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Updated: Mar 17, 2026
Generation of Murine Monoclonal Antibodies by Hybridoma Technology
Published on: January 2, 2017
"Biophysical and colloidal properties govern anti-drug antibody generation: A case study using the tumoricidal
Sanjida Yesmin1, M Monirul Islam2, Md Din Islam1
1Dept. of Biotechnology and Life Science, Faculty of Engineering, Tokyo University of Agriculture and Technology, Nakamachi-2-24-16, Tokyo, 184-8588, Japan.
We investigated how different stress conditions affect the biophysical and colloidal properties of a murine monoclonal antibody (mAb) 4713, whose humanized version is in clinical development, and how these stress-induced properties influence anti-drug antibody (ADA) induction in the Jcl:ICR mouse model. Biophysical analyses showed that non-stressed monomers remained native and non-immunogenic in mice. Stirring and freeze-thawing generated native-like aggregates that retained the tumoricidal activity, whereas heat-induced aggregates were denatured and lost functional activity. Mice immunization study showed that freeze-thawed and non-stressed mAb 4713 elicited no immune response, while heat-induced aggregates were weakly immunogenic and did not result in function-blocking ADA. Conversely, stirred aggregates induced a strong antibody response that completely blocked mAb 4713-mediated cytotoxicity against Raji cells, demonstrating the formation of function-blocking ADA. Together, these findings indicate that aggregate immunogenicity correlates not simply with the presence of aggregates, but with the combined effects of biophysical and colloidal properties, and the specific stress applied. Crucially, by correlating aggregate properties to the induction of function-blocking ADA and the loss of therapeutic activity, rather than immunoglobulin (Ig) G production alone, this work provides a novel framework for assessing clinically relevant risk associated with aggregation.
We investigated how different stress conditions affect the biophysical and colloidal properties of a murine monoclonal antibody (mAb) 4713, whose humanized version is in clinical development, and how these stress-induced properties influence anti-drug antibody (ADA) induction in the Jcl:ICR mouse model. Biophysical analyses showed that non-stressed monomers remained native and non-immunogenic in mice. Stirring and freeze-thawing generated native-like aggregates that retained the tumoricidal activity, whereas heat-induced aggregates were denatured and lost functional activity. Mice immunization study showed that freeze-thawed and non-stressed mAb 4713 elicited no immune response, while heat-induced aggregates were weakly immunogenic and did not result in function-blocking ADA. Conversely, stirred aggregates induced a strong antibody response that completely blocked mAb 4713-mediated cytotoxicity against Raji cells, demonstrating the formation of function-blocking ADA. Together, these findings indicate that aggregate immunogenicity correlates not simply with the presence of aggregates, but with the combined effects of biophysical and colloidal properties, and the specific stress applied. Crucially, by correlating aggregate properties to the induction of function-blocking ADA and the loss of therapeutic activity, rather than immunoglobulin (Ig) G production alone, this work provides a novel framework for assessing clinically relevant risk associated with aggregation.
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