The B7x Immune Checkpoint Pathway: From Discovery to Clinical Trial.

B7x (B7 homolog x, also known as B7-H4, B7S1, and VTCN1) was discovered by ourselves and others in 2003 as the seventh member of the B7 family. It is an inhibitory immune checkpoint of great significance to human disease. Tissue-expressed B7x minimizes autoimmune and inflammatory responses. It is overexpressed in a broad spectrum of human cancers, where it suppresses antitumor immunity. Further, B7x and PD-L1 tend to have mutually exclusive expression in cancer cells. Therapeutics targeting B7x are effective in animal models of cancers and autoimmune disorders, and early-phase clinical trials are underway to determine the efficacy and safety of targeting B7x in human diseases. It took 15 years moving from the discovery of B7x to clinical trials. Further studies will be necessary to identify its receptors, reveal its physiological functions in organs, and combine therapies targeting B7x with other treatments.

Ligand fitting with CCP4.

Crystal structures of protein-ligand complexes are often used to infer biology and inform structure-based drug discovery. Hence, it is important to build accurate, reliable models of ligands that give confidence in the interpretation of the respective protein-ligand complex. This paper discusses key stages in the ligand-fitting process, including ligand binding-site identification, ligand description and conformer generation, ligand fitting, refinement and subsequent validation. The CCP4 suite contains a number of software tools that facilitate this task: AceDRG for the creation of ligand descriptions and conformers, Lidia and JLigand for two-dimensional and three-dimensional ligand editing and visual analysis, Coot for density interpretation, ligand fitting, analysis and validation, and REFMAC5 for macromolecular refinement. In addition to recent advancements in automatic carbohydrate building in Coot (LO/Carb) and ligand-validation tools (FLEV), the release of the CCP4i2 GUI provides an integrated solution that streamlines the ligand-fitting workflow, seamlessly passing results from one program to the next. The ligand-fitting process is illustrated using instructive practical examples, including problematic cases such as post-translational modifications, highlighting the need for careful analysis and rigorous validation.

Structure and cancer immunotherapy of the B7 family member B7x.

B7x (B7-H4 or B7S1) is a member of the B7 family that can inhibit T cell function. B7x protein is absent in most normal human tissues and immune cells, but it is overexpressed in human cancers and often correlates with negative clinical outcome. The expression pattern and function of B7x suggest that it may be a potent immunosuppressive pathway in human cancers. Here, we determined the crystal structure of the human B7x immunoglobulin variable (IgV) domain at 1.59 Å resolution and mapped the epitopes recognized by monoclonal antibodies. We developed an in vivo system to screen therapeutic monoclonal antibodies against B7x and found that the clone 1H3 significantly inhibited growth of B7x-expressing tumors in vivo via multiple mechanisms. Furthermore, the surviving mice given 1H3 treatment were resistant to tumor rechallenge. Our data suggest that targeting B7x on tumors is a promising cancer immunotherapy and humanized 1H3 may be efficacious for immunotherapy of human cancers.

The costimulatory molecule B7-H4 promote tumor progression and cell proliferation through translocating into nucleus.

B7-H4, a member of B7 family, is a transmembrane protein and inhibits T-cells immunity. However, in a variety of tumor cells, B7-H4 was detected predominantly in intracellular compartments with unknown mechanism and functions. In this study, we analyzed B7-H4 expression and subcellular distribution by immunohistochemistry in renal cell carcinoma (RCC) tissues. B7-H4 protein was detected on the membrane, in the cytosol and/or in the nucleus in tumor tissues. The membrane and nuclear expression of B7-H4 was significantly correlated with the tumor stages of RCC. Moreover, the membrane localization of B7-H4 was inversely correlated with the intensity of tumor infiltrates lymphocyte (TILs), whereas no association was observed between nuclear expression of B7-H4 and the density of TILs status. We further identified that B7-H4 is a cytoplasmic-nuclear shuttling protein containing a functional nuclear localization sequence (NLS) motif. A point mutation of B7-H4 NLS motif blocked the leptomycin B -induced nuclear accumulation of B7-H4. HEK293 cells stably expressing B7-H4 NLS mutant exhibited more potent inhibition in T-cell proliferation and cytokine production through increasing its surface expression compared with wild-type B7-H4 transfected cells owing to their increased surface expression. Most importantly, overexpression of wild-type B7-H4 in HEK293 cells enhanced tumor cell proliferation in vitro and tumorigenicity in vivo, promoted G1/S phase transition. The regulation of cell cycle by wild-type B7-H4 was partialy due to upregulation of Cyclin D 1 and Cyclin E. A mutation of B7-H4 NLS motif abolished the B7-H4-mediated cell proliferation and cell cycle regulation. Furthermore, B7-H4 wild-type confers chemoresistance activity to RCC cell lines including Caki-1 and ACHN. Our study provides a new insight into the functional implication of B7-H4 in its subcellular localization.