Poly-L-ornithine with an average molecular weight of 32K was reacted with
beta-amanitin hydroxysuccinimide
ester to form an
amide-linked
toxin conjugate. Loading of the polymeric chain with
amanitin was high, corresponding to up to 35% of the total weight. To this
amatoxin vehicle we attached a targeting molecule, human recombinant leucine-21
epidermal growth factor (hrEGFL), via a
disulfide-containing linker moiety. A typical average stoichiometry of the hrEGFL labeled
toxin conjugate was (L-Orn)164(beta-amanitin)19(COC2H4SSC2H4CO-hrEGFL)2. The affinity for
EGF receptors of hrEGFL bound in this conjugate was tested by using A 431 cells. The affinity was eight times lower than that of unsubstituted hrEGFL but regarded as high enough for studying specific toxicity effects with cells bearing
EGF receptors. We found that
beta-amanitin in the labeled conjugate was able to inhibit the growth of A 431 cells at a concentration of 28 nM, 80 times lower than for native
beta-amanitin and 20 times lower than for
poly-L-ornithine-bound
beta-amanitin without the hrEGFL label. The approximately 20-fold enhancement of cytotoxicity suggests a specific internalization of the
toxin conjugate mediated by the
hormone label. This idea is supported by the fact that also in another transformed fibroblast cell line, with an increased though smaller number of
EGF receptors than A 431 cells, the corresponding enhancement of cytotoxicity was demonstrable but less pronounced (7-fold). The
hormone-mediated increase in cytotoxicity of
EGF labeled
poly-L-ornithine-
beta-amanitin conjugates, combined with their moderate toxicity in the mouse, encourages further examination of such compounds in
tumor model systems in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)