CGT Innovative Therapy - Lentivirus-packaging CAR-T immune cells

CGT Innovative Therapy - Lentivirus-packaging CAR-T immune cells

Immune cell therapy is an innovative cancer treatment that enhances the body's immune system to target and eliminate cancer cells. Key forms include cytokine-induced killer cell therapy, T-cell therapy, and CAR-T cell therapy. CAR-T therapy involves reprogramming T cells to express chimeric antigen receptors (CAR) that specifically target tumor cells, showing significant success in hematological cancers like leukemia and lymphoma.

Keywords: CAR-T, Lentiviral, Lentiviral Vector, Tracer Lentivirus

Preparation of CAR-T Cell Therapy

In general, the key steps to prepare CAR-T cells are as follows:
                                                                         

                                         Figure Source: Dana-Farber Cancer Institute-CAR T-cell clinical trials help multiple myeloma patients return to health

1. 1.Sorting (T-Cell Isolation)

The first step in preparing CAR-T cells is to isolate T-cells from the white blood cells in the patient's peripheral blood. Techniques such as immunomagnetic bead separation or flow cytometry are typically used to extract CD3+-rich T-cells from the blood. To ensure cell purity, the sorting process needs to be very precise, ensuring that a sufficient number of high-quality T-cells are obtained.

2. 2.Stimulation (T-Cell Activation)

The extracted T-cells are activated in vitro, usually using a CD3/CD28 T-Cells Activation /Expansion Kit. Activated T cells enter an active state, preparing them for gene transfection. Through this process, T cell function is enhanced, contributing to subsequent genetic modification and amplification.

3. 3.Transfection (Gene editing)

Activated T cells undergo gene transfection to express a chimeric antigen receptor (CAR). This process typically uses lentiviral vector to introduce CAR genes into T cells. Lentivirus is an effective gene transduction tool, with the advantage of stably integrating the target gene (such as the CAR gene) into the cell genome. By infecting T cells and transfecting the CAR gene into T cell DNA, lentivirus enables T cells to express CARs long-term. The CAR gene includes a single-chain variable fragment (scFv) of an antibody that enables T cells to recognize specific antigens on the surface of tumor cells and initiate an immune response by activating signaling regions of T cells. As shown in Figure 2. Lentiviral transfection has the characteristics of high efficiency and stability, so that CAR gene can be expressed in T cells for a long time.

                                                                                                     Figure Source: Singapore CAR-T cell therapy 

4. 4.Amplification (Cell Proliferation)

After transfection, CAR-T cells enter the amplification stage, where growth factors such as IL-2 are added to promote cell proliferation. The purpose of this process is to ensure that a sufficient number of CAR-T cells are available for eventual infusion into the patient. The amplification process must be conducted under sterile conditions to guarantee cell quality and quantity.

5. 5.Quality Control (QC)
6. Before reinfusion into the patient, quality control is crucial during the preparation process. Prepared CAR-T cells undergo strict testing to ensure their purity, activity, transfection efficiency, and compliance with treatment standards. Additionally, testing for cell contamination or adverse reactions is necessary to ensure the safety and therapeutic efficacy of the cells after reinfusion into the patient.

Importance of lentivirus transfection in immune cells

Lentiviral transfection technology is crucial in immune cell therapy, especially for gene editing and cell engineering. Lentiviral vectors, derived from modified HIV-1, are widely used due to their ability to integrate genes into the host genome, ensuring stable, long-term expression. They can infect both dividing and non-dividing cells, and their safety has been improved by modifying key genes to prevent replication. These vectors are now commonly used in gene therapy, gene editing, and cell biology research for their efficiency and durability in gene delivery.

                                       Figure Source: obiosh.com/product/ Schematic diagram of lentivirus packaging and lentivirus transfection process in 293T cells

1.Efficient gene transduction

Lentiviral vector can efficiently deliver target genes, such as the CAR gene, into immune cells and integrate these genes stably into the cellular genome, ensuring that the CAR gene is continuously expressed during cell division and proliferation. This high-efficiency transduction capability is fundamental to the success of immunotherapy.

2.Lower Immunogenicity  

Compared to other viral vectors, lentiviruses have a lower immunogenicity. As a result, when lentiviruses are used to transduce immune cells, the patient's immune system generates a relatively weaker immune response to the virus. This helps to minimize adverse reactions, particularly in the long-term survival of cells after infusion, thereby preventing excessive immune rejection.

3.High Adaptability to Immune Cells  

Lentiviral vector are capable of efficiently transducing various types of immune cells, particularly T cells and NK cells. Through lentiviral transduction, immune cells can acquire tumor-specific recognition abilities, thereby enhancing their capacity to target and kill tumor cells.

4.Precision of Transduced Effector Cells in Target Cell Recognition  

In immunotherapy, transduced effector cells refer to immune cells that, after transduction, express CAR and exert anti-tumor activity. Lentiviral vectors can precisely deliver the CAR gene into these effector cells, enabling them to recognize and target tumor cells. Target cells are the tumor cells that are identified and destroyed by these immune cells. Through lentiviral transduction, immune cells can enhance their specificity in recognizing and killing tumor cells, thereby improving therapeutic efficacy.

In this context, IPHASE, a leader in in vitro research reagents, has developed a Lentivirus Packaging Kit and Lentivirus Concentration Reagent to simplify the complexity of transduction for immunotherapy customers and improve the success rate of stable gene transfection. These products offer a one-stop solution for customers.

IPHASE Lentivirus Products

The IPHASE Lentivirus Packaging Kit contains all the essential reagents required for lentivirus packaging, significantly simplifying the experimental process and saving time and effort. This kit includes:

• Lentivirus Packaging Plasmid Mix
• Transfect Reagent
• eGFP Plasmid
• IPHASE Lentivirus Concentration Reagent

This kit truly streamlines the experiment! With no need for complicated reagent preparation, lentivirus packaging can be efficiently completed by simply following the included instructions. The unique advantages of the kit include short packaging time, high viral titer, and easy operation, making it ideal for beginners in lentivirus packaging. Each set of reagents undergoes strict quality control to ensure accurate, reliable, and reproducible results, significantly improving transfection efficiency and the stability of viral vectors, thereby providing potent technical support for immune cell preparation.

Additionally, to help customers save costs, IPHASE has also developed and produced the Lentivirus Concentration Reagent, offering a quick and simple concentration solution. By simply mixing the lentivirus supernatant with the concentration reagent, followed by a brief incubation and centrifugation using a standard centrifuge, lentiviral particles can be rapidly concentrated. This process does not require an ultracentrifuge, making it a convenient and cost-effective option for most laboratory users.

In brief, the IPHASE Lentivirus Concentration Reagent not only significantly improves transfection efficiency but also provides high-quality viral vector support for immune cell preparation. It is an essential product for routine experiments, preclinical research, and other application areas.

In summary, immunotherapy, particularly CAR-T cell and CAR-NK cell therapies, represents a new direction in modern cancer treatment. Through precise processes such as cell sorting, activation, transduction, expansion, and quality control, scientists can prepare efficient and safe immune cell products for patients, enabling targeted cancer treatment. Lentiviral transduction, as a core technology, plays a critical role in the preparation of CAR-T and CAR-NK cells due to its high efficiency and stability. With continuous technological advancements, immunotherapy holds the potential to offer innovative treatment options for more patients, providing greater clinical value. In the future, immunotherapy is expected to bring revolutionary therapeutic hope to more cancer patients.

To meet the needs of gene transduction, expression, and disease research, IPHASE offers Lentivirus Packaging Kits, Lentivirus Concentration Reagents, and tracer lentiviruses. These tracer lentiviruses are packaged with Green Fluorescent Protein (GFP), Red Fluorescent Protein mCherry, and Luciferase, enabling easy tracking of target cells both in vitro and in vivo.

Besides,IPHASE can also offers two pre-packaged tracer lentiviruses: GFP & Luciferase and mCherry & Luciferase. These tracers can be selected or combined based on experimental needs, streamlining workflows and enhancing efficiency for real-time tracking and monitoring of target cells.

IPHASE Immunotherapy Related Products

In addition to lentivirus-related products used in CAR-T or CAR-NK therapies, IPHASE, as a leader in in vitro research reagents, has also made significant contributions to support innovations in CGT (Cell and Gene Therapy) and other fields.  IPHASE has developed and produced leukocytes and PBMCs (Peripheral Blood Mononuclear Cells) isolated through leukapheresis, along with various immune cell isolation kits, to support the rapid development of immunotherapy.

IPHASE products offer the following advantages, and a Certificate of Analysis (COA) verified by IPHASE is available for each batch.

Safety  

IPHASE products are sourced from healthy donors/animals that have undergone thorough pre-screening, with negative results for viral testing (HIV-1/2, HBV, HCV, Syphilis).

Compliance  

IPHASE provides informed consent forms signed by the donors, with clear and traceable source verification.

Professionalism  

The products provided by IPHASE, obtained through leukapheresis, are handled by trained professionals. Each batch is accompanied by a corresponding COA, and the products are transported under a strict cold chain to ensure safety, rapid delivery, and quality assurance.

Customer Service  

IPHASE offers high-quality after-sales service, ensuring a smooth and uninterrupted experience throughout the trial process.