Potentiating Antibody Therapy by Targeting Complement on Cancer Cells




Carstens, Elizabeth Jane

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BACKGROUND: Monoclonal antibodies (mAb) are a key component of treatment regimens for hematologic malignancies, but mAb-induced antigen loss on tumor cells can lead to treatment failure. Loss of cell surface CD20 can occur in the treatment of lymphoid malignancies with anti-CD20 antibodies (mAbs) (eg rituximab, ofatumumab) through trogocytosis. This is a frustrated form of phagocytosis, where the target CD20 antigen is removed with a piece of target cell plasma membrane by the immune effector cell, thus creating "escape variants", which are no longer sensitive to the anti-CD20 therapy. In a clinical trial we initiated with anti-CD20 mAb ofatumumab in chronic lymphocytic leukemia (CLL), we observed that these escape variants carried covalently bound complement activation fragments, especially C3d. Indeed, C3d opsonized CLL cells persisted for weeks in circulation. (Beurskens et al., 2012). At final restaging after combined ofatumumab and chemotherapy treatment, many patients had CD20 negative, but C3d positive disease. This suggests that a single mAb is insufficient to deplete cancer cells due to antigen escape. OBJECTIVE: C3d may constitute a neoantigen that could be exploited to re-target cells that have escaped from anti-CD20 mAb therapy. METHODS: To target complement opsonized cells we generated a human IgG1 mouse chimera mAb specific to C3d that is not competed by full length C3 in serum. To test whether targeting C3d can eliminate escape variants after anti-CD20 therapy, we collected blood samples from CLL patients before (Day 1) and 24 hours after administration of ofatumumab (Day2). To demonstrate that anti-C3d targeting was able to effectively circumvent antigen loss in vitro, we tested the anti-C3d mAbs ability to perform complement dependent cytotoxicity (CDC), antibody dependent cytotoxicity (ADCC), apoptosis and phagocytosis on Day 2 CLL cells. To test CDC, Day 2 CLL and previously treated CD20+ cell lines were incubated with anti-C3d mAb and normal human serum and stained for cell death. Similarly, ADCC was tested by coincubating Day 2 cells with an NK cell line in the presence of mAb. To evaluate phagocytosis, CLL cells were incubated with macrophages for six hours in the presence of antibody and imaged using flow cytometry to evaluate degree of internalization. We also tested the efficacy of the anti-C3d mAb in vivo, using two mouse models. First, we transferred peripheral blood mononuclear cells obtained from CLL patients on Day 2 into NSG mice. Mice were treated with either isotype antibody, ofatumumab or anti-C3d mAb. Mice were sacrificed and tumor burden was quantified in peripheral blood and spleen. To evaluate impact on survival, we subcutaneously xenografted HBL2 cells, a CD20+ mantle cell lymphoma (MCL) line, into SCID mice. All mice received an injection of human C3 and either anti-CD20 mAb (rituximab or ofatumumab) alone, anti-CD20 and anti-C3d mAb or isotype control. Caliper measurements of the tumor longest dimension and survival were measured. RESULTS: Anti-C3d mAb did not bind CLL cells obtained pre-treatment but bound cells obtained on Day 2. Day 2 CLL was effectively killed through CDC, NK cell mediated ADCC, and phagocytosis but not apoptosis. Phagocytosis of Day 2 CLL cells in response to anti-C3d treatment was two-fold higher than that observed on Day 1 CLL treated with ofatumumab. Importantly, non B lymphocytes were neither bound nor killed by the anti-C3d mAb, consistent with the highly targeted and selective deposition of C3d on CD20+ cells by ofatumumab in vivo. Our anti-C3d mAb effectively reduced tumor burden in both peripheral blood and spleen of the mice relative to mice treated with isotype control in the CLL primary NSG model. Our anti-C3d antibody extended time to tumor development and also prolonged survival in the MCL model differentially from CD20 targeting alone. CONCLUSION: Collectively, our results identify C3d as a marker for leukemic cells that have escaped in vivo antibody treatment and provide proof of principle evidence for the clinical utility of C3d-targeting. In essence, we present an approach to utilize endogenous complement molecules as targets for immunotherapy. This approach relies on prior treatment with monoclonal antibodies that activate the complement cascade and thus covalently link complement component C3d to tumor cells. We conclude that anti-C3d mAbs can potentiate the anti-tumor activity of complement-fixing antibodies and eliminate antigen loss variants that survive after antibody therapy.

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