ABCD_AF632, ABCD_AQ775, ABCD_AQ776, ABCD_AS298, ABCD_AS299, ABCD_AS300 and ABCD_RC105 antibodies recognize the human PD-1 protein by ELISA

Abstract

The recombinant antibodies ABCD_AF632, ABCD_AQ775, ABCD_AQ776, ABCD_AS298, ABCD_AS299, ABCD_AS300 and ABCD_RC105 detect by ELISA the human protein PD-1.

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 eight 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 study reports the ability of the antibodies ABCD_AF632, ABCD_AQ775, ABCD_AQ776, ABCD_AS298, ABCD_AS299, ABCD_AS300, and ABCD_RC105 to detect human PD-1 by ELISA.

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_RC105 (RC105) (ABCD nomenclature, http://web.expasy.org/abcd/) were produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/) as minibodies with the antigen-binding (scFv or VHH) portion fused to a rabbit IgG Fc (see Table 1 for clone names, formats, and references). RC105 was discovered by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies) by screening a semi-synthetic human phage display library (Novimmune) against PD1. 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 or VHH-Fc of each antibody. Supernatants (~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
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
RC105	RC105	Nanobody	This study

Antigen: All antibodies were tested against the extracellular domain of PD-1 fused to a Twin-Strep-tag® (IBA Lifesciences; PD1-TST). The Twin-Strep-tagged extracellular domain of the human protein TIGIT (UniProt: C9J0B0; TIGIT-TST) was used as a negative control.

Protocol: The whole procedure was carried out at room temperature. Antigens were immobilized on MaxiSorp 96-well plates (Nunc #44-2404-21) for 45 min. As a saturation agent, 50 μL of PBS-BSA 3% (w/v) was added, followed by 20 minutes of incubation. Each well was rinsed three times with 100 μL of washing buffer (PBS + 0.5% (w/v) BSA + 0.05% (w/v) Tween20), then incubated for 30 minutes with 50 µL of antibody-containing supernatant serially diluted in washing buffer (Fig. 1). After rinsing 3 times (100 µL washing buffer), wells were incubated with horseradish peroxidase-coupled goat anti-rabbit IgG (Sigma #A8275, dilution 1:1000, 50 μL per well) for 20 min. After 5 rinses, Tetramethylbenzidine (TMB) substrate (Sigma #T5569) was added (50 μL per well). The reaction was stopped by the addition of 25 μL of 2M H₂SO₄. The absorbance (OD) was measured at 450 nm, and the absorbance at 570 nm was subtracted.

Results & Discussion

As illustrated on Figure 1 (Fig. 1) antibodies AF632, AQ775, AQ776, AS298, AS299, AS300 and RC105 exhibited concentration-dependent binding to the PD-1 protein. Antibody AA679 did not recognize the PD-1 protein by ELISA. None of the antibodies could bind the negative control TIGIT, confirming the specificity of their binding.

Fig. 1. AF632, AQ775, AQ776, AS298, AS299, AS300 and RC105 bound specifically to PD-1 protein, but not to the negative control peptide (TIGIT) (shown only for AS299) the other background curves were superimposed), as detected by ELISA.

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.