Bispecific antibodies consist of two small-chain fragments variable (scFv) of the antibody that has binding affinities to different target antigens. T cell engagers are a class of bispecific antibodies that selectively recruit and activate T cytotoxic cells in the tumor tissue and mediate cytotoxicity. In T cell engagers, one scFv domain is designed to bind to CD3 molecules in the T cell, and the second scFv domain binds to a tumor-associated antigen expressed in the tumor tissue. Certain tumor-associated antigens are known to express in specific cancer types. These tumor-associated antigens have been targeted for the treatment of these specific cancers. These tumor antigens include CD19, BCMA, PSMA, HER2, TROP2, etc.
Bispecific antibodies, similar to any protein therapeutics, can evoke the generation of anti-drug antibodies. Therefore, detection and monitoring of anti-drug antibodies is an important component of immunogenicity assessment. The anti-drug antibody assays are developed with the following considerations:
1) MSD Bridging Assay Format
2) Mitigation of Target Interference
3) Mitigation of Drug Interference
1) MSD Bridging Assay Format:
Bridging assay format is a widely used assay format for the detection of the total antibodies. In the bridging assay format, the biotinylated drug serves as a capture reagent and the ruthenylated drug serves as a detection reagent. Briefly, 0.5 ug/ml of biotin-drug and 0.5 ug/ml of ruthenium drug are mixed with the sample (1:10 to 1:20 MRD; diluted in assay diluent) and incubated for 60 minutes at room temperature. After incubation, 50 microliters of sample mixture (sample + biotin-drug+ ruthenium drug) is transferred to streptavidin-coated MSD plate and incubated for 60 minutes. Next, the plate is washed three times with 1X DPBS-0.5% Tween 20. 150 ul of MSD read buffer is added and read in MSD Quickplex.
1.1) Screening, Confirmation, and Titer Step
The total anti-drug antibody assay is performed in screening, confirmation, and titer steps. The screening step identifies the potential positive samples. Screening cut point (at 95th percentile) is used to distinguish between positive samples and negative samples.
The confirmatory assay identifies the true positive from the potential positive sample obtained from the screening step. For the confirmatory step, the samples are pre-incubated with excess drug (10 ug/ml to 100 ug/ml) and the assay is performed. The drug-specific ADA binds to the ADA in the sample and results in signal inhibition in the assay. The confirmation cut point (at 99 percentile) is used to identify the true positive from the total positive obtained in the screening step.
In the titer step, the samples are diluted in the assay matrix to determine the dilution that is just above the titer cut point (at 99 percentile). This allows the semiquantitative determination of ADA present in the clinical samples.
1.2) Domain Specificity Characterization
Domain specificity characterization of ADA present in the clinical is performed to determine the immunodominant epitope of the multidomain therapeutic protein. Two approaches are widely adopted for the characterization of domain specificity.
1.2.1) Confirmation/specificity testing with scFv fragments: This is an extension of the confirmatory steps of ADA assay. Instead of the drug, each scFv domain component is preincubated with the sample at molar excess concentration. The percent inhibition of the signal in presence of the scFv component is used to determine the specificity. As in the confirmation step, the specificity cutpoint is determined for each domain of the drug to determine the domain specificity of the ADA in the sample. Domain specificity testing provides information on the presence or absence of ADA against specific domains. This approach does not provide information on the magnitude of the ADA response.
1.2.2) Domain-specific detection assay: This approach is utilized for drug that has higher immunogenicity risk and associated safety concerns. The titer values obtained for each domain can be used to evaluate the magnitude of the domain-specific ADA response and its association with the safety and efficacy of the drug.
1.3) Mitigation of Target Interference
Target (Tumor-Associated Antigen) interference is one of the major concerns for the bioanalytical assays for the monoclonal antibodies, ADCs, and T cell engagers immunotherapies. The circulating target bind to the drug and can yield false-positive result in ADA assay. The target may fare present in monomeric, dimeric, and multimeric forms and therefore has its unique challenge. Use of the MSD bridging format is preferred for the reduction of monomeric targets. In the bridging assay format, the bivalent proteins are required for the bridge formation between capture and detection reagent and yield the assay signal. Therefore, the interference from the monomeric target is less of a concern in the bridging assay format.
Interference from dimer & trimer requires different mitigation strategies. Heat treatment can dissociate the dimeric proteins and mitigate interference. The samples are rapidly heated at 60 to 65 degrees celsius for 30-60 seconds and allow dimers to dissociate. This reduces the target interference in the ADA assay.
Another approach is the sequestration of the circulating target with the addition of antibodies, ligands, or receptors into the sample. This approach has been also used to investigate and confirm that the ADA, but not the target, is yielding positive results in the clinical assay.
1.4) Mitigation of Drug Interference
High drug-tolerant ADAs are vital for reporting the incidence and magnitude of the ADA in clinical studies. Circulating drug competes with the capture and detection reagent binding to ADA and thereby yield in the false-negative results or suppressed titer value. The drug interference strategies include acid dissociating, ACE, SPEAD or BEAD method are described elsewhere (click here)
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