A SINGLE-CHAIN IMMUNOTOXIN AGAINST CARCINOEMBRYONIC ANTIGEN THAT SUPPRESSES GROWTH OF COLORECTAL-CARCINOMA CELLS

We have engineered an anti-carcinoembryonic antigen (CEA) single-chain immunotoxin derived from humanized anti-CEA antibody (hMN14) and a tr uncated Pseudomonas exotoxin (PE), PE40, The purified anti-CEA immunot oxin (hMN14(FV)-PE40) was first measured for binding affinity against a CEA-positive colorectal carcinoma cell line and compared with its pa rental IgG and the monovalent Fab fragment. The K-a of sFv-PE40, Fab, and IgG were 5 x 10(7), 6 x 10(7), and 3 x 10(8) M-1, respectively. Th ere was no significant affinity loss by conversion of Fab to the singl e-chain Fv, but these monovalent forms were 5-6-fold reduced in affini ty compared with the parental IgG, In cytotoxicity assays, the hMN14(F v)-PE40 showed specific growth suppression of CEA-expressing colon can cer cell lines MIP-CEA (high CEA) and LS174T (moderate CEA) with IC(50 )s of 12 ng/ml (0.2 nM) and 69 ng/ml (1.1 nM), These IC(50)s correlate d inversely with the surface expression of CEA, such that 50% killing was equivalent for each cell type when expressed in toxin molecules bo und/cell (3000-5000), The presence of soluble CEA up to 1000 ng/ml did not affect the cytotoxicity against CEA-expressing cells, with 50% su ppression only at 4000 ng/ml that correlated with the binding K-d of t he single-chain Fv, The stability of the hMN14(Fv)-PE40 molecule at 37 degrees C was confirmed by bioassay and by lack of aggregation. Our h MN14(Fv)-PE40 may be clinically useful for tumors with high CEA expres sion without affecting normal tissues with low or absent CEA, even in patients with high soluble antigen levels.