TIM-3 Bioassay

Measure the Ability of Biologics to Block Immune Checkpoint Signals

The TIM-3 Bioassay is a bioluminescent reporter 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 TIM-3. TIM-3 (CD366, HAVCR2) is an immune checkpoint receptor expressed on a subset of activated T cells, regulatory T cells (Tregs), macrophages and dendritic cells. TIM-3 has the capacity to inhibit or costimulate T-cell receptor (TCR) signaling in different in vitro systems.

The TIM-3 Bioassay consists of two cell lines:

TIM-3 Effector Cells: Human T cells genetically engineered to express human TIM-3 and a NanoLuc® luciferase reporter driven by T cell activation pathway-dependent response elements
TIM-3 Target Cells: MHCII-positive human cell line

When the two cell types are co-cultured, TIM-3 Target Cells provide low-level stimulation to the TIM-3 Effector Cells through the TCR. The combined stimulation of the TCR and TIM-3 induces promoter-mediated luciferase activation and luminescence. Adding a TIM-3 blocking antibody prevents TIM-3 signaling and reduces promoter-mediated luminescence.

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

Assay Components

The thaw-and-use format of the TIM-3 Bioassay (Cat.# JA2211, JA2215) offers convenience and minimizes variability. The assay consists of two cell lines: A genetically engineered cell line, TIM-3 Effector Cells, that expresses human TIM-3 and a NanoLuc® luciferase reporter driven by T cell activation pathway-dependent response elements; and TIM-3 Target MHCII-positive human cell line.

The assay is also available in CPM format (Cat.# JA2222) 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

The TIM-3 Bioassay measures the potency and stability of antibodies and other biologics that block TIM-3 signaling.

TIM-3 Bioassay Representation — **Representation of the TIM-3 Bioassay.** The bioassay consists of two cell lines, TIM-3 Effector Cells and TIM-3 Target Cells. When cocultured, TIM-3 augments TCR pathway-activated luminescence of NanoLuc® luciferase. Adding anti-TIM-3 antibody blocks TIM-3 binding to ligands, including phosphatidylserine (PS), resulting in reduced TCR pathway activation, which can be detected in a dose-dependent manner by addition of Bio-Glo-NL™ Reagent and quantitation with a luminometer.

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 TIM-3 Bioassay Shows Precision, Accuracy and Linearity.

Parameter	Results
Accuracy	Percent Expected Relative Potency	Percent Recovery
	50	102.6
	70	100.2
	100	97.7
	130	99.8
	150	107.7
Repeatability (% CV)	100% (Reference)	12.9
Intermediate Precision (% CV)		13.4
Linearity (r²)		0.904
Linearity (y = mx + b)		y = 1.078x – 3.277
A 50–150% theoretical potency series of Control Ab, Anti-TIM-3, was analyzed in triplicate in three independent experiments performed on three days by two analysts. Bio-Glo-NL™ Reagent was added and luminescence quantified using the GloMax® Discover System. Data were analyzed and relative potencies calculated after parallelism determination using JMP® software. Data were generated using thaw-and-use cells.

TIM-3 Bioassay MOA and Biologics Specificity — **The TIM-3 Bioassay reflects the mechanism of action (MOA) and specificity of biologics designed to block TIM-3 signaling.** TIM-3 Effector Cells were incubated with TIM-3 Target Cells in the presence of serial titrations of blocking Abs as indicated: Control Ab, Anti-TIM-3 (Cat.# K1210), anti-LAG-3 (Cat.# K1150), anti-PD-1 (nivolumab), anti-CTLA-4 (ipilimumab) or anti-TIGIT blocking antibodies. After a 20-hour induction, Bio-Glo-NL™ Reagent was added and luminescence quantified using the GloMax® Discover System. Data were fitted to a four-parameter logistic curve using GraphPad Prism® software. Data were generated using thaw-and-use cells.

TIM-3 Bioassay Depends on Phosphatidylserine — **TIM-3 Bioassay is dependent on PS but not galectin-9.** The TIM-3 Bioassay was performed using Control Ab, Anti-TIM-3 (Cat.# K1210), or commercial antibodies against PS (Clone 1H6) and galectin-9 (Clone 9M1-3). After a 20-hour induction, Bio-Glo-NL™ Reagent was added and luminescence quantified using the GloMax® Discover System. Data were fitted to a four-parameter logistic curve using GraphPad Prism® software. Data were generated using thaw-and-use cells. Blockade of PS resulted in suppression of reporter activity similar to that seen with anti-TIM-3, while blockade of galectin-9 showed little effect, alone or in combination with anti-PS.

TIM-3 Bioassay is available in 1X and 5X sizes.
Control Ab, Anti-TIM-3, is available separately (Cat.# K1210).
TIM-3 Bioassay is also available in CPM format (Cat.# JA2222).

Protocols

Complete Protocol

TIM-3 Bioassay Technical ManualPDF (1714 KB) – English

TIM-3 Bioassay, Propagation Model, Technical ManualPDF (1238 KB) – English

What's in the box?

Item	Part #	Size
TIM-3 Effector Cells	J313A	1 × 0.5ml
TIM-3 Target Cells	J314A	1 × 0.9ml
Fetal Bovine Serum	J121A	1 × 4ml
RPMI 1640 Medium	G708A	1 × 36ml
Bio-Glo-NL™ Luciferase Assay Buffer	J308A	1 × 10ml
Bio-Glo-NL™ Luciferase Assay Substrate	J309A	1 × 200μl

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-NL™ 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. Nos. 8,557,970, 8,669,103, 9,777,311, 9,840,730, 9,951,373, 10,233,485, 10,633,690, 10,774,364, 10,844,422, 11,365,436, 11,661,623, 11,667,950; European Pat. Nos. 2456864, 2635595, 2990478, 3181687, 3409764; Japanese Pat. Nos. 6038649, 6155424, 6227615, 6374420, 6539689; and other patents and patents pending.

Patent Pending.

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
TIM-3 Effector Cells	J313A	5 × 0.5ml
TIM-3 Target Cells	J314A	5 × 0.9ml
Fetal Bovine Serum	J121A	5 × 4ml
RPMI 1640 Medium	G708A	5 × 36ml
Bio-Glo-NL™ Luciferase Assay Buffer	J308A	5 × 10ml
Bio-Glo-NL™ Luciferase Assay Substrate	J309A	5 × 200μl

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-NL™ 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. Nos. 8,557,970, 8,669,103, 9,777,311, 9,840,730, 9,951,373, 10,233,485, 10,633,690, 10,774,364, 10,844,422, 11,365,436, 11,661,623, 11,667,950; European Pat. Nos. 2456864, 2635595, 2990478, 3181687, 3409764; Japanese Pat. Nos. 6038649, 6155424, 6227615, 6374420, 6539689; and other patents and patents pending.

Patent Pending.

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
TIM-3 Effector Cells (CPM)	J306A	2 × 1ml
TIM-3 Target Cells (CPM)	J307A	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-NL™ 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. Nos. 8,557,970, 8,669,103, 9,777,311, 9,840,730, 9,951,373, 10,233,485, 10,633,690, 10,774,364, 10,844,422, 11,365,436, 11,661,623, 11,667,950; European Pat. Nos. 2456864, 2635595, 2990478, 3181687, 3409764; Japanese Pat. Nos. 6038649, 6155424, 6227615, 6374420, 6539689; and other patents and patents pending.

Patent Pending.

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
Control Ab, Anti-TIM-3	K121A	1 × 100μg

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

Posters

Other Resources

Frequently Used With

Bio-Glo-NL™ Luciferase Assay System

Bioluminescent detection reagent qualified for use with bioassays that utilize NanoLuc® luciferase.

J3081, J3082, J3083

GloMax® Discover System

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

GM3000

TIM-3 Bioassay

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