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  4. Accelerating CAR-T Cell Therapy Research

By using the body's own immune system to kill tumor cells, immune cell therapy is anticipated to be a breakthrough strategy to cure many types of cancer. Among them, chimeric antigen receptor T-cell immunotherapy (CAR-T) has achieved remarkable results in the treatment of various hematological malignancies. As of 2022, seven CAR-T cell therapy products have been launched globally, including three in China.

The long journey of every CAR-T cell therapy begins with steady steps in performing basic research designed for repeatability. The research process generally includes CAR molecule design, lentiviral packaging, CAR-T cell construction, in vitro and in vivo killing assay, cytotoxicity tests, and more. In order to build effective and stable CAR-T cells, many factors need to be fully considered, such as rational design of CAR molecular structure, the exploration of T cell transfection conditions, identification of CAR-T cells, and confirmation of purity and potency.

Based on over 16 years of research experience developing cell and animal models for tumor immunology research, Cyagen offers a one-stop service platform for in vivo and in vitro CAR-T cell therapy research. Available to researchers worldwide, our platform includes scFv sequence screening, CAR molecular design, and full-process services to develop the foundational research for your CAR-T cell therapy. These comprehensive services help streamline the efficiency and accuracy of CAR-T/NK research and related fields. You are welcome to contact us at 800-921-8930 or email to animal-service@cyagen.com for free project consultations.

One-stop Service Platform for CAR-T
Cell Therapy Research
Viral vector manufacturing
  • CAR design
  • Lentiviral packaging and optimization
  • CAR-T cell construction
Tumor Model Construction Platform
  • Target cell construction
  • C-NKG Severe Immunodeficient Mouse Model
  • CDX mouse tumor model
  • Humanized model of PBMC/HSC immune system
In vitro pharmacodynamic evaluation
  • In vitro killing function evaluation of CAR-T cells
  • Cytokine detection
  • Functional experiments such as cell proliferation/apoptosis/migration/invasion
 In vivo pharmacodynamic evaluation
  • Construction of CDX C-NKG mouse and CDX PBMC/HSC C-NKG mouse models
  • Pharmacodynamic evaluation of CAR-T/TCR-T/NK/TIL cells
  • Immunohistochemistry (IHC), HE staining, lymphocyte infiltration analysis
Project Type Project Technical Difficulties Service Advantages of Cyagen's Platform
Target cell construction Difficulty in selecting target cell types A wide range of tumor cells & overexpressing CHO/HEK293 cells in stock
The expression level of the target is not high enough Experience of 500+ stably transformed cell lines construction projects
CAR vector design
and lentivirus
Lack of experience in CAR molecular structure design Extensive experience in designing CAR molecules of different targets
Low virus titer and difficult purification High-quality lentiviral vector and virus packaging, published by multiple peer-reviewed papers
CAR-T/NK cell construction Difficulty in culturing T/NK cells Over 16+ years of primary cell culture experience
It is time-consuming to assess the transfection conditions of T/NK cells; the identification of cells after transfection is difficult An optimized protocol for fully summarizing the transfection parameters of human/murine immune cells
In vitro & in vivo killing assay Difficult to choose evaluation indicators Mature in vitro & in vivo pharmacodynamic experimental platform
Data analysis Extensive experience of both in vitro and in vivo assays, supported by successful publications and IND submissions
案例展示 Case Presentation: Validation Data
CAR molecular design and lentiviral packaging
Preparation of CAR-T cells
In vitro efficacy evaluation

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.