Carlos Chavez

Identification and elimination of target-related matrix interference in a neutralizing anti-drug antibody assay

Biopharmaceuticals administered to the human body have the potential to trigger the production of anti-drug (also called anti-therapeutic) antibodies (ADA) that can neutralize the therapeutic activity. For antibody therapeutics, cell-based neutralizing ADA assays are frequently used to evaluate ADA in clinical studies. We developed a method to detect neutralizing antibodies against MEDI-575, a fully human IgG2κ antagonistic antibody against PDGFR-α. We evaluated three assay formats, two of which measured late responses, cell proliferation and apoptosis, whereas the third assay detected an early signaling event, phosphorylation of PDGFR-α. Measuring phosphorylation provided a superior assay window and therefore was developed as a neutralizing ADA (NAb) assay. Matrix interference, however, was significant, and could be identified to be caused by PDGF-AA and PDGF-AB, apparently the two most abundant ligands of PDGFR-α present in human serum samples. A simple pre-treatment step, addition of an inhibitory antibody to PDGF-A, a subunit present in PDGF-AA and PDGF-AB, was found to eliminate matrix interference, increasing assay reliability and sensitivity. We integrated the pre-treatment step into assay development and qualified a robust NAb assay.

An immunoinhibition approach to overcome the impact of pre-existing antibodies on cut point establishment for immunogenicity assessment of moxetumomab pasudotox.

Immunogenicity can impact PK, PD, efficacy and safety of biopharmaceuticals, and is often evaluated as a secondary objective in clinical studies. Methods to detect anti-drug antibodies (ADA) and neutralizing ADA (NAb) are semi-quantitative and utilize cut points to determine positive or negative samples. Assay cut points are established by the statistical analysis of treatment-naïve subject specimens that are assumed ADA and NAb-negative. Pre-existing antibodies to various biopharmaceuticals have been observed in treatment-naïve subjects and may artificially elevate the cut point, resulting in compromised assay sensitivities, inaccuracy in immunogenicity reporting and ultimately misleading assessment of the impact of immunogenicity on clinical outcomes. Although several approaches such as removal of pre-existing antibody samples or increasing the sample dilution could be used for cut point establishment to mitigate impact of pre-existing antibodies, they each have limitations, especially when a high prevalence of pre-existing antibodies is observed. Here we describe an innovative approach used to establish cut points for ADA and NAb assays of moxetumomab pasudotox (moxetumomab), a recombinant anti-CD22 immunotoxin, to which a high prevalence of pre-existing antibodies was observed. In order to overcome the challenges associated with this high prevalence and prevent establishment of an artificially elevated cut point, we developed an immunoinhibition approach that allowed generation of pseudo ADA and NAb-negative populations for cut point determination. Immunoinhibition was performed by adding excess moxetumomab (for ADA) or a non-CD22 binding PE38-containing immunotoxin, CAT-5001 (for NAb), to treatment-naive samples prior to evaluating samples for cut point establishment. This approach successfully eliminated pre-existing antibody activity in treatment-naive samples, enabling establishment of more accurate ADA and NAb assay cut points. A comparative analysis of the clinical immunogenicity results using cut points derived with immunoinhibition and without immunoinhibition (conventional method) demonstrated that the immunoinhibition approach markedly improved detection sensitivity and accuracy of immunogenicity characterization in patient samples. This innovative approach provides an alternative, practical solution for immunogenicity assay cut point establishment when biopharmaceuticals have a high prevalence of pre-existing antibodies.

Evaluation of assay interference and interpretation of CXCR4 receptor occupancy results in a preclinical study with MEDI3185, a fully human antibody to CXCR4.

Receptor occupancy (RO) assays provide a means to measure the direct interaction of therapeutics with their cell surface targets. Free receptor assays quantify cell‐surface receptors not bound by a therapeutic while total receptor assays quantify the amount of target on the cell surface.

Role of infiltrating CD8 lymphocytes, microsatellite instability and h. pylori level in gastric cancer.

e16096Background: Modulation of immune system has demonstrated tumor response in Gastric Cancer. Tumor infiltrating lymphocytes and microsatellite instability are predictive biomarkers for response...