Purpose: Immunogenicity is well recognized by both the biopharmaceutical industry and health agencies as a safety concern, and it is thus a requirement and expectation that appropriate assays are employed to evaluate immunogenic responses to protein therapeutics throughout the drug development process. Nanobodies are modular proteins wherein two or more peptide modules are fused together to realize the benefits of simultaneously targeting different pathways. In the oncoimmunotherapy field, bispecific nanobodies offer the potential to differentiate from other monoclonal antibody drugs, as the advantage of targeting two immunomodulatory checkpoints simultaneously, the small size, and lack of Fc-mediated effector functions may result in an optimal efficacy/tolerability balance.
Methods: Biotin-labeled MK-Drug and sulfo-Tag-labeled anti Monkey IgG binds to anti-MK-drug antibodies in samples forming immunocomplexes. The biotin-labeled MK-drug in the immunocomplex binds to streptavidin on the MSD plate surface and TAG-MK-drug gives out electrochemiluminescent signal when read at MSD plate reader. The ECL signal is proportional to the concentration of anti-MK drug antibodies in the samples.
Results: The assay was capable of detecting 171 ng/mL (upper 99th percentile) drug-specific ADA. In the presence of 200 ng/mL of drug, 15 ng/mL of the ADA positive control could readily be detected. Samples were tested from 10 individual rhesus monkey serum. Also included were five samples that were grossly hemolyzed. All samples were spiked with anti-MK-drug antibody at the LPC concentrations and tested in the screening assay. In the screening assay, ten out of ten (100%) blank samples were below the cut point. Results for ten out of ten (100%) samples spiked at the LPC concentration were above or equal to the established cut point. Also included in the selectivity assessment were five samples that were grossly hemolyzed. All five hemolyzed samples, fortified and non-fortified, passed along with the pooled serum. No impact on matrix selectivity assessment was observed due to hemolysis of the samples.
Conclusion: The unique structural characteristics of this moiety present unique challenges for anti-drug antibody (ADA) assay development. To minimize the interference from binding of non-ADA serum factors to the nanobody molecule, instead of the traditional bridging ADA assay format, we developed a sandwich anti-drug antibody assay to detect monkey ADA (IgG) for the nanobody molecule in the Rhesus Monkey Serum. However, since a generic mouse anti monkey IgG antibody was used as a detection antibody, the challenge of this assay is very high background. Multiple combined approaches (different salt concentrations, different pHs, different blocking buffers) were tested to lower the background. Finally, 10% horse serum was identified and can reduce the NC counts to ~100 ECL. The results demonstrated the method to be sensitive, specific, and selective. This assay has been qualified and has supported a GLP toxicology study.
Dong Geng– Sr. Director, Legend Biotech, Piscataway, New Jersey