Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

Area of Science:

• Immunology
• Oncology
• Biochemistry

Background:

• Programmed death-1 (PD-1) is a co-inhibitory receptor that tumors exploit to evade immune responses.
• Anti-PD-1 antibodies are a breakthrough in cancer immunotherapy, restoring immune cell function.
• Soluble PD-1 (sPD-1) has been detected in circulation, but a clinical assay is needed.

Purpose of the Study:

• To develop and validate a clinical assay for quantifying soluble PD-1 (sPD-1) protein.
• To establish a reliable method for measuring sPD-1 levels in biological samples.
• To support research into sPD-1 function and anti-PD-1 drug development.

Main Methods:

• Developed and validated a protein assay for sPD-1 using a purified recombinant human PD-1 reference material.
• Validated the assay according to biotherapeutic pharmacokinetic assay standards.
• Investigated potential matrix effects in human sera and confirmed assay specificity against anti-PD-1 antibodies and ligands.

Main Results:

• The sPD-1 assay demonstrated a lower limit of quantitation (LLOQ) of 100 pg/mL and a dynamic range of 100-10,000 pg/mL.
• Assay precision was ≤15% (intra- and inter-assay), with a bias of ≤10%.
• The assay specifically measures total sPD-1 and is not affected by nivolumab, PD-L1, or PD-L2.

Conclusions:

• A robust and specific assay for quantifying soluble PD-1 (sPD-1) protein has been successfully developed and validated.
• This assay provides a crucial tool for studying sPD-1's role in health and disease.
• The validated assay will facilitate the development and clinical application of anti-PD-1 cancer immunotherapies.