Therapy of B-Cell Malignancies by Anti-HLA-DR Humanized Monoclonal Antibody, IMMU-114, Is Mediated through Hyperactivation of ERK and JNK MAP Kinase Signaling Pathways.

HLA-DR is currently under investigation as a target for antibody (mAb) therapy of B-cell malignancies. IMMU-114, a humanized IgG4 form of the murine anti-HLA-DR mAb, L243, recognizes a conformational epitope in the a-chain of HLA-DR. We have previously reported that IMMU-114 lacks effector-cell functions while displaying potent antitumor activity against B-cell lymphomas in vitro and in vivo. Here, we further investigated the cytotoxic effects of IMMU-114 on leukemia, lymphoma, and multiple myeloma (MM) cell lines, as well as clinical specimens of CLL, and explored the signaling pathways involved.

Binding and cytotoxicity of IMMU-114 was examined on a panel of NHL, AML, MCL, ALL, and CLL cell lines. Induction of apoptosis (Annexin V binding), ROS generation (dihydoehidium staining), changes in mitochondrial membrane potential (ψM, TMRE staining) and effects on the ERK1/2 and JNK pathways (immunoblot analyses) were evaluated on selected cell lines. The effects of various inhibitors on IMMU-114-mediated apoptosis also were determined. Studies were performed in tumor-bearing SCID mice.

High expression of HLA-DR was detected on the cell surface of all MCL, ALL, HC, CLL, and NHL cell lines tested, as well as 2/3 AML and 5/6 MM cell lines. IMMU-114 was cytotoxic to 2/2 MCL, 2/2 CLL, 2/4 ALL, 1/1 HC, 2/2 NHL and 2/2 MM cell lines without any added anti-Fc second crosslinking antibody. IMMU-114 was consistently more cytotoxic than anti-CD20 mAbs on these cell lines, as well as on rituximab-resistant variants of Raji and SU-DHL-4. Despite positive staining, AML cell lines, Kasumi-3 and GDM-1, were not killed by IMMU-114. Four of 6 CLL patient samples expressed HLA-DR at markedly higher levels than CD20, with the remaining 2 showing similar HLA-DR and CD20 expression. Approximately 60% cytotoxicity was obtained after incubation with IMMU-114 in CLL patient cells. Induction of apoptosis as well as changes in ψM and ROS correlated with sensitivity of cell lines to IMMU-114-mediated cytotoxicity. Time-course analyses demonstrated that IMMU-114 induced approximately 46% change of ψM and 24% enhancement of ROS in as little as 30 min in Raji cells. These changes were abrogated in the presence of ROS inhibitor, N-acetyl cysteine. Immunoblot analyses revealed that IMMU-114 induces phosphorylation of ERK and JNK mitogen-activated protein (MAP) kinases in all cells defined as IMMU-114-sensitive by the cytotoxicity assays, but not IMMU-114-resistant cell lines. The p38 pathway was found to be constitutively active in these cell lines. Inhibition of ERK, JNK, or ROS by their respective inhibitors decreased apoptosis, although the inhibition was not complete when any single inhibitor was used. This could be due to activation of multiple pathways, since inhibition of 2 or more pathways by specific inhibitors abolishes the apoptosis induced by IMMU-114. Activation (5-fold increases of phosphorylation) of ERK1/2 and JNK signaling pathways also was observed in CLL patient samples following IMMU-114 incubation. Further studies revealed caspase-independent apoptosis and activation of apoptosis-inducing factor (AIF) mediated by IMMU-114. The therapeutic activity of IMMU-114 was evaluated in vivo in 3 NHL models, WSU-FSCCL, Namalwa, and Raji. IMMU-114 was more effective than anti-B-cell mAbs such as rituximab, in all models. For example, in WSU-FSCCL, IMMU-114 treatment yielded 100% long-term survival. Although rituximab also induced significant responses compared to untreated mice (median survival time of 87.5 vs. 36 days for untreated controls, P<0.0001), no long-term survivors were seen.

IMMU-114 is cytotoxic to a variety of hematological malignancies, and its potent activity can be related to high antigen expression of target cells and hyperactivation of ERK and JNK pathways upon binding to HLA-DR. Clinical evaluation of IMMU-114 is planned.

Gupta:Immunomedics Inc.: Employment. Stein:Garden State Cancer Center: Employment. Cardillo:Immunomedics Inc.: Employment. Chang:Immunomedics Inc.: Employment. Goldenberg:Immunomedics Inc.: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.