PRODUCT REFERENCES
| Product name | Reference | Description | |
|---|---|---|---|
CAR T Detection | Human PE-CD19 | RUO | 25 test | Go to shop | |
CAR T Detection | Human PE-CD19 | RUO | 100 test | Go to shop |
CAR T-cells therapy is based on the extraction of patient T-cells, the introduction of a Chimeric Antigen Receptor (CAR) specific to a protein present on cancer cells (e.g. CD19) and the consequently reinfusion of these modified cells, now called CAR T-cells, into the patient to recognise the target cells and eliminate them.
This therapy has gained relevance due to its efficacy, and detecting CAR T-cells tools has become a critical need, both to check that they have been correctly modified, and to monitor them after the reinfusion, since they have a limited life span.
Figure 1: Basic scheme of CAR T-Cell therapy. (A) Own T-Cell does not recognize cancer cell. (B) T-Cell extraction. (C) Modification of T-Cell with CAR. (D) CAR T-Cell cultivation and harvesting. (E) Harvested CAR T-Cells are reinfused into the patient. (F) CAR T-Cell recognizes and eliminates cancer cell.
A differential solution
Identify CAR T-cells from other populations with High Resolution, even with difficult samples
Fluorochrome compatible with
any Flow Cytometer (R-PE)
Compatible with panels of
more than 30 antibodies
There are some reagents for identifying and quantifying CAR T cells, based on fluorescent labeled (PE or FITC) recombinant CD19. However, current reagents have limitations in separating the desired populations with sensitivity, making it more difficult for the analysts to provide an accurate and reliable result.
Those reagents that overcome this problem have the disadvantage of involving more complex and time-consuming protocols, using an amplification reagent in addition to the protein. It is therefore necessary to introduce new simple, straightforward and easy to use reagents that are able to clearly discriminate the CAR T-Cells from the rest of populations for a more reliable analysis.
Figure 2: FACS (Fluorescent-Activated Cell Sorting) protocol. Immunostep vs competitors. Other competitors require more steps and reagents (competitor 1) or longer protocols (competitor 2). Immunostep combines the simplicity with the speed, providing analysts with an easier protocol.
Detection of CAR T-Cells
To meet these needs, we have developed a single reagent for direct and specific recognition of CAR T-Cells with high resolution. This reagent allows the specialists to confirm the successful modification of T-Cells in a quality control prior infusion into the patient and to monitor them after this step.
TECHNOLOGY
Recombinant Fc-Tag CD19 protein (CD19p) has been labeled, by a proprietary process, with R-Phycoerythrin fluorochrome (R-PE or PE), obtaining a conjugate that is able to be recognized by CAR T-Cells and separates them efficiently without any secondary reagent (Immunostep, ref. CD19-0212).
Other manufacturers have similar products based on this fluorochrome-protein complex but have limitations in the separation due to bioconjugation techniques and/or the fluorochrome used.
BrightStepTM: our proprietary labeling technology
This cutting-edge technology is set to redefine CAR-T cell detection. With BrightStep, we’ve improved the art of labeling, ensuring the maintenance of conformation and natural modifications of the protein. This means unparalleled accuracy without compromising the integrity of the sample.
Verified by rigorous flow cytometry testing, Brightstep offers higher performance in specificity and sensitivity. You can trust in its reliability to deliver precise results every time.
Furthermore, with batch-to-batch consistency and reproducibility, you can rely on BrightStep for ensuring reliable outcomes for your research and diagnostics needs.
Experience the future of CAR-T cell detection with BrightStep – where innovation meets accuracy.
Figure 4: Representation of the T-Cell modification to CAR T-Cell and the CAR structure of Yescarta and Kymriah. The T-Cell is transfected with a viral vector containing the information to express the CAR. This is composed of an extracellular domain that specifically recognizes the CD19 antigen, designed from an antibody (FMC63), and other transmembrane and intracellular domains with signaling and co-stimulatory functions that increase the efficiency of the T-Cell recognition and activation.
COMPARISON BETWEEN SOLUTIONS
The fundament of flow cytometry is binding the reagent to a specific population and discriminating it by the reagent fluorescence. The reagent targets the CAR in the engineered T cell, so the separation depends on the level of expression of the CAR and the discriminatory ability of the reagent. The reagent identifies the specific population by his fluorescence, but it is not so important the intensity of that fluorescence, measured as MFI (Median Fluorescence Intensity), but the ability to discriminate between the positive population (CAR T-Cells) and the negative populations (rest of the cells).
Figure 5: A bar graph representing the ratio between Immunostep and other two competitors. In sample C, the most difficult discrimination (academic CAR T-Cells), Immunostep shows highest difference, providing reliable result.
It means that it is necessary the greatest difference between both populations MFI; this value is known as Stain Index (SI), the greater SI the better discrimination. A highly fluorescent reagent is needed when the amount of target molecule is low but sometimes also increases the negative MFI, getting worse the SI. Best reagents have high SI with a moderate or high fluorescence to identify CAR T-Cells, even with low CAR expression.
Figure 6: Stain Index, value to estimate the level of discrimination between positive and negative populations.
STAIN INDEX = (Median of Positive – Median of Negative) / (SD of Negative * 2).
