As we discussed in the last blog, antibodies have become an important part of the therapeutic landscape. During the pre-clinical and clinical evaluation stages, detection of the antibody drug is critical to evaluate dose, toxicity, and efficacy. Remember, the antibody drug has been developed to “look” identical, or as much as possible, to natural human antibodies in order to prevent adverse effects in patients. This poses a particular problem in the development of assays to characterize the drug, as it requires the specific detection of a single antibody (the drug), typically at a concentration in the low ng/mL range, among a sea of circulating natural IgG antibodies typically around 9-12 mg/mL.
II. Anti-Idiotype Characterization
A single antibody with a unique specificity to an individual epitope is defined as an idiotype. An antibody that has been developed to recognize an individual idiotype is referred to as an anti-idiotype (an overview of idiotypes and anti-idiotypes is discussed here). Therefore, if we want to recognize a specific antibody drug, we need to develop anti-idiotype antibodies, which can then be used in specific immunoassays to characterize and evaluate the drug.
During the hybridoma development process, it is critical to select idiotype-specific antibodies and not general anti-IgG antibodies (this should be factored into the hybridoma selection criteria). Once these have been identified, specific assays can be carried out to define the characteristics of the anti-idiotypes, which are fundamental to an effective downstream design of antibody drug assays.
Most of the antibody drug immunoassays require high-quality idiotype-specific capture antibodies. Matched pair screening will help to assess the
ability to identify good capture and detector antibody pairs, including the use of general anti-human IgG Fc antibodies (Figure 1). These assays will help to define antibodies that will perform best in anti-drug immunoassays as well as provide insight into the specificity and sensitivity of the anti-idiotype monoclonal antibodies.
There are several different categories of anti-idiotype antibodies: blocking, partial blocking, and non-blocking. Depending on the purpose of the anti-idiotype, specific types of anti-idiotypes may be important. Assessment of the blocking capacity (Figure 2) is often antigen-specific, in general, the drug target can be coated on an ELISA plate and then anti-idiotype (mouse monoclonal) and antibody drug (human/humanized recombinant antibody) are mixed together and tested for the ability of the antibody drug to bind to the target.
III. Anti-Idiotype PK & ADA Immunoassay
Anti-idiotype antibodies are most commonly used for pharmacokinetic (PK) assays. PK assays are critical in defining the absorption rates, distribution, half-life, and excretion rate of the antibody drug. These assays are used to inform dosing and toxicity at the preclinical and clinical stages of antibody drug functional assessment. The ability to specifically detect and measure the antibody drug from serum samples is imperative for the ability to develop a sensitive PK immunoassay. Developing this assay will be dependent on the need to measure total drug or circulating free drug, therefore non-blocking (total drug) or blocking (free drug) anti-idiotypes need to be identified (Figure 3). These antibodies must be sensitive capture antibodies, while the method of detection can be varied.
Anti-Drug Assays (ADA) are typically used to measure the immunogenicity of the antibody drug. Even though the drug is humanized to look as much like a natural human antibody, specific features (including CDR components, glycosylation, and aggregation) can result in the generation of an antibody response to the antibody drug. This can result in unwanted side effects and reduced efficacy, so it is important to monitor these features. These assays are typically in the format of a bridging assay (Figure 4) and use the patient sera as the “bridging antibody”. Positive controls for this assay are necessary and therefore anti-idiotype antibodies are generated for this purpose. In most cases polyclonal, rather than monoclonal, antibody sera is used. Typically rabbit polyclonals are generated and may be purified for use in this assay. Potentially several monoclonal anti-idiotype antibodies could be mixed together to generate a polyclonal mix to be used as a positive control in the ADA.
The ability to develop and identify high-quality antibodies for PK and ADA development are imperative to the evaluation and monitoring of antibody drug therapies. These critical reagents need to be fully characterized in order to design the appropriate immunoassays. At BBI, we have the experience and expertise to assist in the design, development, and characterization of anti-idiotype antibodies (both polyclonal and monoclonal) for all of your PK and ADA immunoassays.