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Direct in vivo measurement of targeted binding in a human tumor xenograft
1Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston 02114, USA.
Abstract:
Binding is crucial to the function of most biologically active molecules, but difficult to quantify directly in living tissue. To this end, fluorescence recovery after photobleaching was used to detect the immobilization of fluorescently labeled ligand caused by binding to receptors in vivo. Measurements of mAb affinity to target antigen within human tumor xenografts revealed a saturable binding isotherm, from which an in vivo carcinoembryonic antigen density of 0.56 nmol/g (5.0 x 10(5)/cell) and an association constant of Ka < or = 4 x 10(7) M(-1) were estimated. The present method can be adapted for in vivo studies of cell signaling, targeted drugs, gene therapy, and other processes involving receptor-ligand binding.
Insights
This study introduces a novel method using fluorescence recovery after photobleaching to quantify in vivo receptor-ligand binding. The technique successfully measured antibody affinity and antigen density in human tumors, offering new insights into biological interactions.
Area of Science:
- Biophysics
- Molecular Biology
- Oncology
Background:
- Quantifying molecular binding in living tissues is challenging.
- Receptor-ligand interactions are fundamental to biological processes.
Purpose of the Study:
- To develop and validate a method for direct in vivo measurement of receptor-ligand binding.
- To quantify the binding affinity of monoclonal antibodies (mAbs) to their target antigens in human tumor xenografts.
Main Methods:
- Utilized fluorescence recovery after photobleaching (FRAP) to detect ligand immobilization due to receptor binding.
- Applied the method to measure mAb affinity and estimate antigen density in vivo.
Main Results:
- Demonstrated a saturable binding isotherm for mAb-antigen interactions in human tumor xenografts.
- Estimated in vivo carcinoembryonic antigen density at 0.56 nmol/g (5.0 x 10^5 cells) and an association constant (Ka) <= 4 x 10^7 M^-1.
Conclusions:
- Fluorescence recovery after photobleaching is a viable technique for in vivo quantification of receptor-ligand binding.
- The method has broad applicability for studying cell signaling, targeted drug delivery, and gene therapy in vivo.