October 1, 2014 (Vol. 34, No. 17)
Addressing Human mAb Drug Safety and Efficacy Concerns
Drug safety and efficacy are key concerns for manufacturers during the long, complex, and costly drug development process. Regulatory agencies now expect that immune responses caused by drug treatment are monitored during preclinical development and clinical trials. Immune responses leading to the generation of antidrug antibodies (ADAs) can cause adverse events, but the most common problem is loss of efficacy, when ADAs bind the drug and neutralize its activity or speed up its biological elimination.
ADAs are detected using immunoreactivity assays, such as radioimmunoassays, surface plasmon resonance, or enzyme-based solid-phase immunoassays. An initial sensitive screening assay is needed to identify whether samples are positive for ADAs. Subsequent tests may include quantification of these antibodies, confirmation of positives by spiking drug into the sample, and isotyping to distinguish between various immunoglobulin subclasses. Sensitivity, reproducibility, precision, and recovery are important requirements.
Special Considerations for mAb Drugs
The challenges of developing optimized and validated assays are intensified for the class of fully human monoclonal antibodies. When a human or humanized antibody drug is administered into a patient, the drug becomes hidden within the high concentration of very similar antibodies in human serum. Typically, the concentration of the antibody drug can be a million times lower than the serum antibody concentration, making the drug difficult to detect and quantify. Consequently, preclinical and clinical development assays for these antibody drugs require highly specific as well as highly sensitive detection reagents to measure their pharmacokinetics (PK) and immune responses in human subjects.
Most therapeutic monoclonal antibodies developed today are human or humanized, which means that the most likely immunogenic epitopes for the induction of ADAs lie within the hypervariable, complementarity determining regions (CDR) that provide the majority of the binding contacts. The idiotype represents the variable part of an antibody, including the unique antigen binding site, and the combination of epitopes within the idiotype (i.e., the idiotopes) is unique for each antibody (Figure 1).
When one antibody binds to one idiotope of another antibody it is referred to as an anti-idiotypic antibody (anti-ID). These highly specialized anti-idiotypic antibodies can be used in a PK assay to measure the drug level in patient samples and as a positive control or reference standard in any immune response (IR) assay (Figure 2).
Traditional sources of such PK and IR assay antibodies include rodent monoclonal anti-idiotypic antibodies and primate polyclonal serum. However, these traditional methods tend to be laborious, and may result in antibodies that are poor models of clinical responses, with optimal affinity difficult to achieve. Other challenges can include obtaining material consistently and in the required quantity and quality, as for example when using human sera from early clinical trials as a source of ADA reference standard.
Overcoming the Limitations of Traditional Methods
Advances in genetic engineering have made it possible to create recombinant monoclonal antibodies using sophisticated antibody libraries and well-tested phage display technology. Such antibodies overcome the unpredictability and inflexibility of traditional hybridoma technology, and offer significant advantages for generating highly specific and sensitive assays for the drug development process.
Using this technology for anti-idiotypic antibody development, guided selection is carried out on the drug in the presence of isotype subclass-matched antibodies as blockers, to avoid enrichment of specificities that bind to other regions of the antibody drug and to ensure idiotope specificity. Selection performed in the presence of human serum avoids matrix effects later in the assay. To represent the variation in human responses to a drug, using phage-display technology, a panel of anti-idiotypic antibodies that have high and low reactivity to the target antibody can be made by adjusting the selection criteria.
The guided selection method also allows isolation of rare specificities, such as drug-target complex binders that can be used to quantify bound drug, as opposed to free drug levels (Figure 3, Type 3). These complex-specific antibodies bind neither the antibody drug nor the drug target when on their own, and thus can be used to detect bound therapeutic antibodies directly. This sensitive tool allows the developer to design superior assays for analysis of levels of bound drug, as opposed to measuring free drug only (Figure 2B). More sensitive and more robust assays that avoid the bridging format can be built, leading to improved assays for pharmacokinetic studies.
In addition to the neutralizing (Figure 3, Type 1) and complex-specific antibodies, specialized selection strategies enable the generation of an antibody that binds to an idiotope outside the antigen binding site of the drug (Figure 3, Type 2). This antibody is not inhibitory and can be used to detect both free and bound drug in serum. Using a combination of the different types of drug-specific antibodies offers the assay developer enhanced flexibility and better overall information about the availability and the state of the drug antibody.
Using HIghly Specific Antibodies to Improve Assays
A group of researchers was looking for a way to quantitate levels of the humanized monoclonal antibody drug natalizumab, which is of the isotype IgG4. The drug antibodies undergo half-antibody exchange in vivo with other antibodies of the same isotype, randomizing the Fab arms. The result is molecules made up of one drug heavy-light chain pair coupled to a heavy-light chain pair of unknown specificity. The ability to select high specificity anti-idiotypic antibodies provided the solution.
The successful strategy involved the design of two ELISAs, which employed antibodies selected from a panel of anti-idiotypic antibodies generated using phage-display technology. One ELISA measured total antibody drug, including intact and exchanged molecules, the second measured only intact antibody drug. The anti-idiotypic antibodies were utilized instead of the antigen to capture antibody drug. Validation experiments showed that the assays were specific, accurate, and precise, and the strategy has enabled full assessment of levels of the drug in vivo.
Another group of researchers used recombinant phage-display technology to generate anti-idiotypic antibodies to the monoclonal antibodies to human IL-6 and IL-13, to act as robust and highly selective detection and reference reagents for PK and IR assays. The anti-idiotypic antibodies were selected in the presence of human serum and a framework matched antibody to ensure CDR binding exclusivity. The resulting panel of high-affinity, fully human antibodies satisfied the developers’ desired parameters for their preclinical and clinical assays, with the added benefit of a secure, consistent, and long-term supply.
Generation of anti-idiotypic antibodies using antibody libraries and phage-display technology produces fully human antibodies with exquisite binding specificity that can be used in assays for both early- and late-stage development, avoiding continual assay validation. The in vitro production method confers consistency of reproduction and a guaranteed long-term supply, allowing faster optimization of PK and IR assays, and effective management of the resources and costs associated with the complex assay development process.