Flow cytometric cell-based assay to preselect antibody constructs for radionuclide conjugation

Area of Science:

• Bioconjugation Chemistry
• Radiopharmaceutical Development
• Immunotherapy

Background:

• Radiolabeled antibodies are crucial for targeted cancer therapy and imaging.
• Antibody labeling with radionuclides can compromise their binding affinity.
• Developing reliable assays to evaluate antibody conjugate binding is essential.

Purpose of the Study:

• To establish a flow cytometry-based assay for assessing the binding capacity of Cetuximab conjugates.
• To identify promising Cetuximab conjugates for radiolabeling.
• To validate the binding affinity of radiolabeled Cetuximab conjugates.

Main Methods:

• Developed a flow cytometric assay using EGFR-expressing cell lines (FaDu, SAS) and an isotype control.
• Determined non-saturating concentrations for Cetuximab and its conjugates.
• Calculated relative antibody affinities and performed competitive radioactive binding assays.

Main Results:

• The flow cytometry assay effectively evaluated conjugate binding capacity.
• One out of eleven Cetuximab conjugates showed comparable binding to unconjugated Cetuximab.
• The selected conjugate, radiolabeled with Yttrium-90, demonstrated unaltered binding affinity.

Conclusions:

• The flow cytometry assay is a reliable method for assessing antibody conjugate binding.
• Covalent modification with CHX-A"-DTPA and subsequent radiolabeling did not affect Cetuximab's binding affinity.
• [(90)Y]Y-CHX-A"-DTPA-Cetuximab is suitable for preclinical testing in cancer therapy.