Monitoring PD-1-Blocking Antibodies Bound to T Cells Derived from a Drop of Peripheral Blood

Summary

We developed a simple flow cytometry assay for evaluating the binding of PD-1–blocking antibodies to T cells, requiring only a drop of peripheral blood from cancer patients.

Abstract

Immune checkpoint inhibitors, including PD-1–blocking antibodies, have significantly improved treatment outcomes in various types of cancer. The pharmacological efficacy of these immunotherapies is long lasting, extending even beyond the discontinuation of their injections, due to persistent blood concentrations. Here we developed a simple flow cytometry assay to evaluate the T cell binding status of the PD-1–blocking antibodies nivolumab and pembrolizumab. Like a glucose test, this assay requires just a single drop of peripheral blood. Visualizing antibody binding on T cells is more reliable than measuring antibody blood concentrations. In addition, if necessary, we can potentially analyze many distinctive immune-related markers on T cells bound to PD-1–blocking antibodies. Thus, this is a simple and minimally invasive strategy to analyze the pharmacological effect of PD-1–blocking antibodies in cancer patients.

Introduction

PD-1–blocking antibodies have become the standard choice for treatment of various types of cancer, including non-small cell lung cancer (NSCLC)^1,2,3,4. They show a remarkable therapeutic effect in a subset of cancer patients who have not responded to conventional cytotoxic chemotherapies. However, immune checkpoint inhibitors (ICIs), which include PD-1–blocking antibodies, can cause a unique and distinct spectrum of adverse events, termed immune-related adverse events (irAEs)⁵. Although irAEs can affect al....

Protocol

Sampling was performed during routine clinical procedures. All human samples were obtained after informed consent was provided by the subjects, in accordance with the Declaration of Helsinki and with the approval of the ethical review board of the Graduate School of Medicine, Osaka University, Japan (15383 and 752).

1. Whole Blood Sample Preparation and Staining

1. Collect whole blood samples into blood collection tubes containing ethylene diamine tetra-acetic acid (EDTA).
   NOTE: Blood collection can be performed using either a regular needle or a blood lancet.
2. Transfer 20 µL of whole blood samples to 5 mL roun....

Results

The gating strategy and flow cytometry analysis (Figure 1) can detect PD-1–blocking antibody binding to T cells obtained from a drop of NSCLC patient peripheral blood. Before PD-1–blocking antibody is administered, no human IgG4-positive CD8 or CD4 T cells are present, and PD-1 expression can be confirmed by a PD-1–detecting antibody (EH12.1) (Figure 2A). After nivolumab or pembrolizumab administration, IgG4 (nivolumab, pembrol.......

Discussion

In this article, we report a method using a flow cytometer to detect PD-1–blocking antibodies bound to T cells derived from a drop of peripheral blood, which we originally developed for nivolumab detection¹⁰. Although this technique is very simple and easy to perform, two important points should be noted in order to obtain accurate results. One is that to detect PD-1 molecules, an appropriate antibody that competes with nivolumab and pembrolizumab should be used. This issue was evaluated in .......

Materials

Name	Company	Catalog Number	Comments
10X RBC Lysis Buffer (Multi-species)	Thermo Fisher Scientific	00-4300-54	50 mL
APC/Cyanine7 anti-human CD4 Antibody	BioLegend	300518	Clone RPA-T4
BD FACS Canto II Flow Cytometer	BD
Brilliant Violet 510 anti-human CD8a Antibody	BioLegend	301048	Clone RPA-T8
Dulbecco's Phosphate Buffered Saline	nacalai tesque	14249-95	500 mL
Falcon Round-Bottom Polystyrene Tubes	STEMCELL Technologies	352058	5 mL
FcR Blocking Reagent, human	Miltenyi Biotec	130-059-901	2 mL
FLOWJO	BD
Gibco Fetal Bovine Serum	Thermo Fisher Scientific	12676029	500 mL
Mouse IgG1 monoclonal - Isotype control	abcam	ab81200
Mouse monoclonal Anti-Human IgG4 Fc	abcam	ab99825	Clone HP6025
Pacific Blue Mouse Anti-Human CD3	BD	558117	Clone UCHT1
PE-Cy7 Mouse anti-Human CD279 (PD-1)	BD	561272	Clone EH12.1
PE-Cy7 Mouse IgG1 κ Isotype Control	BD	557646