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How High Sensitivity Immunogenicity Assays Overcome the Challenges of Anti-Drug Antibody (ADA) Detection

Drug immunogenicity and the detection of anti-drug antibodies (ADA) have an important role in the drug discovery process for potential new therapeutics. The clinical effects of these immune responses can affect pharmacokinetics, pharmacodynamics, safety, or efficacy. High sensitivity assays for detection and analysis of ADA formation can play a crucial role in any therapeutic protein product development program and speed up ADA assay development. The most common challenges faced by ADA assay development scientists are a result of the detection limits of technologies that are currently available. See how high sensitivity immunogenicity assays, such as those with ultrasensitive Single Molecule Counting (SMC®) technology, help make detecting ADA that would have been previously undetectable, easier.

What Is Immunogenicity?

Immunogenicity is the term used to describe an immune response from a substance such as a biologic or a vaccine. All biological therapeutics have the potential to induce an immune-mediated response ranging from benign to severe adverse effects. These effects can encompass diminished clinical efficacy of the biotherapeutic being administered to hypersensitivity, allergic reactions, or even cytokine storms. The factors that influence immunogenicity are described in Table 1.

Table 1. Factors influencing immunogenicity.

What Are Anti-Drug Antibodies (ADA)?

Anti-drug antibodies (ADA) are antibodies elicited from therapeutics and they are used to measure immunogenicity. It is important to assess the immunogenicity risk of potential biotherapeutics in producing neutralizing and non-neutralizing ADA, especially in clinical phases of drug development. The effects of these neutralizing and non-neutralizing anti-drug antibodies can encompass diminished clinical efficacy of the biotherapeutic. ADA induced by biologic therapeutics often impact drug pharmacokinetics (PK), pharmacodynamic (PD) responses, clinical efficacy, and patient safety.

FDA Immunogenicity Guidelines

Consequently, regulatory agencies are looking to understand the implications of immunogenicity and are directing the industry to integrate programs for immunogenicity risk management starting in early phase drug development in clinical and pre-clinical trials.

The Federal Drug Administration (FDA) and pharmaceutical experts in the area of immunogenicity testing have published guidelines for the design and optimization of immunoassays used in the detection of antibodies against biopharmaceutical drug products in patient samples in the absence of drug and more importantly, when drug is present. It is recommended that the initial screening assay be able to detect all relevant immunoglobulin (Ig) isotypes 1:

  • For non-mucosal routes of administration and in the absence of a risk of anaphylaxis, the relevant ADA isotypes are IgM and IgG.
  • For mucosal routes of administration, IgA isotype ADAs are also relevant.
  • Also, for therapeutic protein products where there is a high risk for anaphylaxis or where anaphylaxis has been observed, results from antigen-specific IgE assays may be informative.

Recommended Sensitivity Limits

The FDA recommends that screening and confirmatory IgG and IgM ADA assays achieve a sensitivity of at least 100 nanograms per milliliter (ng/mL) although a limit of sensitivity greater than 100 ng/mL may be acceptable depending on risk and prior knowledge. However, observations have been made of patients developing persistent ADA responses having levels lower than 100 ng/mL. This data suggests that concentrations below 100 ng/mL may be associated with clinical events 2,3.

Assays developed to assess IgE ADA should have sensitivity in the high picograms per milliliter (pg/mL) to low ng/mL range for therapeutic protein products where there is a high risk for anaphylaxis or where anaphylaxis has been observed. Results from antigen-specific IgE assays may be informative 1. The increased sensitivity recommendation is based on the current state of science observed in the FDA’s filings as well as publicly available studies.

Why Is Sensitivity Important in Immunogenicity Testing?

A more sensitive detection method is important in immunogenicity testing because it may lead to earlier detection of a primary immune response or detection of IgG4, which the FDA can request on a case-by-case basis. Early detection of these immune responses can save time, cost, and reduce the risk of adverse events by catching them before moving to the next stage of clinical trials.

Traditionally ELISAs or electrochemiluminescence (ECL) have been used to identify the presence of ADA. Though effective for detection, ELISA methods often fail to adequately measure specific antibody responses in the presence of circulating protein therapeutics due to the limitation on sensitivity.

According to the FDA, ADA assays must be sensitive enough to detect low levels of ADA before they impact PK, PD, safety, or efficacy 1. This is why the industry is shifting toward ultrasensitive detection technology in immunogenicity testing. High sensitivity ADA assays offer a magnitude fold increase in sensitivity over current existing technologies, especially with regard to multivalent IgM ADA binding to the antigen, where spatial restriction can prevent binding of the detecting reagent and IgE, which is found in low circulating concentrations.

How Does Assay Sensitivity Impact Immunogenicity Testing?

Assay sensitivity impacts immunogenicity testing in various ways. The benefits of ultrasensitive assays in immunogenicity testing include overcoming matrix interference issues and reducing drug/target interference. Researchers can use high sensitivity immunoassay technology to overcome the challenges of their immunogenicity assays. One example is where a drug product given in low/sub nM concentrations and ADA levels of 10 ng/mL can be expected to clear or neutralize all of the given drug. The extra sensitivity can detect ADA that might otherwise be missed by traditional technologies.

Additionally, drug tolerance at 100 ng/mL can sometimes be poor. By having the option to increase dilution and still detect ADA, drug tolerance and matrix effect can be improved by overcoming traditional sensitivity limitations. This can also be advantageous in reducing the need for pre-treatments such as acid dissociation.

Also, low sample volumes in mouse studies or difficult to obtain human matrixes, such as spinal fluid, make these samples critical to conserve to maximize results. The option to dilute while still measuring ADA enables precious samples to be maximized, reducing time and costs.

How Ultrasensitive SMC® Technology Improves Immunogenicity Testing

Ultrasensitive Single Molecule Counting (SMC®) technology allows you to detect ADA which would previously have been undetectable. The SMC® technology can support all phases of immunogenicity testing using digital counting on the SMCxPRO® instrument with the benefits of precision and flexibility. The SMC® technology’s capabilities can be used to overcome the challenges encountered with immunogenicity assays while uncovering data previously difficult to obtain and interpret.

These capabilities include:

  • Overcoming matrix interference issues using dilution and sensitivity, reducing the need for assay modification
  • Reducing circulating drug or target interference, using the SMC® technology’s improved sensitivity to bias assay kinetics towards labeled drug
  • Detecting early IgM response and low concentration IgE response, which are traditionally hard to detect

Two solid-phase assay formats are available, a plate-based option and a bead-based option.

Developing ADA Assays

SMC® technology enables the development of ADA assays by labeling the drug with capture and detection reagents and utilizing buffer reagents to develop and optimize the assays. The technology allows for the ability to develop a homogenous species-independent assay format that is simple, easy to design, and easy to verify. This assay format is often referred to as a “bridging assay” since the ADA acts as a bridge between the drug labeled capture and detection (Figure 1).

Diagram of a bridging assay format for anti-drug antibody detection in sample.

Figure 1.Bridging assay format for anti-drug antibody detection in sample. The immunocomplex drug is Alexa fluor and biotin conjugated and is captured on a magnetic streptavidin bead.

By using a 642 nm laser focused 250 μm above the base of an Aurora plate, a rotating objective scans through the free-floating suspension exciting fluorochromes as they pass through the interrogation space. A low noise avalanche photodiode (APD) counts individual photons as they are emitted (Figure 2). The focused interrogation space of acquisition reduces cross talk from well to well, flare from meniscus diffusion of light, as well as inherent interference from turbid solutions.

Representation of the single molecule counting of Alexa-conjugated drug as it traverses through interrogation window through the use of Single Molecule Counting (SMC®) technology.

Figure 2.Counting of Alexa-conjugated drug as it traverses through interrogation window.

Advantages of SMC® Technology in ADA Detection

SMC® advantages for the detection of anti-drug antibodies include:

  • Ultrasensitive down to pg/mL detection for low-affinity ADA
  • Wide dynamic range for detection of high-affinity ADA with minimal matrix interference
  • All ADA subtypes can be detected including IgM (traditionally difficult to detect) and IgE (present in very low concentrations)
  • Tolerance to high drug concentrations in sample
  • Reduced wash steps for detection of low-affinity antibodies and reduced assay time
  • Enhanced sensitivity allows the use of dilution to overcome matrix interference and drug tolerance issues without the need for assay modifications such as acid dissociation
  • Plate-based assay format for ease of transfer from an ECL assay
  • Bead-based assay format for enhanced sensitivity or extra dilution capability to overcome interference issues through dilution
  • Integrated software package that is easy to use with flexible data interpretation, 21 CFR part II compliant, and LIMS compatible

Simple Workflow for Anti-Drug Antibody Assay Development

Upon completion of the derivatization of the drug for use as capture and detection, the workflow for the bead-based ADA assay development is as follows and shown in Figure 3:

  • Offline Sample Incubation
    • ADA in sample is incubated for 2 hours or overnight
  • Complex Capture
    • Complex is captured onto blocked beads
    • Wash to remove unbound antibodies
  • Elution
    • Complex is dissociated, beads are magnetically separated, and the eluate is transferred to read plate
  • Single Molecule Counting
    • Rotating laser scans the sample
    • Alexa-conjugated drug is excited, and photons generated are counted by an APD
Illustration of the typical immunogenicity ADA bead-based assay workflow.

Figure 3.Illustration of the typical immunogenicity ADA bead-based assay workflow. A bridging immunoassay complex is captured onto beads. Then, the complex is disassociated from the bead and the eluate is read on the SMCxPRO® instrument. A. Offline sample incubation, B. Complex capture, C. Elution, D. Single molecule counting.

In developing an immunogenicity assay, optimization is required to fully verify the immunological system being studied. Considerations such as those listed below can be easily studied with the SMC® technology platform:

  • Drug tolerance
  • Cut point/matrix tolerance
  • Sensitivity/dynamic range of the assay
  • Reproducibility

Further Optimization

Further optimization of different variables can take place to produce the most effective assay for the immunogenicity assessment of a therapeutic protein. These include:

  • Drug concentration (capture and detection reagent)
  • Assay diluents (to mitigate human anti-mouse antibody or other interfering factors)
  • Sample volume
  • Number of wash steps
  • Incubation time
  • Standard / sample diluent
  • Determination of minimum required dilution (MRD)
  • Evaluation of drug interference/tolerance

Drug Tolerance

Drug tolerance is an important consideration in immunogenicity and is a challenge that researchers face where the ability to quantify ADA in matrix is reduced in the presence of high drug concentration as result of competition. In bridging assays of this type, it is important to minimize the amount of free (unlabeled capture or detection reagent) drug to quantify and drive the equilibrium in favor of quantifying ADA in samples. Several methods have been used to overcome this challenge, which include acid dissociation. By using ultrasensitive technology such as the SMC® platform, better sensitivity can help overcome this by simple dilution, thereby eliminating the need for acid dissociation.

SMC® technology offers a 10-fold improvement in sensitivity over the current gold standard assay, electrochemiluminescence immunoassay (ECLIA) as shown in Figure 4.

Graph showing the SMC® assay vs ECLIA comparison.

Figure 4.SMC® assay vs ECLIA comparison. The sensitivity improved 10-fold over the traditional ECLIA method from 195 ng/mL to 20 ng/mL.

SMC® technology also does not show evidence of creating the hook effect that is a concern with immunoassays as shown in Figure 5. The hook effect, also known as the prozone effect, is when there is an excessive amount of analyte that causes falsely low results.

Graph showing the hook effect and sensitivity.

Figure 5.Hook effect and sensitivity. The current ADA assay demonstrated no evidence of hook effect up to 100,000 ng/mL with low sensitivity to pg/mL level.

The improved sensitivity of SMC® technology helps to better analyze drug tolerance. This improved sensitivity may lead to early detection of primary ADA response prior to class type switching and affinity maturation.

Further Implications of Immunogenicity: Adaptive Immune Response Assessment

The adaptive immune response to pathogens can involve different immunoglobulin isotypes. Screening assays do not necessarily need to identify isotypes but need to be capable of binding multiple relevant classes or sub-classes. A number of isotypes play a major role in the immunogenic response. For instance:

  • IgE-specific assays may be informative for products with a history of high risk of anaphylaxis
  • IgG4-specific assays may be informative for products that are chronically administered, or on erythropoietin-treated patients with pure-red cell aplasia
  • IgE and IgG4-specific assays may be requested on a case-by-case basis by the FDA due to hypersensitivity
    • The complement cascade can also be mediated by IgM and IgG

These responses ultimately lead to the generation of an inflammatory response through the formation of anaphylatoxins, such as C1q, C4a, C3a, and C5a. Engagement of FcR or CR (complement receptor) on cells, through immune complex cross-linking, results in the production of chemokines and growth factors that have a cascade effect on the trafficking and growth of T and B cells. This leads to the release of cytokines and chemokines (such as IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, IL-21, IFN-g) which ultimately leads to tissue damage. See an example of this type of assessment using our MILLIPLEX® multiplex kits.

Related Products

The SMC® Immunogenicity Assay Development Kit is designed to guide and assist you with the development process. Our experienced application scientists can also help design, verify, and train end-users to fully utilize the platform. 

Combining our immunoassay portfolio to study the impact on the immunogenicity of a therapeutic can provide great insights into the mechanism of the response. The SMC® technology can offer increased sensitivity which may assist in the detection of low-affinity antibodies and lead to earlier detection of primary ADA response, overcome matrix effects, and may reduce drug tolerance. MILLIPLEX® multiplex kits can also offer insights into the mechanism of the immune response and help to further understand the immune complex-mediated responses to ADA.

References

1.
Immunogenicity Testing of Therapeutic Protein Products —Developing and Validating Assays for Anti-Drug Antibody Detection. [Internet]. Center for Drug Evaluation and Research: US Food and Drug Administration. .[cited 23 Apr 2020]. Available from: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/immunogenicity-testing-therapeutic-protein-products-developing-and-validating-assays-anti-drug.
2.
Plotkin SA. 2010. Correlates of Protection Induced by Vaccination. Clin Vaccine Immunol. 17(7):1055-1065. https://doi.org/10.1128/cvi.00131-10
3.
Zhou L, Hoofring SA, Wu Y, Vu T, Ma P, Swanson SJ, Chirmule N, Starcevic M. 2013. Stratification of Antibody-Positive Subjects by Antibody Level Reveals an Impact of Immunogenicity on Pharmacokinetics. AAPS J. 15(1):30-40. https://doi.org/10.1208/s12248-012-9408-8
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