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2000
Volume 22, Issue 2
  • ISSN: 1573-4110
  • E-ISSN: 1875-6727

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.

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Detection of Anti-drug Antibodies (ADAs) to an Antibody-drug Conjugate (ADC) PYX-201 in Human Plasma Using a Novel Electrochemiluminescence (ECL) Immunoassay
Current Analytical Chemistry 22, 347 (2026); https://doi.org/10.2174/0115734110349489241107072651
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  • Article Type:     Research Article
Keyword(s): ADA; ADC; ECL; extra domain B fibronectin; PYX-201; validation

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