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Recombinant, ETA'-based CD64 immunotoxins: improved efficacy by increased valency, both in vitro and in vivo in a
1Department of Pharmaceutical Product Development, Fraunhofer IME, Forckenbeckstrasse 6, 52074 Aachen, Germany.
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').
Objectives:
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.
