CD276 Promotes an Inhibitory Tumor Microenvironment in Hepatocellular Carcinoma and is Associated with Poor Prognosis.

Background: CD276 is an emerging immune checkpoint molecule that has been implicated in various cancers. However, its specific role in hepatocellular carcinoma (HCC) remains unclear. This study examined the impact of CD276 on patient prognosis and the tumor microenvironment (TME). Methods: The Cancer Genome Atlas (TCGA) database was utilized to evaluate CD276 expression in HCC and the association between CD276 and immune indicators was also analyzed. The signaling pathways correlated with CD276 expression were identified by gene set enrichment analysis (GSEA). Different algorithms were used to assess immune cell infiltration. The effect of CD276 knockdown on HCC cell phenotypes and its relationship with macrophage polarization was examined using the cell counting kit 8 (CCK-8) assay and co-culture system. Results: CD276 was upregulated in HCC and associated with unfavorable clinical outcomes. Hgh CD276 expression was associated with enrichment of the G2/M checkpoint, E2F targets, and mitotic spindles. CD276 expression was correlated with the infiltration of immune cells, including high level of tumor-associated macrophages and low levels of CD8+ T cells. Knockdown of CD276 decreased HCC cell proliferation and increased apoptosis. CD276 silencing in HCC cells and co-culture with THP-1-derived macrophages had a regulatory effect on macrophage polarization and macrophage-mediated cell proliferation and migration. Conclusion: CD276 expression in HCC is associated with unfavorable clinical outcomes and may contribute to the development of an immunosuppressive microenvironment. Specifically, CD276 was associated with alterations in immune cell infiltration, immune marker expression, and macrophage polarization during HCC progression, suggesting its potential as a prognostic indicator and promising target for immunotherapeutic intervention in HCC.

Rapidly-manufactured CD276 CAR-T cells exhibit enhanced persistence and efficacy in pancreatic cancer.

BACKGROUND: Pancreatic cancer is one of the most lethal malignancies and the lack of treatment options makes it more deadly. Chimeric Antigen Receptor T-cell (CAR-T) immunotherapy has revolutionized cancer treatment and made great breakthroughs in treating hematological malignancies, however its success in treating solid cancers remains limited mainly due to the lack of tumor-specific antigens. On the other hand, the prolonged traditional manufacturing process poses challenges, taking 2 to 6 weeks and impacting patient outcomes. CD276 has recently emerged as a potential therapeutic target for anti-solid cancer therapy. Here, we investigated the efficacy of CD276 CAR-T and rapidly-manufactured CAR-T against pancreatic cancer. METHODS: In the present study, CD276 CAR-T was prepared by CAR structure carrying 376.96 scFv sequence, CD8 hinge and transmembrane domain, 4-1BB and CD3ζ intracellular domains. Additionally, CD276 rapidly-manufactured CAR-T (named CD276 Dash CAR-T) was innovatively developed by shortening the duration of ex vitro culture to reduce CAR-T manufacturing time. We evaluated the anti-tumor efficacy of CD276 CAR-T and further compared the functional assessment of Dash CAR-T and conventional CAR-T in vitro and in vivo by detecting the immunophenotypes, killing ability, expansion capacity and tumor-eradicating effect of CAR-T. RESULTS: We found that CD276 was strongly expressed in multiple solid cancer cell lines and that CD276 CAR-T could efficiently kill these solid cancer cells. Moreover, Dash CAR-T was successfully manufactured within 48-72 h and the functional validation was carried out subsequently. In vitro, CD276 Dash CAR-T possessed a less-differentiated phenotype and robust proliferative ability compared to conventional CAR-T. In vivo xenograft mouse model, CD276 Dash CAR-T showed enhanced anti-pancreatic cancer efficacy and T cell expansion. Besides, except for the high-dose group, the body weight of mice was maintained stable, and the state of mice was normal. CONCLUSIONS: In this study, we proved CD276 CAR-T exhibited powerful activity against pancreatic cancer cells in vitro and in vivo. More importantly, we demonstrated the manufacturing feasibility, acceptable safety and superior anti-tumor efficacy of CD276 Dash CAR-T generated with reduced time. The results of the above studies indicated that CD276 Dash CAR-T immunotherapy might be a novel and promising strategy for pancreatic cancer treatment.

GAS-Luc2 Reporter Cell Lines for Immune Checkpoint Drug Screening in Solid Tumors.

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.

CD276 enhances sunitinib resistance in clear cell renal cell carcinoma by promoting DNA damage repair and activation of FAK-MAPK signaling pathway.

OBJECTIVE: This study aimed to explore the effect of CD276 expression on the sunitinib sensitivity of clear cell renal cell carcinoma (ccRCC) cell and animal models and the potential mechanisms involved. METHODS: CD276 expression levels of ccRCC and normal samples were analyzed via online databases and real-time quantitative PCR (RT-qPCR). CD276 was knocked down in ccRCC cell models (sunitinib-resistant 786-O/R cells and sunitinib-sensitive 786-O cells) using shRNA transfection, and the cells were exposed to a sunitinib (2 µM) environment. Cells proliferation was then analyzed using MTT assay and colony formation experiment. Alkaline comet assay, immunofluorescent staining, and western blot experiments were conducted to assess the DNA damage repair ability of the cells. Western blot was also used to observe the activation of FAK-MAPK pathway within the cells. Finally, a nude mouse xenograft model was established and the nude mice were orally administered sunitinib (40 mg/kg/d) to evaluate the in vivo effects of CD276 knockdown on the therapeutic efficacy of sunitinib against ccRCC. RESULTS: CD276 was significantly upregulated in both ccRCC clinical tissue samples and cell models. In vitro experiments showed that knocking down CD276 reduced the survival rate, IC50 value, and colony-forming ability of ccRCC cells. Knocking down CD276 increased the comet tail moment (TM) values and γH2AX foci number, and reduced BRCA1 and RAD51 protein levels. Knocking down CD276 also decreased the levels of p-FAK, p-MEK, and p-ERK proteins. CONCLUSION: Knocking down CD276 effectively improved the sensitivity of ccRCC cell and animal models to sunitinib treatment.

The bispecific B7H3xCD3 antibody CC-3 induces T cell immunity against bone and soft tissue sarcomas.

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.

B7-H3 Expression in Breast Cancer and Brain Metastasis.

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.

Incorporating IL7 receptor alpha signaling in the endodomain of B7H3-targeting chimeric antigen receptor T cells mediates antitumor activity in glioblastoma.

CAR-T-cell therapy has shown promise in treating hematological malignancies but faces challenges in treating solid tumors due to impaired T-cell function in the tumor microenvironment. To provide optimal T-cell activation, we developed a B7 homolog 3 protein (B7H3)-targeting CAR construct consisting of three activation signals: CD3ζ (signal 1), 41BB (signal 2), and the interleukin 7 receptor alpha (IL7Rα) cytoplasmic domain (signal 3). We generated B7H3 CAR-T cells with different lengths of the IL7Rα cytoplasmic domain, including the full length (IL7R-L), intermediate length (IL7R-M), and short length (IL7R-S) domains, and evaluated their functionality in vitro and in vivo. All the B7H3-IL7Rα CAR-T cells exhibited a less differentiated phenotype and effectively eliminated B7H3-positive glioblastoma in vitro. Superiority was found in B7H3 CAR-T cells contained the short length of the IL7Rα cytoplasmic domain. Integration of the IL7R-S cytoplasmic domain maintained pSTAT5 activation and increased T-cell proliferation while reducing activation-induced cell death. Moreover, RNA-sequencing analysis of B7H3-IL7R-S CAR-T cells after coculture with a glioblastoma cell line revealed downregulation of proapoptotic genes and upregulation of genes associated with T-cell proliferation compared with those in 2nd generation B7H3 CAR-T cells. In animal models, compared with conventional CAR-T cells, B7H3-IL7R-S CAR-T cells suppressed tumor growth and prolonged overall survival. Our study demonstrated the therapeutic potential of IL7Rα-incorporating CAR-T cells for glioblastoma treatment, suggesting a promising strategy for augmenting the effectiveness of CAR-T cell therapy.

Bioinformatic Analysis and Clinical Case Studies Identify CD276 as a Promising Diagnostic Biomarker for Clear Cell Renal Cell Carcinoma.

OBJECTIVE: This study aimed to explore the relationship between CD276 and clear cell renal carcinoma (ccRCC) and assess the diagnostic value of CD276 in ccRCC. METHODS: Expression levels of CD276 in ccRCC and para-cancer tissues were compared and analyzed retrospectively using data obtained from TCGA and GEO databases. The clinical data was analyzed prospectively. Immunohistochemistry and RT-PCR analyses were used to analyze the expression of CD276 at the mRNA and protein levels. These analyses compared the expression between ccRCC tissues and para-cancer tissues obtained from 70 patients with ccRCC. Next, ELISA was used to analyze peripheral blood samples from 70 patients with ccRCC and 72 healthy individuals, facilitating the differentiation of ccRCC patients from normal controls. Finally, we utilized the Kaplan-Meier method to generate ROC curves for assessing the diagnostic value of CD276 for ccRCC. RESULTS: Analysis of TCGA and GEO data revealed that the mRNA expression of CD276 was higher in ccRCC tissues than in para-cancer tissues (P < .05). Clinical validation using IHC and RT-PCR confirmed that the expression of CD276 was higher in ccRCC tissues than in para-cancer tissues, both at the mRNA and protein levels (P < .05). ELISA demonstrated that the expression of CD276 was higher in ccRCC patients than in normal individuals, and patients with a higher pathological grade showed higher expression of CD276 in the peripheral blood than those with a lower pathological grade (P < .05). ROC curves drawn from the above three datasets demonstrated that CD276 had a high diagnostic value for ccRCC (AUC = .894, .795, .938, respectively). CONCLUSION: The expression of CD276 was higher in ccRCC tissues and positively associated with the pathological grade. Therefore, CD276 may serve as a molecular biomarker for ccRCC prediction.

Pro-cancer role of CD276 as a novel biomarker for clear cell renal cell carcinoma.

OBJECTIVE: Renal cell carcinoma (RCC) is a common malignant tumor with a high incidence in males and the elderly, and clear cell RCC (ccRCC) is the most common RCC subtype. ccRCC is highly metastatic with a poor prognosis. Therefore, it is crucial to obtain a detailed understanding of the molecular mechanism of ccRCC and to identify suitable biomarkers to realize early diagnosis and improve prognosis. METHODS: We analyzed data from the Cancer Genome Atlas, investigated the overall differential expression of CD276 in ccRCC, and evaluated the influence of CD276 on patient survival and prognosis. We also performed gene set enrichment analysis (GSEA) and pathway enrichment analysis and investigated cell infiltration and drug responsiveness to further assess the regulatory effect of CD276 on ccRCC. Furthermore, we verified CD276 expression in RCC cell lines and control cell lines. RESULTS: The CD276 expression level in ccRCC samples was higher than that in corresponding samples adjacent to the tumors. Moreover, high CD276 expression levels were positively correlated with poor prognosis in patients with RCC. GSEA revealed that CD276 was significantly involved in immune-related pathways, and the level of CD276 expression was confirmed as associated with immune cell infiltration to some extent. Notably, some drugs were predicted to act on CD276, and this was confirmed by molecular docking. Furthermore, high levels of CD276 expression in RCC cell lines were verified. CONCLUSION: CD276 expression was significantly increased in ccRCC tissues and cells and positively correlated with patient prognosis. CD276 is a potential prognostic biomarker of ccRCC. Overall, this study provides a potential therapeutic strategy for ccRCC.

Efficacy of the induced pluripotent stem cell derived and engineered CD276-targeted CAR-NK cells against human esophageal squamous cell carcinoma.

Introduction: Chimeric antigen receptor natural killer (CAR-NK) cells have been found to be successful in treating hematologic malignancies and present potential for usage in solid tumors. Methods: In this study, we created CD276-targeted CAR-expressing NK cells from pluripotent stem cells (iPSC CD276-targeted CAR-NK cells) and evaluated their cytotoxicity against esophageal squamous cell carcinoma (ESCC) using patient-specific organoid (PSO) models comprising of both CD276-positive and CD276-negative adjacent epithelium PSO models (normal control PSO, NC PSO) as well as primary culture of ESCC cell models. In addition, in vitro and in vivo models such as KYSE-150 were also examined. iPSC NK cells and NK-free media were used as the CAR-free and NK-free controls, respectively. Results: The positive CD276 staining was specifically detected on the ESCC membrane in 51.43% (54/105) of the patients of all stages, and in 51.35% (38/74) of stages III and IV. The iPS CD276-targeted CAR-NK cells, comparing with the iPS NK cells and the NK-free medium, exhibited specific and significant cytotoxic activity against CD276-positive ESCC PSO rather than CD276-negative NC PSO, and exhibited significant cytotoxicity against CD276-expressing cultured ESCC cells, as well as against CD276-expressing KYSE-150 in vitro and in BNDG mouse xenograft. Discussion: The efficacy of the iPSC CD276-targeted CAR-NK cells demonstrated by their successful treatment of CD276-expressing ESCC in a multitude of pre-clinical models implied that they hold tremendous therapeutic potential for treating patients with CD276-expressing ESCC.

Clinicopathological features and prognostic value of CD276 expression in female reproductive system malignancies: A meta-analysis.

The relationship between CD276 and malignancies of the female reproductive system has previously been controversial. The purpose of this study was to evaluate the predictive value of CD276 expression in clinicopathological features and prognosis of female reproductive system malignant tumors through meta-analysis. PubMed, Embase, Cochrane Library, Web of Science, CNKI and Wanfang databases were searched for studies published up to December 2022 on the role of CD276 expression in the clinicopathological features and prognosis of female reproductive system malignancies. STATA 14.0 was used for meta-analysis. A total of 10 studies were included, involving 840 patients with malignant tumors of the female reproductive system. The results showed that in terms of clinicopathological features: CD276 expression was closely related to lymph node status [OR = 2.33， 95 %CI = 1.32-4.11， P = 0.003], tumor differentiation [OR = 2.15， 95 %CI = 1.27-3.63, P = 0.004], and FIGO stage [OR = 2.58， 95 %CI = 1.44-4.61, P = 0.001] of reproductive system malignant tumors. In terms of prognosis: CD276 expression is strongly associated with shorter OS in patients with female reproductive system malignancies [HR = 3.33, 95 % CI = 1.36-8.15, P = 0.01]. CD276 may be a new target for immunotherapy and a biomarker for predicting poor prognosis of female reproductive system malignancies.

Comprehensive analysis of PSME3: from pan-cancer analysis to experimental validation.

PSME3 plays a significant role in tumor progression. However, the prognostic value of PSME3 in pan-cancer and its involvement in tumor immunity remain unclear. We conducted a comprehensive study utilizing extensive RNA sequencing data from the TCGA (The Cancer Genome Atlas) and GTEx (Genotype-Tissue Expression) databases. Our research revealed abnormal expression levels of PSME3 in various cancer types and unveiled a correlation between high PSME3 expression and adverse clinical outcomes, especially in cancers like liver cancer (LIHC) and lung adenocarcinoma (LUAD). Functional enrichment analysis highlighted multiple biological functions of PSME3, including its involvement in protein degradation, immune responses, and stem cell regulation. Moreover, PSME3 showed associations with immune infiltration and immune cells in the tumor microenvironment, indicating its potential role in shaping the cancer immune landscape. The study also unveiled connections between PSME3 and immune checkpoint expression, with experimental validation demonstrating that PSME3 positively regulates CD276. This suggests that PSME3 could be a potential therapeutic target in immunotherapy. Additionally, we predicted sensitive drugs targeting PSME3. Finally, we confirmed in both single-factor Cox and multiple-factor Cox regression analyses that PSME3 is an independent prognostic factor. We also conducted preliminary validations of the impact of PSME3 on cell proliferation and wound healing in liver cancer. In summary, our study reveals the multifaceted role of PSME3 in cancer biology, immune regulation, and clinical outcomes, providing crucial insights for personalized cancer treatment strategies and the development of immunotherapy.

CD276 promotes epithelial-mesenchymal transition in esophageal squamous cell carcinoma through the TGF-ß/SMAD signaling.

OBJECTIVE: Aberrant expression of CD276 has been reported in malignant tumors. However, the exact role and mechanisms of CD276 influence the progression of esophageal squamous cell carcinoma (ESCC) still need to be understood. METHODS: Bioinformatics analysis of data from The Cancer Genome Atlas and Gene Expression Omnibus databases, along with immunohistochemistry staining, was used to explore the expression patterns of CD276 in ESCC. Cell counting kit-8 and Transwell assays were employed to evaluate the effects of CD276 expression on tumor cell proliferation and motility. Western blotting and Transwell assays were used to explore the potential pathways through which CD276 mediates the progression of ESCC. Moreover, the in vivo role of CD276 in tumor progression was investigated by establishing a lung metastasis mouse model. RESULTS: A significant upregulation of CD276 was observed in ESCC tissues compared to adjacent tissues. The inhibition of CD276 had no evident impact on ESCC cell proliferation but notably hindered their migratory and invasive properties and the expression of epithelial-mesenchymal transition (EMT) markers. Inversely, overexpressing CD276 led to an upregulation of EMT markers, underscoring the capacity of CD276 to amplify the motility of ESCC cells. Furthermore, CD276 was found to enhance the migratory and invasive abilities of ESCC cells by activating the TGF-ß/SMAD signaling but not the PI3K/AKT pathway. In vivo studies demonstrated that CD276 facilitates pulmonary metastasis. CONCLUSION: CD276 is significant upregulation in ESCC tissues and facilitates the EMT process in ESCC cells via the TGF-ß/SMAD signaling, thus promoting the progression of ESCC.

PathoGraph: An Attention-Based Graph Neural Network Capable of Prognostication Based on CD276 Labelling of Malignant Glioma Cells.

Computerized methods have been developed that allow quantitative morphological analyses of whole slide images (WSIs), e.g., of immunohistochemical stains. The latter are attractive because they can provide high-resolution data on the distribution of proteins in tissue. However, many immunohistochemical results are complex because the protein of interest occurs in multiple locations (in different cells and also extracellularly). We have recently established an artificial intelligence framework, PathoFusion which utilises a bifocal convolutional neural network (BCNN) model for detecting and counting arbitrarily definable morphological structures. We have now complemented this model by adding an attention-based graph neural network (abGCN) for the advanced analysis and automated interpretation of such data. Classical convolutional neural network (CNN) models suffer from limitations when handling global information. In contrast, our abGCN is capable of creating a graph representation of cellular detail from entire WSIs. This abGCN method combines attention learning with visualisation techniques that pinpoint the location of informative cells and highlight cell-cell interactions. We have analysed cellular labelling for CD276, a protein of great interest in cancer immunology and a potential marker of malignant glioma cells/putative glioma stem cells (GSCs). We are especially interested in the relationship between CD276 expression and prognosis. The graphs permit predicting individual patient survival on the basis of GSC community features. Our experiments lay a foundation for the use of the BCNN-abGCN tool chain in automated diagnostic prognostication using immunohistochemically labelled histological slides, but the method is essentially generic and potentially a widely usable tool in medical research and AI based healthcare applications.

B7-H3 Inhibitors in Oncology Clinical Trials: A Review.

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.

Protocol of a first-in-human clinical trial to evaluate the safety, tolerability, and preliminary efficacy of the bispecific CD276xCD3 antibody CC-3 in patients with colorectal cancer (CoRe_CC-3).

Introduction: Colorectal cancer (CRC) is the third most common cancer worldwide in men and women. In the metastasized stage, treatment options and prognosis are limited. To address the high medical need of this patient population, we generated a CD276xCD3 bispecific antibody termed CC-3. CD276 is expressed on CRC cells and on tumor vessels, thereby allowing for a "dual" anticancer effect. Methods and analysis: This first-in-human clinical study is planned as a prospective multicenter trial, enrolling patients with metastatic CRC after three lines of therapy. During the dose-escalation part, initially, an accelerated titration design with single-patient cohorts is employed. Here, each patient will receive a fixed dose level (starting with 50 µg for the first patient); however, between patients, dose level may be increased by up to 100%, depending on the decision of a safety review committee. Upon occurrence of any adverse events (AEs) grade ≥2, dose-limiting toxicity (DLT), or reaching a dose level of ≥800 µg, the escalation will switch to a standard 3 + 3 dose design. After maximum tolerated dose (MTD) has been determined, defined as no more than one of the six patients experiencing DLT, an additional 14 patients receive CC-3 at the MTD level in the dose-expansion phase. Primary endpoints are incidence and severity of AEs, as well as the best objective response to the treatment according to response evaluation criteria in solid tumors (RECIST) 1.1. Secondary endpoints include overall safety, efficacy, survival, quality of life, and pharmacokinetic investigations. Ethics and dissemination: The CD276xCD3 study was approved by the Ethics Committee of the Medical Faculty of the Heinrich Heine University Düsseldorf and the Paul-Ehrlich-Institut (P00702). Clinical trial results will be published in peer-reviewed journals. Trial registration numbers: ClinicalTrials.cov Registry (NCT05999396) and EU ClinicalTrials Registry (EU trial number 2022-503084-15-00).

Targeted therapies in retinoblastoma: GD2-directed immunotherapy following autologous stem cell transplantation and evaluation of alternative target B7-H3.

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.

B7-H3/CD276 and small-cell lung cancer: What's new?

Immunotherapy revolutionized the treatment landscape of several cancers, including small-cell lung cancer (SCLC), with a huge number of practice-changing trials, and becoming a new frontier for their management. The addition of an anti-PD-L1, atezolizumab or durvalumab, to platinum/etoposide regimen became the standard of care for first-line therapy of extensive-stage (ES)-SCLC with the 12 months median survival exceeded for the first time. Nevertheless, most patients show primary or acquired resistance to anti-PD-L1 therefore new promising therapeutic immune-targets are under clinical investigation in several solid tumors. Among these, B7-H3, also known as CD276, is a member of the B7 family overexpressed in tumor tissues, including SCLC, while showing limited expression in normal tissues becoming an attractive and promising target for cancer immunotherapy. B7-H3 plays a dual role in the immune system during the T-cell activation, acting as a T-cell costimulatory/coinhibitory immunoregulatory protein, and promoting pro-tumorigenic functions such as tumor migration, invasion, metastases, resistance, and metabolism. Immunohistochemistry, flow cytometry, and immunofluorescence were the most used methods to assess B7-H3 expression levels and validate a possible relationship between B7-H3 staining patterns and clinicopathological features in lung cancer patients. To date, there are no clinically available therapeutics/drugs targeting B7-H3 in any solid tumors. The most promising preliminary clinical results have been reported by DS7300a and HS-20093, both are antibody-drug conjugates, that are under investigation in ongoing trials for the treatment of pretreated SCLC. This review will provide an overview of B7-H3 and corresponding inhibitors and the clinical development in the management of SCLC.

COL10A1 promotes tumorigenesis by modulating CD276 in pancreatic adenocarcinoma.

BACKGROUND: Pancreatic adenocarcinoma (PAAD) is a lethal malignant tumour. Further study is needed to determine the molecular mechanism and identify novel biomarkers of PAAD. METHODS: Gene expression data from the GSE62165 microarray were analysed with the online software Morpheus to identify differentially expressed genes (DEGs). The STRING database was used to generate a protein‒protein interaction (PPI) network for these DEGs. Hub genes were identified with Cytoscape. COL10A1 expression in PAAD was analysed via the GEPIA database. COL10A1 expression in pancreatic cancer cell lines was measured by using qRT‒PCR. The LinkedOmics database was utilized to perform survival analysis of pancreatic adenocarcinoma patients grouped based on COL10A1 expression level. CCK-8, wound healing, and Transwell assays were used to study the role of COL10A1 in pancreatic cancer cell viability, migration, and invasion. Differentially expressed genes that were related to COL10A1 in PAAD were analysed via the LinkedOmics portal. After COL10A1 was knocked down, CD276 expression was assessed by western blotting. RESULTS: COL10A1 was identified as one of the hub genes in PAAD by bioinformatics analysis of the GSE62165 microarray with Morpheus, the STRING database and Cytoscape. GEPIA revealed elevated expression of COL10A1 in PAAD samples vs. normal samples. COL10A1 expression was also increased in pancreatic cancer cells vs. control cells. Survival analysis of PAAD patients via LinkedOmics revealed that high expression of COL10A1 was associated with a poorer prognosis. Knockdown of COL10A1 inhibited the proliferation, migration, and invasion of cells in functional assays. Furthermore, mechanistic studies indicated that CD276 was a target of COL10A1 and that knockdown of COL10A1 decreased CD276 expression. Overexpression of CD276 in cells reversed COL10A1 knockdown-induced repression of proliferation and migration. CONCLUSIONS: Our research suggests that COL10A1 promotes pancreatic adenocarcinoma tumorigenesis by regulating CD276. This study provides new insight into biomarkers and possible targets for pancreatic cancer treatment.