A chlorambucil-anti-CEA conjugate cytotoxic for human colon adenocarcinoma cells in vitro

The use of chlorambucil (CBL) for the chemotherapeutic treatment of cancer is often limited because of undesirable side effects due to its lack of specificity for cancer cells. Systemic effects such as renal toxicity, marrow aplasia, pulmonary fibrosis and gastrointestinal disorders may be eliminated or greatly reduced by increasing the specificity of the drug towards tumour cells. Carcinoembryonic antigen (CEA), an oncofoetal protein produced by some human cancer cells (Gold & Friedman, 1965), is extensively used as a clinical cancer marker for the follow-up of treated patients. We have already reported that this protein could be a target for daunorubicin-anti-CEA conjugates (Belles-Isles & Page, 1981; Page et al., 1981). Many authors have already described a method to adsorb chlorambucil to antitumour antibodies (Blakeslee & Kennedy,1974; Guclu et al., 1976), but in the previous applications of chlorambucilantibody conjugates, a certain percentage of the reported activity could have been due to the presence of a non-covalently bound aggregate of the drug (Blakeslee et al., 1975). The other method described for the covalent coupling of CBL (Tai et al., 1979) could lead to polymerisation of the carrier and needed a 2 h incubation in an aqueous solution. We present here a new method for the covalent binding of chlorambucil which allows a rapid coupling to proteins without any significant polymerisation. This was obtained using the isocyanate derivative of chlorambucil: 3-{4-[bis(2chloroethyl)amino]phenyl}propyl-1-isocyanate. The conjugate was separated from the remaining free drug by gel filtration on Sephadex G-25 (Pharmacia Fine Chemicals) and the drug:protein ratio was determined by a photofluorometric method to assay the alkylating activity of CBL (Bernier et al., 1983). This ratio could be varied by mixing various proportions of the drug derivative and of the antibody; in the assay conditions reported a ratio of 25 moles of drug per mole of antibody was used. The cytotoxic effects of chlorambucil-anti-CEA conjugates on LoVo cells (CEA producing human colon carcinoma cells) were evaluated by the inhibition of colony formation as already described (Emond & Page, 1982). Figure 1 illustrates that for any concentration of drug used, the highest inhibition of colony formation was obtained with the covalent drug-antibody conjugate. Also the antibodies were neither cytolytic nor cytostatic for these cells in vitro. The concentration required to obtain a 50% inhibition of colony formation (ID 50) was much lower for the conjugate than for the free drug, the physical mixture of both agents or chlorambucil conjugated to non-specific antibodies (anti-alphafoetoprotein). Experiments on non-CEA producing cells (CCL6; human amnions) showed the specificity of the CBL-anti-CEA conjugate for CEA producing cell lines (Table I). The contact period study (Figure 2) shows that a rapid binding of the CBL-anti-CEA conjugate allows a much higher pharmacological activity when compared to equimolar concentrations of the free drug.