title: Isolation, production and characterization of fully human monoclonal antibodies directed to Plasmodium falciparum MSP10.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany. dominika.maskus@rwth-aachen.de.|Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Germany. dominika.maskus@rwth-aachen.de.
authors.forename: Dominika J
authors.initials: DJ
authors.lastname: Maskus
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany. susanne.bethke@ime.fraunhofer.de.
authors.forename: Susanne
authors.initials: S
authors.lastname: Bethke
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany. melanie.seidel@ime.fraunhofer.de.
authors.forename: Melanie
authors.initials: M
authors.lastname: Seidel
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany. stephanie.kapelski@ime.fraunhofer.de.|Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Germany. stephanie.kapelski@ime.fraunhofer.de.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Kapelski
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany. oaddai-mensah.chs@knust.edu.gh.|Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Germany. oaddai-mensah.chs@knust.edu.gh.|Faculty of Allied Health Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. oaddai-mensah.chs@knust.edu.gh.
authors.forename: Otchere
authors.initials: O
authors.lastname: Addai-Mensah
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany. alexander.boes@ime.fraunhofer.de.
authors.forename: Alexander
authors.initials: A
authors.lastname: Boes
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany. gueven.edgue@ime.fraunhofer.de.
authors.forename: Güven
authors.initials: G
authors.lastname: Edgü
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany. holger.spiegel@ime.fraunhofer.de.
authors.forename: Holger
authors.initials: H
authors.lastname: Spiegel
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany. andreas.reimann@ime.fraunhofer.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Reimann
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany. rainer.fischer@ime.fraunhofer.de.|Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Germany. rainer.fischer@ime.fraunhofer.de.
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany. stefan.barth@ime.fraunhofer.de.|Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering at RWTH Aachen University and Hospital, Aachen, Germany. stefan.barth@ime.fraunhofer.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Barth
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany. torsten.klockenbring@ime.fraunhofer.de.
authors.forename: Torsten
authors.initials: T
authors.lastname: Klockenbring
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany. rolf.fendel@ime.fraunhofer.de.|Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Germany. rolf.fendel@ime.fraunhofer.de.|Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering at RWTH Aachen University and Hospital, Aachen, Germany. rolf.fendel@ime.fraunhofer.de.
authors.forename: Rolf
authors.initials: R
authors.lastname: Fendel
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D000953:Antigens, Protozoan / Q000276:immunology*; D006801:Humans; D010963:Plasmodium falciparum / Q000737:chemistry / Q000276:immunology*; D015800:Protozoan Proteins / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D015800:Protozoan Proteins; D011994:Recombinant Proteins