CAR-T cell therapy, also known as Chimeric Antigen Receptor T-cell therapy, is a revolutionary immunotherapy. CAR-T therapy involves the use of genetic engineering techniques to introduce the chimeric antigen receptor (CAR) gene into the patient's T cells. The CAR is composed of a single-chain variable fragment (scFv), which can recognize and bind to specific antigens on the surface of tumor cells. When CAR-T cells recognize tumor antigens, they activate the cytotoxic function of T cells, releasing cytotoxins such as perforin and granzymes, directly killing tumor cells. In addition, CAR-T cells can also activate other immune cells by releasing cytokines, enhancing the overall anti-tumor response. As an emerging immunotherapy, CAR-T cell therapy has shown great potential and broad application prospects in the field of cancer treatment.

Introduction to CAR Structure
The structure of CAR consists of three main parts: the extracellular domain, the transmembrane domain, and the intracellular signaling domain.

  
Extracellular Domain
√ Antigen recognition region: It usually composed of a single-chain variable fragment (scFv), which is formed by connecting the variable heavy chain (VH) and variable light chain (VL) of an antibody through a flexible linker. The scFv endows CAR with the ability to specifically recognize and bind to target antigens.
√ Hinge region: It cConnects the scFv and the transmembrane domain, providing structural flexibility and stability. The length and source of the hinge region (such as IgG hinge or CD8α/CD28 extracellular domain) can affect the function of CAR.

  
Transmembrane Domain
Function: It anchors CAR on the cell membrane, ensuring stable expression of CAR. The design of the transmembrane domain has an important impact on the expression level and stability of CAR.

Intracellular Signaling Domain
√Signaling transduction domain: It usually contains the CD3ζ chain, providing T-cell receptor activation signals.
√Costimulatory domain: To enhance the activation and persistence of CAR-T cells, the second-generation CAR introduced costimulatory domains, such as CD28 or 4-1BB. These costimulatory signals help improve the proliferation and effector function of CAR-T cells.

Illustration of CAR Structure

 
G4S linker and Whitlow/218 Linker are the two most common flexible linkers.

√ G4S linker is a commonly used flexible amino acid chain, usually composed of glycine (Gly, G) and serine (Ser, S), with the sequence (GGGGSGGGGSGGGGS), abbreviated as (G4S)3. This sequence does not lead to the formation of specific secondary structures, and is therefore widely used in protein fusion. The main function of G4S linker is to connect different protein domains, providing structural flexibility to reduce the mutual influence between different domains. In CAR cell therapy, G4S linker is used to connect the variable heavy chain (VH) and variable light chain (VL) in the single-chain variable fragment (scFv), forming a flexible structure. This design helps improve the recognition ability and function of CAR cells.
√ Whitlow/218 Linker mainly used to connect the variable heavy chain (VH) and variable light chain (VL) in the single-chain variable fragment (scFv). Its sequence is based on the research published by Whitlow et al. in 1993, aiming to improve the stability and functionality of scFv. The specific sequence of Whitlow/218 Linker is GSTSGSGKPGSGEGSTKG. In CAR cell therapy, the use of Whitlow/218 Linker can increase the affinity of scFv for tumor antigens. This is very important for the targeting ability and therapeutic effect of CAR-T cells, because higher affinity means that CAR-T cells can more effectively recognize and bind to tumor cells. It also helps reduce the aggregation and degradation of scFv, thereby improving the stability and functionality of CAR-T cells. This is crucial for maintaining the persistence and activity of CAR-T cells in the body.

Comparison of different CAR detection methods

Rabbit recombinant monoclonal antibodies specifically targeting G4S linker and Whitlow/218 Linker can be used for the detection and analysis of CAR-T and other cells containing these linkers. This is very useful in the research and development process of CAR-T and other cell therapies. Starter Bio launched G4S linker and Whitlow/218 Linker FACS antibodies for CAR positive rate detection and functional assessment.

Performance

Flow cytometric analysis ScFv based CAR containing a G4S linker transfected 293T (Human embryonic kidney epithelial cell) (Right panel) or 293T (Left panel) was stained with SDT G4S Linker Recombinant Rabbit mAb (Alexa Fluor® 488 Conjugate) at 0.1 μg/test. Flow cytometry and data analysis were performed using BD FACSymphony™ A1 and FlowJo™ software.

Flow cytometric analysis ScFv based CAR containing a  Whitlow/218 linker transfected 293T (Human embryonic kidney epithelial cell, Right panel) or 293T (Left panel) was stained with SDT Whitlow/218 Linker Recombinant Rabbit mAb (Alexa Fluor® 647 Conjugate) at 0.01 μg/test. Flow cytometry and data analysis were performed using BD FACSymphony™ A1 and FlowJo™ software.

Whitlow/218 Linker antibody Products list

G4S Linker antibody Products list