title: A single-chain immunotoxin against carcinoembryonic antigen that suppresses growth of colorectal carcinoma cells.
abstract: We have engineered an anti-carcinoembryonic antigen (CEA) single-chain immunotoxin derived from humanized anti-CEA antibody (hMN14) and a truncated Pseudomonas exotoxin (PE), PE40. The purified anti-CEA immunotoxin (hMN14(Fv)-PE40) was first measured for binding affinity against a CEA-positive colorectal carcinoma cell line and compared with its parental IgG and the monovalent Fab fragment. The Ka of sFv-PE40, Fab, and IgG were 5 x 10(7), 6 x 10(7), and 3 x 10(8) M(-1), respectively. There was no significant affinity loss by conversion of Fab to the single-chain Fv, but these monovalent forms were 5-6-fold reduced in affinity compared with the parental IgG. In cytotoxicity assays, the hMN14(Fv)-PE40 showed specific growth suppression of CEA-expressing colon cancer cell lines MIP-CEA (high CEA) and LS174T (moderate CEA) with IC50s of 12 ng/ml (0.2 nM) and 69 ng/ml (1.1 nM). These IC50s correlated inversely with the surface expression of CEA, such that 50% killing was equivalent for each cell type when expressed in toxin molecules bound/cell (3000-5000). The presence of soluble CEA up to 1000 ng/ml did not affect the cytotoxicity against CEA-expressing cells, with 50% suppression only at 4000 ng/ml that correlated with the binding Kd of the 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 hMN14(Fv)-PE40 may be clinically useful for tumors with high CEA expression without affecting normal tissues with low or absent CEA, even in patients with high soluble antigen levels.
authors.affiliation: Division of Hematology-Oncology, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Akamatsu
authors.forename: J C
authors.initials: JC
authors.lastname: Murphy
authors.forename: K F
authors.initials: KF
authors.lastname: Nolan
authors.forename: P
authors.initials: P
authors.lastname: Thomas
authors.forename: R J
authors.initials: RJ
authors.lastname: Kreitman
authors.forename: S O
authors.initials: SO
authors.lastname: Leung
authors.forename: R P
authors.initials: RP
authors.lastname: Junghans
mesh_terms: D036002:ADP Ribose Transferases; D000915:Antibody Affinity; D000918:Antibody Specificity; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D001427:Bacterial Toxins; D002272:Carcinoembryonic Antigen / Q000096:biosynthesis / Q000276:immunology*; D002452:Cell Count / Q000187:drug effects; D015179:Colorectal Neoplasms / Q000276:immunology / Q000473:pathology / Q000628:therapy*; D005098:Exotoxins / Q000627:therapeutic use*; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry / Q000627:therapeutic use*; D014407:Tumor Cells, Cultured; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000970:Antineoplastic Agents; D001427:Bacterial Toxins; D002272:Carcinoembryonic Antigen; D005098:Exotoxins; D000922:Immunotoxins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A