CTLA-4 Blockade Bioassay

Measure the ability of biologics to block immune checkpoint signals

CTLA-4 (Cytotoxic T-lymphocyte-associated protein 4), also known as CD152, is an immune inhibitory receptor constitutively expressed on regulatory T cells (Tregs) and upregulated in activated T cells. CTLA-4 plays a critical role in regulating immune responses to tumor antigens and autoantigens. When CTLA-4 expression is upregulated on the surface of T cells, the T cells bind B7 with a higher avidity, and thus outcompete the positive co-stimulatory signal from CD28. In addition, engagement of CTLA-4 by either of its ligands, CD80 (B7-1) or CD86 (B7-2) on an adjacent antigen presenting cell (APC) inhibits CD28 co-stimulation of T cell activation, cell proliferation and cytokine production.

The CTLA-4 Blockade Bioassay reflects the mechanism of action (MOA) of biologics designed to block the interaction of CTLA-4 with its ligands, CD80 and CD86.

Control Ab, Anti-CTLA-4, is a blocking antibody that shows blocking activity of CTLA-4 with its ligands CD80 and CD86. It is intended for use as a control with the CTLA-4 Blockade Bioassay, which can be used to measure the potency and stability of antibodies and other biologics designed to block the CTLA-4/CD80 and CD86 interaction.

Assay Components

The assay consists of two genetically engineered cell lines:

CTLA-4 Effector Cells: Jurkat T cells stably expressing human CTLA-4 and a luciferase reporter driven by a native promoter which responds to TCR/CD28 activation

aAPC/Raji Cells: Raji cells expressing an engineered cell surface protein designed to activate cognate TCRs in an antigen-independent manner and endogenously expressing CTLA-4 ligands CD80 and CD86

When the two cell types are co-cultured, CTLA-4 competes with CD28 for their shared ligands, CD80 and CD86, and thus inhibits CD28 pathway activation and promoter-mediated luminescence. Addition of an anti-CTLA-4 antibody blocks the interaction of CTLA-4 with its ligands CD80 and CD86 and results in promoter-mediated luminescence.

The CTLA-4 Blockade Bioassay is also available in CPM format, containing cryopreserved cells that can be thawed, propagated and banked for long-term use.

The Workflow

Traditional Method

Standard bioassay workflow with cultured cells

'Cells as reagents' Method

Improved bioassay workflow with thaw-and-use cells

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 CTLA-4 Blockade 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 CTLA-4.

14535ma-w-2 — **Representation of the CTLA-4 Blockade Bioassay.** The bioassay consists of two genetically engineered cell lines, CTLA-4 Effector Cells and aAPC/Raji Cells. **Panel A.** When co-cultured, the CTLA-4/CD80 and CD86 interaction inhibits CD28 pathway-activated luminescence. The addition of anti-CTLA-4 antibody blocks the CTLA-4/CD80 and CD86 interaction, thereby re-establishing CD28 pathway-activated luminescence, which 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 non-CTLA-4-expressing Effector Cells (Cat.# J1631), activation also induces luminescence by activation of the CD28 pathway but in a manner independent of anti-CTLA-4 antibody.

Assay Performance Data

Accuracy and Precision are critical to bioassays and the development of biologics. The CTLA-4 Blockade 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 the assay to provide measured values that are proportional to the concentration of the sample.

The CTLA-4 Blockade Bioassay Shows Precision, Accuracy and Linearity.

Parameter	Results
Accuracy	% Expected Relative Potency	% Recovery
	50	105.3
	70	96.7
	130	108.4
	150	112.3
Repeatability (% CV)	100% (Reference)	6.7
Intermediate Precision (% CV)		10.2
Linearity (r²)		0.991
Linearity (y = mx + b)		y = 1.175x – 11.45
A 50–150% theoretical potency series of ipilimumab (CTLA-4 blocking antibody) 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 CTLA-4 Blockade Bioassay reflects the mechanism of action (MOA) and specificity of biologics designed to block the CTLA-4/CD80 and CD86 interaction.

The CTLA-4 Blockade Bioassay measures the inhibitory activity of clinical grade anti-CTLA-4 blocking antibodies.

14536ma-w-2 — CTLA-4 Effector Cells were incubated with aAPC/Raji Cells in the absence or presence of anti-CTLA-4, anti-PD-1, anti-TIGIT or anti-HER2 blocking antibodies, as indicated.

14543ma-wa — CTLA-4 Effector Cells and aAPC/Raji Cells were incubated with increasing concentrations of anti-CTLA-4 antibody, ipilimumab, for 6 hours.

Product Kit Formats and Related Products

Thaw-and-Use format kits are available in 1X and 5X sizes.
Cell Propagation Model format is also available (Cat.# JA1400).
Control Ab, Anti-CTLA-4, is available separately (Cat.# JA1020).

Protocols

Complete Protocol

CTLA-4 Blockade Bioassay Technical ManualPDF (1143 KB) – English

CTLA-4 Blockade Bioassay Propagation Model Technical ManualPDF (1230 KB) – English

What's in the box?

Item	Part #	Size
CTLA-4 Effector Cells	J158A	1 × 0.8ml
aAPC/Raji Cells	J159A	1 × 0.8ml
Fetal Bovine Serum	J121A	1 × 4ml
RPMI 1640 Medium	G708A	1 × 36ml
Bio-Glo™ Luciferase Assay Buffer	G719A	1 × 10ml
Bio-Glo™ Luciferase Assay Substrate	G720A	1 × 1 vial

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

Not For Medical Diagnostic Use.

Storage Conditions

Please see the Protocol for detailed storage conditions.

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.

U.S. Pat. No. 8,008,006.

Patent Pending.

U.S. Pat. No. 10,077,244 and other patents.

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.

Product cannot be used for proficiency testing.

What's in the box?

Item	Part #	Size
CTLA-4 Effector Cells	J158A	5 × 0.8ml
aAPC/Raji Cells	J159A	5 × 0.8ml
Fetal Bovine Serum	J121A	5 × 4ml
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 SDS

Certificate of Analysis

Search by lot number

Use Restrictions

Not For Medical Diagnostic Use.

Storage Conditions

Please see the Protocol for detailed storage conditions.

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.

U.S. Pat. No. 8,008,006.

Patent Pending.

U.S. Pat. No. 10,077,244 and other patents.

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.

Product cannot be used for proficiency testing.

What's in the box?

Item	Part #	Size
CTLA-4 Effector Cells (CPM)	J146A	2 × 1ml
aAPC/Raji Cells (CPM)	J148A	2 × 1ml

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

Not For Medical Diagnostic Use.

Storage Conditions

Please see the Protocol for detailed storage conditions.

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 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.

U.S. Pat. No. 8,008,006.

Patent Pending.

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.

Product cannot be used for proficiency testing.

What's in the box?

Item	Part #	Size	Concentration
Control Ab, Anti-CTLA-4	JA102A	1 × 100μg	1mg/ml

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

Not For Medical Diagnostic Use.

Storage Conditions

Please see the Protocol for detailed storage conditions.

Resources

Articles

Measuring Blockade of Immune Checkpoint Targets with Promega Bioassays and GloMax® Discover

CTLA-4 Blockade Bioassay

Measure Potency and Stability of Biologics Designed to Block CTLA-4 Interactions

Measure the ability of biologics to block immune checkpoint signals

Assay Components

The Workflow

The Assay Principle

Assay Performance Data

The CTLA-4 Blockade Bioassay reflects the mechanism of action (MOA) and specificity of biologics designed to block the CTLA-4/CD80 and CD86 interaction.

The CTLA-4 Blockade Bioassay measures the inhibitory activity of clinical grade anti-CTLA-4 blocking antibodies.

Product Kit Formats and Related Products

Protocols

Complete Protocol

Specifications

What's in the box?

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Patents and Disclaimers

Specifications

What's in the box?

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Patents and Disclaimers

Specifications

What's in the box?

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Patents and Disclaimers

Specifications

What's in the box?

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Resources

Articles

Posters

Other Resources

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Frequently Used With

Bio-Glo™ Luciferase Assay System

GloMax® Discover System

RealTime-Glo™ MT Cell Viability Assay

CellTiter-Glo® 2.0 Cell Viability Assay

pHAb Reactive Dyes

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