Ingelheim: Employment. Kuepcue:Boehringer Ingelheim: Employment. Jacobi:Boehringer Ingelheim: Employment. Konopitzky:Boehringer Ingelheim: Employment. Hirt:Boehringer Ingelheim: Employment. Reichardt:Boehringer Ingelheim: Employment. Borges:Boehringer Ingelheim: Employment.
Rapid Death of Follicular B Cells and Burkitt Lymphoma Cells Effectuated by Xbp1s
