Baseline Severe Acute Respiratory Syndrome Viral Load Is Associated With Coronavirus Disease 2019 Severity and Clinical Outcomes: Post Hoc Analyses of a Phase 2/3 Trial.

BACKGROUND: Elucidating the relationship between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load and clinical outcomes is critical for understanding coronavirus disease 2019 (COVID-19). METHODS: The SARS-CoV-2 levels were analyzed by quantitative real-time polymerase chain reaction (RT-qPCR) of nasopharyngeal or oropharyngeal swab specimens collected at baseline, and clinical outcomes were recorded over 60 days from 1362 COVID-19 hospitalized patients enrolled in a multicenter, randomized, placebo-controlled phase 2/3 trial of sarilumab for COVID-19 (ClinicalTrials.gov NCT04315298). RESULTS: In post hoc analyses, higher baseline viral load, measured by both RT-qPCR cycle threshold and log10 copies/mL, was associated with greater supplemental oxygenation requirements and disease severity at study entry. Higher baseline viral load was associated with higher mortality, lower likelihood of improvement in clinical status and supplemental oxygenation requirements, and lower rates of hospital discharge. Viral load was not impacted by sarilumab treatment over time versus placebo. CONCLUSIONS: These data support viral load as an important determinant of clinical outcomes in hospitalized patients with COVID-19 requiring supplemental oxygen or assisted ventilation.

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.

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.

Bridging immunogenicity assays for IgG4 therapeutics: mitigating interference from Fc-Fc interactions.

AIM: A bridging immunogenicity assay for a human IgG4 mAb therapeutic was transferred to an automation system to increase throughput. However, background signal increased five- to six-fold during the 6- to 8-h run. RESULTS: Noncovalent Fc contacts formed between labeled IgG4 drugs in reagent solutions stored during the automation run. This generated substantial background signal, reducing assay sensitivity by approximately sixfold. Fc interactions also significantly impacted the confirmation assay. Fc contacts formed between labeled and unlabeled drug, significantly increasing signal inhibition (∼7-70%) in the 6-h run. CONCLUSION: Storing labeled antibody solutions separately and combining them immediately before adding to samples reduced interference from Fc interactions. Preincubation time for reagent solutions should be strictly controlled for anti-drug antibody assays with IgG4 drugs to avoid false-positive results.

Matrix interference from Fc-Fc interactions in immunoassays for detecting human IgG4 therapeutics.

BACKGROUND: An assay measuring an IgG4 biotherapeutic in human serum used a drug-specific monoclonal antibody (mAb) capture reagent and an antihuman IgG4 mAb as detection reagent. However, serum IgG4 binding to the capture mAb via Fc-interactions was detected by the anti-IgG4 mAb, causing high background. RESULTS: Two approaches were developed to minimize background; incorporating a mild acid sample preparation step or using the Fab of the capture antibody. Either strategy improved signal:noise dramatically, increasing assay sensitivity >20-fold. Biophysical analyses of antibody domains indicated that noncovalent Fc oligomers could inhibit the background. CONCLUSION: Matrix interference from human IgG4 binding to the capture mAb was reduced with a Fab fragment of the drug-specific capture antibody or by incorporating a mild acid sample treatment into the assay.