ABCD_AF632, ABCD_AQ775, ABCD_AQ776, ABCD_AS298, ABCD_AS299 and ABCD_AS300 antibodies label the human PD-1 protein by immunofluorescence

Abstract

The recombinant antibodies ABCD_AF632, ABCD_AQ775, ABCD_AQ776, ABCD_AS298, ABCD_AS299 and ABCD_AS300 detect by immunofluorescence 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), 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 seven anti–PD-1 antibodies from the ABCD database (Lima et al., 2020) for testing in an immunofluorescent 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 six antibodies ABCD_AF632, ABCD_AQ775, ABCD_AQ776, ABCD_AS298, ABCD_AS299 and ABCD_AS300 to detect the human protein PD-1 by immunofluorescence. Antibody ABCD_AA679 does not recognize the human protein PD-1 by immunofluorescence.

Materials & Methods

Antibodies: ABCD_AA679 (AA679), ABCD_AF632 (AF632), ABCD_AQ775 (AQ775), ABCD_AQ776 (AQ776), ABCD_AS298 (AS298), ABCD_AS299 (AS299), ABCD_AS300 (AS300) and ABCD_AF291 (AF291) ABCD nomenclature, http://web.expasy.org/abcd/) were produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/) and produced as minibodies with the antigen-binding scFv portion fused to a rabbit IgG Fc. AF291 anti-HA antibody (Lima and Cosson, 2020) was fused to a mouse 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 (~5 to 100 mg/L) were collected after 4 days.

Table 1. Clone names, formats and references 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
AQ775	GY-5	scFv	Chen et al., 2019
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 procedure was carried out at room temperature. HeLa cells were fixed with phosphate-buffered saline (PBS) + 4% paraformaldehyde (w/v) (Applichem, #A3013) for 20 min, and blocked with PBS + 40 mM ammonium chloride (NH4Cl) (Applichem, #A3661) for 5 min. Cells were then permeabilized in PBS + 0.2% saponin (v/v) (Sigma, #S-7900) for 5 min, washed once (10 min) with PBS + 0.2% (w/v) bovine serum albumin (PBS-BSA), and incubated for 20 min with the recombinant antibodies (2,5 mg/L in PBS-BSA). After 3 washes (5 min) with PBS-BSA, cells were incubated for 20 min in PBS-BSA with fluorescent secondary antibodies: AlexaFluor-488-coupled anti-mouse-immunoglobulin G (IgG) antibodies (ThermoFisher #A11029, dilution 1/400) and AlexaFluor-647-coupled anti-rabbit-IgG antibodies (ThermoFisher #A21245, dilution 1/400). After 3 washes (5 min) with PBS-BSA, cells were mounted on slides (Menzel-Gläser, 76x26 mm) with Möwiol + 2.5% (w/v) DABCO (Fluka, #33480). Pictures

Results & Discussion

The transfected HA-tagged PD-1 protein was simultaneously detected using anti–PD-1 antibodies and the previously characterized anti-HA antibody AF291, used here as a positive control (Lima and Cosson, 2020). Antibodies AF632, AQ775, AQ776, AS298, AS299, and AS300 specifically labeled the transmembrane PD-1 protein at the surface of transfected HeLa cells (Fig. 1). The labeling obtained with these antibodies largely colocalized with the signal from the anti-HA antibody (Fig. 1). In contrast, no labeling was observed with antibody AA679, confirming that this antibody recognizes another target. Importantly, no signal was detected in non-transfected cells (NT), confirming the specificity of the observed staining (Fig. 1).

Figure 1. Specific binding of AF632, AQ775, AQ776, AS298, AS299 and AS300 antibodies detected by immunofluorescence. HA-tagged PD-1 proteins were detected in transfected HeLa cells using anti-PD-1 antibodies together with the anti-HA antibody AF291. No signal was observed in non-transfected (NT) cells or with the AA679 antibody. Scale bar: 10 μm.

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.