EurJ Cmctr Clin Od, Vol. 25. No. 4, pp. 633-640, 1989. Printed in Great Britain 0277~5379/89$3.00 + 0.00 CQ I989 Pqamon Press plc Doxorubicin-Anti-carcinoembryonic Antigen Immunoconjugate Activity In Vitro VERNON J. RICHARDSON,* CHRISTOPHER H.J. FORD, GEORGIA TSALTAS and M.E. GALLANT Oncology Research, Memorial University of Newfoundland and The Newfoundland Cancer Treatment and Research Foundation, Health Sciences Centre, St John’s, Newfoundland, Canada A 1 B 3V6 Abstract-An in vitro model consisting of a series of 11 human cancer cell lines with varying den.@ of expression of membrane carcinoembryonic antigen (CEA) has been used to evaluate conjugates of doxorubtin (Adriamycin@) covalentb linked by a carbodiimide method to goat polyclonal antibodies and mouse manoclonal antibodies to CEA. Conjugates were produced which retained both antigen binding and drug cytotoxicity. lcso values were determined for free drug, free drug mixed with unconjugated antibodies andfor the immunoconjugates. Cell lines that were very sensitive to free drug (lcso < 100 ng/ml) were also found to be high4 sensitive to conjugated drug and similarly cell lines resistant to drug (q. > 1000 nglml) were also resistant to conjugated drug. Although there was no correlation between CEA expression and conjugates ef&aq, competitive inhibition studies using autologous antibody to block conjugate binding to cells indicated immunoconju- gates speczficity for the CEA target. INTRODUCTION DOXORUBICIN is an extremely potent anti-cancer drug with response rates in a range of human tumours including lung, stomach, breast, sarcomas, some leukaemias and lymphomas. However, like all anti-cancer drugs it is not selective for just cancer cells and its use is limited by its toxicity to normal tissues such as bone marrow, mucous membranes and the muscle tissue of the heart. The drug’s effect on the first two of these tissues is usually only temporary and they soon recover; however, the toxic effects on the heart are much more serious because they are cumulative and chronic. These effects are all dose dependent and severely restrict the maximum amount of drug that can be adminis- tered [l]. Two methods of approach are being used to try and overcome the toxic limitations imposed by this drug: firstly, the drug has been chemically modified in a search for less cardiotoxic derivatives [2] and secondly, drug carrier molecules are being tested which alter the tissue distribution of the drug and reduce the quantity of drug reaching the heart [3]. We have opted to use the latter approach and have selected anti-CEA antibodies as our carrier system, not just to potentially reduce the cardiac uptake of the drug but also to increase specificity by targeting the drug to the cancer cells which express CEA on their surface membranes. CEA is a glycoprotein Accepted 3 November 1988. *Corresponding author. 633 found associated with many cancers including those of the lung, breast, colon, stomach and ovaries [4] (i.e. many that are also sensitive to doxorubicin). Anthracyclines have been covalently conjugated to immunoglobulin carriers using a number of tech- niques: directly [5], indirectly via small spacer molecules [6] or indirectly via secondary carrier macromolecules [7-91. We chose to initiate our studies using a direct conjugation method using a carbodiimide linkage procedure [5]. We have linked doxorubicin to both goat polyclonal (Pab) and to mouse monoclonal (Mab) ( 11-285-l 4) anti-CEA antibodies by this technique. This monoclonal anti- body has been well characterized by us and by other groups and has been used for immunoscintigraphy in animals [lo], cancer patients [ 1 l] and as a drug carrier in vitro [ 121 and in vivo with human tumour xenografts in nude mice [ 131. The objective of the present study has been to evaluate the in vitro characterisics and efficacy of doxorubicin-anti-CEA antibody immunoconju- gates on human tumour cell lines with a range of expression of the target antigen, CEA. MATERIALS AND METHODS CEA CEA was affinity purified from colonic carcinoma liver metastases as previously reported [ 141. Anti-CEA antibodies Mouse monoclonal ( 1 l-285 14) anti-CEA anti- bodies were produced from mouse ascites and pur-