There is a wealth of information about monoclonal antibody (MAb) specificity and function on fixed tissues, yet little is known about formation and release of antigen-antibody complexes and their functional behavior in vivo.
We analyzed the pathway of radiolabeled MAbs directed against melanoma-associated antigens by radioimmunoelectron microscopy (RIEM) on metabolically active cells of the melanoma cell lines SK-MEL-28, MeWo and Colo 38 at different time intervals. In parallel, binding and release of MAbs were investigated by the radioantibody binding assay (RBA). Both procedures gave essentially concordant results.
Preferentially stable binding of immune complexes (ICs) to the cell surface after 30 and 120 min was shown for the MAb L10. Internalization was demonstrated for the MAb M.2.9.4. At the ultrastructural level, direct evidence of this phenomenon was obtained by visualization of radioactivity within the cytoplasm after 120 min. In the RBA this process was indicated by resistance of bound MAbs to acid buffer desorption. RIEM pointed to different transport mechanisms: constitutive internalization by endocytotic vesicles, or receptor-mediated endocytosis by coated vesicles. Shedding was indicated for the MAb R24 by release of the ICs from the cell membrane. It was demonstrated that stable fixation of ICs on the cell surface or modulation by internalization led to high accumulation rates, while shedding of antigen-antibody complexes resulted in a low accumulation of the MAb in tumor cells.
Assuming that the potential of MAbs for clinical application is determined by the biological behavior of antigen-antibody complexes, these methods are suitable for demonstration of antigenic modulation by MAbs and eventually enable us to predict the localization, penetration and distribution pattern of individual MAbs in the melanoma patient.
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This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 136 “Krebsforschung”
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Tilgen, W., Matzku, S., Kaufmann, I. et al. Modulation of melanoma-associated antigens by monoclonal antibodies as visualized by radioimmunoelectron microscopy and radioantibody binding assay. Arch Dermatol Res 279, S116–S126 (1987). https://doi.org/10.1007/BF00585934
- Antigenic modulation
- Monoclonal antibodies
- Radioimmunoelectron microscopy
- Radioantibody binding assay