For CAR-T cell therapy, the CAR molecule is the key to the effectiveness of the treatment. The CAR
molecular structure includes antibody single-chain variable regions and hinge regions that recognize
tumor antigens, as well as intracellular co-stimulatory domains and signaling domains. The
rationality of its design will affect the killing effect, in vivo persistence, and security etc.
Therefore, for different tumor therapeutic targets, we need to design reasonable and effective CAR
molecules and package them into CAR lentiviruses for subsequent construction of CAR-T cells.
CD19 antigen lentiviral packaging and titer detection
The lentivirus purification solution expressing CD19 antigen was serially diluted (0.01, 0.1, 1, 10
μl), and added to the same number of 293T cells respectively. After 72 hours, the positive rate of
293T cells was detected by flow cytometry, and then calculated according to the following formula
for Transduction titer of a virus:
Figure 1. Detection results of CD19 antigen lentivirus titer.
As shown in Figure 1, 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: 1.4×10^8
Tu/ml.
Construction of CD19-293T stably transfected cell line
We transfected 293T cells with the above-mentioned, successfully prepared CD19 virus, and obtained
CD19-293T cells with a positive rate of almost 100% after multiple rounds of screening. The positive
rate test results are shown in the figure below.
Figure 2. Detection results of the positive rate of CD19-293T stably transformed cell lines.
CD19-CAR lentivirus packaging and titer detection
We built a second-generation classic CAR molecule designed for the CD19 target into a lentiviral
vector, and packaged CD19-CAR lentivirus. The virus titer was detected by the above method, and the
calculated viral titer was: 4.33×10^8 Tu /ml.
Figure 3. Molecular structure of CD19 CAR, in which the VH and VL sequences are derived from the CD19 antibody FMC63 clone.
Figure 4. Detection results of CD19 CAR lentivirus titer.
Other lentiviral infections: effects on different cells
Figure 5. Effects on other cells infected with lentivirus.
After completing the packaging of the CAR virus, the next step is to transfect T cells to obtain
CAR-T cells. We have established two different high-efficiency T cell preparation methods, both of
which can produce high-purity T cells (the proportion of CD3+ cells can reach more than 98%), but
the proportions of different subtypes of T cells expanded by the two are quite different.
The T cells amplified by Method 1 are mainly CD8+ T cell subtypes, while Method 2 can amplify T cell
populations with small differences in the proportions of CD8+ T cells and CD4+ T cells. Both
approaches provide a solid foundation for studying the role of different subtypes of T cells.
Figure 6. Experimental results of T cell phenotype analysis
Lentiviruses (LVs) are an important gene
delivery tool for constructing CAR-T cells. Many factors will affect the positive rate of CAR-T
cells, such as differences in T cell activation and expansion methods, transfection times, and more.
We have successfully established an efficient CAR-T cell production process based on lentiviral
transfection through optimization of various experimental conditions.
The figure below shows the detection results of the positive rate of CD19 CAR-T cells. The scFv
derived from the CD19 antibody FCM63 clone is used in this structure, so the transfection efficiency
can be detected by the anti-FMC63 scFv antibody. The results showed that the positive rate of CD19
CAR-T cells could reach 45.59%, indicating that CD19 CAR-T cells with a high positive rate were
successfully constructed.
Figure 7. Detection results of positive rate of CD19 CAR-T cells.
Once the CAR-T cells are constructed, relevant drug efficacy evaluations can be carried out. Usually,
different effect-to-target ratios are used to observe the killing effect of CAR-T cells on target
cells. We incubated CD19 CAR-T cells and T cells with Nalm6 cells at different effect-target ratios
for 48 hours, and detected tumor cell apoptosis by flow cytometry.
As shown in the figure, compared with the T cell group, CD19 CAR-T cells exhibited a significantly
enhanced specific cytotoxic effect on Nalm6 cells under different effect-to-target ratio conditions.
Figure 8. Experimental results of FMC63 CAR-T cells killing Nalm6 tumor cells.
A. The detection results of the positive rate of CD19 antigen on the surface of Nalm6 cells;
B. The results of CD19 CAR-T cells killing Nalm6 cells;
C. Detection results of IFN-γ content in the culture supernatant of Nalm6 cells killed by CD19 CAR-T
cells.
Case Presentation: Validation Data
