u n o- ncol m o m g i y pd1/pd-l1 binding assay · inhibitor tag1-pd-l1 tag2-pd1 anti-tag1-...

TECHNICAL NOTE PD1/PD-L1 BINDING ASSAY ABSTRACT Programmed cell death protein 1 (PD1) is an immune checkpoint receptor that regulates T cell response. Its ligand, programmed death-ligand 1 (PD-L1) is commonly over-expressed on tumor cell surface. When PD1 is bound with PD-L1, T cell response is suppressed, contributing to tumor immune resistance. Checkpoint inhibitors blocking PD1/PD-L1 complex formation are generating considerable interest in cancer immunotherapy. The HTRF PD1/PD-L1 Binding Assay is designed to measure the interaction between PD1 and PD-L1 proteins. Utilizing HTRF (Homogeneous Time-resolved Fluorescence) technology, the assay enables simple and rapid characterization of compound and antibody blockers in a high throughput format. INHIBITION TEST 2 µL compound/antibody 4 µL of PD-L1-Tag1 4 µL of PD1-Tag2 Incubate 15 min @ RT 5 µL of anti-Tag1-Eu 3+ 5 µL of anti-Tag2-XL665 or 10 µL of pre-mixed Anti-tag detection reagents Incubate 2 hours @ RT Reagents should be dispensed in the following order: • 2 µL compound/antibody or diluent buffer. • 4 µL PD-L1-Tag1. • 4 µL PD1-Tag2. Incubate at RT for 15min. • 5 µL of anti-Tag1-Eu 3+ and 5 µL of anti-Tag2-XL665 or 10 µL of pre-mixture of two conjugates. Seal the plate and incubate at RT for 2 hours. Remove the plate sealer and read the ﬂuorescence emission. Inhibitor Tag1-PD-L1 Tag2-PD1 Anti-Tag1- Cryptate Anti-Tag2- XL665 Diluent Detection buffer Sample 2 µL 4 µL 4 µL 5 µL 5 µL Positive control 4 µL 4 µL 5 µL 5 µL 2 µL Negative control 4 µL 5 µL 5 µL 6 µL Cryptate control 5 µL 10 µL 5 µL Buffer control 10 µL 10 µL For 96 & 384-well low volume plates (20 µL). I M M U N O - O N C O L O G Y

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T E C H N I C A L N O T E

PD1/PD-L1 BINDING ASSAY

ABSTRACT Programmed cell death protein 1 (PD1) is an immune checkpoint receptor that regulates T cell response. Its ligand, programmed death-ligand 1 (PD-L1) is commonly over-expressed on tumor cell surface. When PD1 is bound with PD-L1, T cell response is suppressed, contributing to tumor immune resistance. Checkpoint inhibitors blocking PD1/PD-L1 complex formation are generating considerable interest in cancer immunotherapy. The HTRF PD1/PD-L1 Binding Assay is designed to measure the interaction between PD1 and PD-L1 proteins. Utilizing HTRF (Homogeneous Time-resolved Fluorescence) technology, the assay enables

simple and rapid characterization of compound and antibody blockers in a high throughput format.

INHIBITION TEST

2 µL compound/antibody

4 µL of PD-L1-Tag1

4 µL of PD1-Tag2

Incubate

15 min @ RT

5 µL of anti-Tag1-Eu3+ 5 µL of anti-Tag2-XL665

or 10 µL of pre-mixed Anti-tag detection reagents

Incubate

2 hours @ RT

Reagents should be dispensed in the following order:

• 2 µL compound/antibody or diluent buffer.

• 4 µL PD-L1-Tag1.

• 4 µL PD1-Tag2.

Incubate at RT for 15min.

• 5 µL of anti-Tag1-Eu3+ and 5 µL of anti-Tag2-XL665 or 10 µL of pre-mixture of two conjugates.

Seal the plate and incubate at RT for 2 hours.

Remove the plate sealer and read the fluorescence emission.

Inhibitor Tag1-PD-L1 Tag2-PD1 Anti-Tag1- Cryptate

Anti-Tag2- XL665 Diluent Detection buffer

Sample 2 µL 4 µL 4 µL 5 µL 5 µL

Positive control 4 µL 4 µL 5 µL 5 µL 2 µL

Negative control 4 µL 5 µL 5 µL 6 µL

Cryptate control 5 µL 10 µL 5 µL

Buffer control 10 µL 10 µL

For 96 & 384-well low volume plates (20 µL).

IMM

UNO-ONCOLOG

Page 2: U N O- NCOL M O M G I Y PD1/PD-L1 BINDING ASSAY · Inhibitor Tag1-PD-L1 Tag2-PD1 Anti-Tag1- Cryptate Anti-Tag2- XL665 Diluent Detection buffer Sample 2 µL 4 µL 4 µL 5 µL 5 µL

FOR MORE INFORMATION Europe and other countries +33(0)466 796 705 U.S. and Canada 1-888-963-4567 China +86 10 8080 9288 Visit www.htrf.com to find a list of our regional distributors

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PD1/PD-L1 BINDING ASSAY

In order to determine apparent Kd values, PD-L1 was titrated from 0 to 50 nM at a saturating concentration of PD1 at 200 nM. The Kd of PD1 was obtained by titrating from 0 to 400 nM at the saturating concentration of PD-L1 at 20 nM. The two proteins were incubated for 2 hours at room temperature.

Apparent Kd values were calculated by fitting specific signal to One-site specific binding curve.

Specific signal for each ligand concentration = Total binding - Non specific binding

5 nM PD-L1 and 50 nM PD1 were chosen around apparent Kd values for inhibition tests.

INHIBITION OF PD1/PD-L1

The inhibitory effects of small molecules, human and mouse blocking antibodies of PD1 and PD-L1 were tested at 5 nM PD-L1 and 50 nM PD1. An assay window around 10 was obtained.

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