Recombinant Human PD1-Fc Fusion Protein

Soluble human PD-1 (CD279) fused to an IgG1 Fc domain

Potential applications of soluble hPD1-Fc protein

Protein description

InvivoGen offers hPD1-Fc, a soluble human PD-1 (CD279) chimera protein generated by fusing the N-terminal extracellular domain of human PD-1 (aa 24-167) to the N-terminus of a human IgG1 Fc domain with a TEV (Tobacco Etch Virus) sequence linker.
hPD1-Fc has been produced in CHO cells and purified by affinity chromatography. It has an apparent molecular weight of ~62 kDa on an SDS‑PAGE gel.

PD-1 background

Programmed cell death 1 (PD-1), also known as cluster of differentiation 279 (CD279) is a transmembrane protein expressed at the cell surface of activated and exhausted conventional T cells. PD-1 is an inhibitory immune checkpoint that prevents T cell overstimulation and host damage. PD-1 interaction with its ligands PD-L1 and PD-L2 induces inhibition of TCR signaling [1].

 More details

Applications

• Screening of high-affinity anti-human PD-1 monoclonal antibodies by ELISA
• Screening of anti-human PD-L1 monoclonal antibodies using competition assays

Quality control

• Size and purity confirmed by SDS PAGE
• Protein validated by ELISA using Anti-hPD1-Ni-hIgG1 and Anti-hPD1-Pem-hIgG1 mAbs
• Protein validated by FACS using Raji-APC-hPD-L1 cells

References

1. Juneja, V.R. et al. 2017. PD-L1 on tumor cells is sufficient for immune evasion in immunogenic tumors and inhibits CD8 T cell cytotoxicity. J Exp Med 214, 895-904.
2. Kythreotou, A. et al. 2018. PD-L1. J Clin Pathol 71, 189-194.

Figures

SDS-PAGE analysis of the hPD1-Fc protein. 0.5 µg of the fusion protein was loaded on a 12% Mini-PROTEAN® TGX Stain-Free™ Precast Gels (Bio-Rad). Detection was performed as per the manufacturer’s instructions.

Human PD-L1 cell surface detection using hPD1-Fc. ~5 x 10⁵ Raji-APC-hPD-L1 cells were incubated with 2 µg of hPD1-Fc for 45 min at 4°C. Cells were then washed and incubated with 1 µg of mouse anti-human IgG Fc antibody coupled to PE for 1h at 4°C. Cell surface staining was analyzed by flow cytometry.

ELISA detection of hPD1-Fc with Anti-hPD1 mAbs. hPD1-Fc fusion protein (1 µg/ml) was coated on ELISA plates overnight. A 2-fold serial dilution of Anti-hPD1-hIgG1 Nivolumab biosimilar (red curve) or Pembrolizumab biosimilar (orange curve), or Anti-β-Gal-hIgG1 control mAb (grey curve) was performed for the capture step. An HRP-labeled anti-human κ light chain antibody (1/1000 dilution) and the HRP substrate OPD (o-phenylenediamine dihydrochloride) were used for the detection step. Absorbance was read at 490 nm.

Specifications

Protein construction: N-terminal extracellular domain of human PD-1 (aa 24-167) with a C-terminal human IgG1 Fc tag 

Accession sequence: XP_006712636.1

Species: Human

Source: CHO cells

Tag: C-terminal human IgG1 Fc

Total protein size: 388 a.a (secreted form)

Molecular weight: ~62 kDa (SDS-PAGE)

Purification: Protein G affinity chromatography

Purity: >97% (SDS-PAGE)

Quality control:

• The protein has been validated by ELISA upon incubation with  Anti-hPD1-Ni-hIgG1 and Anti-hPD1-Pem-hIgG1.
• The protein has been validated by flow cytometry using Raji-APC-hPD-L1 cells
• ​The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cellular assays.

Contents

• 50 µg lyophilized hPD1-Fc
• 1.5 ml endotoxin-free water

 The product is shipped at room temperature.

 Store lyophilized product at -20°C.

 Resuspended protein is stable for up to 1 month when stored at 4°C, and 1 year when stored at -20°C

Avoid repeated freeze-thaw cycles.

Details

Immune checkpoint signal: PD-1 and PD-L1

— PD-1 (programmed cell death 1; also known as CD279) is a type I transmembrane protein expressed at the cell surface of activated and exhausted conventional T cells. PD-1 is an inhibitory immune checkpoint that prevents T-cell overstimulation and host damage. PD-1 interaction with its ligands PD-L1 and PD-L2 induces inhibition of TCR signaling [1].

— PD-L1 (programmed cell death ligand 1; also known as CD274 or B7-H1) is a transmembrane protein expressed at the cell surface of hematopoietic and non-hematopoietic cells and is induced by pro-inflammatory cytokines, such as in the tumor microenvironment  [1].  PD-L1 is one ligand for PD-1, an inhibitory immune checkpoint receptor that is expressed by activated and exhausted T cells. PD-1:PD-L1 interaction induces inhibition of TCR signaling, thereby preventing T-cell overstimulation and host damage [1].

PD-L1 expression is an immune evasion mechanism exploited by various malignancies and is generally associated with poorer prognosis [2]. Specifically, over-expressed PD-L1 on tumor cells and tumor-infiltrating immune cells, such as macrophages, can bind to PD-1 on cytotoxic T cells, and ultimately inhibit the anti-tumor T cell response [3, 4]. Thus, due to PD-L1’s instrumental role in immune evasion by cancer cells, there are numerous inhibitors in development as promising immuno-oncology therapies. Notably, Atezolizumab (also known as MPDL3280A), a fully humanized IgG1 (N298A) mAb that blocks the interaction of PD-L1 with PD-1 and induces anti-tumor immune reactivation, has been approved by the FDA for combinational use in the treatment of lung and breast cancer [3, 5].

References

1. Ribas A. and Wolchock J.D., 2018. Cancer immunotherapy using checkpoint blockade. Science. 359:1350-55.
2. Sun, C. et al. 2018. Regulation and Function of the PD-L1 Checkpoint. Immunity 48, 434-452. 
3. Kythreotou, A. et al. 2018.  PD-L1. J Clin Pathol 71, 189-194.
4. Lau, J. et al. 2017. Tumour and host cell PD-L1 is required to mediate suppression of anti-tumour immunity in mice. Nat Commun 8, 14572.
5. Heimes, A.S. & Schmidt, M. 2019. Atezolizumab for the treatment of triple-negative breast cancer. Expert Opin Investig Drugs 28, 1-5.