Chimeric antigen receptor T (CAR-T) cell therapy, as a new type of precision targeted therapy, has completely changed the pattern of cancer immunotherapy in the 21st century and has become one of the most cutting-edge and popular directions in medical research today. CAR-T therapy transforms the patient's own T cells through genetic engineering, enabling them to accurately identify and attack cancer cells, especially in blood tumors such as leukemia and lymphoma, showing amazing efficacy, bringing hope and improved quality of life to more and more patients.

Basic principles of CAR-T cell therapy

CAR-T cell therapy is a highly personalized tumor treatment method. Its core is to introduce chimeric antigen receptor (CAR) genes that can specifically recognize tumor antigens into patient T cells through genetic engineering technology to transform them into CAR-T cells. These modified T cells can accurately identify and efficiently kill tumor cells expressing specific antigens, thereby achieving targeted treatment of tumors.

The production and preparation process of CAR-T mainly includes the initial separation and enrichment of T cells, T cell activation, amplification, CAR gene transfer using viral or non-viral vector systems, in vitro CAR-T cell amplification, and the final terminal process and cryopreservation of the manufactured cell products.

Figure 1. Manufacturing steps of CAR-T cells. (Chen R, et al., 2023)

The key role of lentiviral packaging in the preparation of CAR-T cells

In the preparation of CAR-T cells, stable transfection is the prerequisite for the stable expression of CAR in effector T cells. At present, the mainstream method for gene transduction of CAR-T is still viral transduction, mainly γ-retrovirus and lentivirus. However, in recent years, due to the risk of inserting tumors, the inability to infect non-dividing cells, and low viral titers, γ-retroviral transduction has gradually been replaced by lentiviral transduction in clinical trials. Therefore, lentiviral packaging technology plays an indispensable role in the preparation of CAR-T cells.

The specific process is as follows: First, T cells are isolated from the patient's peripheral blood and activated to make them in a state that is easy to accept exogenous genes. Subsequently, the CAR gene is introduced into the T cell using a lentiviral vector. Since the lentiviral vector can efficiently integrate exogenous genes, the stable expression of the CAR gene in T cells is ensured, thereby successfully preparing CAR-T cells.

Characteristics and advantages of lentiviral vectors

Lentiviral vectors (LVs) originate from the genus lentivirus in the retroviridae family, and its prototype is human immunodeficiency virus (HIV). In order to make it safe for gene therapy, scientists have modified the HIV-1 genome. The HIV-1 genome is packaged in different plasmids, and its self-replication ability is removed by co-transfection of cells, thereby constructing a lentiviral vector with only single infection ability. The lentiviral vector genome is a single-stranded positive-strand RNA. After its genome enters the cell, it is reversed into DNA by the reverse transcriptase it carries in the cytoplasm to form a DNA pre-integration complex. After entering the cell nucleus, the DNA is integrated into the cell genome. The integrated DNA is transcribed into mRNA, returned to the cytoplasm, and expressed the target gene or fragment.

The currently widely used four-plasmid system, composed of packaging plasmids, envelope plasmids, transfer plasmids, and auxiliary plasmids, has greatly improved the safety and application range of lentiviral vectors. Its structural characteristics are as follows:

- Envelope protein (such as VSV-G): determines the scope and efficiency of viral infection;

- Genome: carries the target gene (CAR sequence) and necessary packaging elements (such as LTR, Ψ packaging signal);

- Safety modification: The third-generation lentiviral vector eliminates pathogenic genes through a four-plasmid system (transfer plasmid + packaging plasmid + envelope plasmid + auxiliary plasmid) to ensure that it has no replication ability.

The advantages of lentiviral vectors are as follows:

• Efficient transfection: Lentiviral vectors can infect a variety of cell types such as dividing and non-dividing cells, and also have high transduction efficiency for "fortresses" such as primary cells, stem cells and undifferentiated cells that are difficult to conquer with traditional transfection technology.
• Persistent expression: The unique integration mechanism of lentiviral vectors can integrate exogenous genes into the genome of host cells to achieve long-term and stable expression of genes.
• Large capacity: Lentiviral vectors can carry larger gene fragments and can meet the needs of different gene therapies. Especially in the treatment of complex gene defect diseases, it has irreplaceable advantages.
• High safety: The modified lentiviral vector does not have the ability to replicate and will not cause viral infection.

Production and preparation of lentiviral vectors

As one of the core links of CAR-T, the quality of lentiviral vectors has a direct impact on cell products and their therapeutic effects. The production of lentiviral vectors is a complex process that requires multiple steps such as cell culture, virus packaging and virus purification. Its standardized process includes:

1. Cell culture system

- Selection of cell lines: HEK293T cell lines are usually used for large-scale lentiviral production. This cell line has significant advantages such as rapid growth, easy transfection and high virus yield. It can proliferate rapidly under suspension culture conditions, providing an ideal host cell for large-scale production of lentiviruses.

- Evolution of culture methods: Traditional adherent culture methods are widely used in small-scale studies, but have many limitations in large-scale production. With the development of technology, suspension serum-free culture has gradually become the mainstream. Due to its clear chemical composition and lack of serum, this culture method can not only effectively reduce production costs and contamination risks, but also simplify the culture and subsequent purification steps, providing strong support for the large-scale expansion of lentiviral production.

2. Virus packaging process

- Plasmid transfection: When HEK293T cells are cultured to a suitable density, the virus packaging plasmid, envelope plasmid and transfer plasmid carrying the CAR gene are introduced into the cells by co-transfection. During the transfection process, various plasmids work together to start the lentivirus packaging process.

- Virus production and harvesting: During the several days of culture after transfection, HEK293T cells will use their own biosynthetic mechanism to produce a large number of lentivirus particles. These particles are released into the culture medium, and the supernatant containing lentivirus can be harvested through preliminary treatment methods such as centrifugation and filtration.

3. Virus purification and quality control

- Purification method: In order to obtain high-purity lentivirus vectors, ultracentrifugation, ion exchange chromatography (AEX) or affinity chromatography are often used to remove impurities, cell debris and unpackaged plasmids in the culture medium, thereby improving the purity and quality of lentivirus vectors.

- Quality control indicators: The quality control of lentiviral vectors covers multiple aspects, including virus titer determination, purity analysis (detection of host cell proteins, DNA residues, etc.), activity evaluation (verification of its transduction efficiency to target cells and CAR gene expression) and safety testing (such as sterility testing, endotoxin testing, etc.) to ensure that lentiviral vectors meet clinical application standards.

As the core link of CAR-T cell therapy, lentiviral packaging technology plays a vital role in the field of tumor immunotherapy. In-depth research on the biological basis and production process of lentiviral packaging and overcoming the current challenges are of far-reaching significance for promoting the development of CAR-T cell therapy technology and even the progress of the entire field of tumor immunotherapy.