Molecular modification of a recombinant anti-CD3 epsilon-directed immunotoxin by inducing terminal cysteine bridging enhances anti-GVHD efficacy and reduces organ toxicity in a lethal murine model

Immunotoxin (IT) therapy shows potential for selectively eliminating GVHD-c ausing T cells in vivo, but the field has been hampered by toxicity. Previo usly, we showed that a genetically engineered IT consisting of a single-cha in protein, including the anti-CD3sFv spliced to a portion of diphtheria-to xin (DT390) has anti GVHD effects, but pronounced organ toxicity common to this class of agent. A recombinant DT390 anti-CD3sFv protein previously sho wn to have anti-GVHD activity was modified to reduce its filtration into ki dney by genetically inserting a cysteine residue downstream of the sFv moie ty at the c-terminus of the protein, This modification produced an intermol ecular disulfide bridge, resulting in a bivalent, rather than a monovalent IT, termed SS2, that selectively inhibited T-cell proliferation in vitro. A lthough monomer and SS2 were similar in in vitro activity, SS2 had a superi or therapeutic index in vivo with at least a-fold more being tolerated with reduced kidney toxicity, Most importantly, in a lethal model of GVHD, 40 m u g SS2 given for 1 day, protected 100% of the mice from lethal GVHD for 3 months, whereas the maximum tolerated dose (MTD) of monomer protected only 33%, To our knowledge, this is the first time disulfide bonded ITs have bee n created in this way and this simple molecular modification may address se veral problems in the IT field because it (1) markedly increased efficacy c uring mice of GVHD after a single daily treatment, (2) markedly decreased o rgan toxicity, (3) increased the tolerated dosage, and (4) created a therap eutic window where none existed before. (C) 2000 by The American Society of Hematology.