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What is CAR T-Cell Therapy?

With the development of biotechnology, a type of treatment in which a patient’s T cells (a type of immune system cell) are gene engineered to attack cancer cells. T cells are taken from a patient’s blood. Then the gene for a special receptor that binds to a certain protein on the patient’s cancer cells is added in the laboratory. The special receptor is called a chimeric antigen receptor (CAR). Large numbers of the CAR T cells are grown in the laboratory and given to the patient by infusion. CAR T-cell therapy is being studied in the treatment of some types of cancer. Also called chimeric antigen receptor T-cell therapy. Check Emily Whitehead’s amazing cancer fight story with CAR T-cell therapy here.

What’s the mechanism of CAR T-Cell Therapy?

CAR is mainly composed of a cell membrane extracellular antigen binding region and an intracellular signal transduction region through a hinge region and a transmembrane region. The extracellular antigen binding domain can specifically recognize and bind to a target cell surface antigen. It is derived from a single-chain variable region(scFv) of a monoclonal antibody. The intracellular signal transduction domain is mainly composed of a costimulatory signal and a CD3zeta chain of T cell receptor (TCR). After CAR-T cells binding to the target cell antigen through the scFv, the intracellular signal transduction region transmits signals to the T cell, thereby activates T cell, which in turn results secretion of perforin, granzymes and interferons to kill cancel cell.

Could R19M be used on current CAR T-cell therapies?

The FDA has approved two CAR T-cell therapies in 2018, namely Kymriah of Novartis and Yescarta of Gilead. Both of them are directed against CD19-positive B-cell tumors, using the same antigen-binding region, the scFv derived from the mouse monoclonal antibody FMC63. So our R19M  and other detection products are compatible with these established CAR T-Cell therapies.

Why accurate detection of CAR T-cells is important?

Generation of CAR-T cell requires transfection of the CAR gene into T cell via a viral or non-viral system. When the CAR is expressed on the membrane of T cell, CAR T-cell then has the activity of recognizing and killing the target cells. Therefore, accurate detection of CAR-positive T cells is a key step in the quality control of CAR T-Cell therapy, and an important criteria in clinical dose control, process monitoring and assistant diagnosis.

What are the current CAR T-cells detection methods?

There are two common types of detection methods: detection of CAR gene-positive T cells(qPCR) and detection of CAR protein-positive T cells (FACS).

  • Quantitative PCR (qPCR) is widely used for detecting CAR genepositive T cells. The CAR gene integrated in the genome, as well as the copy number of the CAR gene, can be detected by qPCR.
    However, this method is difficult to accurately reflect T cells expressing surface CAR because of possible gene silencing. More seriously, the qPCR method cannot distinguish between CAR positive T cells and CAR-positive B cells, making it impossible to eliminate the risk of treatment and recurrence induced by CARB cells. In addition, the qPCR method does not meet the need for simultaneous detection of
    multiple surface markers of T cells (CD4, CD8, etc.), which further limits the application of this technique.
  • Fluorescent activated cell sorting (FACS), as a specialized type of flow cytometry, provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. With FACS, the positive rate of CAR transfection could be detected. There are currently methods detecting different
    domain of CAR, including CD19 antigen or anti-scFv antibodies, binding to CAR scFv; anti-Fab antibodies or Protein L, targeting light chains framework or hinge region. Among these method, detecting antigen-binding sites has a best specificity for the CAR positive rate, which is ideal for quality control testing and non-clinical research considerations for CAR-T Cell Therapy Products.

Which FACS detection reagent for CAR T-Cell are available. 

There are several reagents, including anti-mouse IgG (Fab’)2, Protein L, CD19/Fc, and mouse monoclonal antibody 136.20.1.

  • Among them, antimouse IgG (Fab’)2 and Protein L demonstrated poor specificity of anti-CD19 CAR, unable to distinguish different CARs generated
    from mouse antibodies.
  • The sensitivity of CD19/Fc for detecting anti-CD19 CAR is low, since the affinity of FMC63 scFv to CD19 is lower than the affinity of the intact antibody to CD19.
  • Antibody 136.20.1, binding to FMC63 scFv, shows great specificity. Its
    sensitivity is close to 0.1%, that is, one anti-CD19 CAR positive
    cell is detected in 1,000 cells. However, when applied to clinical
    assistant diagnosis, the sensitivity is still low.
  • R19M(patent pending, developed by Cytoart)is a high affinity rabbit monoclonal antibody specifically binding to the anti-CD19 scFv that was derived from the FMC63. Its robust specificity and sensitivity allows this antibody to be a perfect tool for flow cytometry and other
    immunostaining applications.
  • R19P, our new ultra-high affinity rabbit anti-mouse polyclonal antibody, push the detection limit to 1/100,000, which means we can even detect 1 positive T-cell among 100,000 cells.