Application of Flow Cytometry in Preclinical CAR-T Development

Chimeric Antigen Receptor T-Cell (CAR-T) Immunotherapy uses genetic engineering technology to transform autologous or allogeneic T cells into new killer cells targeting tumor-specific antigens, by which remarkable results have been achieved in the treatment of hematological malignancies. The preclinical research of CAR-T cell immunotherapy is a key and lengthy step in drug development, which includes target discovery, antibody development, single-chain variable fragment (scFv) screening, CAR-T cell construction, CAR-T cell anti-tumor activity in vitro, evaluation of anti-tumor activity of CAR-T cells in vivo, and more.

Flow cytometry is a technology that can count and sort tiny particles suspended in a fluid. Due to its high efficiency, simplicity, and accuracy, it has been widely used in all aspects of CAR-T cell therapy. Let's take a look at the application of flow cytometry in the pre-clinical development of CAR-T.

1. Detection of expression of target antigen

CAR-T cells accurately recognize the scFv of antigens on tumor cells and play a killing role, so screening ideal target antigens is an important step. To select highly specific target antigens, it is necessary to detect the expression abundance of target antigens in all cells and to select for antigens that are highly expressed in tumor cells but not expressed (or lowly expressed) in normal cells, so as to avoid the toxic side effects of CAR-T cell therapy as much as possible. Flow cytometry has an important application in this process. For example, in the following report, the author detected the distribution of the target antigen B cell maturation antigen (BCMA) by flow cytometry; they found that BCMA was highly expressed on normal and tumor plasma cells (PCs) cells, while it was not expressed or lowly expressed in normal tissues.

                                              Figure 1. The expression abundance of target antigen detected by flow cytometry.^[1]

2. Virus titer detection

Before the clinical development of CAR-T, it is necessary to construct a tumor cell model with CAR-T cells and antigen overexpression for subsequent evaluation of the anti-tumor ability of CAR-T cells. Generally speaking, the above-mentioned stable cell model can be efficiently constructed by infecting the corresponding cell line with a lentivirus capable of encoding the target sequence. During this process, the titer of lentivirus is strictly controlled. Flow cytometry is one of the important means to detect the titer of lentivirus activity. The following is an example to introduce the application of flow cytometry in the detection of lentivirus titer.

Flow Cytometry Detection of CD19 Lentivirus Titer

To detect the titer of the CD19 antigen lentivirus, add the purified lentivirus that can encode the CD19 antigen in four gradients of 0.01, 0.1, 1, and 10 μl to the previously inoculated 293T cells. Perform flow cytometry with the 293T cells after 72 hours to detect the positive rate of CD19 antigen (that is, the percentage of 293T cells infected by lentivirus to the total number of cells), and then calculate the transduction titer of lentivirus according to the following formula:

A: Indicates the proportion of positive cells in the 0.1μl group

B: Indicates the proportion of positive cells in the 0.01μl group

N: the number of cells when the virus was added

V: Indicates the added volume of virus in group A

                                           Figure 2. Detection results of CD19 antigen lentivirus titer (Data source: Cyagen)

As shown in the figure, the number of CD19 antigen-positive cells gradually increased with the increase of the transfected virus gradient, indicating that the lentivirus has good infectious activity, and the virus titer was calculated according to the above formula: 1.4×10⁸ Tu/ml.

Similarly, the FMC63 CAR lentivirus titer was detected by the above method, and the calculated virus titer was about 4.33×10⁸ Tu/ml.

                     
                                          Figure 3. FMC63 CAR lentivirus titer detection results (Data source: Cyagen)

3. Analysis of CAR-T cell phenotype and CAR positive rate (or antigen positive rate of overexpressed antigen cell model)

For the constructed CAR-T cells, the phenotype and the positive rate of CAR molecules’ infectivity need to be tested before functional verification. Flow cytometry can effectively detect the marker proteins on the surface of CAR-T cells and the positive rate of CAR molecules across all cells. Similarly, antigen expression may also be detected via flow cytometry for the antigen overexpression cell model. Below, we introduce real examples to demonstrate relevant research applications of flow cytometry in CAR-T cell phenotype and positive rate of cells.

▶ Detect the phenotype of T cells and the positive rate of CD19 CAR-T cells

The phenotypes of T cells prepared by the two methods were analyzed with antibodies, and the results showed that both methods could obtain high-purity T cells, but the proportions of different subtypes of T cells amplified by the two methods were quite different. The T cells amplified by Method1 are mainly CD8+T cell subtypes, while Method2 can amplify T cell populations with small differences in the proportions of CD8+T cells and CD4+T cells. The role of subtypes of T cells lays a solid foundation.

At the same time, we transfected T cells amplified by Method1 with CD19 CAR lentivirus to construct CD19 CAR-T cells, and used flow cytometry to detect the expression efficiency of CAR protein. The results showed that the positive rate of CD19 CAR-T could reach 45.59% , indicating that we successfully constructed CD19 CAR-T cells.

A: 

B: 


                   

Figure 4. T cell phenotype analysis and FMC63 CAR-T cell positive rate detection.

A. The proportion of T cells and the results of phenotype analysis on the tenth day of PBMC activation and expansion.

B. Detection results of CAR positive rate of FMC63 CAR-T cells. (Data source: Cyagen)

▶ Construction of quality control CD19-293T cell line

293T cells were infected with CD19 lentivirus to construct a 293T cell line stably expressing CD19; the high expression of its antigen CD19 was detected by flow cytometry.


 Figure 5. Flow cytometric detection of CD19 antigen expression in CD19-293T stably transfected cell line (Data source: Cyagen)

4. In vitro detection of the killing effect of CD19 CAR-T cells

Mixing CAR-T cells with tumor cells or constructed antigen-overexpressing cell models, and detecting the death rate of the latter can reflect the killing effect of CAR-T cells. Among them, the apoptosis of tumor cells can be detected by flow cytometry. As in the example below:

CD19 CAR-T cells and T cells were co-incubated with Nalm6 cells at different effect-target ratios for 48 hours, and then the apoptosis of tumor cells was detected by flow cytometry. As shown in the figure, compared with the T cell group (control), CD19 CAR-T cells exhibited a significantly enhanced specific cytotoxic effect on Nalm6 cells under different effect-to-target ratio conditions. At the same time, the results of cytokine content detection in the cell culture supernatant also showed that the secretion of CD19 CAR-T cell factor IFN-γ after co-incubation with tumor cells was significantly enhanced compared with that of T cells.

A:

B: 

C:

Figure 6. The results of CD19 CAR-T cells killing Nalm6 tumor cells.

A. The detection results of CD19 antigen positive rate on the surface of Nalm6 cells;

B-C. The results of FMC63 CAR-T cells killing Nalm6 tumor cells and supernatant cytokine detection experiments. (Data source: Cyagen)

5. Monitoring the therapeutic effect of CAR-T cells in vivo

After verifying the phenotype and killing effect of CAR-T cells in vitro, in vivo experiments can be carried out. CAR-T cells are injected into tumor mouse models (such as: NSG, C-NKG, NOG, etc.), and then peripheral blood (PB) samples of different days are collected and analyzed by flow cytometry to monitor proliferative activity, cell phenotype, and killing ability of CAR-T cells in vivo.

6. Conclusions

CAR-T therapy is one of the most promising technologies in the field of tumor treatment, and it is a treatment method that can "cure" cancer: having shown the ability to eradicate advanced lymphomas and leukemias. Its preclinical research needs to go through many important steps, and each step requires strict quality control standards.Flow cytometry has already provided its own advantages and characteristics, but with the ongoing development of related technology throughout recent years, it will continue to be more widely used in all aspects of CAR-T cell therapy research and development.

Cyagen's One-Stop Solution For Cell Therapy Facilitates Preclinical Research On Tumor Immune Cell Therapy

In the preclinical research of CAR-T and other cell therapy, the selection of cell models and animal models is very important. Clarifying the purpose of the research and establishing effective experimental models and study protocols will make the research more effective. Cyagen provides a one-stop service for cell therapy, from antibody screening to stable strain construction, cell/animal model establishment to pharmacodynamic evaluation, to better accelerate the research and development process of CAR-T and other cell therapies. If you would like more information, please contact us for consultation.

Reference:

Bu DX, Singh R, Choi EE, et al. Pre-clinical validation of B cell maturation antigen (BCMA) as a target for T cell immunotherapy of multiple myeloma. Oncotarget. 2018;9(40):25764-25780.