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Bacterial and viral infections cause a huge burden to healthcare settings worldwide, and mortality rates associated with infectious microorganisms have remained high in recent decades. Despite tremendous efforts and resources worldwide to explore diagnostic biomarkers, rapid and easily assayed indicators for the diagnosis of bacterial and viral infections remain a challenge. B7 homolog 3 (B7-H3), a member of the B7 family of immunoregulatory proteins, is overexpressed in patients with septicemia, meningitis, pneumonia, and hepatitis. Therefore, B7-H3 could be used as a potential clinical indicator and therapeutic target for bacterial and viral infections caused by H. pylori, S. pneumoniae, M. pneumoniae, hepatitis B virus (HBV), viral hemorrhagic septicemia virus (VHSV), respiratory syncytial virus (RSV), and human immunodeficiency virus (HIV). Moreover, the interplay between infectious microorganisms and B7-H3 and exploration of the functional roles of the B7-H3 molecule could aid in the development of novel strategies for disease diagnosis and immunotherapy. [ABSTRACT FROM AUTHOR]

Purpose: Targeted therapy development in soft tissue sarcoma (STS) has been burdened by the heterogeneity of this group of rare tumors. B7 homolog 3 protein (B7-H3) is a molecule in the same family as programmed death-ligand 1 (PD-L1). It has limited expression in noncancerous tissues and is overexpressed in many cancers, making it an attractive target for cancer therapy, and clinical trials targeting B7-H3 are actively underway. While available data demonstrate high expression levels of B7-H3 in individual sarcoma subtypes, its expression patterns across STS subtypes are not well described. The purpose of this study was to characterize the expression patterns of B7-H3 in STS. Patients and methods: This retrospective analysis evaluated STS tumor specimens from patients with a variety of different subtypes. Specimens were evaluated by immunohistochemistry (IHC) for expression and staining pattern of B7-H3 both in tumors and in associated vasculature. Results: Specimens from 153 sarcoma patients included 15 different STS subtypes. B7-H3 was broadly expressed in 97% of samples (95% CI 0.93–0.99) and 69.2% demonstrated high levels of B7-H3 expression (95% CI 0.61–0.76). No significant association between B7-H3 positivity or expression level and prior treatment(s), tumor size, tumor grade, or patient age. B7-H3 positivity in vessels was found in 94.7% (145/153) of samples. In tumors that had been previously assessed for PD-L1 and PD-1, there was no correlation between B7-H3 positivity or expression and the positivity or expression level of PD-L1 or PD-1. Conclusion: These data show high levels of B7-H3 positivity across soft tissue sarcoma subtypes, suggesting its feasibility as a therapeutic target for future sarcoma treatments. Future clinical trials are needed to evaluate whether targeting B7-H3 can provide clinical benefit to help patients with sarcoma. [ABSTRACT FROM AUTHOR]

Background: High‐intensity focused ultrasound (HIFU) is a promising minimally invasive treatment for liver cancer; however, its efficacy is often limited by the attenuation of ultrasonic energy. This study investigates the effectiveness of B7‐H3‐targeted microbubbles (T‐MBs) in enhancing HIFU ablation of liver cancer and explores their potential for clinical translation. Methods: T‐MBs and isotype control microbubbles (I‐MBs) were synthesized through the conjugation of biotinylated anti‐B7‐H3 antibody and isotype control antibody to the microbubble surface, respectively. Contrast‐enhanced ultrasound imaging was performed to compare the accumulation of T‐MBs and I‐MBs in liver cancer at various time points. The efficacy of T‐MBs in enhancing HIFU treatment was evaluated by measuring the immediate tumor ablation rate and long‐term tumor growth suppression. Additionally, the induced antitumor immune response was assessed through cytokine quantification in serum and tumor tissue, along with immunofluorescence staining conducted on days 1, 3, and 7 post‐treatment. Results: T‐MBs demonstrated superior liver cancer‐specific accumulation, characterized by higher concentrations and prolonged retention compared to I‐MBs. The combination of T‐MBs with HIFU resulted in significantly enhanced tumor ablation rates and superior tumor growth suppression. Post‐treatment analysis revealed a gradual uptick in cytokine levels within the tumor microenvironment, along with progressive infiltration of antitumor immune cells. Conclusion: T‐MBs effectively enhance the therapeutic efficacy of HIFU for liver cancer treatment while simultaneously promoting an antitumor immune response. These findings provide a strong experimental foundation for the clinical translation of ultrasound molecular imaging combined with HIFU as a novel approach for tumor therapy. [ABSTRACT FROM AUTHOR]

Background: Breast cancer (BC) is the most common malignancy in women. Immunotherapy has revolutionized treatment options in many malignancies, and the introduction of immune checkpoint inhibition yielded beneficial results also in BC. However, many BC patients are ineligible for this T cell-based therapy, others do not respond or only briefly. Thus, there remains a high medical need for new therapies, particularly for triple-negative BC. CD276 (B7-H3) is overexpressed in several tumors on both tumor cells and tumor vessels, constituting a promising target for immunotherapy. Methods: We analyzed tumor samples of 25 patients using immunohistochemistry to assess CD276 levels. The potential of CC-3, a novel bispecific CD276xCD3 antibody, for BC treatment was evaluated using various functional in vitro assays. Results: Pronounced expression of CD276 was observed in all analyzed tumor samples including triple negative BC. In analyses with BC cells, CC-3 induced profound T cell activation, proliferation, and T cell memory subset formation. Moreover, treatment with CC-3 induced cytokine secretion and potent tumor cell lysis. Conclusion: Our findings characterize CD276 as promising target and preclinically document the therapeutic potential of CC-3 for BC treatment, providing a strong rationale for evaluation of CC-3 in BC patients in a clinical trial for which the recruitment has recently started. [ABSTRACT FROM AUTHOR]

Neuroblastoma (NB) is a solid, neuroendocrine pediatric solid tumor with divergent clinical behavior. Patients with high-risk diseases have poor prognoses despite complex multimodal therapy, which requires the search for new therapeutic approaches. Chimeric antigen receptor T cells (CAR-T) have led to dramatic improvements in the survival of cancer patients, most notably those with hematologic malignancies. Early-phase clinical trials of CAR-T cell therapy for NB have proven safe and feasible, but limited clinical efficacy. At the same time, multiple experimental and preclinical studies have shown that the most common in clinical trials single 2nd or 3rd generation CAR structure is not sufficient for a complete response in solid tumors. Here, we review the recent advances and future perspectives associated with engineered receptors, including several antigens binding, armored CAR-T of 4th and 5th generation and CAR-T cell combination strategies with other immunotherapy. We also summarize the results and shortcomings of ongoing clinical trials of CAR-T therapy for NB. [ABSTRACT FROM AUTHOR]

Chordoma is a rare bone tumor that frequently recurs after surgery, and the prognosis is poor with current treatments. This study aimed to identify potential novel immunotherapeutic targets for chordomas by identifying target proteins in clinical samples as well as tumor microenvironmental factors to enhance efficacy. Fourteen chordoma samples were analyzed by single-cell RNA sequencing, and B7–H3 and IL-7 were identified as potential targets and potentiators, respectively. B7–H3-targeted chimeric antigen receptor T (CAR-T) cells and B7–H3 CAR-T cells expressing IL-7 were synthesized and their anti-tumor activity evaluated in vitro, including in primary chordoma organoid models. The B7–H3 CAR-T/IL-7 therapy showed enhanced cytotoxicity and prolonged duration of action against tumor cells. Additionally, IL-7 modulated favorable subpopulations of cultured CAR-T cells, diminished immune checkpoint expression on T-cell surfaces, and enhanced T-cell functionality. The incorporation of IL-7 molecules into the B7–H3 CAR structure augmented CAR-T-cell function and improved CAR-T-cell efficacy, thus providing a novel dual therapeutic strategy for chordoma treatment. [ABSTRACT FROM AUTHOR]

Background: B7‐H3 protein is an important regulator of the adaptive immune response in human tumorigenesis. 4‐1BB is a co‐stimulatory receptor expressed on activated CD8+ T cells, and regulates T cell immunity. Here, we investigated the role of B7‐H3 in the growth and invasion of nasopharyngeal carcinoma (NPC) and the effect of its interaction with 4‐1BB on tumor immunity. Methods: Short hairpin (sh) RNA was designed to knock down B7‐H3 expression in NPC cells. NPC cells with stable knockdown of B7‐H3 were established and injected into nude mice. The effects of B7‐H3 on cell proliferation, apoptosis, and epithelial‐to‐mesenchymal transition (EMT) were detected by the CCK8 assay, flow cytometry, TUNEL assay, and western blot analysis. The migration and invasion abilities were determined using the Transwell assay and scratch assay. Co‐immunoprecipitation (Co‐IP) assays were performed to study the interaction between B7‐H3 and 4‐1BB. Anti‐4‐1BB antibody was used in a co‐culture system and xenograft mice to study the effect of 4‐1BB on NPC development. Results: NPC cells transfected with sh‐B7‐H3 showed a higher rate of apoptosis, slower growth rate, impaired migration, and less EMT in vitro. Xenograft mice with stable knockout of B7‐H3 had lower tumor burdens, and the stripped tumors had lower rates of cell proliferation, higher rates of apoptosis, and less EMT in vivo. Additionally, decreased B7‐H3 expression was positively correlated with interferon‐γ, tumor necrosis factor‐α, and 4‐1BB+CD8+ tumor‐infiltrating lymphocytes. Co‐IP studies showed that B7‐H3 interacts with 4‐1BB. Also, the inhibitory effects of sh‐B7‐H3 on NPC tumor growth, invasion, and tumor immunity could be alleviated by the anti‐4‐1BB antibody both in vivo and in vitro. Conclusion: Our findings suggest that B7‐H3 may accelerate tumor growth, tumor cell invasion, and EMT, and interact with 4‐1BB to produce CD8+ T cell exhaustion that inhibits tumor immunity. B7‐H3 might serve as a novel target for treating NPC. [ABSTRACT FROM AUTHOR]

Recent studies have indicated that combining oncolytic viruses with CAR-T cells in therapy has shown superior anti-tumor effects, representing a promising approach. Nonetheless, the localized delivery method of intratumoral injection poses challenges for treating metastatic tumors or distal tumors that are difficult to reach. To address this obstacle, we employed HSV-1-infected CAR-T cells, which systemically delivery HSV into solid tumors. The biological function of CAR-T cells remained intact after loading them with HSV for a period of three days. In both immunocompromised and immunocompetent GBM orthotopic mouse models, B7-H3 CAR-T cells effectively delivered HSV to tumor lesions, resulting in enhanced T-cell infiltration and significantly prolonged survival in mice. We also employed a bilateral subcutaneous tumor model and observed that the group receiving intratumoral virus injection exhibited a significant reduction in tumor volume on the injected side, while the group receiving intravenous infusion of CAR-T cells carrying HSV displayed suppressed tumor growth on both sides. Hence, CAR-THSV cells offer notable advantages in the systemic delivery of HSV to distant tumors. In conclusion, our findings emphasize the potential of CAR-T cells as carriers for HSV, presenting significant advantages for oncolytic virotherapy targeting distant tumors. [ABSTRACT FROM AUTHOR]

The prognosis of thyroid cancer in patients varies significantly based on different pathological types or distinct clinical situations. Investigating the expression of immune checkpoint molecules PD-L1 and B7-H3 in high-risk thyroid cancer and their correlation with clinicopathological features and prognosis will contribute to the development of novel therapeutic strategies. A retrospective sample of 202 patients with thyroid cancer who underwent surgery at the Cancer Hospital of the Chinese Academy of Medical Sciences was collected, including 33 cases of anaplastic thyroid cancer (ATC), 21 cases of differentiated thyroid cancer (DTC) with distant metastasis (DM), 7 cases of differentiated high-grade thyroid carcinoma (DHGTC), and 109 cases of aggressive subtypes of papillary thyroid carcinoma (PTC) (including 28 cases of tall cell PTC, 31 cases of diffuse sclerosing PTC, 20 cases of solid PTC, 15 cases of columnar cell PTC, and 15 cases of hobnail PTC). In the control group, there were 32 cases of classic PTC. The differences in protein expression between PD-L1 and B7-H3 in several high-risk thyroid cancers and normal tissues and controls were compared by immunohistochemical staining, and the clinicopathological features and prognostic relevance were statistically analyzed. The expression of PD-L1 in ATC (P < 0.001), tall cell PTC (P = 0.031), and DHGTC (P = 0.003) was significantly higher than that in classic PTC. The expression of B7-H3 in ATC (P < 0.001), DTC with DM (P = 0.001), diffuse sclerosing PTC (P = 0.013), columnar cell PTC (P = 0.007), solid PTC (P < 0.001), hobnail PTC (P < 0.001), and DHGTC (P < 0.001) was significantly higher than that in classic PTC. In ATC, PD-L1 expression correlated significantly with extrathyroidal extension (ETE) (P = 0.027) and B7-H3 expression correlated significantly with male patients (P = 0.031) and lymph node metastasis (LNM) (P = 0.026). The positive expression of B7-H3 (P = 0.041) was an independent risk factor for disease progression in ATC. B7-H3 positive expression (P = 0.049), PD-L1 positive expression (P = 0.015), and tumor diameter ≥ 2 cm (P = 0.038) were independent risk factors for disease progression in patients with DTC with DM. PD-L1 positive expression (P = 0.019) and tumor diameter ≥ 2 cm (P = 0.018) were independent risk factors for disease progression in patients with aggressive subtypes of PTC. B7-H3 and PD-L1 are expected to be effective prognostic indicators for patients with aggressive thyroid cancer, which can help in optimization of individualized treatment strategies. Immunotherapy targeting these two molecules may provide new and complementary ideas for the treatment of high-risk/refractory thyroid cancer. [ABSTRACT FROM AUTHOR]

Abstract Purpose Targeted therapy development in soft tissue sarcoma (STS) has been burdened by the heterogeneity of this group of rare tumors. B7 homolog 3 protein (B7-H3) is a molecule in the same family as programmed death-ligand 1 (PD-L1). It has limited expression in noncancerous tissues and is overexpressed in many cancers, making it an attractive target for cancer therapy, and clinical trials targeting B7-H3 are actively underway. While available data demonstrate high expression levels of B7-H3 in individual sarcoma subtypes, its expression patterns across STS subtypes are not well described. The purpose of this study was to characterize the expression patterns of B7-H3 in STS. Patients and methods This retrospective analysis evaluated STS tumor specimens from patients with a variety of different subtypes. Specimens were evaluated by immunohistochemistry (IHC) for expression and staining pattern of B7-H3 both in tumors and in associated vasculature. Results Specimens from 153 sarcoma patients included 15 different STS subtypes. B7-H3 was broadly expressed in 97% of samples (95% CI 0.93–0.99) and 69.2% demonstrated high levels of B7-H3 expression (95% CI 0.61–0.76). No significant association between B7-H3 positivity or expression level and prior treatment(s), tumor size, tumor grade, or patient age. B7-H3 positivity in vessels was found in 94.7% (145/153) of samples. In tumors that had been previously assessed for PD-L1 and PD-1, there was no correlation between B7-H3 positivity or expression and the positivity or expression level of PD-L1 or PD-1. Conclusion These data show high levels of B7-H3 positivity across soft tissue sarcoma subtypes, suggesting its feasibility as a therapeutic target for future sarcoma treatments. Future clinical trials are needed to evaluate whether targeting B7-H3 can provide clinical benefit to help patients with sarcoma.

Abstract Background Breast cancer (BC) is the most common malignancy in women. Immunotherapy has revolutionized treatment options in many malignancies, and the introduction of immune checkpoint inhibition yielded beneficial results also in BC. However, many BC patients are ineligible for this T cell-based therapy, others do not respond or only briefly. Thus, there remains a high medical need for new therapies, particularly for triple-negative BC. CD276 (B7-H3) is overexpressed in several tumors on both tumor cells and tumor vessels, constituting a promising target for immunotherapy. Methods We analyzed tumor samples of 25 patients using immunohistochemistry to assess CD276 levels. The potential of CC-3, a novel bispecific CD276xCD3 antibody, for BC treatment was evaluated using various functional in vitro assays. Results Pronounced expression of CD276 was observed in all analyzed tumor samples including triple negative BC. In analyses with BC cells, CC-3 induced profound T cell activation, proliferation, and T cell memory subset formation. Moreover, treatment with CC-3 induced cytokine secretion and potent tumor cell lysis. Conclusion Our findings characterize CD276 as promising target and preclinically document the therapeutic potential of CC-3 for BC treatment, providing a strong rationale for evaluation of CC-3 in BC patients in a clinical trial for which the recruitment has recently started.

Background: Glioblastoma multiforme (GBM) is the most lethal primary brain tumor for which novel therapies are needed. Recently, chimeric antigen receptor (CAR) T cell therapy has been shown to be effective against GBM, but it is a personalized medicine and requires high cost and long time for the cell production. CAR-transduced natural killer (NK) cells can be used for "off-the-shelf" cellular immunotherapy because they do not induce graft-versus-host disease. Therefore, we aimed to analyze the anti-GBM effect of CAR-T or NK cells targeting B7-H3, which is known to be highly expressed in GBM. Methods: CAR-T cells targeting B7-H3 were generated using previously reported anti-B7-H3 scFv sequences. Cord blood (CB)-derived NK cells transduced with the B7-H3 CAR were also generated. Their anti-GBM effect was analyzed in vitro. The antitumor effect of intracranial injection of the B7-H3 CAR-T or NK cells was investigated in an in vivo xenograft model with patient-derived GBM cells. Results: Both B7-H3 CAR-T cells and CAR-NK cells exhibited marked cytotoxicity against patient-derived GBM cells in vitro. Furthermore, intracranial injection of CAR-T cells and CAR-NK cells targeting B7-H3 resulted in a significant antitumor effect against patient-derived GBM xenografts. Conclusion: Not only CAR-T cells but also CB-derived CAR-NK cells targeting B7-H3 may have the potential to eliminate GBM cells. [ABSTRACT FROM AUTHOR]

Abstract Although most differentiated thyroid carcinoma has a clinically favorable prognosis, some of specific types of thyroid cancer (such as anaplastic thyroid carcinoma and advanced papillary thyroid carcinoma) show fatal outcomes and require novel treatments. Immunotherapy is a promising avenue for the treatment of advanced thyroid carcinoma. B7-H3 (B7 homolog 3 protein) and ICAM-1 (intercellular adhesion molecule 1), as two important immune checkpoints (ICPs), is becoming hopeful target spots for immunotherapy. A growing amount of evidence has suggested that B7-H3 and ICAM-1 are upregulated in papillary thyroid carcinoma. However, their expression level in specific types of thyroid cancer remains largely unclear. In the present study, we explored the expression level of B7-H3 and ICAM-1 in different types of thyroid carcinoma. In the groups of the TCGA cohort, both B7-H3 and ICAM-1 mRNA were highly expressed in thyroid carcinoma. Furthermore, the patients with Stage2, 61-80y, Follicular thyroid papillary carcinoma and N0 had lower B7-H3 and ICAM-1 mRNA expression. In the groups of our cohort, PTCs and ATCs showed frequently moderate to strong expression of B7-H3 and ICAM-1 protein expression. The significant relevance of B7-H3 staining score with ICAM-1 staining score was observed in TCGA database and our cohort, which might open avenues for the combination therapy in advanced thyroid cancer.

Immune checkpoint blockade therapy has demonstrated significant therapeutic efficacy in certain cancer types; however, the impact of dietary restriction remains scarcely reported in this context. This study aimed to investigate the influence of dietary restriction on anti‐PDL‐1 therapy and the interplay of immune cells within this context. Using an anti‐PDL‐1 regimen combined with dietary restrictions, tumor progression was assessed in LLC‐bearing mice. Flow cytometry was employed to analyze immune cell infiltration and differentiation levels within the tumor microenvironment. The expression of mTORC1/B7‐H3 in tumors subjected to dietary restriction was also examined. LLC tumors with elevated B7‐H3 expression were validated in mice to determine its inhibitory effect on immune cell proliferation and differentiation. A CD3/B7‐H3 chimeric antibody was developed for therapeutic intervention in B7‐H3 overexpressing tumors, with subsequent T cell responses assessed through flow cytometry. Dietary restriction potentiated the effect of anti‐PDL1 therapy by suppressing the intratumorally mTORC1/B7‐H3 axis. In vivo experiments demonstrated that elevated B7‐H3 expression in tumors reduced infiltration and activation of CD8 + T cells within the tumor, while it did not affect tumor‐infiltrating Tregs. In vitro studies revealed that high B7‐H3 expression influenced the proliferation and activation of CD8 + T cells within a Coculture system. The constructed CD3/B7‐H3 chimeric antibody prominently activated TCR within B7‐H3 overexpressing tumors and impeded tumor progression. The findings suggest that dietary restriction enhances the efficacy of immune checkpoint blockade by modulating the intratumoral mTORC1/B7‐H3 axis. [ABSTRACT FROM AUTHOR]

B7-H3 is a member of the B7 superfamily and a putative inhibitory immune checkpoint molecule. Several early-phase clinical trials have reported promising anti-tumor activity and safety of anti-cancer drugs targeting B7-H3, suggesting that it may be a promising target for a potential next-generation immune checkpoint inhibitor. Despite ongoing clinical studies, most B7-H3-targeted drugs being currently investigated rely on direct cytotoxicity as their mechanisms of action rather than modulating its function as an immune checkpoint, at least in part due to its incompletely understood immune regulatory function. Recent studies have begun to elucidate the role of B7-H3 in regulating the tumor microenvironment (TME). Emerging evidence suggests that B7-H3 may regulate the interferon-STAT1 axis in the TME and promote immune suppression. Similarly, increasing evidence shows B7-H3 may be implicated in promoting M1 to M2 polarization of tumor-associated macrophages (TAMs). There is also accumulating evidence suggesting that B7-H3 may play a role in the heterotypic fusion of cancer stem cells and macrophages, thereby promoting tumor invasion and metastasis. Here, we review the recent advances in the understanding of B7-H3 cancer immunobiology with a focus on highlighting its potential role in the interferon priming of TAMs and the heterotypic fusion of TAMs with cancer stem cells and suggest future direction in elucidating its immune checkpoint function. [ABSTRACT FROM AUTHOR]

In recent years, in the development of emerging immunotherapy, B7‐H3 is also termed as CD276 and has become a novel chimeric antigen receptor (CAR)‐T target against glioma and other tumours, and aroused extensive attention. However, B7‐H3 has three isoforms (2, 3 and 4Ig) with the controversial expression and elusive function in tumour especially glioma. The current study mainly focuses on the regulatory factors and related mechanisms of generation of different B7‐H3 isoforms. First, we have determined that 2Ig is dominant in glioma with high malignancy, and 4Ig is widely expressed, whereas 3Ig shows negative expression in all glioma. Next, we have further found that RNA binding protein annexin A2 (ANXA2) is essential for B7‐H3 isoform maintenance, but fail to determine the choice of 4Ig or 2Ig. RNA methyltransferase NOP2/Sun RNA methyltransferase 2 (NSUN2) and 5‐methylcytosine reader Y‐box binding protein 1 (YBX1) facilitate the production of 2Ig. Our findings have uncovered a series of factors (ANXA2/NSUN2/YBX1) that can determine the alternative generation of different isoforms of B7‐H3 in glioma. Our result aims to help peers gain a clearer understanding of the expression and regulatory mechanisms of B7H3 in tumour patients, and to provide better strategies for designing B7H3 as a target in immunotherapy. [ABSTRACT FROM AUTHOR]

Objective: To investigate the regulatory role of miR-17-5p on B7-H3 and its immunomodulatory role in the develop-ment of colorectal cancer. Methods: Twenty-five pairs of human colorectal cancer tissues and adjacent normal tissues were collected， and the expression levels of miR-17-5p， B7-H3 and PD-L1 were analyzed by real-time fluorescence quantitative PCR， protein immunoblotting and immunohistochemical assays. Human colorectal cancer HCT-116 cells were divided into four groups: miR-NC group， miR-17-5p mimic group， si-NC group and si-B7-H3 group. The growth， invasive ability and apoptosis rate of HCT-116 cells were ana-lyzed by cell colony formation assay， Transwell assay and flow cytometry. The regulatory effect of miR-17-5p on B7-H3 was analyzed by luciferase reporter gene assay. The levels of IL-2， IL-4， IL-6， IL-10， IFN-γ and TNF-α cytokines in the cell culture medium were analyzed by ELISA. Results: Compared with paracancerous tissues， miR-17-5p expression was decreased and B7-H3 expression was increased in colorectal cancer tissues. miR-17-5p mimic and si-B7-H3 were able to reduce the number of HCT-116 cell colony forma-tion and invasion， promote HCT-116 cell apoptosis， and decrease the levels of IL-2， IL-4， IL-6， IL-10， IFN-γ and TNF-α cytokines in culture medium. In addition， miR-17-5p mimic and si B7-H3 were able to inhibit PD-L1 expression in HCT-116 cells. Conclusion: miR-17-5p can targeting inhibit B7-H3 expression， affect the immunomodulatory role in colorectal cancer development， and inhibit metastasis and promote apoptosis of colorectal cancer cells. [ABSTRACT FROM AUTHOR]

Simple Summary: We evaluated a novel proprietary murine B7-H3 antibody 8H8 for flow cytometric analyses of patients with acute myeloid leukemia (AML). The results showed substantial B7-H3 expression in a significant proportion of AML cases, particularly in the M5 group and in those with intermediate or poor risk profiles. The correlation between high B7-H3 expression and poorer overall and progression-free survival suggests that B7-H3 may serve as an additional promising negative prognostic marker to be considered in treatment decisions in AML. Despite recent advances in immunophenotyping, the prognosis of acute myeloid leukemia (AML) is still mainly estimated using age and genetic markers. As the genetic heterogeneity of AML patients is high, flow cytometry-based classification with appropriate biomarkers can efficiently complement risk stratification and treatment selection. An increased expression of B7-H3 (CD276), an immune checkpoint protein, has been reported and associated with poor prognosis. However, the available data are limited and heterogeneous. Here, we used a novel, proprietary murine anti-B7-H3 8H8 antibody for the flow cytometric analysis of B7-H3 expression in AML blasts from 77 patients. Our antibody reliably detected substantial B7-H3 expression in 62.3% of AML patients. B7-H3 expression was higher in the monocytic French–American–British (FAB) M5 group and in intermediate and poor risk patients according to the European Leukemia Network. Using receiver operating characteristics (ROCs), we identified a specific fluorescence intensity cut-off of 4.45 to discriminate between B7-H3high and B7-H3low expression. High B7-H3 expression was associated with shorter overall survival (OS) and progression-free survival (PFS). In conclusion, we have developed a novel B7-H3 antibody that serves as a new tool for the detection of B7-H3 expression in AML and may help to facilitate risk stratification and treatment selection in AML patients. [ABSTRACT FROM AUTHOR]

B7-H3 (CD276), an immune checkpoint molecule, is overexpressed in various types of cancer and their tumor vasculature, demonstrating significant associations with adverse clinical outcomes. In addition to its well-known immune functions, B7-H3 exhibits dual co-stimulatory/co-inhibitory roles in normal physiology and the tumor microenvironment. The non-immune functions of B7-H3 in tumor cells and the tumor vasculature, including promoting tumor cell anti-apoptosis, proliferation, invasion, migration, drug resistance, radioresistance, as well as affecting cellular metabolism and angiogenesis, have increasingly gained attention from researchers. Particularly, the co-expression of B7-H3 in both tumor cells and tumor endothelial cells highlights the higher potential and clinical utility of therapeutic strategies targeting B7-H3. This review aims to summarize the recent advances in understanding the non-immune functions of B7-H3 in tumors and provide insights into therapeutic approaches targeting B7- H3, focusing on its co-expression in tumor cells and endothelial cells. The aim is to establish a theoretical foundation and practical reference for the development and optimization of B7-H3-targeted therapies. [ABSTRACT FROM AUTHOR]

Although most differentiated thyroid carcinoma has a clinically favorable prognosis, some of specific types of thyroid cancer (such as anaplastic thyroid carcinoma and advanced papillary thyroid carcinoma) show fatal outcomes and require novel treatments. Immunotherapy is a promising avenue for the treatment of advanced thyroid carcinoma. B7-H3 (B7 homolog 3 protein) and ICAM-1 (intercellular adhesion molecule 1), as two important immune checkpoints (ICPs), is becoming hopeful target spots for immunotherapy. A growing amount of evidence has suggested that B7-H3 and ICAM-1 are upregulated in papillary thyroid carcinoma. However, their expression level in specific types of thyroid cancer remains largely unclear. In the present study, we explored the expression level of B7-H3 and ICAM-1 in different types of thyroid carcinoma. In the groups of the TCGA cohort, both B7-H3 and ICAM-1 mRNA were highly expressed in thyroid carcinoma. Furthermore, the patients with Stage2, 61-80y, Follicular thyroid papillary carcinoma and N0 had lower B7-H3 and ICAM-1 mRNA expression. In the groups of our cohort, PTCs and ATCs showed frequently moderate to strong expression of B7-H3 and ICAM-1 protein expression. The significant relevance of B7-H3 staining score with ICAM-1 staining score was observed in TCGA database and our cohort, which might open avenues for the combination therapy in advanced thyroid cancer. [ABSTRACT FROM AUTHOR]

Simple Summary: Lung squamous cell carcinoma (LSCC) is refractory to various therapies for non-small cell cancer. The expression and significance of B7-H3 in the tumor microenvironment (TME) and its relationship with other immune checkpoint molecules have not yet been investigated. We used high-throughput quantitative multiplex immunohistochemistry to examine B7-H3 expression in the TME. We investigated the relationship between B7-H3 expression and prognosis as well as changes in the TME with B7-H3 expression. The correlation between B7-H3 and programmed cell death-ligand 1 (PD-L1) expression in single cells was also examined. A quantitative analysis of protein expression in macrophages and cancer cells revealed that PD-L1-positive cells expressed higher levels of B7-H3 than that of PD-L1-negative cells. Our findings demonstrate a correlation between B7-H3 and PD-L1 expression in the same cells, indicating that therapies targeting B7-H3 could provide additional efficacy in patients refractory to PD-L1-targeting therapies. Lung squamous cell carcinoma (LSCC) is refractory to various therapies for non-small cell cancer; therefore, new therapeutic approaches are required to improve the prognosis of LSCC. Although immunotherapies targeting B7 family molecules were explored as treatments for several cancer types, the expression and significance of B7-H3 in the tumor microenvironment (TME) and its relationship with other immune checkpoint molecules have not yet been investigated in detail. We used high-throughput quantitative multiplex immunohistochemistry to examine B7-H3 expression in the TME. We investigated the relationship between B7-H3 expression and prognosis as well as changes in the TME with B7-H3 expression using 110 surgically resected pathological specimens retrospectively. We examined the correlation between B7-H3 and programmed cell death-ligand 1 (PD-L1) expression in single cells. High B7-H3 expression in tumor cells was associated with a better prognosis and a significant increase in the number of CD163+PD-L1+ macrophages. Quantitative analysis revealed that there is a positive correlation between B7-H3 and PD-L1 expression in tumor and stromal cells, as well as in intratumoral tumor-infiltrating lymphocytes and tumor-associated macrophages in the same cells. CD68+, CD163+, and CK+ cells with PD-L1+ phenotypes had higher B7-H3 expression compared to PD-L1− cells. Our findings demonstrate a correlation between B7-H3 and PD-L1 expression in the same cells, indicating that therapies targeting B7-H3 could provide additional efficacy in patients refractory to PD-L1-targeting therapies. [ABSTRACT FROM AUTHOR]

ABSTRACT Background High‐intensity focused ultrasound (HIFU) is a promising minimally invasive treatment for liver cancer; however, its efficacy is often limited by the attenuation of ultrasonic energy. This study investigates the effectiveness of B7‐H3‐targeted microbubbles (T‐MBs) in enhancing HIFU ablation of liver cancer and explores their potential for clinical translation. Methods T‐MBs and isotype control microbubbles (I‐MBs) were synthesized through the conjugation of biotinylated anti‐B7‐H3 antibody and isotype control antibody to the microbubble surface, respectively. Contrast‐enhanced ultrasound imaging was performed to compare the accumulation of T‐MBs and I‐MBs in liver cancer at various time points. The efficacy of T‐MBs in enhancing HIFU treatment was evaluated by measuring the immediate tumor ablation rate and long‐term tumor growth suppression. Additionally, the induced antitumor immune response was assessed through cytokine quantification in serum and tumor tissue, along with immunofluorescence staining conducted on days 1, 3, and 7 post‐treatment. Results T‐MBs demonstrated superior liver cancer‐specific accumulation, characterized by higher concentrations and prolonged retention compared to I‐MBs. The combination of T‐MBs with HIFU resulted in significantly enhanced tumor ablation rates and superior tumor growth suppression. Post‐treatment analysis revealed a gradual uptick in cytokine levels within the tumor microenvironment, along with progressive infiltration of antitumor immune cells. Conclusion T‐MBs effectively enhance the therapeutic efficacy of HIFU for liver cancer treatment while simultaneously promoting an antitumor immune response. These findings provide a strong experimental foundation for the clinical translation of ultrasound molecular imaging combined with HIFU as a novel approach for tumor therapy.

Bacterial and viral infections cause a huge burden to healthcare settings worldwide, and mortality rates associated with infectious microorganisms have remained high in recent decades. Despite tremendous efforts and resources worldwide to explore diagnostic biomarkers, rapid and easily assayed indicators for the diagnosis of bacterial and viral infections remain a challenge. B7 homolog 3 (B7-H3), a member of the B7 family of immunoregulatory proteins, is overexpressed in patients with septicemia, meningitis, pneumonia, and hepatitis. Therefore, B7-H3 could be used as a potential clinical indicator and therapeutic target for bacterial and viral infections caused by H. pylori, S. pneumoniae, M. pneumoniae, hepatitis B virus (HBV), viral hemorrhagic septicemia virus (VHSV), respiratory syncytial virus (RSV), and human immunodeficiency virus (HIV). Moreover, the interplay between infectious microorganisms and B7-H3 and exploration of the functional roles of the B7-H3 molecule could aid in the development of novel strategies for disease diagnosis and immunotherapy.

Abstract Background B7‐H3 protein is an important regulator of the adaptive immune response in human tumorigenesis. 4‐1BB is a co‐stimulatory receptor expressed on activated CD8+ T cells, and regulates T cell immunity. Here, we investigated the role of B7‐H3 in the growth and invasion of nasopharyngeal carcinoma (NPC) and the effect of its interaction with 4‐1BB on tumor immunity. Methods Short hairpin (sh) RNA was designed to knock down B7‐H3 expression in NPC cells. NPC cells with stable knockdown of B7‐H3 were established and injected into nude mice. The effects of B7‐H3 on cell proliferation, apoptosis, and epithelial‐to‐mesenchymal transition (EMT) were detected by the CCK8 assay, flow cytometry, TUNEL assay, and western blot analysis. The migration and invasion abilities were determined using the Transwell assay and scratch assay. Co‐immunoprecipitation (Co‐IP) assays were performed to study the interaction between B7‐H3 and 4‐1BB. Anti‐4‐1BB antibody was used in a co‐culture system and xenograft mice to study the effect of 4‐1BB on NPC development. Results NPC cells transfected with sh‐B7‐H3 showed a higher rate of apoptosis, slower growth rate, impaired migration, and less EMT in vitro. Xenograft mice with stable knockout of B7‐H3 had lower tumor burdens, and the stripped tumors had lower rates of cell proliferation, higher rates of apoptosis, and less EMT in vivo. Additionally, decreased B7‐H3 expression was positively correlated with interferon‐γ, tumor necrosis factor‐α, and 4‐1BB+CD8+ tumor‐infiltrating lymphocytes. Co‐IP studies showed that B7‐H3 interacts with 4‐1BB. Also, the inhibitory effects of sh‐B7‐H3 on NPC tumor growth, invasion, and tumor immunity could be alleviated by the anti‐4‐1BB antibody both in vivo and in vitro. Conclusion Our findings suggest that B7‐H3 may accelerate tumor growth, tumor cell invasion, and EMT, and interact with 4‐1BB to produce CD8+ T cell exhaustion that inhibits tumor immunity. B7‐H3 might serve as a novel target for treating NPC.

Abstract Background Radionuclide molecular imaging can be used to visualize the expression levels of molecular targets. Affibody molecules, small and high affinity non-immunoglobulin scaffold-based proteins, have demonstrated promising properties as targeting vectors for radionuclide tumour imaging of different molecular targets. B7-H3 (CD276), an immune checkpoint protein belonging to the B7 family, is overexpressed in different types of human malignancies. Visualization of overexpression of B7-H3 in malignancies enables stratification of patients for personalized therapies. Affinity maturation of anti-B7-H3 Affibody molecules as an approach to improve the binding affinity and targeting properties was recently investigated. In this study, we tested the hypothesis that a dimeric format may be an alternative option to increase the apparent affinity of Affibody molecules to B7-H3 and accordingly improve imaging contrast. Results Two dimeric variants of anti-B7-H3 Affibody molecules were produced (designated ZAC12*-ZAC12*-GGGC and ZAC12*-ZTaq_3-GGGC). Both variants were labelled with Tc-99m (99mTc) and demonstrated specific binding to B7-H3-expressing cells in vitro. [99mTc]Tc-ZAC12*-ZAC12*-GGGC showed subnanomolar affinity (KD1=0.28 ± 0.10 nM, weight = 68%), which was 7.6-fold higher than for [99mTc]Tc-ZAC12*-ZTaq_3-GGGC (KD=2.1 ± 0.9 nM). Head-to-head biodistribution of both dimeric variants of Affibody molecules compared with monomeric affinity matured SYNT-179 (all labelled with 99mTc) in mice bearing B7-H3-expressing SKOV-3 xenografts demonstrates that both dimers have lower tumour uptake and lower tumour-to-organ ratios compared to the SYNT-179 Affibody molecule. Conclusion The improved functional affinity by dimerization does not compensate the disadvantage of increased molecular size for imaging purposes.

Abstract Background Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a poor prognosis. Current treatment options are limited and often ineffective. CAR T cell therapy has shown success in treating hematologic malignancies, and there is growing interest in its potential application in solid tumors, including GBM. However, current CAR T therapy lacks clinical efficacy against GBM due to tumor-related resistance mechanisms and CAR T cell deficiencies. Therefore, there is a need to improve CAR T cell therapy efficacy in GBM. Methods We conducted large-scale CRISPR interference (CRISPRi) screens in GBM cell line U87 MG cells co-cultured with B7-H3 targeting CAR T cells to identify genetic modifiers that can enhance CAR T cell-mediated tumor killing. Flow cytometry-based tumor killing assay and CAR T cell activation assay were performed to validate screening hits. Bioinformatic analyses on bulk and single-cell RNA sequencing data and the TCGA database were employed to elucidate the mechanism underlying enhanced CAR T efficacy upon knocking down the selected screening hits in U87 MG cells. Results We established B7-H3 as a targetable antigen for CAR T therapy in GBM. Through large-scale CRISPRi screening, we discovered genetic modifiers in GBM cells, including ARPC4, PI4KA, ATP6V1A, UBA1, and NDUFV1, that regulated the efficacy of CAR T cell-mediated tumor killing. Furthermore, we discovered that TNFSF15 was upregulated in both ARPC4 and NDUFV1 knockdown GBM cells and revealed an immunostimulatory role of TNFSF15 in modulating tumor-CAR T interaction to enhance CAR T cell efficacy. Conclusions Our study highlights the power of CRISPR-based genetic screening in investigating tumor-CAR T interaction and identifies potential druggable targets in tumor cells that confer resistance to CAR T cell killing. Furthermore, we devised targeted strategies that synergize with CAR T therapy against GBM. These findings shed light on the development of novel combinatorial strategies for effective immunotherapy of GBM and other solid tumors.

Abstract Background CAR T cell therapy is a promising approach to improve outcomes and decrease toxicities for patients with cancer. While extraordinary success has been achieved using CAR T cells to treat patients with CD19-positive malignancies, multiple obstacles have so far limited the benefit of CAR T cell therapy for patients with solid tumors. Novel manufacturing and engineering approaches show great promise to enhance CAR T cell function against solid tumors. However, similar to single agent chemotherapy approaches, CAR T cell monotherapy may be unable to achieve high cure rates for patients with difficult to treat solid tumors. Thus, combinatorial drug plus CAR T cell approaches are likely required to achieve widespread clinical success. Methods We developed a novel, confocal microscopy based, high-content screen to evaluate 1114 FDA approved drugs for the potential to increase expression of the solid tumor antigen B7-H3 on the surface of osteosarcoma cells. Western blot, RT-qPCR, siRNA knockdown and flow cytometry assays were used to validate screening results and identify mechanisms of drug-induced B7-H3 upregulation. Cytokine and cytotoxicity assays were used to determine if drug pre-treatment enhanced B7-H3-CAR T cell effector function. Results Fifty-five drugs were identified to increase B7-H3 expression on the surface of LM7 osteosarcoma cells using a novel high-content, high-throughput screen. One drug, ingenol-3-angelate (I3A), increased B7-H3 expression by up to 100%, and was evaluated in downstream experiments. Validation assays confirmed I3A increased B7-H3 expression in a biphasic dose response and cell dependent fashion. Mechanistic studies demonstrated that I3A increased B7-H3 (CD276) mRNA, total protein, and cell surface expression via protein kinase C alpha activation. Functionally, I3A induced B7-H3 expression enhanced B7-H3-CAR T cell function in cytokine production and cytotoxicity assays. Conclusions This study demonstrates a novel high-content and high-throughput screen can identify drugs to enhance CAR T cell activity. This and other high-content technologies will pave the way to develop clinical trials implementing rational drug plus CAR T cell combinatorial therapies. Importantly, the technique could also be repurposed for an array of basic and translational research applications where drugs are needed to modulate cell surface protein expression.

Abstract Background B7-H3 has been implicated in clinical pathological features and prognosis across various cancer types, suggesting its potential as a cancer biomarker. Nevertheless, consensus remains elusive regarding its clinical-pathological and prognostic significance in bladder cancer. To address this gap, we conducted a systematic review and meta-analysis. Methods We systematically searched PubMed, Embase, Web of Science, Cochrane, and CNKI databases from their inception up to October 6, 2022. We evaluated the literature’s quality using the Newcastle-Ottawa Scale. We performed meta-analysis using Review Manager 5.3 and STATA 12.0, synthesizing data and calculating odds ratios (ORs) or hazard ratios (HRs) with corresponding 95% confidence intervals (CIs). Results After applying eligibility criteria and conducting assessments, we included data from 8 studies, encompassing 1622 bladder cancer patients. Bladder tumor tissues exhibited significantly elevated B7-H3 protein expression compared to normal bladder tissues. Elevated B7-H3 expression was notably associated with patient age, tumor infiltration, and recurrence in bladder cancer. However, no significant correlations were observed with other clinical characteristics. Our pooled HR analysis indicated no significant association between B7-H3 expression and overall survival in bladder cancer patients. Conclusion Our meta-analysis unveils the complex role of B7-H3 in bladder cancer progression. It appears to be directly involved in tumor infiltration and recurrence but cannot definitively serve as a prognostic biomarker for bladder cancer. To validate these findings, further well-designed studies, encompassing larger sample sizes and diverse racial backgrounds, are warranted. PROSPERO registration No. CRD42022364688.

Sarcomas are rare and heterogeneous malignancies that are difficult to treat. Approximately 50% of patients diagnosed with sarcoma develop metastatic disease with so far very limited treatment options. The transmembrane protein B7-H3 reportedly is expressed in various malignancies, including different sarcoma subtypes. In several cancer entities B7-H3 expression is associated with poor prognosis. In turn, B7-H3 is considered a promising target for immunotherapeutic approaches. We here report on the preclinical characterization of a B7-H3xCD3 bispecific antibody in an IgG-based format, termed CC-3, for treatment of different sarcoma subtypes. We found B7-H3 to be expressed on all sarcoma cells tested and expression on sarcoma patients correlated with decreased progression-free and overall survival. CC-3 was found to elicit robust T cell responses against multiple sarcoma subtypes, resulting in significant activation, release of cytokines and effector molecules. In addition, CC-3 promoted T cell proliferation and differentiation, resulting in the generation of memory T cell subsets. Finally, CC-3 induced potent target cell lysis in a target cell restricted manner. Based on these results, a clinical trial evaluating CC-3 in soft tissue sarcoma is currently in preparation. [ABSTRACT FROM AUTHOR]

Neuroblastoma (NB), a common solid tumour in young children originating from the sympathetic nervous system during embryonic development, poses challenges despite therapeutic advances like high‐dose chemotherapy and immunotherapy. Some survivors still grapple with severe side effects and drug resistance. The role of lncRNA NUTM2A‐AS1 has been explored in various cancers, but its function in drug‐resistant NB progression is unclear. Our study found that NUTM2A‐AS1 expression in cisplatin‐resistant NB cells increased in a time‐ and dose‐dependent manner. Knockdown of NUTM2A‐AS1 significantly improved NB cell sensitivity to cisplatin and inhibited metastatic abilities. Additionally, we identified B7‐H3, an immune checkpoint‐related protein, as a NUTM2A‐AS1‐associated protein in NB cells. NUTM2A‐AS1 was shown to inhibit the protein degradation of B7‐H3. Moreover, NUTM2A‐AS1 modulated immune evasion in cisplatin‐resistant NB cells through B7‐H3. Furthermore, NUTM2A‐AS1 expression in cisplatin‐resistant NB cells was transactivated by NR1D1. In summary, our results unveil the molecular or biological relationship within the NR1D1/NUTM2A‐AS1/B7‐H3 axis in NB cells under cisplatin treatment, providing an intriguing avenue for fundamental research into cisplatin‐resistant NB. [ABSTRACT FROM AUTHOR]

Background: Radionuclide molecular imaging can be used to visualize the expression levels of molecular targets. Affibody molecules, small and high affinity non-immunoglobulin scaffold-based proteins, have demonstrated promising properties as targeting vectors for radionuclide tumour imaging of different molecular targets. B7-H3 (CD276), an immune checkpoint protein belonging to the B7 family, is overexpressed in different types of human malignancies. Visualization of overexpression of B7-H3 in malignancies enables stratification of patients for personalized therapies. Affinity maturation of anti-B7-H3 Affibody molecules as an approach to improve the binding affinity and targeting properties was recently investigated. In this study, we tested the hypothesis that a dimeric format may be an alternative option to increase the apparent affinity of Affibody molecules to B7-H3 and accordingly improve imaging contrast. Results: Two dimeric variants of anti-B7-H3 Affibody molecules were produced (designated ZAC12*-ZAC12*-GGGC and ZAC12*-ZTaq_3-GGGC). Both variants were labelled with Tc-99m (99mTc) and demonstrated specific binding to B7-H3-expressing cells in vitro. [99mTc]Tc-ZAC12*-ZAC12*-GGGC showed subnanomolar affinity (KD1=0.28 ± 0.10 nM, weight = 68%), which was 7.6-fold higher than for [99mTc]Tc-ZAC12*-ZTaq_3-GGGC (KD=2.1 ± 0.9 nM). Head-to-head biodistribution of both dimeric variants of Affibody molecules compared with monomeric affinity matured SYNT-179 (all labelled with 99mTc) in mice bearing B7-H3-expressing SKOV-3 xenografts demonstrates that both dimers have lower tumour uptake and lower tumour-to-organ ratios compared to the SYNT-179 Affibody molecule. Conclusion: The improved functional affinity by dimerization does not compensate the disadvantage of increased molecular size for imaging purposes. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a poor prognosis. Current treatment options are limited and often ineffective. CAR T cell therapy has shown success in treating hematologic malignancies, and there is growing interest in its potential application in solid tumors, including GBM. However, current CAR T therapy lacks clinical efficacy against GBM due to tumor-related resistance mechanisms and CAR T cell deficiencies. Therefore, there is a need to improve CAR T cell therapy efficacy in GBM. Methods: We conducted large-scale CRISPR interference (CRISPRi) screens in GBM cell line U87 MG cells co-cultured with B7-H3 targeting CAR T cells to identify genetic modifiers that can enhance CAR T cell-mediated tumor killing. Flow cytometry-based tumor killing assay and CAR T cell activation assay were performed to validate screening hits. Bioinformatic analyses on bulk and single-cell RNA sequencing data and the TCGA database were employed to elucidate the mechanism underlying enhanced CAR T efficacy upon knocking down the selected screening hits in U87 MG cells. Results: We established B7-H3 as a targetable antigen for CAR T therapy in GBM. Through large-scale CRISPRi screening, we discovered genetic modifiers in GBM cells, including ARPC4, PI4KA, ATP6V1A, UBA1, and NDUFV1, that regulated the efficacy of CAR T cell-mediated tumor killing. Furthermore, we discovered that TNFSF15 was upregulated in both ARPC4 and NDUFV1 knockdown GBM cells and revealed an immunostimulatory role of TNFSF15 in modulating tumor-CAR T interaction to enhance CAR T cell efficacy. Conclusions: Our study highlights the power of CRISPR-based genetic screening in investigating tumor-CAR T interaction and identifies potential druggable targets in tumor cells that confer resistance to CAR T cell killing. Furthermore, we devised targeted strategies that synergize with CAR T therapy against GBM. These findings shed light on the development of novel combinatorial strategies for effective immunotherapy of GBM and other solid tumors. [ABSTRACT FROM AUTHOR]

Nanobodies have emerged as promising tools in biomedicine due to their single-chain structure and inherent stability. They generally have convex paratopes, which potentially prefer different epitope sites in an antigen compared to traditional antibodies. In this study, a synthetic phage display nanobody library was constructed and used to identify nanobodies targeting a tumor-associated antigen, the human B7-H3 protein. Combining next-generation sequencing and single-clone validation, two nanobodies were identified to specifically bind B7-H3 with medium nanomolar affinities. Further characterization revealed that these two clones targeted a different epitope compared to known B7-H3-specific antibodies, which have been explored in clinical trials. Furthermore, one of the clones, dubbed as A6, exhibited potent antibody-dependent cell-mediated cytotoxicity (ADCC) against a colorectal cancer cell line with an EC50 of 0.67 nM, upon conversion to an Fc-enhanced IgG format. These findings underscore a cost-effective strategy that bypasses the lengthy immunization process, offering potential rapid access to nanobodies targeting unexplored antigenic sites. [ABSTRACT FROM AUTHOR]

Brain metastasis is a significant challenge for some breast cancer patients, marked by its aggressive nature, limited treatment options, and poor clinical outcomes. Immunotherapies have emerged as a promising avenue for brain metastasis treatment. B7-H3 (CD276) is an immune checkpoint molecule involved in T cell suppression, which is associated with poor survival in cancer patients. Given the increasing number of clinical trials using B7-H3 targeting CAR T cell therapies, we examined B7-H3 expression across breast cancer subtypes and in breast cancer brain metastases to assess its potential as an interventional target. B7-H3 expression was investigated using immunohistochemistry on tissue microarrays of three clinical cohorts: (i) unselected primary breast cancers (n = 347); (ii) brain metastatic breast cancers (n = 61) and breast cancer brain metastases (n = 80, including a subset of 53 patient-matched breast and brain metastasis cases); and (iii) mixed brain metastases from a range of primary tumours (n = 137). In primary breast cancers, B7-H3 expression significantly correlated with higher tumour grades and aggressive breast cancer subtypes, as well as poorer 5-year survival outcomes. Subcellular localisation of B7-H3 impacted breast cancer-specific survival, with cytoplasmic staining also correlating with a poorer outcome. Its expression was frequently detected in brain metastases from breast cancers, with up to 90% expressing B7-H3. However, not all brain metastases showed high levels of expression, with those from colorectal and renal tumours showing a low frequency of B7-H3 expression (0/14 and 2/16, respectively). The prevalence of B7-H3 expression in breast cancers and breast cancer brain metastases indicates potential opportunities for B7-H3 targeted therapies in breast cancer management. [ABSTRACT FROM AUTHOR]

Background: B7-H3 has been implicated in clinical pathological features and prognosis across various cancer types, suggesting its potential as a cancer biomarker. Nevertheless, consensus remains elusive regarding its clinical-pathological and prognostic significance in bladder cancer. To address this gap, we conducted a systematic review and meta-analysis. Methods: We systematically searched PubMed, Embase, Web of Science, Cochrane, and CNKI databases from their inception up to October 6, 2022. We evaluated the literature's quality using the Newcastle-Ottawa Scale. We performed meta-analysis using Review Manager 5.3 and STATA 12.0, synthesizing data and calculating odds ratios (ORs) or hazard ratios (HRs) with corresponding 95% confidence intervals (CIs). Results: After applying eligibility criteria and conducting assessments, we included data from 8 studies, encompassing 1622 bladder cancer patients. Bladder tumor tissues exhibited significantly elevated B7-H3 protein expression compared to normal bladder tissues. Elevated B7-H3 expression was notably associated with patient age, tumor infiltration, and recurrence in bladder cancer. However, no significant correlations were observed with other clinical characteristics. Our pooled HR analysis indicated no significant association between B7-H3 expression and overall survival in bladder cancer patients. Conclusion: Our meta-analysis unveils the complex role of B7-H3 in bladder cancer progression. It appears to be directly involved in tumor infiltration and recurrence but cannot definitively serve as a prognostic biomarker for bladder cancer. To validate these findings, further well-designed studies, encompassing larger sample sizes and diverse racial backgrounds, are warranted. PROSPERO registration: No. CRD42022364688. [ABSTRACT FROM AUTHOR]

Neuroblastoma (NB), which mainly originates from the adrenal gland, is one of the most common tumors in infants and young children. Abnormal B7 homolog 3 (B7-H3) expression has been reported in human NB, although its mechanism of action and precise role in NB are still unclear. The present study was performed to explore the role of B7-H3 in glucose metabolism in NB cells. Our findings showed that B7-H3 expression was increased in NB samples, and markedly promoted the migration and invasion of NB cells. B7-H3 silencing decreased the migration and invasion of NB cells. Moreover, B7-H3 overexpression also increased tumor proliferation in the human NB cell xenograft animal model. B7-H3 silencing reduced NB cell viability and proliferation, while B7-H3 overexpression had the opposite effects. Furthermore, B7-H3 increased PFKFB3 expression, resulting in increased glucose uptake and lactate production. This study suggested that B7-H3 regulated the Stat3/c-Met pathway. Taken together, our data showed that B7-H3 regulates NB progression by increasing glucose metabolism in NB. [ABSTRACT FROM AUTHOR]

B7-H3 (B7 homology 3 protein) is an important transmembrane immunoregulatory protein expressed in immune cells, antigen-presenting cells, and tumor cells. Studies reveal a multifaceted role of B7-H3 in tumor progression by modulating various cancer hallmarks involving angiogenesis, immune evasion, and tumor microenvironment, and it is also a promising candidate for cancer immunotherapy. In colorectal cancer (CRC), B7-H3 has been associated with various aspects of disease progression, such as evasion of tumor immune surveillance, tumor-node metastasis, and poor prognosis. Strategies to block or interfere with B7-H3 in its immunological and non-immunological functions are under investigation. In this study, we explore the role of B7-H3 in tumor plasticity, emphasizing tumor glucose metabolism, angiogenesis, epithelial-mesenchymal transition, cancer stem cells, apoptosis, and changing immune signatures in the tumor immune landscape. We discuss how B7-H3-induced tumor plasticity contributes to immune evasion, metastasis, and therapy resistance. Furthermore, we delve into the most recent advancements in targeting B7-H3-based tumor immunotherapy as a potential approach to CRC treatment. [ABSTRACT FROM AUTHOR]

Despite intensive therapies, pediatric patients with relapsed or refractory solid tumors have poor outcomes and need novel treatments. Immune therapies offer an alternative to conventional treatment options but require the identification of differentially expressed antigens to direct antitumor activity to sites of disease. B7-H3 (CD276) is an immune regulatory protein that is expressed in a range of malignancies and has limited expression in normal tissues. B7-H3 is highly expressed in pediatric solid tumors including osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, Wilms tumor, neuroblastoma, and many rare tumors. In this article we review B7-H3-targeted chimeric antigen receptor (B7-H3-CAR) T cell therapies for pediatric solid tumors, reporting preclinical development strategies and outlining the landscape of active pediatric clinical trials. We identify challenges to the success of CAR T cell therapy for solid tumors including localizing to and penetrating solid tumor sites, evading the hostile tumor microenvironment, supporting T cell expansion and persistence, and avoiding intrinsic tumor resistance. We highlight strategies to overcome these challenges and enhance the effect of B7-H3-CAR T cells, including advanced CAR T cell design and incorporation of combination therapies.

Abstract Breast cancer (BC) is the second-leading factor of mortality for women globally and is brought on by a variety of genetic and environmental causes. The conventional treatments for this disease have limitations, making it difficult to improve the lifespan of breast cancer patients. As a result, extensive research has been conducted over the past decade to find innovative solutions to these challenges. Targeting of the antitumor immune response through the immunomodulatory checkpoint protein B7 family has revolutionized cancer treatment and led to intermittent patient responses. B7-H3 has recently received attention because of its significant demodulation and its immunomodulatory effects in many cancers. Uncontrolled B7-H3 expression and a bad outlook are strongly associated, according to a substantial body of cancer research. Numerous studies have shown that BC has significant B7-H3 expression, and B7-H3 induces an immune evasion phenotype, consequently enhancing the survival, proliferation, metastasis, and drug resistance of BC cells. Thus, an innovative target for immunotherapy against BC may be the B7-H3 checkpoint. In this review, we discuss the structure and regulation of B7-H3 and its double costimulatory/coinhibitory function within the framework of cancer and normal physiology. Then we expound the malignant behavior of B7-H3 in BC and its role in the tumor microenvironment (TME) and finally focus on targeted drugs against B7-H3 that have opened new therapeutic opportunities in BC.

B7-H3 is a transmembrane receptor highly prevalent on malignant cells and plays an important role in adaptive immunity that is not fully elucidated. Targeted B7-H3 inhibitors, including antibody-drug conjugates, radioimmunotherapy, and monoclonal antibodies, are a new class of antineoplastic agents showing promising preliminary clinical efficacy, observed with several of these agents against multiple tumor types. Particularly promising treatments are enoblituzumab for prostate cancer, 131I-omburtamab for central nervous system malignancies, and HS-20093 for small-cell lung cancer but further studies are warranted. There are clinical trials on the horizon that have not yet enrolled patients examining chimeric antigen receptor T-cell therapies, bi- and tri-specific killer engagers, and dual-affinity retargeting proteins. These data will be telling of the efficacy of B7-H3 inhibitors in both hematologic and solid malignancies. This study aimed to compile available results of B7-H3 inhibitors in oncology clinical trials.

Abstract Background Lysophosphatidic acids (LPAs) belong to a class of bioactive lysophospholipids with multiple functions including immunomodulatory roles in tumor microenvironment (TME). LPA exerts its biological effects via its receptors that are highly expressed in fibroblasts among other cell types. As cancer‐associated fibroblasts (CAFs) are a key component of the TME, it is important to understand LPA signaling and regulation of receptors in fibroblasts or CAFs and associated regulatory roles on immunomodulation‐related molecules. Methods Cluster analysis, immunoblotting, real‐time quantitative‐PCR, CRISPR‐Cas9 gene editing system, immunohistochemical staining, coculture model, and in vivo xenograft model were used to investigate the effects of LPA‐LPAR1 on B7‐H3 in tumor promotion of CAFs. Results In this study, we found that LPAR1 and CD276 (B7‐H3) were generally highly expressed in fibroblasts with good expression correlation. LPA induced B7‐H3 up‐expression through LPAR1, and stimulated fibroblasts proliferation that could be inhibited by silencing LPAR1 or B7‐H3 as well as small molecule LPAR1 antagonist (Ki16425). Using engineered fibroblasts and non‐small cell lung carcinoma (NSCLC) cell lines, subsequent investigations demonstrated that CAFs promoted the proliferation of NSCLC in vitro and in vivo, and such effect could be inhibited by knocking out LPAR1 or B7‐H3. Conclusion The present study provided new insights for roles of LPA in CAFs, which could lead to the development of innovative therapies targeting CAFs in the TME. It is also reasonable to postulate a combinatory approach to treat malignant fibrous tumors (such as NSCLC) with LPAR1 antagonists and B7‐H3 targeting therapies.

Breast cancer (BC) is the second-leading factor of mortality for women globally and is brought on by a variety of genetic and environmental causes. The conventional treatments for this disease have limitations, making it difficult to improve the lifespan of breast cancer patients. As a result, extensive research has been conducted over the past decade to find innovative solutions to these challenges. Targeting of the antitumor immune response through the immunomodulatory checkpoint protein B7 family has revolutionized cancer treatment and led to intermittent patient responses. B7-H3 has recently received attention because of its significant demodulation and its immunomodulatory effects in many cancers. Uncontrolled B7-H3 expression and a bad outlook are strongly associated, according to a substantial body of cancer research. Numerous studies have shown that BC has significant B7-H3 expression, and B7-H3 induces an immune evasion phenotype, consequently enhancing the survival, proliferation, metastasis, and drug resistance of BC cells. Thus, an innovative target for immunotherapy against BC may be the B7-H3 checkpoint. In this review, we discuss the structure and regulation of B7-H3 and its double costimulatory/coinhibitory function within the framework of cancer and normal physiology. Then we expound the malignant behavior of B7-H3 in BC and its role in the tumor microenvironment (TME) and finally focus on targeted drugs against B7-H3 that have opened new therapeutic opportunities in BC. [ABSTRACT FROM AUTHOR]

Pancreatic cancer is a highly lethal disease with limited treatment options. Hence, there is a considerable medical need for novel treatment strategies. Monoclonal antibodies (mAbs) have significantly improved cancer therapy, primarily due to their ability to stimulate antibody-dependent cellular cytotoxicity (ADCC), which plays a crucial role in their therapeutic efficacy. As a result, significant effort has been focused on improving this critical function by engineering mAbs with Fc regions that have increased affinity for the Fc receptor CD16 expressed on natural killer (NK) cells, the major cell population that mediates ADCC in humans. Here we report on the preclinical characterization of a mAb directed to the target antigen B7-H3 (CD276) containing an Fc part with the amino acid substitutions S239D/I332E to increase affinity for CD16 (B7-H3-SDIE) for the treatment of pancreatic cancer. B7-H3 (CD276) is highly expressed in many tumor entities, whereas expression on healthy tissues is more limited. Our findings confirm high expression of B7-H3 on pancreatic cancer cells. Furthermore, our study shows that B7-H3-SDIE effectively activates NK cells against pancreatic cancer cells in an antigen-dependent manner, as demonstrated by the analysis of NK cell activation, degranulation and cytokine release. The activation of NK cells resulted in significant tumor cell lysis in both short-term and long-term cytotoxicity assays. In conclusion, B7-H3-SDIE constitutes a promising agent for the treatment of pancreatic cancer. [ABSTRACT FROM AUTHOR]

Background: Lysophosphatidic acids (LPAs) belong to a class of bioactive lysophospholipids with multiple functions including immunomodulatory roles in tumor microenvironment (TME). LPA exerts its biological effects via its receptors that are highly expressed in fibroblasts among other cell types. As cancer‐associated fibroblasts (CAFs) are a key component of the TME, it is important to understand LPA signaling and regulation of receptors in fibroblasts or CAFs and associated regulatory roles on immunomodulation‐related molecules. Methods: Cluster analysis, immunoblotting, real‐time quantitative‐PCR, CRISPR‐Cas9 gene editing system, immunohistochemical staining, coculture model, and in vivo xenograft model were used to investigate the effects of LPA‐LPAR1 on B7‐H3 in tumor promotion of CAFs. Results: In this study, we found that LPAR1 and CD276 (B7‐H3) were generally highly expressed in fibroblasts with good expression correlation. LPA induced B7‐H3 up‐expression through LPAR1, and stimulated fibroblasts proliferation that could be inhibited by silencing LPAR1 or B7‐H3 as well as small molecule LPAR1 antagonist (Ki16425). Using engineered fibroblasts and non‐small cell lung carcinoma (NSCLC) cell lines, subsequent investigations demonstrated that CAFs promoted the proliferation of NSCLC in vitro and in vivo, and such effect could be inhibited by knocking out LPAR1 or B7‐H3. Conclusion: The present study provided new insights for roles of LPA in CAFs, which could lead to the development of innovative therapies targeting CAFs in the TME. It is also reasonable to postulate a combinatory approach to treat malignant fibrous tumors (such as NSCLC) with LPAR1 antagonists and B7‐H3 targeting therapies. [ABSTRACT FROM AUTHOR]

B7-H3 is a transmembrane receptor highly prevalent on malignant cells and plays an important role in adaptive immunity that is not fully elucidated. Targeted B7-H3 inhibitors, including antibody-drug conjugates, radioimmunotherapy, and monoclonal antibodies, are a new class of antineoplastic agents showing promising preliminary clinical efficacy, observed with several of these agents against multiple tumor types. Particularly promising treatments are enoblituzumab for prostate cancer, 131Iomburtamab for central nervous system malignancies, and HS-20093 for small-cell lung cancer but further studies are warranted. There are clinical trials on the horizon that have not yet enrolled patients examining chimeric antigen receptor Tcell therapies, bi- and tri-specific killer engagers, and dual-affinity retargeting proteins. These data will be telling of the efficacy of B7-H3 inhibitors in both hematologic and solid malignancies. This study aimed to compile available results of B7-H3 inhibitors in oncology clinical trials. [ABSTRACT FROM AUTHOR]

Background: GD2-directed immunotherapy is highly effective in the treatment of high-risk neuroblastoma (NB), and might be an interesting target also in other high-risk tumors. Methods: The German-Austrian Retinoblastoma Registry, Essen, was searched for patients, who were treated with anti-GD2 monoclonal antibody (mAb) dinutuximab beta (Db) in order to evaluate toxicity, response and outcome in these patients. Additionally, we evaluated anti-GD2 antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in retinoblastoma cell lines in vitro. Furthermore, in vitro cytotoxicity assays directed against B7-H3 (CD276), a new identified potential target in RB, were performed. Results: We identified four patients with relapsed stage IV retinoblastoma, who were treated with Db following autologous stem cell transplantation (ASCT). Two out of two evaluable patients with detectable tumors responded to immunotherapy. One of these and another patient who received immunotherapy without residual disease relapsed 10 and 12 months after start of Db. The other patients remained in remission until last follow-up 26 and 45 months, respectively. In vitro, significant lysis of RB cell lines by ADCC and CDC with samples from patients and healthy donors and anti-GD2 and anti-CD276-mAbs were demonstrated. Conclusion: Anti-GD2-directed immunotherapy represents an additional therapeutic option in high-risk metastasized RB. Moreover, CD276 is another target of interest. [ABSTRACT FROM AUTHOR]

The primary pathological changes of rheumatoid arthritis (RA) include chronic synovial inflammation, bone destruction, and aggressive pannus formation on cartilage, in which angiogenesis plays a critical role. B7-H3, an important immune checkpoint molecule, represents a novel target in tumor therapy and plays a significant role in the pathogenesis of autoimmune diseases. However, its biological mechanism in RA remains unclear. Hematoxylin-eosin (HE) staining and immunohistochemistry were used to explore the histological characteristics and expression of B7-H3, CD34, and vascular endothelial growth factor (VEGF) in patients with RA and collagen-induced arthritis (CIA) mice. ELISA was used to detect VEGF, soluble B7-H3, and disease markers in the peripheral blood of patients. A monoclonal anti-B7-H3 antibody was used to treat CIA mice by blocking B7-H3-mediated signaling. The ELISA and HE staining results showed a positive correlation between the expression of B7-H3 and the degree of joint cavity destruction and pannus formation. B7-H3 expression also correlated with increased expression of the vessel biomarkers CD34 and VEGF. Anti-B7-H3 effectively reduced pannus formation in CIA mice. B7-H3 modulates angiogenic activity in the joint synovium, demonstrating its therapeutic value in the context of RA. • There was a correlation between B7-H3 expression and the vessel biomarkers. • B7-H3-blocking antibody could reduce pannus formation in CIA mice. • B7-H3 could modulate the ability of angiogenesis. [ABSTRACT FROM AUTHOR]

Gastric‐type endocervical adenocarcinoma (GEA) is the second most common subtype of endocervical adenocarcinoma and has a poor prognosis. Anti‐programmed death‐1 and anti‐programmed death‐ligand 1 (PD‐L1) inhibitors have emerged as a major treatment option for GEA; however, data on the expression of other immune checkpoints in GEA are limited. We analyzed the expression of T‐cell immunoglobulin and mucin‐domain containing‐3 (TIM‐3) and B7 homolog 3 protein (B7‐H3) in 58 GEA and investigated their prognostic significance as well as association with PD‐L1 expression and other known prognostic factors. Applying the tumor proportion score (TPS) with a cutoff of 1%, B7‐H3 and TIM‐3 were present in 48.3% and 17.2% of cases, respectively. Applying the combined positive score (CPS) with a cutoff of 1, TIM‐3 expression was present in 70.7% of cases. Moreover, the expression of three checkpoints (B7‐H3, TIM‐3, and PD‐L1) was incompletely overlapping. Patients with B7‐H3 positive tumors (by TPS) or TIM‐3 positive tumors (by TPS) had significantly worse recurrence‐free survival (RFS) and overall survival (OS) (log‐rank). Using CPS, patients with TIM‐3 positive tumors showed significantly worse RFS (log‐rank). Similarly, B7‐H3 positivity (by TPS) and TIM‐3 positivity (by TPS) were associated with worse RFS and OS in univariate analysis. TIM‐3 positivity (by CPS) was associated with worse RFS in univariate analysis and the final Cox multivariate analysis. In conclusion, our results show that (1) B7‐H3 and TIM‐3 are frequently expressed in GEA and their expression overlaps incompletely with PD‐L1; and (2) both B7‐H3 and TIM‐3 are independent negative prognostic markers in GEA. [ABSTRACT FROM AUTHOR]

Immune checkpoints (ICs) are molecules implicated in the fine-tuning of immune response via co-inhibitory or co-stimulatory signals, and serve to secure minimized host damage. Targeting ICs with various therapeutic modalities, including checkpoint inhibitors/monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), and CAR-T cells has produced remarkable results, especially in immunogenic tumors, setting a paradigm shift in cancer therapeutics through the incorporation of these IC-targeted treatments. However, the large proportion of subjects who experience primary or secondary resistance to available IC-targeted options necessitates further advancements that render immunotherapy beneficial for a larger patient pool with longer duration of response. B7-H3 (B7 Homolog 3 Protein, CD276) is a member of the B7 family of IC proteins that exerts pleiotropic immunomodulatory effects both in physiologic and pathologic contexts. Mounting evidence has demonstrated an aberrant expression of B7-H3 in various solid malignancies, including tumors less sensitive to current immunotherapeutic options, and has associated its expression with advanced disease, worse patient survival and impaired response to IC-based regimens. Anti-B7-H3 agents, including novel mAbs, bispecific antibodies, ADCs, CAR-T cells, and radioimmunotherapy agents, have exhibited encouraging antitumor activity in preclinical models and have recently entered clinical testing for several cancer types. In the present review, we concisely present the functional implications of B7-H3 and discuss the latest evidence regarding its prognostic significance and therapeutic potential in solid malignancies, with emphasis on anti-B7-H3 modalities that are currently evaluated in clinical trial settings. Better understanding of B7-H3 intricate interactions in the tumor microenvironment will expand the oncological utility of anti-B7-H3 agents and further shape their role in cancer therapeutics. [ABSTRACT FROM AUTHOR]

Background: Gastric cancer (GC) is one of the most prevalent types of cancer worldwide. B7-H3, an immune checkpoint molecule with promising potential, has been found to be overexpressed in various cancers. CD47 is an anti-phagocytic molecule that interacts with the signal regulatory protein alpha (SIRPα) to affect phagocytes. The relationship between the expression of B7-H3 and CD47, two potential therapeutic targets found in tumor cells, remains unknown. In this study, our objective is to investigate the clinical significance of co-expression of B7-H3 and CD47, as well as the potential therapeutic value of combination therapy in GC. Methods: We utilized immunohistochemistry (IHC) to assess the expression of B7-H3, CD47, CD68, CD86 and CD163 in tissue microarrays obtained from 268 GC patients who underwent surgeries. Western blotting was employed to assess the protein level of B7-H3 and CD47 in GC tissues. The co-localization of B7-H3/CD47 and CD68 in GC tissues was determined using multiplex immunohistochemistry (m-IHC). We further verified the relationship between B7-H3/CD47 and macrophage infiltration via flow cytometry. To estimate the clinical outcomes of patients from different subgroups, we employed the Kaplan–Meier curve and the Cox model. Results: Among the 268 GC cases, a total of 180 cases exhibited positive expression of B7-H3, while 122 cases showed positive expression of CD47. In fresh GC clinical tissues, B7-H3 and CD47 protein level was also higher in tumor tissue than in adjacent normal tissue. Remarkably, 91 cases demonstrated co-expression of B7-H3 and CD47. We observed a significant correlation between B7-H3 expression and tumor stage (P = 0.001), differentiation (P = 0.045), and depth (P = 0.003). Additionally, there was a significant association between B7-H3 and CD47 expression (P = 0.018). The percentage of B7-H3 and CD47 double positive cells in fresh GC tumor tissues were elevated compared with control adjacent tissues regardless of CD45− or CD45+ cells (P = 0.0029, P = 0.0012). Patients with high B7-H3 or CD47 expression had significantly lower overall survival (OS) rates compared to those with low expression levels (P = 0.0176 or P = 0.0042). Surprisingly, patients with combined high expression of B7-H3 and CD47 exhibited a considerably worse prognosis than others (P = 0.0007). Univariate analysis revealed that cases with high expression of B7-H3, CD47, or both had significantly higher hazard ratios (HR) than cases with low expression of these markers. Furthermore, the results of multivariate analysis indicated that B7-H3/CD47 co-expression and CD47 expression alone are independent prognostic factors for overall survival. Moreover, significant correlations were observed between B7-H3 and CD68 expression, CD47 and CD68 expression, as well as B7-H3/CD47 co-expression and CD68 expression in GC patients (P < 0.001, P = 0.003, and P < 0.001). Flow cytometry test showed that the percentage of CD68-positive cells but not CD86-positive cells among B7-H3-positive or CD47-positive immune cells in GC tumor tissue was elevated significantly compared with adjacent tissue. Conclusion: Our findings demonstrated a correlation between B7-H3 expression and CD47 expression in GC patient tissues. Co-expression of B7-H3 and CD47 can serve as an indicator of poor prognosis in GC patients. In GC tumor tissue, but not adjacent tissue, B7-H3 and CD47 expression was accompanied with macrophage infiltration. [ABSTRACT FROM AUTHOR]