Novel Anti-CD37 Small Modular Immunopharmaceutical (SMIP) Induces B-Cell-Specific, Caspase-Independent Apoptosis in Human CLL Cells.

Tru 16.4 is a single chain protein with a modified CD37 binding Fv domain linked to a modified human IgG1 hinge, CH2 and CH3 domains. It is a member of a novel composition class called small modular immunopharmaceuticals (SMIP) construct. CD37 is a stable marker for B-cell chronic lymphocytic leukemia (BCLL) and non-Hodgkins lymphoma (NHL). Although radiolabeled murine anti-CD37 antibody MB-1 has been evaluated in NHL patients and shown to be able to target tumors, efficacy was not established. There has been no evaluation of an unconjugated anti-CD37 antibody in human clinical trials. Here we report that Tru 16.4, binds to CD37 on primary CLL cells surface, and induces caspase-independent apoptosis that is further enhanced by a secondary crosslinking with goat anti-human antibody. The percentage of dead cell population over control based upon Annexin V/PI staining of 12 BCLL patient samples (28.3%, SD 15.4%) is significantly greater than that of isotype control, trastuzumab (3.4%, SD 7.6%). The expression of CD37 on T cells is low, and the exposure of Tru 16.4 under the same conditions does not demonstrate significant binding or induce noticeable apoptosis on normal T cells at 24 hours (0.47%, SD 0.99%). This provides a selective advantage for BCLL antibody therapy. Results from a comparison study utilizing 6 additional patient samples demonstrated that Tru 16.4 induced apoptosis (31.8%, SD 12.2%) is significantly higher than that of the anti-CD20 antibody, rituximab (21.8%, SD 5.25%) and similar to that observed with the anti-CD52 antibody, alemtuzumab (36.6%, SD 19.5%). The cell death induced by Tru 16.4 is dependent upon dose and duration of treatment, indicating 5μg/mL as a saturation concentration and 24 hours as the optimal time for maximal in vitro apoptosis. The degree of CLL cell death was found to be directly proportionate to cell surface membrane expression of CD37, based on nonparametric Spearman rank correlation analysis of the mean fluoresecnce intensity (MFI) after PE-labelled anti-CD37 antibody immunostaining and percentages of Annexin V/PI positive cells, with a correlation factor rs = 0.80 ( P < 0.01). Ongoing studies suggest Tru 16.4 induces intracellular signaling following CD37 antigen ligation and current efforts are focused on identifying the specific death pathways mediated by this treatment. Overall, these findings indicate that Tru 16.4 mediates B-cell specific apoptosis through a novel caspase independent mechanism and supports effort for further clinical investigation in CLL.