Fusion Protein For Anti-Cd19 Chimeric Antigen Receptor Detection

Summary

Researchers at UCLA have developed a fusion protein that can detect immune cells expressing anti-CD19 chimeric antigen receptors with higher specificity and lower background than existing antibodies.

Background

CD19, a glycoprotein specific to the B-cell lineage, is a target of a number of cancer immunotherapies. Clinical trials testing the effectiveness of T cells equipped with anti-CD19 chimeric antigen receptors (CARs) in B-cell malignancy treatments are currently underway. Improving the detection of T cells that carry anti-CD19 CARs would greatly improve the characterization of the cells used for these immunotherapies and increase therapeutic potential.

The detection of CD19 CAR expression by FACS is typically determined through the use of an antibody to either the hinge region or FC portion of the receptor. Detection using commercially available antibodies can result in high levels of background and non-specific binding, especially in blood samples where many lgG expressing cells are present.

Innovation

Dr. Donald Kohn and colleagues have developed a CD19 fusion protein that can be used to detect anti-CD19 CAR+ cells. This fusion protein can be secreted from producer cells (e.g. 293T) and purified from the cell culture supernatant. The purified protein molecule is conjugated with a fluorescent fluorophore and used directly to detect anti-CD19 CAR+ cells in fluorescence-based techniques, such as flow cytometry (also known as fluorescent activated cell sorting, or FACS) or confocal imaging. This fusion protein contains specific domains of CD19 that should allow the detection of all anti-CD19 CAR+ cells irrespective of the antibody binding domain used to construct the CAR. Immune cells with the anti-CD19 CAR will recognize and specifically bind the CD19 fusion protein, positively labelling the cells. The CD19 domain incorporated into the fusion protein binds anti-CD19 CAR in a highly specific manner and enhances the detection of anti-CD19 CAR+ cells.

Applications

• Detection of anti-CD19 CAR+ cells using a fluorescence-based technique, such as FACS or confocal microscopy 
• Quantitative measurement of blood cell phenotype and frequency 

Advantages

• Increase specificity compared to available antibodies to anti-CD19 CAR
• Decrease non-specific binding compared to available anti-CD19 CAR detection antibodies
• Improve cell characterization for immunotherapies
• Increase therapeutic potential of anti-CD19 CAR cell therapies

State Of Development

• The fusion protein has been constructed, expressed, purified and conjugated. 
• The conjugated fusion protein has been shown to detect anti-CD19 CAR expression in T-cells, including immortalized cell lines (Jurkat) as well as in primary cells and in blood samples from mice engrafted with human cells.

Related Materials

• De Oliveira SN … Kohn DB, Hollis RP. A CD19/Fc fusion protein for detection of anti-CD19 chimeric antigen receptors. J Transl Med. 2013;11:23.