Abstract 1490: Potentiating immunotherapy by targeting complement deposited on cancer cell surfaces
Authors: Carstens Elizabeth J. , Skarzynski Martin , Butera Vicent , Lindorfer Margaret , Vire Berengere , Farooqui Mohammed , Rader Christoph , Taylor Ronald , Wiestner Adrian 
Published: July 2016

Abstract
Treatment of lymphoid malignancies with anti-CD20 antibodies (mAbs) can be frustrated by the loss of cell surface CD20 through trogocytosis, creating “escape variants” that are no longer sensitive to the anti-CD20 mAb. In patients with chronic lymphocytic leukemia (CLL) treated with the anti-CD20 mAb ofatumumab, we observed that these CD20 escape variants carried covalently bound C3d complement fragments and that these C3d opsonized CLL cells persisted for weeks in circulation. Therefore, we hypothesized that C3d is a neoantigen that could be exploited to re-target cells that have escaped from anti-CD20 mAb therapy.
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 (day 2). As expected, CLL cells on day 2 had lost CD20 expression and could neither bind, nor be killed by ofatumumab. In contrast, the anti-C3d mAb did not bind CLL cells obtained pre-treatment but bound cells obtained on day 2 with high affinity (kD = 6.7nM) and were effectively killed through CDC, NK cell mediated ADCC, and phagocytosis. 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. Interestingly, when C3d opsonized CLL was exposed repetitively to anti-C3d mAb ex vivo, the amount of cell bound C3d and the fraction of cells killed increased with successive rounds of treatment consistent with an auto-amplification of C3d targeting.
We tested the efficacy of a chimerized anti-C3d mAb in two mouse models. First, we transferred PBMCs obtained from CLL patients on day 2 of ofatumumab treatment (containing the C3d opsonized CD20 escape variants) into NSG mice and three days later injected either isotype control mAb (trastuzumab) or anti-C3d mAb. One injection of anti-C3d mAb effectively reduced tumor burden in both peripheral blood (from 42.5 to 0.59 CLL cells/ul of blood; p&lt;0.01) and spleen (from 574 to 2.19 CLL cells/100,000 splenocytes; p&lt;0.01). Second, we subcutaneously xenografted HBL2 cells, a CD20+ mantle cell lymphoma line, into SCID mice. Mice were treated three days after cell injection with ofatumumab alone, ofatumumab and anti-C3d mAb or isotype control (trastuzumab). Caliper measurements of the tumor dimensions and survival were recorded. The combination of the anti-C3d mAb with ofatumumab extended time to tumor development and prolonged overall survival compared to ofatumumab alone (median survival 88 days vs 22 days, respectively, p&lt;0.02).
We conclude that targeting C3d deposited on cancer cells can eliminate antigen escape variants and potentiate complement fixing antibodies.
Citation Format: Elizabeth J. Carstens, Martin Skarzynski, Vicent Butera, Margaret Lindorfer, Berengere Vire, Mohammed Farooqui, Christoph Rader, Ronald Taylor, Adrian Wiestner. Potentiating immunotherapy by targeting complement deposited on cancer cell surfaces. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1490.