Membrane VEGF Target Cells

Measure Effector Functions and Antibody Binding Affinity

Vascular endothelial growth factor (VEGF) is an important signaling protein that is secreted from epithelial cells, tumor cells and macrophages. It has many functions, including stimulating angiogenesis, increasing vascular permeability, enhancing tumor invasion and survival, and inhibiting antitumor response in regulatory T cells. Because some VEGF isoforms are membrane-bound (mVEGF), the Fc effector function of VEGF-targeted antibodies needs to be characterized, particularly its potential to induce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). However, such studies are hampered by the lack of model cell lines naturally expressing mVEGF.

mVEGF Target Cells are a genetically engineered cell line stably expressing a cleavage-resistant form of VEGF that enforces its surface expression. They are designed to be used as target cells in assays that measure the effector functions, such as ADCC and CDC, of anti-VEGF blockers. In addition, they can be used to measure antibody binding affinity to mVEGF.

Assay Components

Membrane VEGF Target Cells are provided in thaw-and-use format (Cat.# J3351, J3355). These cryopreserved cells are ready to be thawed for use in assays that measure the effector functions of anti-VEGF blockers.

Membrane VEGF Target Cells are also available in Cell Propagation Model (CPM) format (Cat.# J3342). These cryopreserved cells 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.
Easily amenable to high-throughput workflows.

Assay Principle

17870ma — **mVEGF Target Cells used to measure ADCC with the ADCC Reporter Bioassay.** Following engagement with the Fc region of a relevant antibody bound to a Target Cell, ADCC Bioassay Effector Cells expressing the FcγR will transduce intracellular signals, resulting in NFAT-mediated luciferase activity that can be easily quantified.

17871ma — **mVEGF Target Cells used to measure CDC with the CytoTox-Glo™ Assay.** The assay measures dead-cell protease activity, and the amount of luminescence directly correlates with the percentage of cells undergoing cytotoxic stress.

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 ADCC Reporter Bioassay using mVEGF Target Cells Shows Precision, Accuracy and Linearity.

Parameter	Results
Accuracy	% Expected Relative Potency	% Recovery
	50	47.5
	70	71.3
	100	97.2
	140	148.1
	200	211.8
Repeatability (% CV)	100% (Reference)	2.1
Intermediate Precision (% CV)		5.0
Linearity (r²)		0.9981
Linearity (y = mx + b)		y = 1.098x – 7.794
A 50–200% simulated potency series of bevacizumab was analyzed in triplicate in three independent experiments performed on three days by two analysts using mVEGF Target Cells in the ADCC Reporter Bioassay. Bio-Glo™ 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.

17857ma — **The ADCC Bioassay Effector Cells with mVEGF Target Cells measures the activity of the anti-VEGF antibody bevacizumab.** mVEGF Target Cells were added to a 96-well assay plate 18 hours prior to the assay. On the day of the assay, ADCC Effector Cells, Propagation Model (Cat.# G7102), ADCC assay buffer and a titration of bevacizumab were added. After a 24-hour induction at 37°C, Bio-Glo™ Reagent was added and luminescence measured using the GloMax® Discover System. Data were fitted to a four-parameter logistic curve using GraphPad Prism® software. The EC₅₀ was 0.72µg/ml and fold induction was 3.8.

17858ma — **A CDC assay measures the activity of the anti-VEGF antibody bevacizumab.** mVEGF Target Cells were added to a 96-well assay plate 18 hours prior to the assay. On the day of assay, 10% normal human serum complement and a titration of bevacizumab in CDC assay buffer were added. After a 6-hour induction at 37°C, CytoTox-Glo™ Reagent was added and luminescence measured using the GloMax® Discover System. Data were fitted to a four-parameter logistic curve using GraphPad Prism® software. The EC₅₀ for bevacizumab was 2.94µg/ml, fold induction was 6.6 and specific lysis was 25.6%.

Protocols

Complete Protocol

Membrane VEGF Target Cells Technical ManualPDF (2231 KB) – English

Membrane VEGF Target Cells, Propagation Model Technical ManualPDF (2178 KB) – English

What's in the box?

Item	Part #	Size
Membrane VEGF Target Cells	J335A	1 × 0.5ml

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 luminescent assay reagents purchased from Promega for all luminescence-based 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.

Product cannot be used for proficiency testing.

What's in the box?

Item	Part #	Size
Membrane VEGF Target Cells	J335A	5 × 0.5ml

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 luminescent assay reagents purchased from Promega for all luminescence-based 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.

Product cannot be used for proficiency testing.

What's in the box?

Item	Part #	Size
Membrane VEGF Target Cells (CPM)	J334A	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 luminescent assay reagents purchased from Promega for all luminescence-based 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.

Product cannot be used for proficiency testing.

Membrane VEGF Target Cells

Assess the Mechanism of Action of Biologics Designed to Affect VEGF Signaling