PD-1+TIGIT Combination Bioassay
Measure potency and stability of biologics designed to block immune checkpoint signals
- Prequalified according to ICH guidelines
- 96- and 384-well plate formats
- No primary cell culture required
- Supplied in convenient thaw-and-use format; also available in cell propagation model (CPM) format
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Catalog Number: J2211
Catalog Number: J2215
Catalog Number: J2102
Catalog Number: J2051
Catalog Number: J1921
Measure the ability of biologics to block immune checkpoint signals
PD-1 and TIGIT are both immune inhibitory receptors that are expressed on T cells. Engagement of PD-1 by PD-L1 on an adjacent cell inhibits TCR signaling and TCR-mediated proliferation, transcriptional activation and cytokine production. The ligand for TIGIT is CD155. TIGIT negatively regulates NK cell killing and T cell activation by competing with CD226 for binding to CD155 and by directly interfering with CD226 homodimerization.
Preclinical cancer studies suggest that targeted therapies that simultaneously block PD-1 and TIGIT are superior to those that block either pathway alone. The PD-1+TIGIT Combination Bioassay reflects the mechanism of action (MOA) of biologics designed to block the PD-1/PD-L1 and TIGIT/CD155 interactions.
Control Ab, Anti-TIGIT, is a mouse monoclonal antibody that shows blocking activity of TIGIT with its ligand, CD155. It is intended for use as a control with the PD-1+TIGIT Combination Bioassay and the TIGIT/CD155 Blockade Bioassay. These assays can be used to measure the potency and stability of antibodies and other biologics designed to block the TIGIT/CD155 interaction.
TIGIT Negative Cells are for use as a negative control in the TIGIT/CD155 Blockade Bioassay. TIGIT Negative Cells are activated by engagement of the TCR/CD3 complex and costimulatory receptor CD28 co-engagement.
Assay Components
The assay consists of two genetically engineered cell lines:
PD-1+TIGIT Effector Cells: Jurkat T cells engineered to express human PD-1 and human TIGIT with a luciferase reporter driven by a native promoter that can respond to both TCR activation and CD226 co-stimulation
PD-L1+CD155 aAPC/CHO-K1 Cells: CHO-K1 cells engineered to express human PD-L1 and human CD155 with an engineered cell-surface protein designed to activate the TCR complex in an antigen-independent manner
When the two cell types are co-cultured, PD-1 binds to PD-L1 and inhibits TCR-induced activation and promoter-mediated luminescence. In addition, TIGIT inhibits CD226 activation and promoter-mediated luminescence. Addition of an anti-PD-1 Ab or an anti-TIGIT Ab blocks the interaction of PD-1 with PD-L1 or TIGIT with CD155, respectively, and results in a modest enhancement in promoter-mediated luminescence. Addition of antibodies that block both PD-1/PD-L1 and TIGIT/CD155 will result in much higher promoter-mediated luminescence due to synergy between these pathways.
The PD-1+TIGIT Combination Bioassay, Propagation Model, allows propagation and banking of the PD-1+TIGIT Effector Cells. Bio-Glo™ Luciferase Assay System (Cat.# G7940, G7941) is the required reagent for use with the PD-1+TIGIT Combination Bioassay, Propagation Model.
The Workflow
Traditional Method
'Cells as reagents' Method
Standardized Reagents Improve Results
- Thaw-and-use cell format provides significant time and labor savings over traditional methods while reducing variability.
- Kit includes all of the required reagents in standardized formats.
- Easily amenable to high-throughput workflows.
The Assay Principle
The PD-1+TIGIT Combination Bioassay is a bioluminescent cell-based assay that overcomes the limitations of existing assays and can be used to measure the potency and stability of antibodies and other biologics targeting PD-1 and TIGIT.
Representation of the PD-1+TIGIT Combination Bioassay. The bioassay consists of two genetically engineered cell lines, PD-1+TIGIT Effector Cells and PD-L1+CD155 aAPC/CHO-K1 Cells. Panel A. When co-cultured, PD-1 inhibits TCR pathway-activated luminescence, and TIGIT inhibits CD226 pathway-activated luminescence. The addition of anti-PD-1 Ab blocks PD-1 binding to PD-L1, resulting in full TCR pathway activation. The addition of anti-TIGIT Ab blocks the TIGIT/CD155 interaction, thereby re-establishing CD226 pathway-activated luminescence. Blocking of PD-1/PD-L1 and TIGIT/CD155 can be detected in a dose-dependent manner by addition of Bio-Glo™ Reagent and quantitation with a luminometer. Panel B. When co-cultured with Effector Cells that do not express PD-1 or TIGIT (Cat.# J1920), TCR activation and CD226/CD155 induce luminescence.
Assay Performance Data
Accuracy and Precision are critical to bioassays and the development of biologics. The PD-1+TIGIT Combination Bioassay is both accurate and precise. Accuracy is a measure of how close results are to a target or reference value. Precision is a measure of repeatability. This assay was developed and prequalified according to ICH guidelines for accuracy and precision, giving it the performance characteristics required for many applications of a bioassay used for antibody drug discovery research and development.
Accuracy: Measure of ability to replicate a reference or "target" value.
IntraLab Precision: Measure of repeatability from multiple assays of the same homogeneous sample under the same operating conditions over a short time (same analyst; same lab; same day).
Intermediate Precision: Measure of repeatability of multiple assays of the same homogeneous sample with different analysts or equipment or on different days.
Linearity: Ability of your assay to provide measured values that are proportional to the concentration of the sample.
The PD-1+TIGIT Combination Bioassay Shows Precision, Accuracy and Linearity.
| Parameter | Results | |
|---|---|---|
| Accuracy | % Expected Relative Potency | % Recovery |
| 50 | 95.3 | |
| 70 | 105.5 | |
| 140 | 104.9 | |
| 200 | 109.9 | |
| Repeatability (% CV) | 100% (Reference) | 4.1 |
| Intermediate Precision (% CV) | 10.4 | |
| Linearity (r2) | 0.999 | |
| Linearity (y = mx + b) | y = 1.131x + 0.028 | |
| A 50–200% theoretical potency series of a 1:1 ratio of nivolumab (anti-PD-1 Ab) and Control Ab, Anti-TIGIT, was analyzed in triplicate in three independent experiments performed on three days by two analysts. Bio-Glo™ Reagent was added and luminescence quantified using the GloMax® Discover Detection System. Data were analyzed and relative potencies calculated after parallelism determination using JMP® software. Data were generated using thaw-and-use cells. | ||
The PD-1+TIGIT Combination Bioassay reflects the mechanism of action (MOA) and specificity of biologics designed to block the PD-1/PD-L1 and TIGIT/CD155 interaction.
The PD-1+TIGIT Combination Bioassay measures the activity of Control Ab, Anti-PD-1, and Control Ab, Anti-TIGIT.
Product Kit Formats and Related Products
- PD-1+TIGIT Combination Bioassay is available in 1X and 5X sizes.
- PD-1+TIGIT Combination Bioassay is also available in CPM format (Cat.# J2102).
- Control Ab, Anti-TIGIT, is available separately (Cat.# J2051).
- TIGIT Negative Cells are available separately (Cat.# J1921).
Specifications
Catalog Number:
What's in the box?
| Item | Part # | Size |
|---|---|---|
PD-1+TIGIT Effector Cells |
J176A | 1 × 0.5ml |
PD-L1+CD155 aAPC/CHO-K1 Cells |
J184A | 1 × 0.5ml |
Fetal Bovine Serum |
J121A | 1 × 4ml |
Ham's F-12 Medium |
J123A | 1 × 25ml |
RPMI 1640 Medium |
G708A | 1 × 36ml |
Bio-Glo™ Luciferase Assay Buffer |
G719A | 1 × 10ml |
Bio-Glo™ Luciferase Assay Substrate |
G720A | 1 × 1 vial |
SDS
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Use Restrictions
Not For Medical Diagnostic Use.Storage Conditions
Please see the Protocol for detailed storage conditions.
NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE STATEMENT. If the recipient is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the recipient with a full refund.
This product may not be further sold or transferred by the recipient and may be used only by the recipient, and then only for (1) research use, (2) discovery, development and monitoring of biologic drugs and vaccines, (3) quality assurance testing of biologic drugs and vaccines, and (4) product release assays for biologic drugs and vaccines. No other commercial use is allowed. "Commercial use” means any and all uses of this product by recipient for monetary or other consideration, including providing a service, information or data to unaffiliated third parties, and resale of this product for any use. Recipient has no right to propagate, modify, derivatize, genetically engineer or otherwise create variations of the cells or genes stably transfected within the cells. In addition, recipient must use Bio-Glo™ Luciferase Assay System purchased from Promega Corporation for all luminescence assays using this product or contact Promega to obtain a license for use of this product with reagents other than Promega’s. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.
Licensed from Lonza Cologne GmbH under U.S. Pat. Nos. 7,700,357, 8,192,990 and 8,003,389, European Pat. Nos. 1297119, 1522587, 1607484 and 1741778 and other pending and issued patents.
What's in the box?
| Item | Part # | Size |
|---|---|---|
PD-1+TIGIT Effector Cells |
J176A | 5 × 0.5ml |
PD-L1+CD155 aAPC/CHO-K1 Cells |
J184A | 5 × 0.5ml |
Fetal Bovine Serum |
J121A | 5 × 4ml |
Ham's F-12 Medium |
J123A | 5 × 25ml |
RPMI 1640 Medium |
G708A | 5 × 36ml |
Bio-Glo™ Luciferase Assay Buffer |
G719A | 5 × 10ml |
Bio-Glo™ Luciferase Assay Substrate |
G720A | 5 × 1 vial |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
Not For Medical Diagnostic Use.Storage Conditions
Please see the Protocol for detailed storage conditions.
NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE STATEMENT. If the recipient is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the recipient with a full refund.
This product may not be further sold or transferred by the recipient and may be used only by the recipient, and then only for (1) research use, (2) discovery, development and monitoring of biologic drugs and vaccines, (3) quality assurance testing of biologic drugs and vaccines, and (4) product release assays for biologic drugs and vaccines. No other commercial use is allowed. "Commercial use” means any and all uses of this product by recipient for monetary or other consideration, including providing a service, information or data to unaffiliated third parties, and resale of this product for any use. Recipient has no right to propagate, modify, derivatize, genetically engineer or otherwise create variations of the cells or genes stably transfected within the cells. In addition, recipient must use Bio-Glo™ Luciferase Assay System purchased from Promega Corporation for all luminescence assays using this product or contact Promega to obtain a license for use of this product with reagents other than Promega’s. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.
Licensed from Lonza Cologne GmbH under U.S. Pat. Nos. 7,700,357, 8,192,990 and 8,003,389, European Pat. Nos. 1297119, 1522587, 1607484 and 1741778 and other pending and issued patents.
What's in the box?
| Item | Part # | Size |
|---|---|---|
PD-1+TIGIT Effector Cells (CPM) |
J172A | 2 × 1ml |
PD-L1+CD155 aAPC/CHO-K1 Cells (CPM) |
J183A | 2 × 1ml |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
Not For Medical Diagnostic Use.Storage Conditions
Please see the Protocol for detailed storage conditions.
NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE STATEMENT. If the recipient is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the recipient with a full refund.
This product may not be further sold or transferred by the recipient and may be used only by the recipient, and then only for (1) research use, (2) discovery, development and monitoring of biologic drugs and vaccines, (3) quality assurance testing of biologic drugs and vaccines, and (4) product release assays for biologic drugs and vaccines. No other commercial use is allowed. "Commercial use" means any and all uses of this product by recipient for monetary or other consideration, including providing a service, information or data to unaffiliated third parties, and resale of this product for any use. Recipient has no right to modify, derivatize, genetically engineer or otherwise create variations of the cells or genes stably transfected within the cells except that recipient may propagate and store the cells for long-term use. In addition, recipient must use Bio-Glo™ Luciferase Assay System purchased from Promega Corporation for all luminescence assays using this product or contact Promega to obtain a license for use of this product with reagents other than Promega's. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.
Licensed from Lonza Cologne GmbH under U.S. Pat. Nos. 7,700,357, 8,192,990 and 8,003,389, European Pat. Nos. 1297119, 1522587, 1607484 and 1741778 and other pending and issued patents.
What's in the box?
| Item | Part # | Size | Concentration |
|---|---|---|---|
Control Ab, Anti-TIGIT |
J205A | 1 × 100μg | 1mg/ml |
SDS
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Use Restrictions
Not For Medical Diagnostic Use.Storage Conditions
Please see the Protocol for detailed storage conditions.
The transfer of this product is conditioned on the buyer using the purchased product for (i) research use, (ii) discovery, development and monitoring of biologic drugs and vaccines, (iii) quality assurance and quality control testing of biologic drugs and vaccines, and (iv) product release assays for biologic drugs and vaccines conducted by the buyer, including contract research or any fee for service research, and the buyer must not (1) use this product or its components for (1a) any use, indirectly (except as allowed in (iii) and (iv) above) or directly in manufacturing, or (1b) diagnostic, therapeutic or prophylactic purposes; and/or (2) sell or transfer this product or its components for resale, whether or not resold for use in research.
What's in the box?
| Item | Part # | Size |
|---|---|---|
TCR/CD3 Effector Cells (IL-2) |
J129A | 1 × 0.8ml |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
Not For Medical Diagnostic Use.Storage Conditions
Please see the Protocol for detailed storage conditions.
NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE STATEMENT. If the recipient is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the recipient with a full refund.
This product may not be further sold or transferred by the recipient and may be used only by the recipient, and then only for (1) research use, (2) discovery, development and monitoring of biologic drugs and vaccines, (3) quality assurance testing of biologic drugs and vaccines, and (4) product release assays for biologic drugs and vaccines. No other commercial use is allowed. "Commercial use” means any and all uses of this product by recipient for monetary or other consideration, including providing a service, information or data to unaffiliated third parties, and resale of this product for any use. Recipient has no right to propagate, modify, derivatize, genetically engineer or otherwise create variations of the cells or genes stably transfected within the cells. In addition, recipient must use Bio-Glo™ Luciferase Assay System purchased from Promega Corporation for all luminescence assays using this product or contact Promega to obtain a license for use of this product with reagents other than Promega’s. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.
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