As life lies in movement, immunity relies on balance. A healthy individual has immune cells that can easily eradicate the recurring diseased cells in the body, including those potentially evolving to cancerous cells. Usually, the tumors are able to escape the body’s immune surveillance, thus being ignored by T cells, which function as the main force of all the anti-tumor immune systems in the body. The activity of T cells is largely regulated by two types of molecules — inhibitory and activated immune checkpoint molecules. In 2018, American scientist James Allison and Japanese scientist Tasuku Honjo shared the Nobel Prize in physiology or medicine for their pioneering contributions to the research on how CTLA-4 and PD-1 inhibit the negative regulation of T cells.

4-1BB (CD137) belonging to the TNFR superfamily acts as an important activated immune checkpoint molecule on the surface of T cells. CAR-T cells containing 4-1BB signals have been widely applied to miscellaneous targeted cell therapies. Moreover, multiple 4-1BB activated antibodies have already entered clinical trials, including the Urelumab from Bristol-Myers Squibb and Pfizer’s Utomilumab. When used alone or in combination with other tumor therapies, these antibodies dramatically enhance the efficiency of tumor therapies. Therefore, the study on the activation mechanism of 4-1BB, its ligand and activated antibodies can not only clarify its activation mechanism, but also has great value for the development of tumor immunotherapy drugs targeted at 4-1BB.

Recently, Cell Reports reported important findings on the action mechanism by which 4-1BB interacts with ligands and activated antibodies. The research team found that the action mode of 4-1BB and 4-1BBL belongs to the classical TNF-TNFR binding mode — 4-1BBL in the form of trimer binds three mono-4-1BB molecules, indicating the conserved mode in which the monomer 4-1BB binds the ligand. Analysis of the binding mechanism of Utomilumab, a 4-1BB activated antibody, showed that 4-1BBL, the ligand of 4-1BB, overlaps to a certain extent with the epitope of 4-1BB where the activated antibody Utomilumab binds. Furthermore, the experiments on the analysis of interactions between flow cytometry and Octet molecules have further demonstrated that both of them apparently compete to bind to 4-1BB, suggesting that the 4-1BB activation signal mediated by 4-1BBL is blocked during the action of Utomilumab.

By testing the activated T cells, the team discovered that the T cells can express mono-4-1BB and di-4-1BB molecules. In addition, the team also studied the binding mode of the two 4-1BB molecules, particularly the di-4-1BB. Their studies have shown that 4-1BBL binding to 4-1BB on the surface of T cells neither results in extensive cross-linking to 4-1BB nor over-activates downstream signals. The team proposed the action mode in which 4-1BB binds the ligand and activated antibodies, and pointed out that the cross-linking of the ligand and activated antibody mediated 4-1BB is regulated by its unique binding mode, and the ratio of mono-4-1BB and di-4-1BB molecules on the surface of T cells also constitutes an important regulatory factor for its signal activation.

The article’s explanation of 4-1BB, its ligand and activated antibodies is of great significance to understand the molecular basis of 4-1BB molecule activation and the action mechanism of activated antibodies. Meanwhile, this study lays important theoretical foundation for the development of activated immune checkpoint antibody drugs represented by 4-1BB.