(W1030-06-32) An Electrochemiluminescence (ECL)-Based Assay for the Specific Detection of Neutralizing Anti-CAEL-01 Antibodies in Human Serum with a Pre-Treatment Procedure to Eliminate Therapeutic Drug

Purpose: CAEL-101 (Anselamimab) is a chimeric IgG-k monoclonal antibody that recognizes misfolded free immunoglobulin light chains. It has the potential to clear amyloid fibrils from organs in light chain (AL) amyloidosis patients. However, biologics can induce an undesirable immune response, leading to the formation of anti-drug antibodies (ADA), including neutralizing antibodies (NAb). The emergence of neutralizing antibodies against a therapeutic protein can result in treatment failure or inefficacy. Therefore, there is a need to develop robust immunogenicity assays. We developed a bead-based electrochemiluminescence (ECL) assay for the detection of neutralizing anti-CAEL-101 antibodies in human serum.

Methods: The bioanalytical method consists of two main steps: (1) sample pre-treatment to remove the drug present in the samples, and (2) analyzing the purified neutralizing anti-drug antibodies using a bridging immunoassay.  In the pre-treatment step, the serum sample is mixed with biotinylated CAEL-101 (Bt-CAEL-101) and incubated at 37°C overnight. This allows the anti-drug antibodies in the serum to bind to Bt-CAEL-101. After incubation, these anti-drug antibodies bound to Bt-CAEL-101 are captured using streptavidin-labeled magnetic beads, and then eluted from the beads by acid dissociation. In the next step, the eluted anti-drug antibodies are incubated with sulfo-Tag labeled CAEL-101 on a biotinylated target peptide-coated streptavidin MSD plate. If neutralizing antibodies (NAb) are present in the positive controls (PCs) or test samples, they will block the binding of sulfo-Tag labeled CAEL-101 to the biotinylated peptide, leading to a reduction in the ECL signal. This reduction indicates the amount of NAb present in the samples.

Results: During the method development stage, several challenges were encountered, including inconsistency, and lack of purification efficiency and sensitivity. The inconsistency was attributed to the solubility of the biotinylated peptide, which was resolved by using DMSO as a solvent with minimal impact on sensitivity. The purification efficiency was optimized by adjusting the volume of beads and using different types of elution buffers to achieve a minimal 90% recovery rate. To optimize sensitivity, a checkerboard strategy was adopted, evaluating various combinations of coating reagent concentrations, sulfo-Tag labeled CAEL-101 concentrations, and types of magnetic beads. Ultimately, the optimal conditions were determined to be 0.3 µg/mL of biotinylated CAEL-101 and 10 ng/mL of sulfo-Tag labeled CAEL-101, achieving a desired sensitivity of 224.8 ng/mL after purification with Dyno beads. The assay was successfully developed and validated according to regulatory guidance and in-house standard operating procedures. Validation parameters included the cut-point determination, sensitivity, selectivity, intra- and inter-assay precision, drug tolerance, as well as various stability and interference tests. The final cut-point for this assay was established at 0.927 (ratio to negative control). The inter-assay precision of RECL (ratio to NC) for HPC (5000 ng/mL) was 11.4%, for MPC (500 ng/mL) was 7.3%, and for LPC (300 ng/mL) was 4.1%. Drug tolerance was determined to be 100 µg/mL CAEL-101 at 300 ng/mL positive control and 300 µg/mL CAEL-101 at 1000 ng/mL positive control.  Tests for hemolysis effect, lipemic effect, biotin effect, selectivity, and stability all confirmed the robustness of the assay.

Conclusion: A novel magnetic bead-based ECL assay for the specific detection of neutralizing anti-CAEL-01 antibody in human serum was developed with sample pre-treatment to remove therapeutic drug interference, ensuring high specificity and sensitivity.