What are clinically significant anti-drug antibodies and why is it important to identify them.

The FDA has released new draft guidance to standardize how immunogenicity of protein therapeutics is described in product labels. A key aspect to this new guidance is that companies should describe anti-drug antibodies that have clinical significance in addition to reporting ADAs' incidence. Factors to consider when determining clinical significance include if those antibodies have a significant effect on the drug's pharmacokinetics, pharmacodynamics, efficacy, and/or safety.

The immunogenicity of human-origin therapeutic antibodies are associated with V gene usage.

Therapeutic antibodies can elicit unwanted immune responses in a subset of patients, which leads to the production of anti-drug antibodies (ADA). Some of these ADAs have been reported to effect the pharmacokinetics, efficacy and/or safety of the therapeutic antibodies. The sequence diversity of antibodies are generated by VDJ recombination and mutagenesis.

An Anticancer Drug Cocktail of Three Kinase Inhibitors Improved Response to a Dendritic Cell-Based Cancer Vaccine.

Monocyte-derived dendritic cell (moDC)-based cancer therapies intended to elicit antitumor T-cell responses have limited efficacy in most clinical trials. However, potent and sustained antitumor activity in a limited number of patients highlights the therapeutic potential of moDCs. culture conditions used to generate moDCs can be inconsistent, and moDCs generated are less effective than natural DCs.

Clinical Manifestations of an Anti-Drug Antibody Response: Autoimmune Reactions.

Antibodies can be generated against a therapeutic protein upon administration to human subjects. When the therapeutic protein closely mimics one of the subject's endogenous proteins, those antibodies might bind to the endogenous protein in addition to the therapeutic protein. This scenario results when tolerance to the endogenous protein is broken.

Strategic characterization of anti-drug antibody responses for the assessment of clinical relevance and impact.

All therapeutic proteins have the potential to induce anti-drug antibodies (ADA). Clinically relevant ADA can impact efficacy and/or safety of a biological therapeutic. Immunogenicity assessment strategy evaluates binding and neutralizing ADA, and the need for additional characterization (e.

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.

Immunogenicity testing strategy and bioanalytical assays for antibody-drug conjugates.

Background: Immunogenicity testing is an important component of clinical development for large-molecule biotherapeutics. New complex types of large molecules, such as antibody-drug conjugates (ADCs), require careful evaluation of the testing strategy and bioanalytical assays used to monitor the development of antitherapeutic antibodies.

Results: An electrochemiluminescence-based immunoassay for the detection and epitope characterization of anti-ADC antibodies was validated.

Stratification of antibody-positive subjects by antibody level reveals an impact of immunogenicity on pharmacokinetics.

The availability of highly sensitive immunoassays enables the detection of antidrug antibody (ADA) responses of various concentrations and affinities. The analysis of the impact of antibody status on drug pharmacokinetics (PK) is confounded by the presence of low-affinity or low-concentration antibody responses within the dataset. In a phase 2 clinical trial, a large proportion of subjects (45%) developed ADA following weekly dosing with AMG 317, a fully human monoclonal antibody therapeutic.

Immunogenicity assessment in non-clinical studies.

Recent ICH S6 guidance on preclinical safety evaluation of biotechnology derived biopharmaceuticals indicates that testing for anti-drug antibodies is not always required to establish the safety of a protein therapeutic. Most human protein therapeutics will induce a rapid and robust anti-drug antibody response in preclinical studies and the presence of high levels of circulating drug complicates the detection of anti-drug antibodies. The presence of anti-drug antibodies in preclinical studies does not predict if a protein therapeutic will be immunogenic in the clinic.

Development and characterization of a human antibody reference panel against erythropoietin suitable for the standardization of ESA immunogenicity testing.

Recombinant human erythropoietin (EPO) has been used therapeutically for more than two decades in the treatment of anemia. Although EPO is generally well tolerated, in rare cases, patients have developed anti-EPO antibodies that can negatively impact safety and efficacy. Therefore, the detection of antibodies against EPO is a regulatory requirement during clinical development and post-approval.

Epitope characterization of pre-existing and developing antibodies to an aglycosylated monoclonal antibody therapeutic of G1m17,1 allotype.

Allotypes of IgG1 molecules can influence the immunogenicity of therapeutic monoclonal antibodies and may account for the presence of some pre-existing antibodies. An electrochemiluminescent (ECL) bridging immunoassay was used to characterize the binding epitopes of anti-therapeutic antibodies (ATAs) in a Phase 1 single ascending dose clinical trial of a therapeutic aglycosylated IgG1monoclonal antibody (mAb). There was no evidence for ATAs specific for a possible neo-epitope created due to the lack of glycosylation.

Immunogenicity of panitumumab in combination chemotherapy clinical trials.

Background: Panitumumab is a fully human antibody against the epidermal growth factor receptor that is indicated for the treatment of metastatic colorectal cancer (mCRC) after disease progression on standard chemotherapy. The purpose of this analysis was to examine the immunogenicity of panitumumab and to evaluate the effect of anti-panitumumab antibodies on pharmacokinetic and safety profiles in patients with mCRC receiving panitumumab in combination with oxaliplatin- or irinotecan-based chemotherapies.

Methods: Three validated assays (two screening immunoassays and a neutralizing antibody bioassay) were used to detect the presence of anti-panitumumab antibodies in serum samples collected from patients enrolled in four panitumumab combination chemotherapy clinical trials.

Detection of anti-ESA antibodies in human samples from PRCA and non-PRCA patients: an immunoassay platform comparison.

Background: The immunological methods for detecting antibodies to erythropoiesis-stimulating agents (ESAs) differ in assay sensitivity. However, this parameter, routinely determined in clinical assays using a high-affinity non-human polyclonal antibody, gives a one-dimensional assessment of antibody detection. We compare three widely used immunological methods and evaluate the ability of each to detect mature human antibodies and human antibodies characteristic of an early immune response.

Strategy to confirm the presence of anti-erythropoietin neutralizing antibodies in human serum.

Functional cell-based assays are the preferred method to test for the presence of anti-rHuEPO neutralizing antibodies (NAbs). However, due to the unpredictable nature of test serum matrix effects on cell-based assays, confirmatory assays are essential for verifying NAb positive results observed during the course of sample testing. The cell-based assay used for the detection of NAbs described by Wei et al.

Impact of matrix-associated soluble factors on the specificity of the immunogenicity assessment.

Background: Specificity and sensitivity are essential in assays for immunogenicity assessment of biotherapeutics. Nonspecific interactions from excess therapeutic or anti-therapeutic antibody, soluble ligands (e.g.

Rheumatoid factor interference in immunogenicity assays for human monoclonal antibody therapeutics.

Rheumatoid factors (RFs) are endogenous human antibodies that bind to human gamma globulins. RFs demonstrate preferential binding to aggregated gamma globulins and are involved in the clearing mechanism of immune complexes. Immunoassays designed to measure human anti-human antibodies (HAHA) after administration of monoclonal antibody therapeutics are thus vulnerable to interference from RFs.

Identification and inhibition of drug target interference in immunogenicity assays.

A well-designed anti-drug antibody (ADA) immunoassay is critical for appropriately monitoring the immunogenicity profile of a therapeutic protein during its development. AMG 386 is a peptide-Fc fusion protein that inhibits angiogenesis by preventing the interaction of angiopoietins with the Tie2 receptor. In bridging immunoassays for ADA, interference by the drug target, present in the assay sample, can result in false positive antibody detection.

Assessment of immunogenicity of romiplostim in clinical studies with ITP subjects.

Romiplostim is an Fc-peptide fusion protein that activates intracellular transcriptional pathways via the thrombopoietin (TPO) receptor leading to increased platelet production. Romiplostim has been engineered to have no amino acid sequence homology to endogenous TPO. Recombinant protein therapeutics can be at a risk of development of an antibody response that can impact efficacy and safety.

Comparing exponentially weighted moving average and run rules in process control of semiquantitative immunogenicity immunoassays.

The immunogenicity immunoassay validation process ensures development of a robust, reproducible method. However, no matter how well developed, validated, and maintained a method is, in the course of running a large number of samples over time, it is not uncommon to see bad reagents, poorly calibrated equipment, personnel errors, or other unknown and unpredictable factors that have an impact in the performance of the method and quality of the sample results. The immunogenicity immunoassay thus needs to be closely monitored with an internal statistical quality control process overtime to ensure a consistent and reliable output.

A step-wise approach for transfer of immunogenicity assays during clinical drug development.

We designed a three-step statistical approach to transfer bioanalytical assays (ELISA and Biacore) which evaluates the (1) average equivalence between the two labs (2) concordance in individual sample results between the two labs, and (3) long-term stability of assay performance. Each experimental design evaluated the contribution of four critical variables to the overall variability. Two lots of each variable were examined in a controlled experiment.

Assessing specificity for immunogenicity assays.

Developing sensitive and specific bioanalytical assays for measuring the immunogenicity of biological therapeutics has become an integral component of the drug-development process. The strategy for measuring these immune responses involves performing sensitive screening assays that are capable of detecting low levels of both low- and high-affinity antibodies. However, having sensitive assays inherently results in a certain rate of false-positivity.

The development and validation of a sensitive, dual-flow cell, SPR-based biosensor immunoassay for the detection, semi-quantitation, and characterization of antibodies to darbepoetin alfa and epoetin alfa in human serum.

A surface plasmon resonance (SPR)-based biosensor immunoassay was developed and validated using the Biacore 3000 instrument to detect, semi-quantitate, and characterize serum antibodies against darbepoetin alfa (Aranesp) and epoetin alfa (EPOGEN). In this sensitive, dual-flow cell assay, epoetin alfa and darbepoetin alfa are covalently immobilized onto consecutive flow cells of a carboxymethyl dextran-coated sensor chip. Diluted human serum samples are injected sequentially over both surfaces.

Immunogenicity issues in drug development.

Immunogenicity is an important factor that manufacturers must consider as they develop new protein therapeutics. It is important to understand the immunogenicity of new proteins both at the preclinical phase and in the clinical phase of development. This paper provides an overview of the issues that manufacturers should consider including some of the potential reasons that some proteins induce an immune response, a discussion regarding current methodology used to understand immunogenicity, and some examples of marketed protein therapeutics with immunogenicity issues.

Recommendations on risk-based strategies for detection and characterization of antibodies against biotechnology products.

The appropriate evaluation of the immunogenicity of biopharmaceuticals is of major importance for their successful development and licensure. Antibodies elicited by these products in many cases cause no detectable clinical effects in humans. However, antibodies to some therapeutic proteins have been shown to cause a variety of clinical consequences ranging from relatively mild to serious adverse events.

A non-radioactive method for detecting neutralizing antibodies against therapeutic proteins in serum.

The presence of neutralizing antibodies against protein therapeutics continues to cause concern in the biomedical field. These antibodies not only reduce the efficacy of the protein therapeutics, but may also block the normal function of their endogenous counterparts, which can result in serious health risks to the patient. To date, a limited number of in vitro cell-based bioassays for detecting neutralizing antibodies against therapeutic proteins have been developed.