4-1BB Bioassay

Simple Bioassay, Consistent Results

4-1BB (CD137/TNFRSF9), a member of the tumor necrosis factor receptor superfamily, is an inducible co-stimulatory receptor expressed on T cells, natural killer (NK) cells and innate immune cell populations. When present on the cell surface, 4-1BB interacts with 4-1BB ligand (4-1BBL) and induces subsequent cell proliferation and production of interferon gamma (IFNγ) and interleukin-2 (IL-2), particularly in T and NK cells.

Current methods used to measure the activity of biologic drugs targeting 4-1BB rely on assays that are laborious and highly variable due to their reliance on donor primary cells, complex assay protocols and unqualified assay reagents and, as a result, are difficult to establish in a quality-controlled, drug-development setting.

The 4-1BB Bioassay is a biologically relevant, MOA-based assay that can be used to measure the potency and stability of ligands or agonist antibodies that can bind and activate 4-1BB.

Control Ab, Anti-4-1BB, is a mouse anti-human 4-1BB agonist antibody. It can be used as a positive control in the 4-1BB Bioassay in the presence of FcγRIIb CHO-K1 Cells (Cat.# JA2251, JA2255).

Assay Components

The thaw-and-use format of the assay (Cat.# JA2351, JA2355) offers convenience and minimizes variability. The assay consists of a genetically engineered Jurkat T cell line that expresses human 4-1BB and a luciferase reporter driven by a response element that can respond to 4-1BB ligand/agonist antibody stimulation. The 4-1BB Effector Cells included are provided as cryopreserved cells that can be thawed, plated and used in an assay without the need for cell propagation.

The assay is also available in CPM format (Cat.# J2332) containing cryopreserved cells that can be thawed, propagated and banked for long-term use.

Workflow

Standardized Reagents Improve Results

Thaw-and-use 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.

Assay Principle

4-1BB Bioassay schematic showing assay with FcγRIIb-dependent agonist antibody.

4-1BB Bioassay schematic showing assay with FcγRIIb-independent agonist antibody or ligand.

Panel A. Assay with FcγRIIb-dependent agonist antibody. The bioassay consists of two engineered cell lines, 4-1BB Effector Cells and FcγRIIb CHO-K1 Cells. In the presence of FcγRIIb CHO-K1 Cells, the anti-4-1BB antibody can be crosslinked, thereby inducing 4-1BB pathway-activated luminescence. Panel B. Assay with FcγRIIb-independent agonist antibody or ligand. The bioassay consists of one engineered cell line, 4-1BB Effector Cells. In the absence of agonist antibody or 4-1BB ligand, the 4-1BB receptor is not activated and luminescence signal is low. The addition of agonist antibody or 4-1BB ligand induces the 4-1BB pathway-activated luminescence, which can be detected in a dose-dependent manner.

Assay Performance Data

Accuracy and precision are critical to bioassays and the development of biologics. 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 how close results are to 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 4-1BB Bioassay Shows Precision, Accuracy and Linearity.

Parameter	Results
Accuracy	% Expected Relative Potency	% Recovery
	50	106.0
	70	96.2
	140	97.3
	200	103.2
Repeatability (% CV)	100% (Reference)	3.5
Intermediate Precision (% CV)		6.6
Linearity (r²)		0.996
Linearity (y = mx + b)		y = 1.025x – 1.835
A 50–200% theoretical potency series of Control Ab, Anti-4-1BB, 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.

4-1BB Bioassay Sensitivity and MOA Panel A 15367ma-a

4-1BB Bioassay sensitivity and MOA schematic Panel B 15367ma-b

The 4-1BB Bioassay reflects mechanism of action and shows specificity of biologics designed to activate 4-1BB. Panel A. 4-1BB Effector Cells were induced with a serial titration of ligands: GITRL, 4-1BBL and OX40L as indicated. Panel B. 4-1BB Effector Cells were induced with a serial titration of Control Ab, Anti-GITR (Cat.# K1171), Control Ab, Anti-4-1BB (Cat.# K1161), and Control Ab, Anti-OX40 (Cat.# K1191), as indicated, in the presence of FcγRIIb CHO-K1 Cells (Cat.# JA2251, JA2255). For both panels, Bio-Glo™ Reagent was added, and luminescence was quantified using the GloMax® Discover System.

4-1BB Bioassay is available in 1X and 5X sizes.
4-1BB Bioassay is also available in CPM format (Cat.# J2332).
Control Ab, Anti-4-1BB (Cat.# K1161), is available separately.
FcγRllb CHO-K1 Cells (Cat.# JA2251, JA2255) are available separately.

Protocols

Complete Protocol

4-1BB BioassayPDF (2176 KB) – English

4-1BB Bioassay, Propagation ModelPDF (2182 KB) – English

What's in the box?

Item	Part #	Size
4-1BB Effector Cells	J235A	1 × .5ml
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

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Certificate of Analysis

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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
4-1BB Effector Cells	J235A	5 × .5ml
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.

Patent Pending.

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

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

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.

What's in the box?

Item	Part #	Size
4-1BB Effector Cells (CPM)	J233A	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-4-1BB	K116A	1 × 50μg	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.

4-1BB Bioassay

A Simpler, More Consistent 4-1BB Bioassay