PD-1+TIGIT Combination Bioassay

by MyBio Ireland
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J2211

Description

  • Overview
  • Protocols
  • Specifications
  • Resources
  • 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. 

    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. 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 Workflow

    Traditional Method

    12052MA-W-a

    'Cells as reagents' Method

    12052MA-W-b

    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.

    14847ma-w
    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 for Assay Performance Data
    Accurate, but not precise

    Accuracy
    Measure of ability to replicated a reference or "target" value.
    IntraLab Precision
    Precise, but not accurate

    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 Data Target 3
    Accurate and precise

    Intermediate Precision

    Measure of repeatability of multiple assays of the same homogeneous sample with different analysts or equipment or on different days.

    32454752-data-linear 
    Linear

    Linearity

    Ability of your assay to provide measured values that are proportional to the concentration of the sample.

     

    Accuracy

    Expected Relative Potency Recovery
    50% 95.3%
    70% 105.5%
    140% 104.9%
    200% 109.9%

    Repeatability (100% reference, %CV)

    4.1%

    Intermediate Precision (%CV)

    10.4%

    Linearity

    r 2 = 0.999 (50-200% range)
    y = 1.131x + 0.028


    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.

    14848mb-w
    PD-1+TIGIT Effector Cells were incubated with PD-L1+CD155 aAPC/CHO-K1 Cells in the presence of serial titrations of Control Ab, Anti-TIGIT (Cat.# J2051), Control Ab, Anti-PD-1 (Cat.# J1201), anti-PD-L1 Ab (research grade) or anti-CTLA-4 (ipilimumab) blocking Abs as indicated. 
    14922ma-w
    PD-L1+CD155 aAPC/CHO-K1 Cells were plated overnight. The following day, a titration of a 1:1 ratio of either Control Ab, Anti-PD-1, and Control Ab, Anti-TIGIT, or a research-grade anti-PD-L1 Ab and Control Ab, Anti-TIGIT, was added, followed by PD-1+TIGIT Effector Cells. 
  • Specifications

    What's in the box?
    Item Part # Size

    PD-1+TIGIT Effector Cells

    J176A 1 × 1 vial

    PD-L1+CD155 aAPC/CHO-K1 Cells

    J184A 1 × 1 vial

    Fetal Bovine Serum

    J121A 1 × 4ml

    Bio-Glo™ Luciferase Assay Buffer

    G719A 1 × 10ml

    Bio-Glo™ Luciferase Assay Substrate

    G720A 1 × 1 vial

    Ham's F-12 Medium

    J123A 1 × 25ml

    RPMI 1640 Medium

    G708A 1 × 36ml

    Use Restrictions

    Not For Medical Diagnostic Use.

    Patents and Disclaimers

    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.

    Patent Pending.

    U.S. Pat. No. 8,008,006 and European Pat. No. 1341808.

    Product cannot be used for proficiency testing.

    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.

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