CAR Molecule Design and Lentivirus Preparation

CAR Molecule Design

A CAR (chimeric antigen receptor) consists of a signal peptide, a single-chain variable region derived from an antibody, a hinge region, a transmembrane domain, a co-stimulatory molecule structure, and a CD3ζ signal transduction domain. The process of CAR molecule design and lentivirus preparation is an important step in the development of cell therapy and involves several key stages.

 

  • scFv Screening：The affinity and specificity of scFv have a significant impact on the function of CAR molecules, and it is essential to screen for appropriate single-chain variable fragments (scFvs) with functional antigen binding activity.
  • Optimization of Linkers：Design linkers of different lengths to increase the flexibility between VH and VL, thus affecting the binding affinity.
  • Optimization of Hinge Regions：Design hinges of different lengths to increase the spatial flexibility of the antigen binding domain.
  • Selection of Co-stimulatory Molecule Structures：Different co-stimulatory molecules have different properties, 4-1BB and CD28 are the most used co-stimulatory molecules .
  • Reporter Genes：Green fluorescent protein genes or other tag gene can be connected to characterize the successful transduction of the CAR molecule.

 

Figure 1. CAR molecular structure.

 

Category
 Source Function Expression site
Signal peptide

CD8α, GM-CSF, etc.

Direct the CAR molecule to the cell membrane

Extracellular

Antibody scFv
Patents, literature, antibody development

Targeting recognition of tumor antigens

Extracellular

Hinge region

CD8α, IgG1, IgG4, IgGD, etc.
Maintain the relative spatial conformation between scFv and cell membrane

Extracellular

Transmembrane domain

CD8α, CD28, CD4, etc.

Anchor CAR molecule membrane expression

Transmembrane

Co-stimulatory molecules

4-1BB, CD28, CD27, ICOS, etc.

Provide T cell activation second signal

Intracellular

Signaling Molecules

CD3ζ

Provide the first activation signal for T-cells

Intracellular

Table 1. Common elements used in CAR molecule design.

 

Vector Construction

After designing the CAR molecule, it is usually synthesized and subcloned onto a lentiviral shuttle plasmid to generate a CAR expression vector, as shown in the diagram. Additionally, Cyagen offers a variety of in-stock CAR lentiviral plasmids, targeting human CD19, BCMA, GPC3, FAP, HER2, and other antigens, which are constantly updated and expanded.

Figure 2. CAR lentiviral vector structure.

 

Virus Packaging and Purification

Cyagen has extensive experience in preparing lentiviral vectors and provides diverse lentivirus preparation services. Using a highly safe third-generation lentivirus packaging system, Cyagen follows the preparation process outlined in Figure 4.

Figure 3. Lentiviral packaging technology route.

Service Offerings:

 

  • CAR molecule design and vector construction
  • CAR lentivirus production
  • Stable cell line construction for target antigen expression

 

Deliverables:

 

  • Vector construction report
  • Lentivirus quality control report
  • Concentrated lentivirus

 

Customer-Provided Materials:

 

  • Target antigen information
  • Antibody scFv sequence (optional)
  • CAR molecule structure information
  • Required vector information

Why choose Cyagen?

  • Industry-leading expertise in CAR molecule design, providing comprehensive and customized solutions
  • Robust library of CAR lentiviral vectors, delivering fast and top-quality services
  • Specialized stable cell line construction service, ensuring high levels of antigen expression and stability
  • Highly efficient lentivirus production process, yielding high titer and purity products
  • Rigorous quality control standards, ensuring the highest level of product quality and reliability

Case Study

  • CD19 Antigen Lentivirus Titer Detection

The purified lentivirus expressing CD19 antigen was subjected to gradient dilution (four gradients of 0.01, 0.1, 1, and 10 μL) and added to an equal number of 293T cells. After 72 hours, the percentage of 293T cells positive for lentivirus infection (i.e. the percentage of lentivirus-infected 293T cells out of the total number of cells) was determined by flow cytometry to calculate the lentiviral transduction titer.

Figure 4. Lentiviral titration results for CD19 antigen.

 

As shown in the figure, the number of CD19 antigen-positive cells gradually increased with the increase in the viral transduction gradient, indicating that the lentivirus has good infectivity. The viral titer was calculated to be 1.4 × 108 TU/mL.

 

  • Construction of CD19-293T Stable Cell Line

Using the aforementioned CD19 antigen lentivirus to transfect 293T cells, we obtained a highly homogeneous CD19-293T stable cell line with high expression of CD19 antigen after multiple rounds of screening.

Figure 5. CD19 antigen positivity rate detection results in CD19-293T cells.

  • FMC63 CAR Lentiviral Titration

We designed the FMC63 CAR molecule targeting CD19 antigen with the following structure and constructed it on a lentiviral vector for lentivirus preparation. The lentiviral titer of FMC63 CAR was detected using the aforementioned method and was found to be approximately 4.33 × 108 TU/mL.

 

Figure 6. FMC63 CAR molecule structure.

 


Figure 7. FMC63 CAR lentiviral titration results.