Comparison of Platelet-Activating Potential of Bevacizumab+VEGF and Aflibercept+VEGF Complexes
Authors: Meyer Todd , Robles-Carrillo Liza , Desai Hina , Hatfield Meghan , Amaya Mildred , Francis John , Amirkhosravi Ali 
Published: October 2019

Abstract
Abstract 3200
Anti-angiogenic therapy with the monoclonal antibody (mAb) bevacizumab (bev; Genentech) is associated with venous and arterial thrombosis in cancer patients. We recently showed that bev immune complexes (ICs) activate platelets in vitro and cause thrombotic thrombocytopenia in mice transgenic for the human platelet IgG receptor, FcγRIIa (FCGR2A mice; Jax Labs). Previous studies conducted in other labs using wild type mice injected with bevacizumab failed to identify thrombotic side effects; however, wild type mice do not have platelet FcγRIIa IgG receptors. Platelets from human donors or from FCGR2A mice are directly activated when FcγRIIa is clustered by the Fc domains of mAbs that have been assembled into higher order ICs by multivalent antigens, such as was shown for bev (Meyer, et al, J Thromb Haemost, 2009;7:171) and for anti-CD154 ICs (Robles-Carrillo, et al, J Immunol, 2010;185:1577). Aflibercept (aflib, Regeneron/sanofi-aventis, also known as VEGF Trap) is a soluble decoy receptor protein that binds all isoforms of VEGF-A including VEGF165 and inhibits VEGF165 activity. Aflib also binds the related angiogenic molecule PlGF, and like bev has anti-angiogenic activity. The aflib molecule contains the Fc domain of the IgG isotype antibody; however, although VEGF165 efficiently clusters bev into higher order ICs, VEGF165 does not cluster aflib into higher order complexes (Rudge JS, et al, PNAS, 2007;104:18363). This suggests that aflib+VEGF165 should not be able to trigger platelet FcγRIIa activation (because FcγRIIa requires clustering for activation). We therefore hypothesized that aflib+VEGF165 complexes would fail to activate platelet FcγRIIa in vitro or in FCGR2A transgenic mice. For in vitro studies, we chose the washed platelet aggregation method as a measure of IC-induced platelet activation. To assess platelet activation in vivo, we measured IC-induced reductions in the number of circulating platelets (thrombocytopenia) following intravenous injection of ICs into FCGR2A mice. Aflib+VEGF165 or bev+VEGF165 complexes were pre-formed at room temperature in the presence or absence of heparin. (Heparin can enhance bev IC-induced platelet activation. Many cancer patients are routinely exposed to heparin to maintain patency of chemotherapy ports.) In 5 donors, bev+VEGF165 complexes (500nM) rapidly induced full aggregation of human platelets in an FcγRIIa-dependent manner, whereas 500nM aflib+VEGF165 failed to induce aggregation under all conditions tested. Within 5 minutes following intravenous injection of bev+VEGF165, FCGR2A mice (n=10) experienced signs of distress consistent with thrombotic shock, including rapid shallow breathing, hunched posture, and decreased or dysfunctional locomotor activity. Animals receiving aflib complexes (n=10) did not exhibit these symptoms. Platelet counts 10 minutes following IC injection revealed thrombocytopenia in animals receiving bev+VEGF165 but not aflib+VEGF165 complexes. Specifically, animals injected with PBS (vehicle; n=5) had baseline platelet counts of (1173±179;mean±SD), whereas animals receiving bev+VEGF165 had mean platelet counts of 331±217 (with heparin; P < 0.001) and 725±303 (without heparin; P < 0.003). Animals receiving aflib+VEGF165 had mean platelet counts of 966±168 (with heparin) and 878±250 (without heparin), which were not statistically different from baseline animal counts. These results indicate that aflibercept lacks that platelet activating potential observed with bevacizumab, and also suggest that anti-angiogenic therapy with aflibercept may lack the capacity to induce the platelet associated thrombotic side effects observed in patients receiving bevacizumab.

Disclosures:
Meyer: Regeneron Pharmaceuticals, Inc.: Florida Hospital received research funding from Regeneron Pharmaceuticals in support of some of this work.