Preclinical development of a high affinity anti-exatecan monoclonal antibody and application in bioanalysis of antibody-exatecan conjugates

Exatecan, a topoisomerase I inhibitor, is currently utilized as a potent payload in antibody-drug conjugates, significantly enhances the efficacy and safety of these therapeutic agents. In the research of antibody-drug conjugates with exatecan as the payload conjugation, an anti-exatecan antibody serves as a crucial reagent for bioanalysis. In this study, BALB/c mice were immunized with bovine serum albumin conjugate exatecan (BSA-exatecan), and hybridoma technology was employed to screen seven hybridoma cell lines that stably express monoclonal antibodies. After evaluating their binding activity to exatecan, the cell line NO. 8B5-3H6 has been selected based on the EC₅₀ value. The antibody was purified using protein A affinity chromatography, resulting in a mouse anti-exatecan monoclonal antibody with a purity exceeding 99 %. The binding profile with the exatecan demonstrated strong affinity, with an EC₅₀ of 1.382. Bio-Layer Interferometry (BLI) analysis further confirmed the high affinity of this mouse anti-exatecan antibody with a K_D of less than 1 pM. Subsequently a detection method was developed using the mouse anti-exatecan antibody as the coating reagent and mouse anti-human IgG Fab conjugate HRP as the detection reagent. The standard curve and quantification range of the method were established at 31.25 ng/mL to 4000 ng/mL. Validation of accuracy, precision, selectivity, stability, dilution linearity, hook effect, parallelism and specificity were performed in accordance with ICH M10 and FDA bioanalytical method validation guidelines, laying a solid foundation for subsequent toxicological and pharmacokinetic studies of antibody-drug conjugate.