A flow cytometric method of studying the internalization of a monoclonal antibody (Mab) directed against carcinoembryonic antigen (CEA) has been compared with Western blotting, using three human colonic cancer cell lines which express varying amounts of the target antigen. Cell samples incubated for increasing time intervals with fluoresceinated or unlabelled Mab were analyzed using flow cytometry or polyacrylamide gel electrophoresis and Western blotting. SDS/PAGE analysis of cytosolic and membrane components of solubilized cells from the cell lines provided evidence of non-degraded internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 min. Internalized anti-CEA was detected in the case of high CEA expressing cell lines (LS174T, SKCO1). Very similar results were obtained with an anti-fluorescein flow cytometric assay. Given that these two methods consistently provided comparable results, use of flow cytometry for the detection of internalized antibody is suggested as a rapid alternative to most currently used methods for assessing antibody internalization. The question of the endocytic route followed by CEA-anti-CEA complexes was addressed by using hypertonic medium to block clathrin mediated endocytosis.

Key words: carcinoembryonic antigen; bispecific antibody; targeting; doxorubicin; colon cancerMonoclonal antibodies (MAbs) have therapeutic potential on their own, 1 when used as carriers of radioisotopes for immunodiagnosis 2 and radioimmunotherapy 3 or when conjugated to drugs 4,5 or toxins. 6 In a complementary approach, bispecific antibodies (BsMAbs) are being developed as novel bioconjugates 7,8 that recognise a tumour-associated target with one antigen-binding site and a variety of molecules with the other antigen-binding site, including molecules on cells of the immune system, 9 -12 toxins 13,14 and cytokines. 15 We have previously reported production of immunoconjugates of the chemotherapeutic drug doxorubicin (Dox) with an anti-carcinoembryonic antigen (CEA) MAb 16,17 and production of BsMAbs recognising CEA and Dox. 18 Our present objective was to evaluate the efficacy of one of these BsMAbs with human colon cancer cell lines growing in vitro and in a pre-clinical in vivo model. MATERIAL AND METHODS Cell linesHybridoma 26-61-10 is 1 of 7 hybridomas secreting BsMAbs to CEA and Dox. 18 It is grown in RPMI-1640 medium with 10% FBS (GIBCO BRL, Gaithersburg, MD) supplemented with L-glutamine, penicillin and streptomycin to final concentrations of 2 mM, 105 units/ml and 105 g/ml, respectively. Pristane-primed BALB/c mice were inoculated i.p. with 10 6 hybridoma cells, and ascitic fluid was obtained after 2 to 3 weeks. Human colon cancer cell lines LS174T, SKCO1 and COLO320DM were maintained in culture as described previously. 19 BsMAb purificationA method was developed and used for purifying BsMAbs that combined protein A (Pharmacia Biotech, Piscataway, NJ) affinity chromatography and hydroxyapatite HPLC affinity chromatography (Bio Rad, Hercules, CA) in conjunction with selection of fractions for pooling based on dual anti-CEA/anti-Dox activity by ELISA. 20 Flow cytometryCEA expression by human colon cancer cell lines was assessed as described previously. 19 Anti-CEA MAb (hybridoma 11-285-14, one of the fusion partners for BsMAb 26-61-10) and non-specific Ag8 as control (P3x63Ag8, a mouse myeloma that secretes a non-specific IgG1 of the same isotype as 11-285-14) were used to assess CEA expression (both at 10 g/ml) with an EPICS XL (Coulter, Hialeah, FL). Goat anti-mouse Ig-FITC conjugate (Coulter), at a dilution of 1/100, was used for detection. MTT assayThe in vitro sensitivity of the cell lines to Dox or BsMAb ϩ Dox was assessed using the MTT assay, as previously described. 21 Briefly, cell lines were trypsinised and 10 4 cells plated into the wells of microplates, allowed a 24 hr recovery, exposed to Dox concentrations for 24 hr followed by washing, a 24 hr recovery period and then termination of the assay. For the BsMAb assay, cells were exposed to either BsMAb or Ag8 as control (after the first 24 hr recovery period) at 10, 1, 0.1 and 0.01 g/ml for 15 min on ice. Supernatants were then removed, Dox dilutions were added and the assay continued as for the Dox assay. Xenograft experimentsA breeding col...

A flow cytometric method of studying the internalization of a monoclonal antibody (Mab) directed against carcinoembryonic antigen (CEA) has been compared with Western blotting, using three human colonic cancer cell lines which express varying amounts of the target antigen. Cell samples incubated for increasing time intervals with fluoresceinated or unlabelled Mab were analyzed using flow cytometry or polyacrylamide gel electrophoresis and Western blotting. SDS/PAGE analysis of cytosolic and membrane components of solubilized cells from the cell lines provided evidence of non‐degraded internalized anti‐CEA Mab throughout seven half hour intervals, starting at 5 min. Internalized anti‐CEA was detected in the case of high CEA expressing cell lines (LS174T, SKCO1). Very similar results were obtained with an anti‐fluorescein flow cytometric assay. Given that these two methods consistently provided comparable results, use of flow cytometry for the detection of internalized antibody is suggested as a rapid alternative to most currently used methods for assessing antibody internalization. The question of the endocytic route followed by CEA‐anti‐CEA complexes was addressed by using hypertonic medium to block clathrin mediated endocytosis. © 1996 Wiley‐Liss, Inc.