Abstract
Several Methotrexate (MTX)-resistant sublines of the osteogenic sarcoma cell line 791T were derived by continuous selection in the presence of MTX and 12-O-tetradecanoylphorbol-13-acetate (TPA). Studies including assays of the uptake and binding of [3H]MTX and fluoresceinated-MTX, determined that these sublines showed diminished MTX transport, and that none of them appeared to overproduce the MTX-target enzyme dihydrofolate reductase. Conjugates of the anti-791T monoclonal antibody 791T/36 linked to MTX via human serum albumin (HSA) were prepared by Dr M.C. Garnett. These were cytotoxic selectively for cells bearing the 791T/36-defined antigen (gp72), and were found to be as cytotoxic to most of the MTX-resistant 791T sublines as they were to parental 791T cells. Furthermore, an anti-MTX/anti-gp72 bispecific antibody 516 augmented the cytotoxicity of HSA-MTX conjugate to the MTX-resistant 791T variant R120 apparently as efficiently as for parental 791T cells. It is suggested that acquired drug resistance caused by deficient transport mechanisms may be partially or wholly overcome by targeting the drug to a readily-internalised cell surface antigen.
Highlights
Cell lines The human osteogenic sarcoma tumour cell line 791T was grown in monolayer cultures in 90 mm diameter plastic petri dishes (Cell-Cult, Sterilin Limited, Hounslow) in Eagles Minimal Essential Medium (EMEM, Flow Laboratories, Rickmansworth) supplemented with 10% heat-inactivated newborn calf serum (NBCS, Gibco Limited, Basingstoke, Hants)
Monoclonal antibodies Monoclonal antibody 791T/36 is a murine IgG2b antibody, obtained from a hybridoma produced by fusing spleen cells from a mouse immunised against osteogenic sarcoma cell line 791T, with P3-NSl-Ag-4 mouse myeloma (Embleton et al, 1981)
The 791T/48 antibody was of the IgG2b subclass, and was raised against 791T cells, but it recognises an epitope on 791T cells separate from that detected by 791T/36 (Embleton et al, 1981)
Summary
Cell lines The human osteogenic sarcoma tumour cell line 791T was grown in monolayer cultures in 90 mm diameter plastic petri dishes (Cell-Cult, Sterilin Limited, Hounslow) in Eagles Minimal Essential Medium (EMEM, Flow Laboratories, Rickmansworth) supplemented with 10% heat-inactivated newborn calf serum (NBCS, Gibco Limited, Basingstoke, Hants).The MTX-resistant sublines of the 791T line were grown in EMEM plus 10% NBCS, with added MTX at a concentration in which the subline was able to grow at the same rate as the parental cell line.The MTX-resistant Chinese hamster ovary cell line M3000 was kindly provided by Dr A. Cell lines The human osteogenic sarcoma tumour cell line 791T was grown in monolayer cultures in 90 mm diameter plastic petri dishes (Cell-Cult, Sterilin Limited, Hounslow) in Eagles Minimal Essential Medium (EMEM, Flow Laboratories, Rickmansworth) supplemented with 10% heat-inactivated newborn calf serum (NBCS, Gibco Limited, Basingstoke, Hants). Monoclonal antibodies Monoclonal antibody 791T/36 is a murine IgG2b antibody, obtained from a hybridoma produced by fusing spleen cells from a mouse immunised against osteogenic sarcoma cell line 791T, with P3-NSl-Ag-4 mouse myeloma (Embleton et al, 1981). This antibody was affinity purified from ascites fluid on a protein A column eluted with 3 M sodium thiocyanate. The 791T/48 antibody was of the IgG2b subclass, and was raised against 791T cells, but it recognises an epitope on 791T cells separate from that detected by 791T/36 (Embleton et al, 1981)
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