title: HuMab-7D8, a monoclonal antibody directed against the membrane-proximal small loop epitope of CD20 can effectively eliminate CD20 low expressing tumor cells that resist rituximab-mediated lysis.
abstract: BACKGROUND
  Incorporation of the chimeric CD20 monoclonal antibody rituximab in the treatment schedule of patients with non-Hodgkin's lymphoma has significantly improved outcome. Despite this success, about half of the patients do not respond to treatment or suffer from a relapse and additional therapy is required. A low CD20-expression level may in part be responsible for resistance against rituximab. We therefore investigated whether the CD20-expression level related resistance to rituximab could be overcome by a new group of CD20 mAbs (HuMab-7D8 and ofatumumab) targeting a unique membrane-proximal epitope on the CD20 molecule.
  
  
  METHODS
  By retroviral transduction of the CD20 gene into CD20-negative cells and clonal selection of transduced cells a system was developed in which the CD20-expression level is the only variable. These CD20 transduced cells were used to study the impact of rituximab and HuMab-7D8 mediated complement-dependent cytotoxicity. To study the in vivo efficacy of these mAbs an in vivo imaging system was generated by retroviral expression of the luciferase gene in the CD20-positive cells.
  
  
  RESULTS
  We show that HuMab-7D8 efficiently killed CD20(low) cells that are not susceptible to rituximab-induced killing in vitro. In a mouse xenograft model, we observed a comparable increase in survival time between HuMab-7D8 and rituximab-treated mice. Most significantly, however, HuMab-7D8 eradicated all CD20-expressing cells both in the periphery as well as in the bone marrow whereas after rituximab treatment CD20(low) cells survived.
  
  
  CONCLUSIONS
  Cells that are insensitive to in vitro and in vivo killing by rituximab as the result of their low CD20-expression profile may be efficiently killed by an antibody against the membrane-proximal epitope on CD20. Such antibodies should, therefore, be explored to overcome rituximab resistance in the clinic.
authors.affiliation: Department of Immunology, Medical Center Utrecht, Utrecht, the Netherlands. tmeerten@umcutrecht.nl
authors.forename: Tom
authors.initials: T
authors.lastname: van Meerten
authors.forename: Henk
authors.initials: H
authors.lastname: Rozemuller
authors.forename: Samantha
authors.initials: S
authors.lastname: Hol
authors.forename: Petra
authors.initials: P
authors.lastname: Moerer
authors.forename: Mieke
authors.initials: M
authors.lastname: Zwart
authors.forename: Anton
authors.initials: A
authors.lastname: Hagenbeek
authors.forename: Wendy J M
authors.initials: WJ
authors.lastname: Mackus
authors.forename: Paul W H I
authors.initials: PW
authors.lastname: Parren
authors.forename: Jan G J
authors.initials: JG
authors.lastname: van de Winkel
authors.forename: Saskia B
authors.initials: SB
authors.lastname: Ebeling
authors.forename: Anton C
authors.initials: AC
authors.lastname: Martens
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology / Q000494:pharmacology*; D018951:Antigens, CD20 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D045744:Cell Line, Tumor; D003602:Cytotoxicity, Immunologic / Q000187:drug effects* / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007942:Leukemia, Experimental / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D021381:Protein Transport; D000069283:Rituximab; D023041:Xenograft Model Antitumor Assays
chemical_list: D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20; D000939:Epitopes; D049452:Green Fluorescent Proteins; D000069283:Rituximab; D008156:Luciferases