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

Abstract

Abstract. Programmed death-1 (PD-1) protein is a co-inhibitory receptor which negatively regulates immune cell activation and permits tumors to evade normal immune defense. Anti-PD-1 antibodies have been shown to restore immune cell activation and effector function-an exciting breakthrough in cancer immunotherapy. Recent reports have documented a soluble form of PD-1 (sPD-1) in the circulation of normal and disease state individuals. A clinical assay to quantify sPD-1 would contribute to the understanding of sPD-1-function and facilitate the development of anti-PD-1 drugs. Here, we report the development and validation of a sPD-1 protein assay. The assay validation followed the framework for full validation of a biotherapeutic pharmacokinetic assay. A purified recombinant human PD-1 protein was characterized extensively and was identified as the assay reference material which mimics the endogenous analyte in structure and function. The lower limit of quantitation (LLOQ) was determined to be 100 pg/mL, with a dynamic range spanning three logs to 10,000 pg/mL. The intra-and inter-assay imprecision were ≤15%, and the assay bias (percent deviation) was ≤10%. Potential matrix effects were investigated in sera from both normal healthy volunteers and selected cancer patients. Bulk-prepared frozen standards and pre-coated Streptavidin plates were used in the assay to ensure consistency in assay performance over time. This assay appears to specifically measure total sPD-1 protein since the human anti-PD-1 antibody, nivolumab, and the endogenous ligands of PD-1 protein, PDL-1 and PDL-2, do not interfere with the assay.

Soluble programmed cell death protein 1 (sPD‐1) and the soluble programmed cell death ligands 1 and 2 (sPD‐L1 and sPD‐L2) in lymphoid malignancies

“…PD‐1/PD‐L1/PD‐L2 exist in the peripheral blood in soluble forms 20,21 . Soluble PD‐1 (sPD‐1) originates from an alternative splice variant of the PD‐1 gene, whereas soluble PD‐L1 (sPD‐L1) and soluble PD‐L2 (sPD‐L2) are assumed to originate from proteolytic cleavage of membrane PD‐L1/PD‐L2 22‐25 .…”

Serum PD-1 levels measured by ELISA using Nivolumab increased in advanced RCC patients: novel approach to develop companion diagnostics for antibody therapy

“…PD-1 is expressed on tumor-infiltrating lymphocytes and its soluble form is measured in several cancers (Ni et al 2015 ; Rijnders et al 2017 ). We demonstrated that serum PD-1 levels increased in advanced RCC patients, when measured by Nivolumab-based ELISA.…”

Recommendations for Selection and Characterization of Protein Biomarker Assay Calibrator Material

“…Both monomeric sPD-1-His and dimeric sPD-1-Fc were available as recombinant calibrator material. During the development of a sPD-1 electrochemiluminescent (ECL) assay (22), surface plasmon resonance studies showed the capture antibody had a higher binding affinity for sPD-1-Fc than sPD-1-His (0.5 versus 2.8 nM). Consequently, there was approximately a 50-fold difference in concentration-dependent signal when the calibrator curves of both sPD-1-His monomer and sPD-1-Fc dimer were tested (Fig.…”