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Uptake, penetration, and binding of monoclonal antibodies with increasing affinity in human osteosarcoma multicell
M H Hjelstuen1, K Rasch-Halvorsen, O Bruland
1Department of Physics, Norwegian University of Science and Technology, Trondheim, Norway.
Background:
Multicell spheroids from the human osteosarcoma cell line, OHS, were incubated with increasing concentrations of the monoclonal antibodies TP-1, TP-3 and 9.2.27 having different affinities (Ka = 8.5 x 10(8) M-1, 3.4 x 10(9) M-1 and 1.4 x 10(11) M-1, respectively).
Materials And Methods:
Uptake and penetration of the fluorescein labelled antibodies were studied using confocal laser scanning fluorescence microscopy, and antibodies bound per cell were measured using flow cytometry.
Results:
The antibody with highest affinity, 9.2.27, bound to all available binding sites in one cell-layer before reaching the next, whereas TP-3 and TP-1 gradually bound to an increasing number of epitopes of all cells throughout the spheroids. The penetration rate and antibody uptake increased with increasing antibody concentration up to saturating concentrations. 9.2.27 required saturation concentrations to reach the center of the spheroids, whereas TP-1 and TP-3 penetrated the centre when concentrations below saturation were applied.
Conclusion:
The antibodies might be useful in radioimmunotherapy of micrometastases.
Insights
Antibody affinity influences penetration into multicellular spheroids. Higher affinity antibodies saturate binding sites faster, impacting delivery for potential radioimmunotherapy applications.
Area of Science:
- Oncology
- Immunology
- Biotechnology
Background:
- OHS human osteosarcoma cell line spheroids used as a model.
- Monoclonal antibodies TP-1, TP-3, and 9.2.27 with varying affinities were tested.
Purpose of the Study:
- To investigate the impact of monoclonal antibody affinity on penetration and uptake in multicellular spheroids.
- To assess antibody distribution within tumor models.
Main Methods:
- Confocal laser scanning fluorescence microscopy for visualizing antibody penetration.
- Flow cytometry for quantifying antibody binding per cell.
Main Results:
- High-affinity antibody (9.2.27) saturated binding sites in the outer layer before penetrating deeper.
- Lower affinity antibodies (TP-3, TP-1) showed gradual binding throughout spheroids.
- Penetration and uptake increased with antibody concentration, reaching saturation.
Conclusions:
- Antibody affinity significantly affects penetration dynamics in tumor spheroids.
- Potential utility of these antibodies in radioimmunotherapy for micrometastases.