The Immune Checkpoint Pathway - PD-1 Recombinant Protein Collection

Immune checkpoint pathway is the focus of current cancer research, and one of the best-characterized checkpoint proteins is PD-1. T-cell activation is suppressed by the binding that occurs between PD-1 and its ligand PD-L1 and/or PD-L2, and allows cancer cells to escape from the body’s immune surveillance. This kind of mechanism encourages the idea that neutralization of the PD-1 pathway could help in re-engaging the immune system to combat cancer.

In the past few years, multiple lines of anti-PD-1 drugs have been formulated, and many have demonstrated significant potential in recent clinical trials. Patients afflicted with renal cell carcinoma, melanoma, hematological cancers, or non-small cell lung cancer (NSCLC) have been shown to respond positively to this therapy.

ACROBiosystems offers a complete range of PD-1 related products for researchers to study and target the PD-1 pathway.

PD1 Pathway Targets

Key Features

Biotinylated PD-1 Pathway Proteins

ELISA

The assay technique, ELISA, is routinely used for characterizing interactions between proteins. It is usually applied in initial high-throughput screening, or HTS, of compound library. A series of pre-biotinylated PD-1 pathway proteins has been developed by ACROBiosystems. Biotinylated proteins can act as two-way antibodies for capture as well as detection in ELISA assays. Compared to traditional antibodies, the biotinylated proteins show higher specificity and detection sensitivity.

Figure 1. Immobilized human PD-1 (Cat. No. PD1-H5257) at 2 μg/mL (100 μL/well) can bind to biotinylated human PD-L1, His & Fc tag (Cat. No. PD1-H82F3) with a linear range of 0.01—0.25 μg/ml.

Suggested Pairs

	Product Description	Cat. No.	Species	Structure
Pair 1	Human PD-L1 / B7-H1 Protein, Fc Tag, low endotoxin	PD1-H5258
	Biotinylated Human PD-1 / PDCD1, His Tag & Fc Tag, Avi Tag (AviTagTM)	PD1-H82F2
Pair 2	Mouse PD-L1 / B7-H1 Protein, Fc Tag	PD1-M5251
	Biotinylated Mouse PD-1 / PDCD1, Fc Tag, Avi Tag (AviTagTM)	PD1-M82F4
Pair 3	Human PD-1 / PDCD1 Protein, Fc Tag (HPLC-verified), low endotoxin	PD1-H5257
	Biotinylated Human PD-L1 / B7-H1, His Tag & Fc Tag, Avi Tag (AviTagTM)	PD1-H82F3
Pair 4	Mouse PD-1 / PDCD1 Protein, Fc Tag	PD1-M5259
	Biotinylated Mouse PD-L1 / B7-H1, Fc Tag, Avi Tag (AviTagTM)	PD1-M82F5

A PD-1[Biotin]: PD-L1 Inhibitor Screening Kit (Cat. No. EP-101) has been developed to further simplify research processes. This assay uses a simple colorimetric ELISA platform, which determines the binding between the in-house developed biotinylated PD-1 protein and the immobilized human PD-L1 protein. This product is optimized for rapid and high-throughput screenings of putative PD-1 and PD-L1 inhibitors.

Molecule	Cat. No.	Product Description	Species	Size
PD-1 & PD-L1	EP-101	PD-1[Biotin]: PD-L1 Inhibitor Screening Pair		96 tests, 480 tests

Figure 2. Immobilized PD-L1 at 2 μg/mL (100 μL/well) can bind biotinylated human PD-1 with a linear range of 0.038 - 0.6 μg/mL when detected by Streptavidin-HRP.

Figure 3. Inhibition of PD-1-PD-L1 binding by an anti-PD-1 neutralizing antibody is measured by the PD-1 [Biotinylated]: PD-L1 Inhibitor Screening ELISA Assay Pair (Cat. No. EP-101).

FAC Sorting

Biotinylated proteins can be used together with fluorophore-tagged SA for detecting/isolating cells that express specific surface markers. In a US patent titled “ANTI-PD-1 ANTIBODIES AND METHODS OF USE THEREOF” (US patent 20160159905), the authors from Pfizer Inc. applied ACROBiosystems’ biotinylated PD-L1 (Cat. No. PD1-H82F3) as well as biotinylated PD-L2 (Cat. No. PD2-H82F6) to contend with candidate antibodies in a binding assay against PD-1 protein expressing cells. This assay offers measurement for the binding activity of the corresponding antibodies.

Biopanning

Biopanning is progressively becoming one of the best mainstream methods for developing antibodies. AviTag^TM biotinylated proteins can be applied with SA-coated magnetic beads/surfaces in biopanning. This provides a much better option when compared to chemically biotinylated or non-biotinylated proteins because it offers uniformed antigen presentation and higher coating density.

It is worth noting that the Fc tag is not usually preferred for these kinds of experiments because it produces false positive results. ACROBiosystems has developed AviTagTM PD-1 pathway proteins with just His tag attached. These products are recommended specifically for biopanning.

Molecule	Cat. No.	Species	Structure	Purity	Features
PD-1	PD1-H82E4
PD-L1	PD1-H82E5
PD-L2	PD2-H82E8

Cell-Based Assay Verified Proteins

Being a professional supplier of pharmaceutical companies, ACROBiosystems has established a cell-based assay platform to validate the target protein’s activity, which provides cell-based experiment data support for screening drugs.

ACROBiosystems employed human PD-1 overexpressed 293 cells that were developed in-house in flow cytometry assay and cell-based ELISA, and the outcomes demonstrated good performance of PD-L1 (Cat. No. PDI-H5258) and PD-L2 (Cat. No. PD2-H5251) binding to PD-1.

FACS Analysis of PD-L1 or PD-L2 Binding to Cell Surface PD-1

Figure 4. Flow Cytometry assay shows that recombinant human PD-L1 (Cat. No. PD1-H5258) can bind to 293 cell overexpressing human PD-1. The concentration of PD-L1 used is 10 µg/mL.

Figure 5. Flow Cytometry assay shows that recombinant human PD-L2 (Cat. No. PD2-H5251) can bind to 293 cell overexpressing human PD-1. The concentration of PD-L2 used is 1µg/mL.

Validated Applicable for Screening of Neutralizing Antibody

Figure 6. FACS analysis shows that the binding of PD-L1 (Cat. No. PD1-H5258) to 293 overexpressing PD-1 was inhibited by increasing concentration of neutralizing anti-hPD-L1 antibody. The concentration of PD-L1 used is 10 μg/mL. The IC50 is 12.92 μg/mL.

Cell-Based ELISA

Figure 7. Immobilized cell surface PD-1 (5x10⁴ of cells per well) can bind Human PD-L1, Fc Tag (Cat. No. PD1-H5258) with an EC50 of 0.029 μg/mL.

Figure 8.  Immobilized cell surface PD-1 (5x10⁴ of cells per well) can bind Human PD-L2, Fc Tag (Cat. No. PD2-H5251) with an EC50 of 0.018 μg/mL.

Latest Citations

1. Hydrogel dual delivered celecoxib and anti-PD-1 synergistically improve antitumor immunit. Authors: Li Y, et al. Journal: Oncoimmunology Application: ELISA Product: PD1-M5228 Request for Full-text
2. Hererocyclic compounds as immunomodulators. Authors: L Wu, et al. Journal: US20170107216A1 Application: HTRF Binding Assay Product: PD1-H5229
3. Hererocyclic compounds as immunomodulators. Authors: J Li, et al. Journal: US20170145025A1 Application: HTRF Binding Assay Product: PD1-H5229
4. A novel PD-L1-targeting antagonistic DNA aptamer with antitumor effects. Authors: Lai WY, et al. Journal: Mol Ther Nucleic Acids Application: ELISA-based competition assay Product: PD1-H5229
5. Anti-pd-1 antibodies and methods of use thereof. Authors: Yasmina Noubia Abdiche, et al. Journal: US20160159905A1 Application: Flow Cytometry Product: PD1-H82E5
6. Anti-pd-1 antibodies and methods of use thereof. Authors: Yasmina Noubia Abdiche, et al. Journal: US20160159905A1 Application: Flow Cytometry Product: PD2-H82E8

References

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ACROBiosystems

ACROBiosystems is a cornerstone enterprise of the pharmaceutical and biotechnology industries. Their mission is to help overcome challenges with innovative tools and solutions from discovery to the clinic. They supply life science tools designed to be used in discovery research and scalable to the clinical phase and beyond. By consistently adapting to new regulatory challenges and guidelines, ACROBiosystems delivers solutions, whether it comes through recombinant proteins, antibodies, assay kits, GMP-grade reagents, or custom services. ACROBiosystems empower scientists and engineers dedicated towards innovation to simplify and accelerate the development of new, better, and more affordable medicine.

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