The TDAR Assay’s Purpose: Immunotoxicology or Pharmacodynamic Readouts?

The T-Dependent Antibody Response assay (TDAR assay) is an in vivo functional assay originally designed to evaluate the potential immunosuppressive side effect of a drug.

The principle is based on the monitoring of an immune response induced in an animal model of interest by administration of a defined antigen. These molecules were selected on the capacity to induce an immune response involving a maximum number of immune players (including both T and B cells) to access to a full representative picture of the state of the immune system.

Traditionally used Immunogenic Antigens (Ag) are Keyhole Limpet Hemocyanin (KLH), Tetanus Toxoid (TT), or Sheep Red Blood Cells (SRBC). The main readout of the TDAR assay is the measurement of the Ag specific humoral response (both IgM and IgG) occurring at the end of the chain of the immune reaction.

A variant of the TDAR assay is called “modified TDAR,” which uses suboptimal concentrations of Ag in order to assess immuno-activation. The purpose is mainly to evaluate:

  • The pharmacodynamic effect of an immune activator (like immune checkpoint inhibitors)
  • Pharmacodynamic (PD) endpoint
  • Safety endpoint in case of exaggerated PD
Checkpoint inhibitors assess by TDAR

Webinar: Utilizing TDAR Assay to Assess Immune Checkpoint Inhibitors

Discover recent advances in the TDAR assay to evaluate immuno-stimulation in non-human primates. Find out how this model can benefit your drug development program.

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Recommended TDAR Assays by Industry

  • Pharmaceutical

    ICH S8 guideline specified immune function test to identify immuno-suppression or enhancement.

  • Chemistry

    OECD test guideline 443 provides a detailed description of the objectives and procedures of the Extended One-Generation Reproductive Toxicity Study (EOGRTS). The guideline describes three cohorts of F1 animals spanning various developmental and reproductive endpoints.

    Human animal cell under microscope

    TDAR Assay and Splenic Lymphocyte Subpopulation Analysis in EOGRTS

    Share control data on the T cell dependent antibody response assay (TDAR) and splenic lymphocyte subpopulation analysis. TDAR is assessed by immunization of all Cohort 3 with KLH as a model antigen.

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  • Agriculture

    Sheep red blood cell is used in place of KLH. In line with the requirements of the United State Environmental Protection Agency (EPA), the immunogenic antigen SRBC can also be used in TDAR assay analysis. TDAR assay with SRBC is a core requirement of the immunotoxicity guidance outlined by the US EPA in which anti-SRBC IgM levels in rats or mice are measured following exposure to test item for at least 30 days, with SRBC immunization in the final 4-6 days of exposure. Following exposure to the test item and SRBC immunization serum, anti-SRBC IgM levels are measured by ELISA.

TDAR assay: Design and Timepoint

A general outline is given below, however, the exact protocol may vary based on your drug, needs, and provider recommendations:

  • Primary immunization: 1-2 weeks after dosing as required, based on drug development & MOA
  • Secondary immunization: ≥ 21 days after primary immunization
  • Samples for analysis collected: predose, 7, 14, and 21 days post immunization
  • Other study designs available upon request

Our TDAR assay will help you construct the optimal design for your drug development.

Immune response to antigens in a TDAR assay

Schema of the TDAR assay showing mouse immunization and cellular response

TDAR assay: Which Immunogen Do I Use?

We provide various designs for immunosuppression or immunoenhancement across a full range of safety assessment species. We offer the following principle model antigens:

  • TT: Tetanus Toxoid
  • KLH: Keyhole Limpet Hemocyanin
  • SRBC: Sheep Red Blood Cell

KLH is a common choice of antigen for TDAR assay based on the extensive historical database, growing standardization, and experience across multiple labs. Other antigens (e.g., sheep erythrocytes and tetanus toxoid) have also been used in drug development. The choice of species tested, antigen chosen, and conditions of the TDAR assay should be justified based on the assessments required. For example, TT can be used in parallel with KLH as a second (neoantigen). This allows parallel assessment of the primary response (TT) and secondary response (KLH).

Antibodies attacking SARS-CoV-2 virus

Validation of Tdar Assay, In the Presence of an Immunosuppressor or an Immunostimulant, Using Two Antigens

TDAR assay in appropriate species is an appropriate endpoint to assess suppressed or enhanced antibody responses of a test article on nonclinical studies.

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In addition to assessing the humoral response, ELISPOT assay can be performed in parallel to enumerate IFNγ or IL-4-secreting cells both on PBMCs or splenocytes. This allows quantification of alterations in cell mediated immunity during drug exposure.

TDAR Assay: Method Analysis and Data Interpretation

There are multiple methods that can be used such as qualitative (titre based) or quantitative (standard curve) ELISA method. Where high throughput is required, a method can be automated using a robotic system.

TDAR Assay: Antibody Response Over Time

Curve of antibodies: IgM and IgG response over time during TDAR assay at the primary and secondary responses

Descriptive statistics for the TDAR assessment are typically performed. Additional statistics, such as inferential statistics or Area Under the Curve (AUC), can be included to aid in data interpretation.

AUC is calculated using the trapezoidal interpolation and determined for each period of response (each immunization) for each animal and for both IgG and IgM, using the nominal sampling times. For each animal within each period, the time of first occurrence of maximum concentration (Tpeak) and the maximum concentration (PEAK) can be reported.

Parameter

Unit

Description of Parameter

Tpeak

day

Time of first occurrence of maximum concentration

PEAK

µg/mL

Maximum concentration

AUCX-Ydays

(µg.d/mL)

Area under the curve from X days to Y days post-dose (calculated according to the linear trapezoidal rule)

The TDAR assay can be offered in many species to a variety of different immunogens with the aim to evaluate the immunotoxic potential of a drug candidate and the immuno-stimulation of immunomodulatory drugs. Design and timepoint of TDAR assay will be identified in concordance with your needs and experienced suppliers’ recommendations.

Associated publication: The T-Cell-Dependent Antibody Response Assay in Nonclinical Studies of Pharmaceuticals and Chemicals: Study Design, Data Analysis, Interpretation

 

Frequently Asked Questions (FAQs) About TDAR Assays

  • Is it mandatory to include TDAR to demonstrate the pharmacological activity of immune modulators in the framework of tox studies?

    Assessments of immune modulators and potential impacts is a regulatory requirement. The ICH S8 guidance states for potential immunotoxic compounds, “It is recommended that an immune function study be conducted, such as a T-Cell-Dependent Antibody Response assay (TDAR assay)”, and “The TDAR should be performed using a recognized T-cell dependent antigen (e.g., sheep red blood cells (SRBC) or keyhole limpet hemocyanin (KLH)) that results in a robust antibody response.” Moreover, the regulatory filings for anti-PD1/PD-L1 approved products demonstrates that TDAR is part of the package for the majority of them except Atezolizumab and Avelumab. For Avelumab however, it is indicated as a post-marketing requirement that FDA required a TDAR with KLH.

    While these assessments should form part of the overall strategy, drug developers should always take a risk-based approach to determine the most appropriate assessments. It is important that the included assessments are appropriate to the mode of action and the potential risks that this may cause.

  • Can we include TDAR as part of chronic general toxicity studies? Do you use satellite animals for TDAR?

    Considering the fact that immunizations are well tolerated in NHP, TDAR assays can be performed on principal animals of chronic toxicity studies. The key takeaway is that immunization is performed at the steady-state and can be achieved in shorter studies too.

  • Do you recommend different adjuvants for each antigen i.e., TT and KLH?

    We use a commercial vaccine for TT which is already adjuvanted with aluminum hydroxide (ALU). KLH is highly immunogenic by itself and does not require adjuvant to generate a robust humoral immune response. The addition of adjuvant can be used as requested.

  • What kind of other readouts would you recommend for the assessment of cellular immune response?

    Depending on the mechanism of action of the compound, intracellular staining after PBMC or splenocytes recall stimulation with the antigen can be utilized to discriminate subpopulation specific to the Ag (e.g.: CD4+ and CD8+ T cells). Additional functional assays, such as ELISPOT, T cell proliferation, or cytotoxicity assays can also be performed.

  • How is the guideline used by the chemistry industry used to assess potential impact in the immune system?

    The guideline, OECD 443, describes three cohorts of F1 animals spanning various developmental and reproductive endpoints. Among them, Cohort 3 is designed to assess the potential impact of chemical exposure on the developing immune system. TDAR assay (used for that purpose) is assessed by immunization of all Cohort 3, animals with KLH as a model antigen on Day 56 post-partum. Anti-KLH IgM concentrations in serum are measured before and 5 days after immunization using a validated ELISA method.