Isolation, production and characterization of fully human monoclonal antibodies directed to Plasmodium falciparum MSP10

Dominika J Maskus, Susanne Bethke, Melanie Seidel, Stephanie Kapelski, Otchere Addai-Mensah, Alexander Boes, Güven Edgü, Holger Spiegel, Andreas Reimann, Rainer Fischer, Stefan Barth, Torsten Klockenbring, Rolf Fendel

Semi-immunity against the malaria parasite is defined by a protection against clinical episodes of malaria and is partially mediated by a repertoire of inhibitory antibodies directed against the blood stage of Plasmodium falciparum, in particular against surface proteins of merozoites, the invasive form of the parasite. Such antibodies may be used for preventive or therapeutic treatment of P. falciparum malaria. Here, the isolation and characterization of novel human monoclonal antibodies (humAbs) for such applications is described. B lymphocytes had been selected by flow cytometry for specificity against merozoite surface proteins, including the merozoite surface protein 10 (MSP10). After Epstein-Barr virus (EBV) transformation and identification of promising resulting lymphoblastoid cell lines (LCLs), human immunoglobulin heavy and light chain variable regions (Vh or Vl regions) were secured, cloned into plant expression vectors and transiently produced in Nicotiana benthamiana in the context of human full-size IgG1:κ. The specificity and the affinity of the generated antibodies were assessed by ELISA, dotblot and surface plasmon resonance (SPR) spectroscopy. The growth inhibitory activity was evaluated based on growth inhibition assays (GIAs) using the parasite strain 3D7A. Supernatants from two LCLs, 5E8 and 5F6, showed reactivity against the second (5E8) or first (5F6) epidermal growth factor (EGF)-like domain of MSP10. The isolated V regions were recombinantly expressed in their natural pairing as well as in combination with each other. The resulting recombinant humAbs showed affinities of 9.27 × 10(-7) M [humAb10.1 (H5F6:κ5E8)], 5.46 × 10(-9) M [humAb10.2 (H5F6:κ5F6)] and 4.34 × 10(-9) M [humAb10.3 (H5E8:κ5E8)]. In GIAs, these antibodies exhibited EC50 values of 4.1 mg/ml [95% confidence interval (CI) 2.6-6.6 mg/ml], 6.9 mg/ml (CI 5.5-8.6 mg/ml) and 9.5 mg/ml (CI 5.5-16.4 mg/ml), respectively. This report describes a platform for the isolation of human antibodies from semi-immune blood donors by EBV transformation and their subsequent characterization after transient expression in plants. To our knowledge, the presented antibodies are the first humAbs directed against P. falciparum MSP10 to be described. They recognize the EGF-like folds of MSP10 and bind these with high affinity. Moreover, these antibodies inhibit P. falciparum 3D7A growth in vitro.