Bioanalytical Challenges due to Prior Checkpoint Inhibitor Exposure: Interference and Mitigation in Drug Concentration and Immunogenicity Assays.

Monoclonal antibodies (mAbs) are a leading class of biotherapeutics. In oncology, patients often fail on early lines of biologic therapy to a specific target. Some patients may then enroll in a new clinical trial with a mAb specific for the same target. Therefore, immunoassays designed to quantify the current mAb therapy or assess immunogenicity to the drug may be susceptible to cross-reactivity or interference with residual prior biologics. The impact of two approved anti-PD-1 mAbs, pembrolizumab and nivolumab, was tested in several immunoassays for cemiplimab, another approved anti-PD-1 mAb. The methods included a target-capture drug concentration assay, a bridging anti-drug antibody (ADA) assay and a competitive ligand-binding neutralizing antibody (NAb) assay. We also tested bioanalytical strategies to mitigate cross-reactivity or interference in these assays from other anti-PD-1 biologics. Both pembrolizumab and nivolumab cross-reacted in the cemiplimab drug concentration assay. This was mitigated by addition of antibodies specific to pembrolizumab or nivolumab. ADA specific for pembrolizumab and nivolumab did not interfere in the cemiplimab ADA assay. However, pembrolizumab and nivolumab generated a false-positive response in a target-capture NAb assay. Our results demonstrate that similar exogenous pre-existing anti-PD-1 mAbs (biotherapeutics) such as pembrolizumab and nivolumab are detected and accurately quantified in the cemiplimab drug concentration assay. However, once steady state is achieved for the new therapy, prior biologics would likely not be detected. Cross-reactivity and interference in immunoassays from previous treatment with class-specific biotherapeutic(s) pose significant bioanalytical challenges, especially in immuno-oncology.

Drug Removal Strategies in Competitive Ligand Binding Neutralizing Antibody (NAb) Assays: Highly Drug-Tolerant Methods and Interpreting Immunogenicity Data.

Neutralizing anti-drug antibody (NAb) assays often have lower drug tolerance (DT) than trough drug concentrations, potentially under-estimating NAb incidence. To improve DT, drug-specific proteins were coupled to magnetic beads to deplete drug in the sample. To avoid interference from carryover, drug-specific proteins that did not interfere in the NAb assay, such as target or non-blocking anti-drug antibodies, were selected. With the drug depletion step, DT improved by > 10-fold in two competitive ligand binding NAb assays. Analysis of anti-drug antibody positive clinical samples with elevated drug levels demonstrated that NAb incidence was under-estimated without the drug depletion step. However, these NAb-positive samples had low titer and no impact on drug concentrations.

Anti-VEGF drug interference with VEGF quantitation in the R&D systems human quantikine VEGF ELISA kit.

AIM: To evaluate the accuracy of the Quantikine Human VEGF Immunoassay (R&D Systems) in the presence of VEGF inhibitors. MATERIALS & METHODS: Quantikine VEGF ELISA (R&D), anti-VEGF165 mAb (R&D), VEGF165 and aflibercept (Regeneron), ranibizumab and bevacizumab (Genentech). RESULTS: Binding affinity of anti-VEGF165 mAb for VEGF was threefold weaker than aflibercept, but 33- and 40-fold stronger than ranibizumab or bevacizumab. Extended incubation of VEGF complexed with inhibitors led to VEGF dissociation from ranibizumab and bevacizumab, but not aflibercept, and subsequent binding by the immunoassay capture antibody. The immunoassay also detected VEGF:ranibizumab and VEGF:bevacizumab complexes but not VEGF:aflibercept complexes. CONCLUSION: The immunoassay cannot accurately quantitate VEGF in the presence of these VEGF inhibitors as they interfere with the capture and detection of free VEGF.