Detection of neutralizing anti-therapeutic protein antibodies in serum or plasma samples containing high levels of the therapeutic protein.

Neutralizing antibodies against therapeutic proteins can be potentially harmful if the antibody blocks not only the therapeutic activities of the therapeutic protein but also the normal functions of the endogenous counterpart. Detection of the neutralizing anti-therapeutic protein antibodies generally relies on bioassays measuring changes in the biologic activity of the therapeutic protein triggered by the presence of the antibody. Most of the bioassays, particularly the cell-based in vitro assays, fail to detect neutralizing anti-therapeutic protein antibodies when the remaining therapeutic protein level in the assay samples is high. The remaining therapeutic protein, either a free molecule or an immune complex with anti-therapeutic protein antibodies, can inhibit the neutralizing activity of the antibody and prevent detection. We describe the development of a procedure that uses acid dissociation and affinity adsorption to remove therapeutic protein from assay samples. With this procedure, we can detect the presence of neutralizing anti-therapeutic protein antibodies from samples containing high levels of therapeutic protein.

Development of a biosensor-based immunogenicity assay capable of blocking soluble drug target interference.

As with other protein therapeutics, trebananib (AMG 386), an investigational peptide Fc-fusion protein ("peptibody") that inhibits angiogenesis by neutralizing the interaction of angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) with the Tie2 receptor, has the potential to trigger an immune response in cancer patients treated with the therapeutic. An electrochemiluminescence bridging anti-drug antibody (ADA) assay that was utilized to support early-phase clinical trials in the development of trebananib was found to lack adequate sensitivity and drug tolerance in later-phase clinical studies when higher doses of trebananib were administered. Therefore, we developed a surface plasmon resonance (SPR) immunoassay method utilizing a secondary confirmatory detector antibody (goat anti-human IgG F[ab']2) known to cross-react with human IgG and IgM to better assess the potential impact of immunogenicity on the pharmacokinetics, pharmacodynamics, and toxicity of trebananib. The SPR method was more sensitive than the electrochemiluminescence bridging assay because of signal amplification from the confirmatory binding of the detector antibody; drug tolerance was improved since antibody binding avidity does not affect detection on this platform. Despite the inability of the confirmatory detector antibody to bind angiopoietins in protein-free buffer, false-positive ADA results were generated from patient serum samples containing Ang1 and Ang2 through an apparently specific binding between the angiopoietins and the confirmatory detector antibody, likely mediated by the interaction of the angiopoietins with serum immunoglobulins. Addition to the sample diluent of a human antibody that specifically binds to Ang1 and Ang2 with high affinity resulted in a complete block of angiopoietin interference without affecting ADA detection. This biosensor-based assay provides a reliable method for assessing immunogenicity in phase 3 clinical trials.

Comparing ELISA and surface plasmon resonance for assessing clinical immunogenicity of panitumumab.

Evaluation of the immunogenicity of panitumumab, a fully human anti-epidermal growth factor receptor mAb approved for use in colorectal cancer patients, led to the development of two separate immunoassays for the detection of anti-panitumumab Abs. The first immunoassay used a bridging ELISA capable of detecting 10 ng/ml positive control anti-panitumumab Ab. The ELISA incorporated an acid dissociation step to reduce drug interference and tolerated the presence of approximately 100-fold molar excess of drug. During eight clinical trials, the ELISA detected developing Ab responses in 2 of 612 (0.3%) subjects. In one of the ELISA positive subjects, neutralizing Abs were detected using an epidermal growth factor receptor phosphorylation bioassay. The second immunoassay used a Biacore biosensor immunoassay format capable of detecting 1 mug/ml positive control Ab while tolerating the presence of equal molar amounts of drug. Although less sensitive and less tolerant to competing drug in the assay, the Biacore assay detected developing Ab responses in 25 of the 604 (4.1%) subjects. Additionally, the Biacore assay identified eight subjects who developed neutralizing Abs. Mouse mAbs with affinities ranging from 1.1 x 10(-6) to 8.4 x 10(-10) M were used to characterize both assay types. The ELISA was more sensitive for the detection of higher affinity mAbs and detected high-affinity mAbs in the presence of higher molar ratio of drug to mAb. The Biacore assay was more sensitive for detection of lower affinity mAbs and detected low affinity Abs in the presence of higher molar ratios of drug to mAb.