Mechanism for platelet reduction in anti-neuropilin-1 (MNRP1685A)–treated phase I patients.

Abstract

e13598 Background: Neuropilin-1 (NRP1), a high affinity receptor for members of the VEGF family, is expressed on endothelial cells (EC) and tumor cells, and is involved with maturation and remodeling of blood vessels. In two phase I studies, administration of human anti-NRP1 antibody (Ab) MNRP1685A resulted in transient platelet (PLT) reductions in patients. To determine if these PLT reductions were due to PLT production or consumption, we evaluated the binding of MNRP1685A to human megakaryocyte (MK) progenitor cells and PLTs and the impact of MNRP1685A on MK development and PLT function. Methods: MNRP1685A binding to human hematopoietic stem cells (HSC), cultured MKs and PLTs was assessed by Western blot (WB), mass spectrometry and flow cytometry (FACS). MNRP1685A effects on platelets from healthy donors and patients on study were analyzed by FACS for activation markers CD62P and activated GPIIbIIIa (PAC1) and evaluated by aggregometry. MNRP1685A effects on MK development were evaluated by HALO proliferation assay and expression of CD61/41 maturation markers via FACS. To examine PLT binding to EC, fluorescein-labeled PLTs were incubated with TNF-stimulated human umbilical vein endothelial cells (HUVEC) in the presence of MNRP1685A and HUVEC-associated fluorescence was measured. Results: We demonstrate specific CD32 (FCγR2a)-dependent and independent MNRP1685A binding to human HSC, cultured MKs and PLTs by FACS and WB. There was no effect of MNRP1685A on HSC proliferation or maturation to MK. Exposure of MNRP1685A to human PLTs ex vivo or in vivo (phase 1 patients) result in modest PLT activation via a CD32-dependent mechanism as measured by FACS for CD62P and PAC1, as well as increased PLT aggregation compared to a control Ab. An increase of CD32-dependent binding of PLTs to HUVEC in vitro was observed in the presence of MNRP1685A, but not with a control Ab. Conclusions: MNRP1685A specifically binds to NRP1 on the surface of human PLTs and cells in the MK lineage, induces modest CD32-dependent PLT activation and binding to activated endothelial cells. These properties of MNRP1685A can contribute to PLT consumption and may explain decreases in circulating PLT levels in patients dosed with MNRP1685A in phase I clinical studies.