The Non-Hodgkin Lymphomas (NHLs) are a heterogeneous group of malignancies with approximately 85% of NHL belonging to the B-cell lineage. The use of monoclonal antibodies (mAb), including the anti-CD20 therapy rituximab, has become a standard of care for the treatment of many forms of NHL. However, only a subset of patients respond to rituximab and the majority of those eventually relapse after treatment. Additional therapeutic options are necessary for these patients. Here we report the expression of CD37, a lineage-specific B-cell antigen, in different subtypes of B-cell NHL tissues and evaluate the functional activity of an anti-CD37 scFv-Fc fusion protein, known as a SMIP ™ protein (CD37-SMIP) against NHL cells.
Fresh NHL tissues or peripheral blood from leukemia-stage lymphoma patients were used for flow cytometric assay of CD37 expression. Monotypic kappa or lambda IgG light chain positive malignant B-cells were gated for this assay. Immunohistochemical (IHC) staining of formalin fixed paraffin-embedded (FFPE) NHL tissues was performed for CD37 expression. Fresh primary NHL cells were isolated and used as targets in assays to measure antibody dependent cellular cytotoxicity (ADCC) activity as well as direct cell death mediated by CD37-SMIP. The effects of CD37-SMIP on multiple B-cell NHL lines was also determined by measuring cell proliferation, Annexin V staining, caspase3/7 release and changes in mitochondria membrane potentials using JC-1 staining.
Eighteen cases of NHL (follicular lymphoma (2), chronic lymphocytic leukemia/small lymphocytic lymphoma (3), mantle cell lymphoma (5), persistent marginal zone lymphoma (1), diffuse large B-cell lymphoma (1) and other types (6)) were analyzed by flow cytometry. Six of the eighteen cases had a history of rituximab treatment. Flow data showed malignant B-cells from these NHL specimens were strongly positive for CD37, while other cell populations had minimal to low levels of CD37 expression. IHC results on FFPE samples (n=170) indicated 89% (151 of 170 cases) of stained lymphoma tissues were positive for CD37, including follicular lymphoma (80/88), mantle cell lymphoma (39/44), DLBCL (32/38), and marginal zone lymphoma (5/5). Twenty seven B-cell lymphoma cell lines representing MCL, DLBCL, FL, and Burkitt's lymphoma subtypes were evaluated for CD37 expression and functional response to CD37-SMIP. CD37-SMIP mediated cell death in a wide range of NHL cell lines. The response to CD37-SMIP correlated with the levels of CD37 surface expression in NHL cell lines (Pearson analysis, r=0.78) The CD37-SMIP mediated response was rapid, with Annexin V staining and mitochondria depolarization observed in 3 hours; however, no caspase activity was observed at this time point. Two of ten NHL cell lines had modest caspase 3/7 activity at 24 h post treatment. However, caspase inhibitors did not suppress the CD37-SMIP mediated response. ADCC and direct killing activities elicited by SMIP-CD37 on freshly isolated primary NHL cells are under evaluation.
CD37 is highly expressed on majority of B-cell NHL tissues, including those from patients treated with rituximab. CD37-SMIP mediated rapid cell death in NHL cell lines with mitochondria dysfunction but appears to be independent of caspase activity. The expression pattern of CD37 and functional effects of CD37-SMIP supports the targeting of CD37 in B-cell NHL. TRU-016, a humanized SMIP protein specific for CD37, is currently being evaluated in relapsed CLL and NHL patients in a phase 1 trial.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.