ABCD_RC105, RC226, RC227 and RC229 antibodies recognize the human PD-1 protein by ELISA and immunofluorescence

Abstract

In this study we selected by phage display several new antibodies against human PD-1: the ABCD_RC105 nanobody and ABCD_RC226, RC227 and RC229 human antibodies. We then tested them in a variety of immunodetection assays, in parallel with a previously described therapeutic antibody (ABCD_AS298). The newly discovered antibodies recognize the human PD-1 protein in ELISA and immunofluorescence assays and, albeit less efficiently, by western blot. The newly generated recombinant antibodies exhibited performance comparable to that of newly developed animal-derived antibodies.

Introduction

A shift from animal-based to recombinant antibodies serves both scientific and ethical goals. In this report and in a companion article, we describe the performance of fully recombinant antibodies, compared with animal-based antibodies, contributing to ongoing efforts to improve antibody technologies. Human PD-1 (Programmed Cell Death Protein 1; PDCD1; CD279; Uniprot #Q15116) is a type I transmembrane inhibitory receptor primarily expressed on T lymphocytes. It acts as an inhibitory immune checkpoint by suppressing T-cell activation and promoting T-cell apoptosis (Zak et al., 2015). Several therapeutic antibodies have been reported to bind specifically to PD-1, among which ABCD_AS298 (AS298) has proven particularly efficient in diverse experimental procedures (Schaffner et al., 2026). Here we describe several new anti-PD-1 antibodies and compare their reactivity with the AS298 antibody by ELISA, western blot, immunofluorescence and immunoprecipitation.

Materials & Methods

Antigen: For phage display selection as well as for ELISA detection, we used a fusion protein composed of a human IL-2 signal sequence for insertion in the ER followed by the coding sequence of the human PD-1 extracellular domain (amino acids 25 to 170) and fused at its C-terminus to a Twin-Strep-Tag® (IBA Lifesciences, PD-1-TST). The fusion protein was produced and secreted in transiently transfected HEK293 cells and purified from cellular supernatants using MagStrep Strep-Tactin XT beads according to the manufacturer’s instructions (IBA Lifesciences #2-5090-002). For other immunodetection methods, we used HeLa cells expressing transiently a fusion protein composed of the human IL-2 signal sequence for insertion in the ER followed by the coding sequence of the human PD-1 extracellular domain (amino acids 25 to 170) and fused at its C-terminus with the IL-2-RA transmembrane and cytosolic domain followed by a C-terminal HA Tag, as previously described (Marchetti et al., 2023). The resulting membrane-embedded PD-1 (PD-1-TM-HA) is expected to be present at the cell surface as well as in the membrane of endocytic and exocytic intracellular compartments.

Antibodies: One PD-1-binding VHH domain (ABCD_RC105; ABCD nomenclature, http://web.expasy.org/abcd/) as well as three PD-1-binding human scFv (ABCD_RC226, ABCD_RC227 and ABCD_RC229) were selected by phage display using the PD-1-TST protein adsorbed to MagStrep Strep-Tactin XT beads. ABCD_AS298 (therapeutic anti-PD-1) and ABCD_AF291 (anti-HA) were described previously (Schaffner et al., 2026; Keszei & Picard, 2019). These antibodies will be referred to here as RC105, RC226, RC227, RC229, AS298 and AF291. To produce the recombinant antibodies described above, 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 antigen-binding scFv or VHH domain of each antibody fused to a rabbit Fc domain. The synthesized scFv sequences (GeneArt, Invitrogen) correspond to the sequences of the variable regions joined by a peptide linker (GGGGS)₃. Supernatants containing secreted antibodies (~50 mg/L) were collected after 4 days.

ELISA: The whole procedure was carried out at room temperature. Purified PD-1-TST was immobilized on MaxiSorp ELISA plates (ThermoFisher #439454) for 30 min. Each well was rinsed three times with 100 μL of washing buffer (WB: PBS + 0.5% (w/v) BSA + 0.05% (w/v) Tween20), then incubated for 1 hour with 50 µl WB containing the indicated antibody (1, 0.1 or 0.01 µg/ml) (Fig. 1). After rinsing 3 times (100 µl WB), wells were incubated with horseradish peroxidase-coupled goat anti-rabbit IgG (Sigma #A8275, 1:1000, 50 μL per well) for 30 min. After 3 rinses, Tetramethylbenzidine (TMB) substrate (Sigma #T5569) was added (50 μL per well). The reaction was stopped by the addition of 25 μL of 2 M H₂SO₄. The absorbance (OD) was measured at 450 nm, and the absorbance at 570 nm was subtracted.

Western blot: HeLa cells (5 x 10⁶) expressing the PD-1-TM-HA fusion protein, or not, were lysed in 400 µL Lysis buffer (PBS + 0.5% TX100 + protease inhibitors (20 µg/mL leupeptin, 20 µg/mL aprotinin, 18 µg/mL PMSF, 1.8 mg/mL iodoacetamide) for 15 min at 4°C, then centrifuged at 10’000g to remove unlysed material. The cleared cell lysate was then mixed to an equivalent volume of 4x-concentrated non-reducing Sample buffer (20.6% (w/v) sucrose, 100 mM Tris pH 6.8, 10 mM EDTA, 0.1% (w/v) bromophenol blue, 4% (w/v) SDS, no -mercaptoethanol). The proteins were loaded on an SDS-PAGE (10 µL per lane), separated by electrophoresis (200 V, 30 min) on a 4-15% acrylamide gel (Mini-PROTEAN® TGX™ Precast Gel, Bio-Rad #456-1086), and transferred to a nitrocellulose membrane using a dry transfer system for 10 min (iBlot gel transfer device, Invitrogen #IB1001EU). The membranes were blocked overnight at 4°C in PBS containing 0.1% (v/v) Tween20 and 7% (w/v) milk, then washed three times for 15 min in PBS + 0.1% (v/v) Tween20. The membranes were incubated with the indicated anti-PD-1 antibodies (1 µg/mL in PBS-Tween) overnight at 4°C, then washed three times for 15 min. As a control, the anti-HA antibody was used. The membranes were then incubated 1 h with horseradish peroxidase-coupled goat anti-rabbit IgG (Novex, dilution 1:3000) and washed 5 times for 5 min in PBS-Tween. The signal was revealed by enhanced chemiluminescence (ECL) (Amersham Biosciences) using a PXi-4 gel imaging system (Syngene).

Immunoprecipitation: To assess the ability of antibodies to immunoprecipitate the PD-1 protein, individual antibodies (1 µg) were incubated with 50 µL of Protein G-Sepharose (GE Healthcare 17-0618-01) for 1 h at room temperature. The Sepharose resin with the bound antibodies was washed 3 times with 1mL PBS, then incubated for 1 h at 4°C with a cleared lysate of 10⁶ HeLa cells transiently transfected to express the PD-1-TM-HA fusion protein, obtained as described above by lysing 10⁷ cells in 10 mL Lysis buffer. The resin was then washed 5 times at 4°C with PBS+0.1% TX-100, one time with PBS, then resuspended in reducing Sample buffer (containing 6% (v/v) -mercaptoethanol), migrated on an SDS-PAGE gel and transferred to a nitrocellulose membrane. The PD-1-TM-HA protein was revealed using the AF291 (anti-HA) antibody fused to a mouse Fc and a secondary HRP-coupled anti-mouse Fc antibody (Novex, dilution 1:3000).

Immunofluorescence: The whole procedure was carried out at room temperature. Transfected HeLa cells expressing transiently the PD-1-TM-HA fusion protein were rinsed once with PBS, fixed with PBS + 4% paraformaldehyde (w/v) (Applichem, #A3013) for 30 min, and blocked with PBS + 40 mM ammonium chloride (NH₄Cl) (Applichem, #A3661) for 5 min. Cells were then permeabilized in PBS + 0.2% saponin (w/v) (Sigma, #S7900) for 10 min, washed once (5 min) with PBS + 0.2% (w/v) BSA (PBS-BSA), and incubated for 30 min with the rabbit anti-PD-1 antibodies (1 µg/mL) and a mouse AF291 anti-HA. After 3 washes (5 min) with PBS-BSA, cells were incubated for 30 min in PBS-BSA with a secondary anti-rabbit IgG conjugated to AlexaFluor-488 (1:400, Molecular Probes, #A11029) and with an AlexaFluor-647 conjugated anti-mouse IgG (1:400, Molecular Probes, #A11029). After 3 washes (5 min) with PBS-BSA, cells were mounted on slides (Menzel-Gläser, 76x26 mm) with Möwiol (Hoechst) + 2.5% (w/v) DABCO (Fluka, #33480). Pictures were taken with identical settings using a Zeiss LSM700 confocal microscope, with a 63x Neofluar oil immersion objective.

Results

When tested by ELISA, all antibodies bound PD-1 immobilized on ELISA plates efficiently and in a concentration-dependent manner (Fig. 1).

Figure 1. Specific binding of antibodies to PD-1 as detected by ELISA. The AS298 therapeutic antibody, the newly identified RC105 nanobody and newly identified RC226, RC227 and RC229 human antibodies all bound PD-1 efficiently.

We next tested the ability of the same panel of antibodies to bind and immunoprecipitate an HA-tagged transmembrane form of PD-1 (PD-1-TM-HA). For this we used a lysate of HeLa cells expressing PD-1-TM-HA. Each antibody was immobilized on Protein G-Sepharose beads, then incubated with a cell lysate containing PD-1-TM-HA. After washing the beads, the PD-1-TM-HA protein bound to the antibody was migrated on an SDS-PAGE gel, transferred to nitrocellulose and detected using a rabbit anti-HA antibody (Fig. 2). As expected, PD-1-TM-HA was efficiently immunoprecipitated by an anti-HA antibody. It appeared as a mixture of a ≈38 kDa monomer (star) and ≈80 kDa dimer (arrowhead). AS298 efficiently immunoprecipitated PD-1. RC105, RC226 and RC229 did not, while RC227 exhibiteda very weak specific signal (Fig. 2).

Figure 2. Immunoprecipitation of PD-1-TM-HA. PD-1-TM-HA was immunoprecipitated from a lysate of HeLa cells expressing PD-1-TM-HA (PD-1), or not (non-transfected: NT). The immunoprecipitated protein migrated mostly at the expected size of a monomer (≈38 kDa; star) and a smaller fraction as a dimer (≈80 kDa; arrowhead) on an SDS-PAGE gel and was revealed by western blot using an anti-HA antibody (mouse AF291). The size of the MW controls (15-170 kDa) is indicated on the left.

To test the ability of the same panel of antibodies to recognize PD-1 by immunofluorescence, we used HeLa cells expressing PD-1-TM-HA. Cells were fixed, permeabilized and incubated with individual anti-PD-1 antibodies, and then with a fluorescent secondary antibody (Fig. 3; PD-1; green). In order to identify transfected cells, the cells were also labelled with an anti-HA antibody (Fig. 3; Tag HA; red). In the absence of primary antibodies, no signal was observed (Fig. 3A). On the contrary, all antibodies tested recognized efficiently PD-1 (Fig. 3B, C, D, E, F).

Figure 3. Staining of PD-1-TM-HA by double immunofluorescence. HeLa cells expressing a PD-1-TM-HA fusion protein were stained with an antibody against PD-1 (PD-1; green) and an antibody against the HA tag (Tag HA; red). Bar: 10 µm.

Finally, we tested the ability of each antibody to recognize PD-1 by western blot. For this, lysates from cells expressing the PD-1-TM-HA fusion protein were migrated on an SDS-PAGE gel, transferred to nitrocellulose, and incubated with the indicated antibodies (Fig. 4). The AS298 antibody efficiently recognized PD-1-TM-HA, as expected. The newly identified anti-PD-1 antibodies also detected PD-1, but less efficiently, with RC227 as the most efficient of them.

Figure 4. Detection of the PD-1 protein by western blot. Cell lysates containing the PD-1-TM-HA fusion protein (PD-1) or not (Non-transfected, NT) were migrated on an SDS-PAGE gel, transferred to nitrocellulose and revealed with the indicated anti-PD-1 antibody. The size of the MW controls (15-170 kDa) is indicated on the left.

Discussion

In this study, we isolated and characterized a panel of novel recombinant antibodies targeting human PD1 and evaluated their ability to recognize PD1 across a range of commonly used experimental applications. A previously validated therapeutic antibody was included as a positive control for comparison. All antibodies tested detected PD1 by ELISA and immunofluorescence. In western blot analyses, RC105, RC226, RC227, and RC229 also recognized PD1, although with substantially lower efficiency than the control antibody AS298. Similarly, RC227 was able to immunoprecipitate PD1, but again with markedly reduced efficiency relative to AS298. These results are summarized in Figure 5.

Overall, the newly selected recombinant antibodies performed well in assays that preserve the native conformation of PD1, such as ELISA and immunofluorescence, but showed reduced performance in techniques that can disrupt protein folding, including western blotting and immunoprecipitation. This pattern is likely a direct consequence of the phage display selection strategy, which prioritizes binding to the native antigen.

In this study and in the complementary report on CD33 antibodies (Dhungana et al., 2026), we developed fully recombinant antibodies against two targets by phage display and compared their performance with established animal-derived and/or therapeutic antibodies in various experimental applications. These recombinant antibodies achieved performance at least comparable to animal-derived antibodies. Additional steps in their further validation would be to determine their recognition and binding of PD-1 in cells that express the protein naturally, for example macrophages, B lymphocytes, dendritic cells, monocytes, activated T cells, myeloid cells and natural killer cells under conditions of chronic antigen exposure.

The technological progress in this field in recent years is such that replacement of animal use is no longer a theoretical prospect; rather, the development of a wide range of high-quality recombinant antibodies has become a reality. These findings support the continued development of recombinant antibodies for a wide range of applications, and with this goal in mind we review recommendations that have been made over recent years in a complementary Commentary article, which stresses that all stakeholders in research involving antibody use can adopt measures that will reduce research waste, increase replicability and human relevance, and take a more ethical/Three Rs-compliant stance (Bailey et al., 2026).

Figure 5. Summary of anti-PD-1 reactivity in different experimental procedures.

Conflict of interest

MD is supported by funding from ABCD Antibodies SA

Data Availability Statement

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