Preclinical Observations of Systemic and Ocular Antidrug Antibody Response to Intravitreally Administered Drugs.

Intravitreally administered biotherapeutics can elicit local and systemic immune responses with potentially serious clinical consequences. However, little is known about the mechanisms of ocular antidrug immune response, the incidence of ocular antidrug antibodies (ADAs), and the relationship between ocular and systemic ADA levels. Bioanalytical limitations and poor availability of ocular matrices make studies of ocular immunogenicity particularly challenging. We have recently reported a novel bioanalytical ADA assay and shown its applicability for the ADA detection in ocular matrices. In the present study, we used this assay to analyze a large set of preclinical samples from minipig and cynomolgus monkeys treated with different ocular biotherapeutics. We found a significant association between the incidence of ADAs in plasma and ocular fluids after a single intravitreal administration of the drugs. Importantly, none of the animals with ADA-negative results in plasma had detectable ADAs in ocular fluids and systemic ADA response always preceded the appearance of ocular ADAs. Overall, our results suggest the systemic origin of ocular ADAs and support the use of plasma as a surrogate matrix for the detection of ocular ADA response.

Novel bioanalytical method for the characterization of the immune response directed against a bispecific F(ab) fragment.

Aim: The work was aimed at developing a bioanalytical approach to identify immunogenic parts of a bispecific F(ab) fragment and to characterize the immune response seen in a preclinical study. Experimental: The bioanalytical method consists of a set of domain detection assays that use germlined variants of the drug. Results: The method demonstrated that anti-drug antibodies (ADAs) were predominantly directed against both antigen-binding sites of the drug. Conclusion: The method was capable to discriminate between ADAs directed against one of the antigen-binding sites, both sites or the constant domain, allowing for an estimation of the relative binding prevalence for these subunits. The developed approach provides a practical and robust solution for exploratory characterization of ADAs against multidomain biotherapeutics.

Immunogenicity testing of therapeutic antibodies in ocular fluids after intravitreal injection.

AIM: High drug concentrations in ocular fluids after intravitreal administration preclude the use of drug-sensitive immunoassays. A drug-tolerant immunoassay is therefore desirable for immunogenicity testing in ophthalmology. EXPERIMENTAL: Immune complex (IC) antidrug antibody (ADA) assays were established for two species. The assays were compared with the bridging assay in ocular and plasma samples from two preclinical studies. RESULTS: The IC assays showed high drug tolerance, which enabled a reliable ADA detection in ocular fluids after intravitreal administration. The IC assays were superior to the bridging assay in the analysis of ocular fluids with high drug concentrations. CONCLUSION: The IC assay allows a reliable ADA detection in matrices with high drug concentrations, such as ocular fluids.

Fully automated quantification of hepatitis C virus (HCV) RNA in human plasma and human serum by the COBAS AmpliPrep/COBAS TaqMan system.

BACKGROUND: HCV RNA is commonly recognized as key parameter for reliable diagnosis and treatment monitoring of HCV infection. Determination of blood HCV RNA concentrations reduces the pre-seroconversion period in the diagnosis of HCV infection and supports management of interferon alpha-based therapies of chronic HCV infection. OBJECTIVES AND STUDY DESIGN: The COBAS AmpliPrep/COBAS TaqMan HCV Test combines automated extraction of nucleic acids on the COBAS AmpliPrep Instrument with real-time PCR on the COBAS TaqMan Analyzer, thus greatly reducing hands-on time during sample preparation and amplification/detection. The test, which is calibrated to the 1st International HCV WHO Standard, was evaluated for sensitivity, dynamic range, precision, matrix equivalence, genotype inclusivity, interfering substances, diagnostic and analytical specificity, as well as for correlation with two other commercial tests for HCV RNA quantification. RESULTS: The COBAS AmpliPrep/COBAS TaqMan HCV Test demonstrated a >6-log dynamic range of 43-6.90 E+7 IU/mL, a sensitivity (95% hit rate) of at least 15 IU/mL for HCV WHO Standard and a comparable quantification of genotypes 1-6. HCV quantification results were in good correlation with those obtained by the COBAS AMPLICOR HCV MONITOR Test v2.0 and the VERSANT HCV RNA 3.0 test. CONCLUSIONS: The fully automated COBAS AmpliPrep/COBAS TaqMan HCV Test excellently accomplishes the requirements for highly sensitive detection and reliable quantification of HCV in clinical samples and thus improves therapy monitoring and management of HCV infection.

Detection of antidrug antibodies against human therapeutic antibodies lacking Fc-effector functions by usage of soluble Fcγ receptor I.

AIM: Bridging immunoassays for the detection of antidrug antibodies (ADAs) are limited to detection of bivalent molecules and are prone to interference by drug and soluble targets. Hence, alternative approaches for ADA detection are desired. Materials & methods: A novel ADA assay with secondary Fc detection using human soluble Fcγ receptor I (hsFcγRI) was established and compared with standard bridging assay. RESULTS: Both assays showed consistent results in human and cynomolgus monkey samples. In contrast to the bridging assay, the hsFcγRI-based assay was insensitive to the presence of oligomeric targets and appeared to have better drug tolerance. CONCLUSION: The hsFcγRI-based ADA assay can serve as alternative screening assay or as orthogonal confirmation method for preclinical and clinical immunogenicity testing of IgG therapeutics lacking Fc effector functions.