Abstract

Background: PYX-201 is an Antibody-Drug Conjugate (ADC) composed of a fully human IgG1 antibody, a cleavable linker mcValCitPABC, and toxic auristatin payloads Aur0101, with a drug antibody ratio (DAR) of approximately 4. PYX-201 is a promising candidate for oncology treatment because it targets the extra domain B splice variant of fibronectin (EDB + FN), which is expressed at low levels in normal adult tissues while at moderate or high levels in various human solid tumors.

Methods: An electrochemiluminescence (ECL) immunoassay was developed and validated for the detection (screening, confirmatory, and titration) of antibodies to an ADC PYX-201 in human plasma. Anti-PYX-201 antibodies were captured by biotinylated PYX-201 (Bio-PYX-201) and detected by ruthenylated PYX-201 (Ru-PYX-201) on a Meso Sector imager S 600 or 6000 reader.

Results: The screening cut-point factor (SCPF), confirmatory cut-point (CCP), and titration cut-point factor (TCPF) were found to be 1.11, 20.7%, and 1.23, respectively. Sensitivity was determined to be 2.25 ng/mL in the screening assay and 5.34 ng/mL in the confirmatory assay for anti-PYX-201 antibodies. Sensitivity was determined to be 7.70 ng/mL in the confirmatory assay for anti-PYX-201 monoclonal antibody (mAb) antibodies. The positive controls (PCs) were set at the following levels: low positive control (LPC) at 14.0 ng/mL, medium positive control (MPC) at 100 ng/mL, and high positive control (HPC) at 5,000 ng/mL. The drug tolerance was up to 200 μg/mL at the HPC level, up to 100 μg/mL at the MPC level, and 0 μg/mL at the LPC level. The intra-assay percent coefficient of variation (%CV) was ≤ 4.5% for PCs in the screening assay and ≤ 11.5% for PCs in the confirmatory assay. The inter-assay %CV was ≤ 13.6% for PCs in the screening assay and ≤ 19.2% for PCs in the confirmatory assay. No hook effect, hemolysis effect, or lipemia effect was found in this ADA method. Anti-PYX-201 antibodies were found stable in human plasma for at least 24 hours at room temperature or after six freeze/thaw cycles.

Conclusion: Anti-PYX-201 ADA bioanalytical method validation was reported for the first time in any biological matrix. This ADA method has been successfully applied to human sample analysis to support a clinical study.

Keywords: ADA, ADC, PYX-201, extra domain B fibronectin, ECL, validation.