LAG-3/MHCII Blockade Bioassay

Measure the ability of biologics to block immune checkpoint signals

The LAG-3/MHCII Blockade Bioassay is a bioluminescent cell-based assay that measures potency and stability of antibodies and other biologics designed to block the interaction of LAG-3 with its best characterized ligand, major histocompatibility complex II (MHCII). LAG-3, also known as CD223, is an immune checkpoint receptor expressed on activated CD4⁺ and CD8⁺ T cells and natural killer (NK) cells. LAG-3 plays a critical role in regulating immune responses to tumor antigens and autoantigens. Engagement of LAG-3 by MHCII inhibits TCR signaling, cytokine production and proliferation of activated T cells. Therapeutic antibodies designed to block the LAG-3/MHCII interaction show promising results in clinical trials for the treatment of a variety of cancers.

The LAG-3/MHCII Blockade Bioassay reflects the mechanism of action (MOA) of biologics designed to block the interaction of LAG-3 with its ligand, MHCII.

The Control Ab, Anti-LAG-3 is a mouse anti-human LAG-3 antibody that shows blocking activity for LAG-3 with its ligand MHCII. It is intended for use as a control with the LAG-3/MHCII Blockade Bioassay, which can be used to measure the potency and stability of antibodies and other biologics designed to block the LAG-3/MHCII interaction.

TCR Activating Antigen (Cat.# K1201) is an antigen that is presented by MHCII on MHCII APC Cells to specifically activate the TCR on the LAG-3 Effector Cells, designed for use with the LAG-3/MHCII Blockade Bioassay.

Assay Components

The assay consists of two cell lines:

LAG-3 Effector Cells: Jurkat T cells stably expressing human LAG-3 and a luciferase reporter driven by T-cell activation pathway-dependent response elements.

MHC APC Cells: APC Cells presenting TCR Activating Antigen, a proprietary peptide designed to specifically activate the TCR on the LAG-3 Effector Cells.

When the two cell types are co-cultured in the presence of antigen, MHCII on the APC cells presents the Ag to the TCR on the LAG-3 Effector Cells to activate the Effector Cells. LAG-3 on the Effector Cells inhibits TCR signaling and promoter-mediated luciferase activity. Addition of the anti-LAG-3 antibody releases LAG-3 mediated inhibition, resulting in promoter-mediated luminescence.

The LAG-3/MHCII Blockade Bioassay is also available in CPM format, containing cryopreserved cells that can be thawed, propagated and banked for long-term use.

The Workflow

Standard bioassay workflow with cultured cells

Improved bioassay workflow with thaw-and-use cells

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.

The Assay Principle

The LAG-3/MHCII 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 LAG-3.

Assay Performance Data

Accuracy and Precision are critical to bioassays and the development of biologics. The LAG-3/MHCII 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 replicated 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 CTLA-4 Blockade Bioassay Shows Precision, Accuracy and Linearity.

Parameter	Results
Accuracy	% Expected Relative Potency	% Recovery
	50	94.4
	75	94.8
	100	100.3
	100	100.3
	200	99.8
Repeatability (% CV)	100% (Reference)	7.5
Intermediate Precision (% CV)		9.7
Linearity (r²)		0.998
Linearity (y = mx + b)		y = 1.03x – 3.65
A 50–200% theoretical potency series of Control Ab, Anti-LAG-3, 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 LAG-3/MHCII Blockade Bioassay reflects the MOA and specificity of biologics designed to block the LAG-3/MHCII Interaction.

lag-3-mhcii-blockade-bioassay-mechanism-of-action — LAG-3 Effector Cells were incubated with TCR Activating Antigen and MHCII APC cells in the presence of anti-LAG-3, anti-PD-1, anti-CTLA-4 or anti-TIGIT antibodies, as indicated.

The LAG-3/MHCII Blockade Bioassay measures the activity of Control Ab, Anti-LAG-3.

lag-3-mhcii-blockade-bioassay-control-antibody-activity — MHCII APC Cells and TCR Activating Antigen were plated overnight. The following day, a titration of Control Ab, Anti-LAG-3 was added to the plate, followed by LAG-3 Effector Cells, and then incubated for 5 hours.

Product Kit Formats and Related Products

LAG-3/MHCII Blockade Bioassay is available in 1X and 5X sizes.
LAG-3/MHCII Blockade Bioassay is also available in CPM format (Cat.# JA1112).
Control Ab, Anti-LAG-3, is available separately (Cat.# K1150).
TCR Activating Antigen is available separately (Cat.# K1201).

Protocols

Complete Protocol

LAG-3/MHCII Blockade Bioassay Technical ManualPDF (1900 KB) – English

LAG-3/MHCII Blockade Bioassay, Propagation ModelPDF (2078 KB) – English

What's in the box?

Item	Part #	Size
LAG-3 Effector Cells	J249A	1 × 0.5ml
MHCII APC Cells	J250A	1 × 0.5ml
TCR Activating Antigen Stock Solution	K120A	1 × 60μl
DMEM with 4.5g/L Glucose, L-Glutamine, Sodium Pyruvate	J236A	1 × 60ml
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

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.

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
LAG-3 Effector Cells	J249A	5 × 0.5ml
MHCII APC Cells	J250A	5 × 0.5ml
TCR Activating Antigen Stock Solution	K120A	5 × 60μl
DMEM with 4.5g/L Glucose, L-Glutamine, Sodium Pyruvate	J236A	5 × 60ml
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

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.

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
LAG-3 Effector Cells (CPM)	J245A	2 × 1ml
TCR Activating Antigen Stock Solution	K120B	1 × 500μl
MHCII APC Cells (CPM)	J246A	2 × 1ml

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

Not For Medical Diagnostic Use.

Storage Conditions

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.

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-LAG-3	K115A	1 × 100μg	1mg/ml

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

Not For Medical Diagnostic Use.

Storage Conditions

See the Protocol for detailed storage conditions.

What's in the box?

Item	Part #	Size
TCR Activating Antigen Stock Solution	K120B	1 × 500μl

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

Not For Medical Diagnostic Use.

Storage Conditions

See the Protocol for detailed storage conditions.

Resources

Posters

Other Resources

Immune Checkpoint Bioassays Power Combination Immunotherapy

Frequently Used With

GloMax® Discover System

High-performance microplate reader for detecting luminescence, fluorescence and absorbance.

GM3000

pHAb Reactive Dyes

Measure antibody internalization in real time with pH-sensitive fluorescent dyes.

G9841, G9831, G9835, G9845

LAG-3/MHCII Blockade Bioassay

Measure Potency and Stability of Biologics Designed to Block LAG-3 Interactions

Measure the ability of biologics to block immune checkpoint signals

Assay Components

The Workflow

The Assay Principle

Assay Performance Data

The LAG-3/MHCII Blockade Bioassay reflects the MOA and specificity of biologics designed to block the LAG-3/MHCII Interaction.

The LAG-3/MHCII Blockade Bioassay measures the activity of Control Ab, Anti-LAG-3.

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

Specifications

What's in the box?

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Resources

Posters

Other Resources

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Frequently Used With

GloMax® Discover System

pHAb Reactive Dyes

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Europe