Immunogenicity of Cemiplimab: Low Incidence of Antidrug Antibodies and Cut-Point Suitability Across Tumor Types.

The immunogenicity of cemiplimab, a fully human immunoglobulin G4 monoclonal antibody directed against programmed cell death 1, was assessed in patients across multiple tumor types. The development of antidrug antibodies (ADAs) against cemiplimab was monitored using a validated bridging immunoassay. To identify ADA-positive samples in the assay, statistically determined cut points were established by analyzing baseline clinical study samples from a mixed population of different tumor types, and this validation cut point was used to assess immunogenicity in all subsequent studies. Regulatory guidance requires that ADA assay cut points be verified for appropriateness in different patient populations. Thus, for the cemiplimab ADA assay, we evaluated whether each new oncology population was comparable with the validation population used to set the cut point. Assay responses from 2393 individual serum samples from 8 different tumor types were compared with the validation population, using established statistical methods for cut-point determination and comparison, with no significant differences observed. Across tumor types, the immunogenicity of cemiplimab was low, with an overall treatment-emergent ADA incidence rate of 1.9% and 2.5% at intravenous dose regimens of 3 mg/kg every 2 weeks and 350 mg every 3 weeks, respectively. Moreover, no neutralizing antibodies to cemiplimab were detected in patients with ADA-positive samples, and there was no observed impact of cemiplimab ADAs on pharmacokinetics. Study-specific cut points may be required in some diseases, such as immune and inflammatory diseases; however, based on this analysis, in-study cut points are not required for each new oncology disease indication for cemiplimab.

Pre-existing Reactivity to an IgG4 Fc-Epitope: Characterization and Mitigation of Interference in a Bridging Anti-drug Antibody Assay.

Twenty percent of baseline patient samples exhibited a pre-existing response in a bridging anti-drug antibody (ADA) assay for a human IgG4 monoclonal antibody (mAb) therapeutic. In some cases, assay signals were more than 100-fold higher than background, potentially confounding detection of true treatment-emergent ADA responses. The pre-existing reactivity was mapped by competitive inhibition experiments using recombinant proteins or chimeric human mAbs with IgG4 heavy chain regions swapped for IgG1 sequences. These experiments demonstrated that the majority of the samples had reactivity to an epitope containing leucine 445 in the CH3 domain of human IgG4. The pre-existing reactivity in baseline patient samples was mitigated by replacing the ADA assay capture reagent with a version of the drug containing a wild type IgG1 proline substitution at residue 445 without impacting detection of drug-specific, treatment-emergent ADA. Finally, purification on Protein G or anti-human IgG (H + L) columns indicated the pre-existing response was likely due to immunoglobulins in patient samples.

Assay pH and critical reagent optimization in measuring concentrations of a monoclonal antibody and its target.

Aim: To mitigate assay interference in the drug and target assays to support the development of monoclonal antibody REGN-Z. Results: Mild acidic assay conditions and capture and detection antibodies with different affinities and t1/2 under different assay pHs were used to mitigate interference in the total drug and total target assays. A free target assay was also developed using a lower-affinity capture antibody with a much slower association and dissociation rate. The impact of sample incubation, dilution and storage on the accurate detection of the free target was also evaluated. Conclusion: The total drug, total and free target assays can accurately quantitate drug and target concentrations when tested with a subset of clinical study samples.

REGEN-COV® antibody cocktail bioanalytical strategy: comparison of LC-MRM-MS and immunoassay methods for drug quantification.

Aim: In response to the COVID-19 pandemic, Regeneron developed the anti-SARS-CoV-2 monoclonal antibody cocktail, REGEN-COV® (RONAPREVE® outside the USA). Drug concentration data was important for determination of dose, so a two-part bioanalytical strategy was implemented to ensure the therapy was rapidly available for use. Results & methodology: Initially, a liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) assay, was used to analyze early-phase study samples. Subsequently, a validated electrochemiluminescence (ECL) immunoassay was implemented for high throughput sample analysis for all samples. A comparison of drug concentration data from the methods was performed which identified strong linear correlations and for Bland-Altman, small bias. In addition, pharmacokinetic data from both methods produced similar profiles and parameters. Discussion & conclusion: This novel bioanalytical strategy successfully supported swift development of a critical targeted therapy during the COVID-19 public health emergency.

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.