Recent studies highlight the integral role of the interferon gamma receptor (IFNγR) pathway in T cell-mediated cytotoxicity against solid but not liquid tumors. IFNγ not only directly facilitates tumor cell death by T cells but also indirectly promotes cytotoxicity via myeloid phagocytosis in the tumor microenvironment. Meanwhile, full human ex vivo immune checkpoint drug screening remains challenging. We hypothesized that an engineered gamma interferon activation site response element luciferase reporter (GAS-Luc2) can be utilized for immune checkpoint drug screening in diverse ex vivo T cell-solid tumor cell co-culture systems. We comprehensively profiled cell surface proteins in ATCC\'s extensive collection of human tumor and immune cell lines, identifying those with endogenously high expression of established and novel immune checkpoint molecules and binding ligands. We then engineered three GAS-Luc2 reporter tumor cell lines expressing immune checkpoints PD-L1, CD155, or B7-H3/CD276. Luciferase expression was suppressed upon relevant immune checkpoint-ligand engagement. In the presence of an immune checkpoint inhibitor, T cells released IFNγ, activating the JAK-STAT pathway in GAS-Luc2 cells, and generating a quantifiable bioluminescent signal for inhibitor evaluation. These reporter lines also detected paracrine IFNγ signaling for immune checkpoint-targeted ADCC drug screening. Further development into an artificial antigen-presenting cell line (aAPC) significantly enhanced T cell signaling for superior performance in these ex vivo immune checkpoint drug screening platforms.

Immunotherapy aiming at suppressing tumor development by relying on modifying or strengthening the immune system prevails among cancer treatments and points out a new direction for cancer therapy. B7 homolog 3 protein (B7-H3, also known as CD276), a newly identified immunoregulatory protein member of the B7 family, is an attractive and promising target for cancer immunotherapy because it is overexpressed in tumor tissues while showing limited expression in normal tissues and participating in tumor microenvironment (TME) shaping and development. Thus far, numerous B7-H3-based immunotherapy strategies have demonstrated potent antitumor activity and acceptable safety profiles in preclinical models. Herein, we present the expression and biological function of B7-H3 in distinct cancer and normal cells, as well as B7-H3-mediated signal pathways in cancer cells and B7-H3-based tumor immunotherapy strategies. This review provides a comprehensive overview that encompasses B7-H3\'s role in TME to its potential as a target in cancer immunotherapy.

BACKGROUND: Novel immune checkpoint-based immunotherapies may benefit specific groups of prostate cancer patients who are resistant to other treatments.
METHODS: We analyzed by immunohistochemistry the expression of B7-H3, PD-L1/B7-H1, and androgen receptor (AR) in tissue samples from 120 prostate adenocarcinoma patients treated with radical prostatectomy in Spain, and from 206 prostate adenocarcinoma patients treated with radical prostatectomy in Norway.
RESULTS: B7-H3 expression correlated positively with AR expression and was associated with biochemical recurrence in the Spanish cohort, but PD-L1 expression correlated with neither of them. Findings for B7-H3 were validated in the Norwegian cohort, where B7-H3 expression correlated positively with Gleason grade, surgical margins, seminal vesicle invasion, and CAPRA-S risk group, and was associated with clinical recurrence. High B7-H3 expression in the Norwegian cohort was also consistent with positive AR expression.
CONCLUSIONS: These results suggest distinct clinical relevance of the two immune checkpoint proteins PD-L1 and B7-H3 in prostate cancer. Our findings highlight B7-H3 as an actionable novel immune checkpoint protein in prostate cancer.

Single domain antibodies have certain advantages including their small size, high stability and excellent tissue penetration, making them attractive drug candidates. Rabbit antibodies can recognize diverse epitopes, including those that are poorly immunogenic in mice and humans. In the present study, we established a method to isolate rabbit VH single domain antibodies for potential cancer therapy. We immunized rabbits with recombinant human B7-H3 (CD276) protein, made a phage-displayed rabbit VH single domain library with a diversity of 7 × 109, and isolated two binders (A1 and B1; also called RFA1 and RFB1) from phage panning. Both rabbit VH single domains exhibited antigen-dependent binding to B7-H3-positive tumor cell lines but not B7-H3 knockout tumor cell lines. Our study shows that protein immunization followed by phage display screening can be used to isolate rabbit single domain antibodies. The two single domain antibodies reported here may have potential applications in cancer immunotherapy.

Type 1 Diabetes (T1D) is an autoimmune disease resulting from insulin-secreting β-cells mediated by autoreactive T cells. We demonstrated increased level of sB7-H3 in T1D patients than in healthy control group. This result suggests that B7-H3 may be may be a promising biomarker associated with the pathogenesis of T1D.

B7-H3 (CD276) belongs to the B7 family of immunoregulatory proteins and has been implicated in cancer progression and metastasis. In this study, we found that metastatic melanoma cells with knockdown expression of B7-H3 showed modest decrease in proliferation and glycolytic capacity and were more sensitive to dacarbazine (DTIC) chemotherapy and small-molecule inhibitors targeting MAP kinase (MAPK) and AKT/mTOR pathways: vemurafenib (PLX4032; BRAF inhibitor), binimetinib (MEK-162; MEK inhibitor), everolimus (RAD001; mTOR inhibitor), and triciribidine (API-2; AKT inhibitor). Similar effects were observed in melanoma cells in the presence of an inhibitory B7-H3 monoclonal antibody, while the opposite was seen in B7-H3-overexpressing cells. Further, combining B7-H3 inhibition with small-molecule inhibitors resulted in significantly increased antiproliferative effect in melanoma cells, as well as in BRAFV600E mutated cell lines derived from patient biopsies. Our findings indicate that targeting B7-H3 may be a novel alternative to improve current therapy of metastatic melanoma.

CD276 (B7-H3) is a costimulatory molecule that plays a potent role in T cell responses, however, the role of B7-H3 in autoimmune diseases has not been elucidated. We analyzed B7-H3 expression in rheumatoid arthritis (RA) for the first time and found B7-H3 was significantly up-regulated on monocytes in RA patients, while the levels of soluble B7-H3 in serum were lower than in controls (P < 0.0001). These differences correlated with clinical and laboratory disease parameters and informatory factor TNF-α. Through in vitro experiments, we demonstrated that B7-H3 promoted TNF-α secretion. In addition, a new polymorphism variant, B7-H3-T-A-C-T, was identified and shown to be associated with the incidence of RA and the decreased release of sB7-H3. These results suggest that B7-H3 may be a promising biomarker associated with the pathogenesis of RA. Notably, the new B7-H3-T-A-C-T polymorphism variant is associated with RA risk and might be associated with the release of soluble B7-H3.