title: Recombinant, ETA'-based CD64 immunotoxins: improved efficacy by increased valency, both in vitro and in vivo in a chronic cutaneous inflammation model in human CD64 transgenic mice.
abstract: BACKGROUND
  Dysregulated, activated macrophages play a pivotal role in chronic inflammatory diseases such as arthritis and atopic dermatitis. These cells display increased expression of the high-affinity Fcgamma receptor (CD64), making them ideal targets for CD64-specific immunotoxins. We previously showed that a chemically linked immunotoxin, the monoclonal H22-RicinA, specifically eliminated infiltrating activated macrophages and resolved chronic cutaneous inflammation. However, several disadvantages are associated with classic immunotoxins, and we therefore followed a fusion protein strategy to express the antigen-binding site alone (scFv H22) fused to a derivative of Pseudomonas exotoxin A (ETA').
  
  
  OBJECTIVE
  To assess the potential effect of increased valency on efficacy, we produced monovalent [H22(scFv)-ETA'] and bivalent [H22(scFv)(2)-ETA'] versions and evaluated their potential for eliminating activated macrophages both in vitro and in vivo.
  
  
  METHODS
  Both immunotoxins were produced by bacterial fermentation. Binding was assessed by flow cytometry on the monocytic CD64+ cell line U937. Toxicity was analysed by XTT and apoptosis induction by annexin V bioassay. The in vivo effect was tested in a human CD64 transgenic mouse model for cutaneous inflammation.
  
  
  RESULTS
  The cytotoxic effects of both immunotoxins were clearly due to apoptosis with an IC(50) of 140 pmol L(-1) for monovalent and only 14 pmol L(-1) for the divalent version. In vivo treatment with H22(scFv)-ETA' reduced CD64+ activated macrophages to 21% of their initial numbers whereas H22(scFv)(2)-ETA' treatment reduced these cells to 4.8% (P < 0.001).
  
  
  CONCLUSIONS
  These data clearly show increased efficacy due to increased valency of the anti-CD64 immunotoxin. Both recombinant immunotoxins have a low IC(50), making them suitable for the treatment of diseases involving dysregulated, activated macrophages.
authors.affiliation: Department of Pharmaceutical Product Development, Fraunhofer IME, Forckenbeckstrasse 6, 52074 Aachen, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Ribbert
authors.forename: T
authors.initials: T
authors.lastname: Thepen
authors.forename: M K
authors.initials: MK
authors.lastname: Tur
authors.forename: R
authors.initials: R
authors.lastname: Fischer
authors.forename: M
authors.initials: M
authors.lastname: Huhn
authors.forename: S
authors.initials: S
authors.lastname: Barth
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002908:Chronic Disease; D004195:Disease Models, Animal; D005098:Exotoxins / Q000276:immunology / Q000494:pharmacology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology / Q000494:pharmacology; D000922:Immunotoxins / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D007249:Inflammation / Q000503:physiopathology*; D008264:Macrophages / Q000187:drug effects / Q000276:immunology*; D051379:Mice; D008822:Mice, Transgenic; D017452:Receptors, IgG / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000494:pharmacology; D012871:Skin Diseases / Q000188:drug therapy* / Q000276:immunology
chemical_list: D005098:Exotoxins; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins