ORIGINAL ARTICLE Graeme Denton á Kevin Brady á Benny K. C. Lo Andrea Murray á C. Rosamund L. Graves Owen D. M. Hughes á Saul J. B. Tendler Charles A. Laughton á Michael R. Price Production and characterization of an anti-(MUC1 mucin) recombinant diabody Received: 16 September 1998 / Accepted: 2 December 1998 Abstract A recombinant diabody fragment based on the anti-MUC1 monoclonal antibody, C595 has been produced in a bacterial expression system. Substitution of a 7-amino-acid linker sequence (Gly 6 Ser) for the original single-chain (sc)Fv 15-amino-acid linker (Gly 4- Ser) 3, using polymerase-chain-reaction-based strategies, forces variable heavy (V H ) and light (V L ) domains to pair with complementary domains on neighbouring scFv molecules, forming a scFv dimer (diabody). This re- combinant protein shows similar binding characteristics to the parental C595 monoclonal antibody. The ability to bind to MUC1 mucin on carcinoma cell surfaces will allow its potential as a diagnostic and therapeutic re- agent of clinical utility to be investigated. Key words MUC1 á Mucin á Diabody á scFv á Immunotherapy Introduction MUC1 mucins are highly glycosylated glycoproteins expressed on the luminal surfaces of glandular epithelia [8, 23]. Apart from their major physiological functions as protective agents and biological lubricants, they are frequently elevated and/or altered in cancer and thus have potential as tumour markers. In breast carcinomas, for example, their expression is frequently up-regulated and they may be secreted into the circulation. Determi- nation of the levels of MUC1 antigen in the blood has been exploited as a measure of tumour burden and changing levels re¯ect the response to therapy [3, 18]. Novel determinants on mucins from malignant cells have been involved as targets for immune manipulation in the cancer patient since they may induce both hu- moral [12, 27] and cellular [6, 11] responses. The MUC1 glycoprotein is a complex molecule with a protein core containing a large domain of variable numbers of a highly conserved 20-amino-acid-repeat sequence (PDTRPAPGSTAPPAHGVTSA) [7]. Many murine antibodies reactive with the MUC1 mucin have now been produced by immunisation with diverse ma- terials including milk fat globule membranes, tumour cells and isolated mucin preparations. Most, if not all, murine anti-MUC1 antibodies reactive with the protein core, de®ne epitopes of 3, 4 or 5 amino acids within the hydrophilic region, APDTRPAP, of the 20-amino-acid repeat. The antibody C595 is one such antibody. This antibody has been proved to be a reagent of clinical utility. It has been employed in immunoassays for the measurement of circulating mucin in breast cancer pa- tients [5, 26] and has been used for in vivo diagnostic tests in the identi®cation of malignant ovarian tumours by immunoscintigraphy [22, 29]. Also, bladder tumours have been detected by gamma camera imaging following intravesical administration of 111 In-labelled [13] and 67 Cu-labelled C595 [10]. Despite the success of C595 in cancer diagnosis, po- tential problems exist in its use as a therapeutic agent. These include immunogenicity due to its murine origin and poor tumour-penetration characteristics due to its large size (approx. 150 kDa). The former is a particular limitation in therapies where multiple administrations Cancer Immunol Immunother (1999) 48:29±38 Ó Springer-Verlag 1999 G. Denton (&) á K. Brady á A. Murray á B.K.C. Lo C.A. Laughton á M.R. Price Cancer Research Laboratories, School of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK S.J.B. Tendler Laboratory of Biophysics and Surface Analysis, School of Pharmaceutical Sciences, University of Nottingham, Nottingham, UK C.R.L. Graves Department of Surgery, City Hospital, Nottingham, UK O.D.M. Hughes Department of Urology, City Hospital, Nottingham, UK