Background

Appropriately designed pharmacokinetic (PK) assays that are sensitive to the effects of antidrug antibodies (ADAs) on relevant exposures are an alternative strategy for understanding the neutralization potential of ADAs. However, there are currently no guidelines on how to develop such PK assays and how to confirm the functional effects of ADAs on exposure.

On May 31, 2024, researchers from Roche Pharmaceuticals published an article titled "Characterization of anti-drug antibody responses to the T-cell engaging bispecific antibody cibisatamab to understand the impact on exposure" in Frontiers in Immunology. In this study, researchers developed a PK assay based on the mechanism of action (MoA) of the T-cell engaging bispecific antibody cibisatamab. A key monoclonal anti-idiotypic (anti-ID) antibody positive control was used as a surrogate for ADA to assess its impact on exposure in preclinical settings. The results of this study could provide a benchmark for developing appropriate PK assays to understand the impact of potential neutralizing ADA.

Development of a PK analysis method based on the mechanism of action of cibisatamab

Designing an assay format to develop an appropriate PK assay is a key prerequisite for measuring relevant drug exposure. The first important aspect to consider is the drug's mechanism of action. Cibisatamab's mechanism of action is based on simultaneous binding of cibisatamab to CEA/CEACAM5 on tumor cells and the CD3ϵ chain on T cells, leading to T cell activation and subsequent killing of tumor cells. This dual binding capability was exploited in the development of a mechanism-based PK assay. To mimic the effects of ADA on cibisatamab, researchers generated and used monoclonal anti-anti-idiotypic (anti-ID) antibodies as positive controls. These anti-ID antibodies target specific functional binding sites on cibisatamab, allowing for the assessment of functional drug exposure.

These anti-ID antibody reagents target the target binding-associated complementarity determining regions (CDRs) of anti-CEA and the anti-CD3 domain of cibisatamab, quantifying drug concentrations and exposing their free binding moieties to both targets while achieving ultrasensitive detection of <1 ng/ml. In validation and Phase I clinical studies, interassay statistical results for standard and quality control drug concentrations placed the assay accuracy and precision within acceptable standards.

The study found that MoA-based PK assays can detect key target-binding capacity drug exposures and are sensitive to binding interference from an anti-ID ADA positive control.

The researchers evaluated the impact of patient-derived ADA responses on cibisatamab exposure. Increases in cibisatamab ADA titers were associated with decreased cibisatamab exposure in patients with target binding activity (Patient B), whereas drug exposure remained stable over time in ADA-negative patients (Patient A). These data confirm that MoA-based PK assays can be used to link patient-derived ADA responses to exposure.

Characterization of ADA responses

Using an ADA domain detection assay to identify specific ADA-responsive drug domains, we found that the ADA response was primarily directed against the anti-CD3 domain of cibisatamab, and that the anti-CD3 domain had binding specificity with cibisatamab.

By using the ADA immune complex assay to measure both IgM and IgG and to determine the isotype of the ADA response, it was found that at the onset of ADA, IgM triggered a low-titer initial response, whereas at later time points, the response was dominated by a high-titer ADA-IgG response, and drug exposure disappeared. Four weeks after the onset of ADA, IgG detection had reached saturation, IgM detection fell below the critical point, and exposure was lost. The ADA response that resulted in a significant loss of exposure in the patient was driven by ADA-IgG.

Advanced Characterization of Anti-ID Antibodies as ADA Positive Controls

The binding of anti-ID to cibisatamab was analyzed by ELISA and biolayer interferometry, and it was found that all four anti-IDs showed similar binding characteristics to the anti-CD3 domain of cibisatamab.

Specific anti-CD3 domain constructs, which either have functional CDRs in the heavy chain V domain and germline CDRs in the light chain V domain, or vice versa, were purified and used as capture reagents in CDR-specific domain detection assays. Appropriate controls, such as a construct with two functional CDRs in the antibody paratope, were used as positive controls, or germline CDR sequences in the antibody paratope were used as negative controls.

Using ELISA, each anti-ID antibody bound to the HC/LC positive control but not to the negative control construct (germline in HC/LC). Only one anti-ID (anti-ID4) bound to an anti-CD3 domain construct with functional CDRs in the HC, while the other three did not. Another anti-ID antibody (ADA anti-ID2) bound to an anti-CD3 domain constructed with functional CDRs from the LC, while the other three did not. These results were further confirmed by an orthogonal binding readout using biolayer interferometry. In summary, all four monoclonal anti-ID antibodies with similar binding affinities showed strong binding to fully functional CDRs. The anti-ID antibodies target distinct CDR epitopes within the anti-CD3 variable domain, encompassing both the functional CDRs of the heavy chain (HC) and the light chain (LC). Therefore, they may have distinct neutralizing potentials that interfere with cibisatamab binding to its CD3ϵ antigen receptor target.

Characterization of patient-derived ADA

To understand whether patient-derived ADAs were also anti-ID antibodies and targeted functional CDRs, the researchers used identically designed anti-CD3 domain constructs and modified functional CDRs (HC, LC, or both) to analyze the same 10 patient samples collected 4 weeks after ADA onset. They found that patient-derived ADAs were confirmed to be anti-ID antibodies and mainly targeted the complementarity-determining regions (CDRs) of the heavy chain (HC) of the anti-CD3 domain of cibisatamab.

CD3 receptor cell experimental analysis

To analyze the neutralizing potential of four selected anti-ID antibodies as relevant ADA positive controls, researchers used a CD3ϵ antigen receptor-specific reporter cell line and found that all four monoclonal anti-ID antibodies completely abolished CD3ϵ-mediated cell activation. Anti-ID antibodies inhibited the binding of the anti-CD3 IgG drug to the receptor antigen-target. All anti-ID antibodies served as effective ADA positive controls, neutralizing both drug-antigen target binding and CD3ϵ-mediated signaling.

To demonstrate neutralizing interference with CD3-specific ADA responses in patients, a CD3ϵ-specific reporter cell line was used with identical ADA-positive samples from ten selected patients, collected four weeks after ADA development. Patient-derived ADA interfered with drug binding to the CD3ϵ receptor and elicited ADA neutralization potential similar to that of the tested monoclonal anti-ID antibodies. ADA inhibition of drug-target binding was dependent on the ADA-to-drug molar ratio.

Effect of ADA on cibisatamab exposure in clinical trials

In a phase I study using MoA-based PK testing, increases in cibisatamab ADA titers were found to be associated with decreases in cibisatamab exposure, which correlated with target binding capacity.

Summarize

This study developed a PK assay using a monoclonal anti-anti-idiotypic ( anti-ID ) antibody as a positive control. This assay can sensitively detect exposures relevant to drug function and assess the potential neutralization of ADA. MoA-based PK assays are important for assessing the neutralization potential of ADA and guiding clinical development decisions. This study provides a basis for developing appropriately designed PK assays, which is crucial for understanding the impact of ADA neutralization by bispecific antibodies on exposure. MoA-based PK assays are important for assessing the neutralization potential of ADA and guiding clinical development decisions.