Antibody Assays to Assess PK and Immunogenicity

Determining the safety and efficacy of biotherapeutics is a key part of the drug development process. There is a need to develop assays that accurately and reliably detect and quantify immune responses such as those that lead to the generation of anti-drug antibodies.

We spoke to Amanda Turner, Product Manager, Custom Antibody Products at AbD Serotec, a Bio-Rad company, to find out about some of the challenges of developing antibody assays, and the advantages that recombinant monoclonal anti-idiotypic antibodies can offer. 

AM: Can you describe some of the adverse effects of anti-drug antibodies?

Amanda Turner (AT): The most common problem caused by anti-drug antibodies (ADAs) is loss of efficacy, when ADAs bind the drug and neutralise its activity or speed up its biological elimination. Drug safety and efficacy are key concerns for developers and manufacturers, and regulatory agencies now expect that immune responses caused by drug treatment are monitored during preclinical development and clinical trials.

AM: Assessing immunogenicity is an important step in the development of biotherapeutics. What are some of the challenges of developing antibody drug assays?

AT: Assays for antibodies in biological fluids are susceptible to a wide range of technical difficulties, in addition to scientific challenges in their interpretation. A panel of methods is usually necessary for a detailed understanding of the types of antibodies raised against a biotherapeutic protein. ADAs are detected using immunoreactivity assays, such as radioimmunoassays, surface plasmon resonance or enzyme-based solid-phase immunoassays, and the neutralizing capacity of these antibodies is assessed using functional cell-based bioassays or inhibition assays.

AM: What makes humanised monoclonal antibodies particularly difficult to detect and quantify? 

AT: It’s particularly challenging to develop optimised and validated assays for fully human monoclonal antibodies, due to the high excess of human antibodies in serum, in combination with relatively low expected immune response. When a human or humanised antibody drug is administered to a patient, the drug becomes hidden within the high concentration of very similar antibodies in human serum. 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.

Preclinical and clinical development assays for these antibodies require detection reagents that are both highly specific and highly sensitive, in order to measure pharmacokinetics and immune responses in human subjects.

Highly specific and highly sensitive reagents are needed in order to measure drug levels with a pharmacokinetic (PK) assay, that bind to the drug but not to the vast excess of similar immunoglobulin molecules. A well-designed immune response (IR) assay requires a highly specific reference antibody against which to measure an anti-drug response, and for the clinical phases of development it should ideally be one or more fully human antibodies with varying affinities and/or varying immunoglobulin subclasses in order to reflect the natural immune response.

AM: Can you describe some of the ways anti-idiotypic antibodies can be used in preclinical and clinical research assays for antibody drug development?

AT: An anti-idiotypic antibody is a highly specialised antibody, generated to bind specifically to one unique antibody drug. 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. When one antibody binds to one idiotope of another antibody it is referred to as an anti-idiotypic antibody.

Anti-idiotypic antibodies are especially useful for detecting therapeutic monoclonal antibodies, as most antibodies of this type developed today are human or humanised, in which 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. These highly specialised antibodies can be used in PK assays to measure the drug level in patient samples, and as positive controls or reference standards in any IR assay. 

AM: What are some of the limitations of using classic hybridoma technology to generate anti-idiotypic antibodies, and what advantages do recombinant anti-idiotypic monoclonal antibodies offer?

AT: Traditionally, PK and IR assay antibodies have been sourced from rodent monoclonal hybridomas and primate polyclonal serum, although success of the immunisation protocol is not guaranteed and there are ethical considerations for the use of animals, especially primates. Human sera from early clinical trials is an alternative source of ADA reference standard, but it is challenging to obtain material consistently and in the required quantity and quality. This also requires that the assay validation process is continual throughout the clinical development stage.

More recently, phage display-derived human monoclonal antibodies have been found to be equally effective for these applications, and have advantages over these traditional sources. Recombinant monoclonal antibodies created using antibody libraries and well-tested phage display technology 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. For instance, 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. 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. 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 and complex-specific antibodies, specialised selection strategies enable the generation of an antibody that binds to an idiotope outside the antigen binding site of the drug. 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 state of the drug antibody.

AM: Can you tell us a little about AbD Serotec, and your recombinant anti-idiotypic antibody generation?

AT: At AbD Serotec we use a synthetic antibody library and an improved method of phage display to generate anti-idiotypic antibodies. The HUCAL® library consists of 45 billion fully human antibodies in Fab format, and is based on a set of framework master genes with highly diversified CDRs. The genes cover more than 95% of the structural immune repertoire. HuCAL antibodies are expressed from human DNA sequences and are therefore fully human. The technology enables in vitro generation of fully human Fab antibodies or full-length immunoglobulins that bind specifically and with high affinity to just about any given antigen. AbD Serotec’s antibody generation process is highly automated and fast, taking only eight weeks to select, purify and test Fab antibodies from the library. 

Using this technology AbD Serotec has developed a range of ready-made anti-idiotypic antibodies against marketed antibody drugs to support preclinical research, clinical trials and patient monitoring for innovator and biosimilar products. Included in the range are neutralising and non-neutralising antibodies, and also anti-complex antibodies that recognise the drug only when bound to its target. Anti-idiotypic antibody generation is also offered as part of a broad custom service, which also includes affinity maturation, affinity measurement, sandwich pair screening and antigen generation. HuCAL® is a registered trademark of MorphoSys AG.

Amanda Turner was speaking to Anna-Marie MacDonald, Editor for Technology Networks.