ABCD_AF632, ABCD_AQ776, ABCD_AS298, ABCD_AS299 and ABCD_AS300 antibodies label the human PD-1 protein by immuno-cytometry

Abstract

The recombinant antibodies ABCD_AF632, ABCD_AQ776, ABCD_AS298, ABCD_AS299 and ABCD_AS300 detect by immuno-cytometry the human protein PD-1 at the surface of HeLa cells.

Introduction

The Programmed cell death protein 1 (PD-1, UniProt #Q15116) is a transmembrane receptor expressed on activated T, B, and NK cells. Upon engagement with its ligands PD-L1 (CD274) or PD-L2 (PDCD1LG2; CD273), PD-1 transmits inhibitory signals that suppress T-cell activation and cytokine production, thereby contributing to the maintenance of peripheral immune tolerance (Keir et al., 2008). Tumor cells can exploit this pathway to escape immune surveillance, making PD-1 a central target for immune checkpoint blockade therapies (Topalian et al., 2012). In this study, we selected six anti–PD-1 antibodies from the ABCD database (Lima et al., 2020) for testing in an ELISA assay. The clone names, formats, and original references for these antibodies are described in Table 1. Interestingly, ABCD_AA679 (pidilizumab) was wrongly annotated as an anti–PD-1 antibody in the ABCD database. Although pidilizumab was initially reported as targeting PD-1, this interaction could not be conclusively demonstrated. More recent studies suggest that it may instead bind delta-like protein 1 (DLL1) (Albuquerque et al., 2022). This paper reports the ability of five antibodies ABCD_AF632, ABCD_AQ776, ABCD_AS298, ABCD_AS299 and ABCD_AS300 to detect the human protein PD-1 by immuno-cytometry. The lack of signal observed with antibody ABCD_AA679 supports a possible incorrect annotation in the database

Materials & Methods

Antibodies: ABCD_AA679 (AA679), ABCD_AF632 (AF632) ,ABCD_AQ776 (AQ776), ABCD_AS298 (AS298), ABCD_AS299 (AS299) and ABCD_AS300 (AS300) (ABCD nomenclature, http://web.expasy.org/abcd/) were produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/) and produced with the antigen-binding scFv portion fused to a rabbit IgG Fc. The synthesized scFv sequences (GeneArt, Invitrogen) correspond to the sequences of the variable regions joined by a peptide linker (GGGGS)₃. HEK293 suspension cells growing in HEK TF medium (Xell #861-0001, Sartorius), supplemented with 0.1% Pluronic F68 (Sigma #P1300), were transiently transfected with the vector coding for the scFv-Fc of each antibody. Supernatants containing antibodies (~5 to 100 mg/L) were collected after 4 days.

Table 1: Clone names, formats and references and of the antibodies used in this study

ABCD		name	Format	Reference
AA679		pidilizumab	scFv	Hardy et al., 2008
AF632		m107	scFv	Dimitrov et al., 2017
AQ776		GY-14	scFv	Chen et al., 2019
AS298		MH8	scFv	Finlay et al., 2019
AS299		MH4	scFv	Finlay et al., 2019
AS300		MH12	scFv	Finlay et al., 2019

Antigen: We used a fusion protein composed of the extracellular domain of the human PD-1 protein fused to the transmembrane domain of the interleukin 2 receptor alpha subunit (UniProt #P01589), with a C-terminal HA epitope TAG (YPYDVPDYASLRS). The fusion protein was produced in transiently transfected HeLa cells. Non-transfected (NT) HeLa cells were used as a negative control.

Protocol: The whole process was carried out at 4°C. 1x10⁵ transfected or non-transfected HeLa cells were pelleted by centrifugation 5 min at 300g and washed once with washing buffer (PBS + 0.2% BSA (w/v) + EDTA 2 mM). Cells were then incubated for 20 minutes with the tested antibody (5µg/mL) in 400 µL of washing buffer. After two washes in washing buffer and centrifugation 5 min at 300g, cells were resuspended and incubated for 20 minutes in the dark with 400 µL of secondary goat anti-rabbit IgG conjugated to Alexa Fluor 488 diluted 1/400 (Molecular Probe #A11034). After three washes in washing buffer and centrifugation 5 min at 300g, cells were resuspended in 400 µL of washing buffer and analyzed with a CytoFLEX S4 flow cytometer (Beckman Coulter). Cell debris and dead cells were excluded based on forward and side scatter (FSC/SSC) gating. Fluorescence intensity was recorded using the AF-488 channel.

Results & Discussion

Based on the AF488 signal obtained from non-transfected cells, a gating strategy was established to identify immunolabeled cells. Antibodies AF632, AQ776, AS298, AS299, and AS300 successfully detected the PD-1 protein by flow cytometry in transfected HeLa cells. Between 21% and 45% of cells were immunolabeled in the transfected condition, consistent with the expected transfection efficiency (Fig. 1). No signal was detected in non-transfected cells, confirming the specificity of the labeling. Antibody AA679 did not recognize the PD-1 protein supporting the hypothesis that this antibody recognizes another target. (Fig. 1).

Fig.1. Bi-parametric representation of flow cytometry analysis depicting Forward Scatter (FSC) and Alexa Fluor 488 signal. AF632, AQ776, AS298, AS299 and AS300 antibodies labeled HeLa transfected cells (T) overexpressing the PD-1 protein. No signal was detected in non-transfected cells (NT). AA679 did not recognize the PD-1 protein by immuno-cytometry. Transfection efficiency was estimated to be around 35 % (data not shown).

Conflict of interest

Tania Jauslin is an associate-editor of the journal Antibody Reports.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.