In a study published on August 13, 2023 in Nature Communications, a team of researchers identified that the complementarity determining region (CDR) loop in a single chain variable fragment (scFv) can lead to CAR aggregation, which can cause chimeric antigen receptor (CAR) - T cell failure, cell death, and reduced tumor cell reactivity to target antigen expression, resulting in antigen independent activation and potential functional impairment.

CAR-T cell therapy is highly effective for the treatment of blood cancer, and in this cell therapy, the design of CARs is even more important. In this study, the extracellular domain of human interleukin-13 receptor alpha 2 (IL13R alpha 2) was co expressed with BirA biotin ligase using the multiBac expression system. After a series of optimized expression of the receptor protein, purification and protein validation were carried out. A human scFv synthetic library was used for phage display screening, and corresponding scFv antibodies were selected for production through solid-phase screening and binding assay analysis. Suitable antibodies were selected for lentiviral vector construction and packaging through SPR and ELISA assays. The researchers further transduced and cultured T cells, and then examined CAR in these cells using confocal microscopy. The distribution on top. To analyze the behavior of CAR-T cells, researchers evaluated the binding of CAR-T cells to recombinant IL13R α 2, stained T cell surface markers, and studied the expression of CAR on the surface and inside the cells. In addition, they also explored CAR-T cell proliferation and cytokine secretion, and analyzed gene expression in different CAR-T constructs, emphasizing their differences. In vivo experiments involve implanting tumor cells in situ into mice, followed by CAR-T therapy and monitoring tumor growth through in vivo imaging systems (IVIS). Perform statistical analysis using GraphPad Prism and standard significance markers.

In the current study, researchers evaluated the CAR construct structure, its extracellular scFv, linker, and additional domains to understand their impact on CAR-T cell performance. It has been determined that the CDR loop within scFv is responsible for antigen recognition, and certain frameworks within scFv may inadvertently activate CAR signaling, potentially impairing T cell function. The interactions within the CDR loop may further affect the stability of CAR. Small differences in the CDR loop can greatly alter the effectiveness of CAR-T cells, and a new rapid screening method for CAR behavior has been proposed.

The key to CAR T-cell therapy (Chimeric Antigen Receptor T-cell therapy) is to help coordinate the immune response of T cells, modify T cells, insert CAR genes and express them on the surface of T cells as chimeric antigen receptor CAR proteins. CAR can directly recognize antigens on the surface of cancer cells, thereby achieving a specific therapeutic method for identifying and killing cancer cells in the patient's own body. Since 2017, the FDA has approved six CAR-T cell therapies for the treatment of blood cancers such as lymphoma. However, there are still difficulties in treating solid tumors, as the surrounding mediators can prevent CARs from recognizing antigens on the tumor surface. Additionally, solid tumors have heterogeneity, making it difficult to achieve therapeutic effects due to differences in the same cancer type within the body.