Biophysical and Colloidal Properties Govern Anti-Drug Antibody Generation: A Case Study Using the Tumoricidal mAb 4713

Anti-drug antibodies (ADA) are a well-recognized clinical problem, but their sporadic occurrence has limited detailed biophysical characterization of causative factors. We investigated how different stress conditions influence aggregation of a murine monoclonal antibody, mAb 4713, and the resulting potential to induce ADA in mice. Dynamic light scattering (DLS) revealed that stirring, freeze-thaw, and heat stress each generated subvisible aggregates (30-700 nm), whereas non-stressed mAb 4713 remained predominantly monomeric. Stirring- and freeze-thaw-induced aggregates retained native-like secondary and tertiary structures, thermal and biochemical stability, and anti-tumor cytotoxicity, as shown by circular dichroism, tryptophan fluorescence, thermal shift assay, limited proteolysis, and cell death assay. In contrast, heat-induced aggregates displayed complete structural denaturation and loss of cytotoxic activity. Immunization of Jcl: ICR mice demonstrated that heat-induced aggregates were only mildly immunogenic, while freeze-thawed and non-stressed mAb 4713 elicited no detectable immune response, and ADA generated under these conditions did not affect anti-tumor activity. Strikingly, stirred aggregates triggered a strong anti-mAb antibody response, as measured by ELISA, and the resulting antisera completely abolished mAb 4713 cytotoxicity against Raji cells, a Burkitt lymphoma cell line, indicating induction of neutralizing ADA. These results show that aggregate immunogenicity depends not simply on aggregate presence but critically on their structural and colloidal properties and formation pathways, highlighting the need to assess both structural and colloidal properties of aggregates in biopharmaceutical development to mitigate ADA risks.