The application of chimeric antigen receptor (CAR) T-cell therapy in the treatment of leukemia and lymphoma has yielded some significant therapeutic results. In 2012, Emily Whitehead, a 7-year-old girl with acute lymphoblastic leukemia, received the world's first clinical trial of CAR T-cell therapy and was cured. In 2017, the US Food and Drug Administration (FDA) approved the world's first CAR T-cell therapy. Since then, this field has entered a period of rapid development of innovative research In 2021, two drugs that implement CAR T-cell therapy have been approved for marketing in China, priced at 1.2 million to 1.29 million RMB (~$200,000) per injection, which has attracted widespread attention around the world.

This article focuses on the process of developing CAR T-cell therapy and discusses the importance of each preclinical and clinical stage in the research of CAR T-cell therapy.


Figure 1. Emily Whitehead, the world's first leukemia patient who received CAR T-cell therapy and was cured, has been living healthy for 9 years. (Source:


What is CAR T-cell therapy?

Chimeric antigen receptor (CAR) T-cell therapy is a cell-based gene therapy method that enables immune cells (T cells) to find and destroy cancer cells. CAR T-cell therapy needs to draw blood from patients and isolate T cells, activate T cells, and transfect them (usually through viral transfection, but technologies such as CRISPR/Cas9 have also been reported to be used). [1] These procedures enable T cells to express CARs on the cell surface to specifically recognize tumor antigens. Finally, the CAR T-cells are expanded and infused back into the patient for the purpose of treating cancer.


Figure 2. CAR T-cell cancer therapy process (Source:


In recent years, CAR T-cell therapy has been a hot research field. At present, most of the clinical studies are carried out in Europe, America, and China. Current clinical research on CAR T-cell therapy is distributed across all stages, mainly focused in clinical phases I and II, with a smaller proportion in clinical phase III - which includes some drugs that have already been marketed. The CAR-T cell therapy products approved for marketing since 2017 are shown in Table 1; these products are mainly for the treatment of leukemia, non-Hodgkin's lymphoma, and multiple myeloma. China approved two CD19-targeting CAR-T products in June and September 2021, respectively.





Approved time





August 2017

Refractory or relapsed (R/R) acute lymphoblastic leukemia (ALL) , Refractory or relapsed (R/R)  large B-cell lymphoma


Kite (Gilead)


October 2017

Refractory or relapsed (R/R)  large B-cell lymphoma


Kite (Gilead)


July 2020

Relapsed or refractory (R/R)  mantle cell lymphoma


Juno Therapeutic


February 2021

Refractory or relapsed (R/R)  large B-cell lymphoma


BMS/Bluebird bio


March 2021

Adult patients with multiple myeloma

Axicabtagene Ciloleucel

Fosun Kite Biotechnology


June 2021

Adult patients with refractory or relapsed (R/R)  large B-cell lymphoma

Relmacabtagene Autoleucel 

JW Therapeutics


September 2021

Adult patients with relapsed or refractory (R/R) large B-cell lymphoma (LBCL) after two or more lines of systemic therapy

Table 1. List of CAR-T products that have been marketed.


Research Process for CAR T-Cell Therapy Development

1. Target discovery and selection

There are three types of tumor antigens:

(1) Cell-lineage-specific antigens: are expressed in specific cell lineages, such as B-cell lineage-specific antigens CD19 and CD20.

(2) Tumor-specific antigens (TSA): are ideal targets for cancer immunotherapy because they are exclusively expressed by cancer/tumor cells.

(3) Tumor-associated antigens (TAA): are preferentially expressed by tumor cells, but they are also oftentimes found in normal tissues. This is currently the most-studied type of antigen.

For safety, CAR-T cells need to be engineered to specifically attack cancer cells instead of normal cells in the body. Therefore, it’s important to select a target that is highly expressed on tumor cells and tissues, and has (little to) no expression in normal tissues.

Due to the microenvironment in solid tumors and other factors that restrict the efficacy of CAR T-cell therapy, research on CAR T-cell therapy is divided into two parts: hematological tumors and solid tumors. The seven drugs currently on the market that use CAR T-cell therapy are all targeting hematological malignancies, yet hematological malignancies only account for about 10% of all malignancies. [2] This reveals that there is a huge difference in the difficulty of CAR T-cell therapy research between hematological tumors and solid tumors. The most commonly used target in hematological research is CD19, which targets leukemia and lymphoma; followed by B-cell maturation antigen (BCMA), which is used in treatment research for multiple myeloma. Commonly studied targets in solid tumors include Mesothelin, MUC1, and GPC3.


Figure 3. The number of CAR T-cell therapy clinical trials for each target. [3] A. Studies of targets in hematological tumors; B. Studies of targets in solid tumors.


2. CAR molecular design

Specific immunity is the body's third line of defense against pathogens: after pathogens invade the body, they stimulate lymphocytes to produce antibodies, causing a series of immune responses. Scientists combine the properties of antibodies to specifically recognize antigens with the properties of T cell receptor (TCR) to activate T cells, which is the basic principle of CAR molecular design.

At present, the molecular structure of 4 generations of CAR has been defined in scientific research:

  1. The first-generation CAR molecule consists of three parts. The first part is the antigen-recognizing single-chain variable fragment (scFv) from the antibody, the second part is the transmembrane region, the third part is the intracellular signaling domain CD3ζ molecule from endogenous TCR;

  2. The second generation of CAR molecules adds a costimulatory domain on the basis of the first generation. The costimulatory domain is between the transmembrane region and the signaling domain CD3ζ molecule; different costimulatory domains activate CAR T-cells in different directions. Currently, FDA approved CAR T-cell products are all second-generation designs with CD28 or 4-1BB costimulatory domains;

  3. A CAR molecule that contains two costimulatory structures at the same time is called a third-generation CAR; 

  4. Fourth-generation CARs add modifications that enhance T cell function, such as making T cells produce additional proteins (such as cytokines) or possess additional receptors.

Figure 4. Schematic diagram of the molecular structure of the 1-4 generation CAR.  [5]


CAR molecular construction can be adjusted and optimized on the basis of various generations of CAR structures, such as:

  • Selection of scFv: The focus of scFv selection is specificity. If there is a cross-reaction with proteins outside the target, the probability of off-target effects will be higher. Generally, CAR molecules need suitable affinity, too high or too low will affect the effect of CAR T-cells, so it is necessary to further design experiments to screen out CAR molecules with suitable affinity;
  • Optimization of linker region: The linker is the region that connects the variable heavy chain (VH) and light chain (VL) of scFv. The linker usually comes from repeated glycine and serine residues, [4] so its length has optimizable space;
  • Selection of hinge: The hinge region connects the extracellular domain and the transmembrane region that recognizes the antigen, it affects the spatial position of the extracellular domain and the cell membrane, and this region needs to have a certain flexibility to allow scFv to bind antigen;
  • Selection of costimulatory domains: Different costimulatory molecules have different properties. Table 2, from Novartis' website, lists the differences between two common costimulatory domains, 4-1BB and CD28.




Enhanced CAR-T cell expansion and persistence

Enhance early expansion and rapid expansion of CAR-T cells

In vitro experiments have been shown to induce differentiation of central memory T cells and increase proliferative potential

In vitro experiments have shown that it is related to the differentiation of effector memory T cells, providing timely protection

May prevent CAR-T cell exhaustion

Limited persistence and rapid depletion of CAR-T cells

Table 2. Function of co-stimulatory domains 4-1BB and CD28.


3. Construction of CAR T-cells

The construction of CAR T-cells is similar to the construction of stably transfected cell lines. However, T cells are generally in a quiescent state and T cells need to be activated first. After the CAR molecule and vector are designed, the construction of CAR-T cells can be started.

For lentiviral transfection, due to the particularity of immune cells, the viral purity required for lentiviral transduction of T cells is higher than common stable transfected strains. The general construction method of CAR T-cells is as follows: isolate and activate peripheral blood mononuclear cells (PBMCs), transduction with lentivirus after 1-2 days, with the expansion and identification of CAR-T cells carried out later.


Figure 5. CAR T-cell construction process.


Evaluation of the killing effect of CAR-T cells in vitro

In vitro killing experiments of CAR T-cells usually involve co-incubation of CAR T-cells and target cells, alongside a series of evaluations. There are two sources of target cells, one is the tumor cell line that naturally expresses the antigen, and the other is the artificially constructed stably transfected cell line that expresses the positive antigen. The main method of artificially constructing stable transfected strains is via the lentivirus method. After constructing the viral vector, cells (such as 293T cells) are transfected to allow the cells to express or overexpress the antigen naturally.

After the CAR molecule recognizes the antigen, it will release T cell effector responses, including proliferation, cytokine release, metabolic changes, and cytotoxicity. CAR-T cells are thought to exert their cytotoxic functions mainly by secreting granzymes and perforin. [4] Cytokines (such as interferon IFN-γ, interleukin IL-2, etc.) can modulate immune responses and perform different antitumor functions. Common in vitro killing effect evaluation methods include T cell proliferation detection, killing experiment (detecting the degree of lysis of target cells), cytokine detection, etc. Flow cytometry is one of the effective methods to quantitatively detect various cell subsets. Cell counting reagents (Cell Counting Kit-8, CCK-8), MTT, calcein, and real-time label-free cell analysis (RTCA) can be used for detection of killing effects. Cytokine detection experiments commonly use a combination of enzyme linked immunosorbent assay (ELISA) and cytometric bead array (CBA) methods.


In vivo antitumor activity evaluation of CAR T-cells

Generally, in vivo models of CAR-T research will use tumor cell line derived xenografts (CDX) models or human tumor patient derived xenografts (PDX) models, and further studies will also use syngeneic transplantation models and animal models of human immune system reconstruction. In vivo anti-tumor monitoring of CAR-T cells is often used in vivo imaging, so constructing Luci-GFP target cells to express green fluorescent protein is commonly used.

After inoculating the target cells to construct a tumor animal model, the CAR-T cells are reinfused, and then downstream evaluation experiments are performed. Figure 6 lists the commonly used immunodeficient mice, tumor formation methods, and CAR-T cell reinfusion methods for building tumor models. Different tumor formation methods are suitable for different mechanism studies, such as tumor metastasis. Subcutaneous tumor formation can be directly observed and the size of the tumor measured, and in situ inoculation can simulate the real environment of tumor occurrence.


Figure 6. In vivo functional evaluation of CAR-T cells.


The in vivo evaluation mainly focuses on tumor growth monitoring, mouse survival status, and toxicity detection. In vivo imaging of small animals is one of the most commonly used detection methods, which can intuitively reflect the growth of tumor cells in vivo. At the same time, Mean Fluorescence Intensity (MFI) in vivo is also one of the common indicators to evaluate tumor growth. Regular monitoring of the body weight, hair changes, survival period, and proliferation status of T cell subsets in the body of small animals can be used to evaluate the impact of CAR-T cell reinfusion on the animal body.

In addition, pathological tests such as immunohistochemistry or H&E staining can reflect the degree of infiltration of CAR-T cells, tumor cells, or pathological changes in organs and tissues from different dimensions.


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Based on years of research experience in the field of tumor immunity, Cyagen can provide CAR-T and other cell therapy researchers with full-process services from CAR virus preparation, tumor immune cell and animal model construction, to in vitro and in vivo efficacy evaluations. Contact us to discover how we can accelerate the development of your CAR-T and cell therapy research.




[1] Li, C., Mei, H., & Hu, Y. (2020). Applications and explorations of CRISPR/Cas9 in CAR T-cell therapy. Briefings in functional genomics, 19(3), 175-182.

[2] Banerjee, S., Parasramka, M. A., & Paruthy, S. B. (2018). Garcinol: Preclinical Perspective Underpinning Chemo-and Radiosensitization of Cancer. In Role of Nutraceuticals in Cancer Chemosensitization (pp. 297-324). Academic Press.

[3] Wei, J., Guo, Y., Wang, Y., Wu, Z., Bo, J., Zhang, B., ... & Han, W. (2021). Clinical development of CAR T cell therapy in China: 2020 update. Cellular & molecular immunology, 18(4), 792-804.

[4] Larson, R. C., & Maus, M. V. (2021). Recent advances and discoveries in the mechanisms and functions of CAR T cells. Nature Reviews Cancer, 21(3), 145-161.

[5] Singh, A. K., & McGuirk, J. P. (2020). CAR T cells: continuation in a revolution of immunotherapy. The Lancet Oncology, 21(3), e168-e178.