B7-H3 Expression Research Articles

Expression analysis of inhibitory B7 family members in Alzheimer's disease.

Alzheimer's disease (AD) is a global health problem due to its complexity, which frequently makes the development of treatment methods extremely difficult. Therefore, new methodologies are necessary to investigate the pathophysiology of AD and to treat AD. The interaction of immune modulation and neurodegeneration has added new dimensions in current knowledge of AD etiology and offers an attractive opportunity for the discovery of novel biomarkers and therapies. Using quantitative polymerase chain reaction, we compared the expression levels of inhibitory B7 family members (B7-1, B7-2, B7-H1, B7-DC, B7-H3, B7-H4, B7-H5, B7-H7, and ILDR2), as immune regulators, in the peripheral blood of late-onset AD (LOAD) patients (n = 50) and healthy individuals (n = 50). The levels of B7-2, B7-H4, ILDR2, and B7-DC expression were significantly higher in-patient blood samples than in control blood samples. Furthermore, we discovered a substantial positive correlation between all gene expression levels. In addition, the current study indicated that ILDR2, B7-H4, B7-2, and B7-DC might serve as diagnostic biomarkers to identify LOAD patients from healthy persons. The present work provides additional evidence for the significance of inhibitory B7 family members to the etiology of LOAD.

B7H3 increases ferroptosis resistance by inhibiting cholesterol metabolism in colorectal cancer.

Ferroptosis, a newly discovered form of regulated cell death, has been reported to be associated with multiple cancers, including colorectal cancer (CRC). However, the underlying molecular mechanism is still unclear. In this study, we identified B7H3 as a potential regulator of ferroptosis resistance in CRC. B7H3 knockdown decreased but B7H3 overexpression increased the ferroptosis resistance of CRC cells, as evidenced by the expression of ferroptosis-associated genes (PTGS2, FTL, FTH, and GPX4) and the levels of important indicators of ferroptosis (malondialdehyde, iron load). Moreover, B7H3 promoted ferroptosis resistance by regulating sterol regulatory element binding protein 2 (SREBP2)-mediated cholesterol metabolism. Both exogenous cholesterol supplementation and treatment with the SREBP2 inhibitor betulin reversed the effect of B7H3 on ferroptosis in CRC cells. Furthermore, we verified that B7H3 downregulated SREBP2 expression by activating the AKT pathway. Additionally, multiplex immunohistochemistry was carried out to show the expression of B7H3, prostaglandin-endoperoxide synthase 2, and SREBP2 in CRC tumor tissues, which was associated with the prognosis of patients with CRC. In summary, our findings reveal a role for B7H3 in regulating ferroptosis by controlling cholesterol metabolism in CRC.

Evaluation of affinity matured Affibody molecules for imaging of the immune checkpoint protein B7-H3

B7-H3 (CD276), an immune checkpoint protein, is a promising molecular target for immune therapy of malignant tumours. Sufficient B7-H3 expression level is a precondition for successful therapy. Radionuclide molecular imaging is a powerful technique for visualization of expression levels of molecular targets in vivo. Use of small radiolabelled targeting proteins would enable high-contrast radionuclide imaging of molecular targets if adequate binding affinity and specificity of an imaging probe could be provided. Affibody molecules, small engineered affinity proteins based on a non-immunoglobulin scaffold, have demonstrated an appreciable potential in radionuclide imaging. Proof-of principle of radionuclide visualization of expression levels of B7-H3 in vivo was demonstrated using the [99mTc]Tc-AC12-GGGC Affibody molecule. We performed an affinity maturation of AC12, enabling selection of clones with higher affinity. Three most promising clones were expressed with a –GGGC (triglycine-cysteine) chelating sequence at the C-terminus and labelled with technetium-99m (99mTc). 99mTc-labelled conjugates bound to B7-H3-expressing cells specifically in vitro and in vivo. Biodistribution in mice bearing B7-H3-expressing SKOV-3 xenografts demonstrated improved imaging properties of the new conjugates compared with the parental variant [99mTc]Tc-AC12-GGGC. [99mTc]Tc-SYNT-179 provided the strongest improvement of tumour-to-organ ratios. Thus, affinity maturation of B7-H3 Affibody molecules could improve biodistribution and targeting properties for imaging of B7-H3-expressing tumours.

B7-H3 confers stemness characteristics to gastric cancer cells by promoting glutathione metabolism through AKT/pAKT/Nrf2 pathway.

Cancer stem-like cells (CSCs) are a small subset of cells in tumors that exhibit self-renewal and differentiation properties. CSCs play a vital role in tumor formation, progression, relapse, and therapeutic resistance. B7-H3, an immunoregulatory protein, has many protumor functions. However, little is known about the mechanism underlying the role of B7-H3 in regulating gastric cancer (GC) stemness. Our study aimed to explore the impacts of B7-H3 on GC stemness and its underlying mechanism. GC stemness influenced by B7-H3 was detected both in vitro and in vivo . The expression of stemness-related markers was examined by reverse transcription quantitative polymerase chain reaction, Western blotting, and flow cytometry. Sphere formation assay was used to detect the sphere-forming ability. The underlying regulatory mechanism of B7-H3 on the stemness of GC was investigated by mass spectrometry and subsequent validation experiments. The signaling pathway (Protein kinase B [Akt]/Nuclear factor erythroid 2-related factor 2 [Nrf2] pathway) of B7-H3 on the regulation of glutathione (GSH) metabolism was examined by Western blotting assay. Multi-color immunohistochemistry (mIHC) was used to detect the expression of B7-H3, cluster of differentiation 44 (CD44), and Nrf2 on human GC tissues. Student's t -test was used to compare the difference between two groups. Pearson correlation analysis was used to analyze the relationship between two molecules. The Kaplan-Meier method was used for survival analysis. B7-H3 knockdown suppressed the stemness of GC cells both in vitro and in vivo . Mass spectrometric analysis showed the downregulation of GSH metabolism in short hairpin B7-H3 GC cells, which was further confirmed by the experimental results. Meanwhile, stemness characteristics in B7-H3 overexpressing cells were suppressed after the inhibition of GSH metabolism. Furthermore, Western blotting suggested that B7-H3-induced activation of GSH metabolism occurred through the AKT/Nrf2 pathway, and inhibition of AKT signaling pathway could suppress not only GSH metabolism but also GC stemness. mIHC showed that B7-H3 was highly expressed in GC tissues and was positively correlated with the expression of CD44 and Nrf2. Importantly, GC patients with high expression of B7-H3, CD44, and Nrf2 had worse prognosis ( P = 0.02). B7-H3 has a regulatory effect on GC stemness and the regulatory effect is achieved through the AKT/Nrf2/GSH pathway. Inhibiting B7-H3 expression may be a new therapeutic strategy against GC.

Influence of ADT on B7-H3 expression during CRPC progression from hormone-naïve prostate cancer.

Androgen deprivation therapy (ADT) is the standard care for advanced prostate cancer (PCa) patients. Unfortunately, although tumors respond well initially, they enter dormancy and eventually progress to fatal/incurable castration-resistant prostate cancer (CRPC). B7-H3 is a promising new target for PCa immunotherapy. CD276 (B7-H3) gene has a presumptive androgen receptor (AR) binding site, suggesting potential AR regulation. However, the relationship between B7-H3 and AR is controversial. Meanwhile, the expression pattern of B7-H3 following ADT and during CRPC progression is largely unknown, but critically important for identifying patients and determining the optimal timing of B7-H3 targeting immunotherapy. In this study, we performed a longitudinal study using our unique PCa patient-derived xenograft (PDX) models and assessed B7-H3 expression during post-ADT disease progression. We further validated our findings at the clinical level in PCa patient samples. We found that B7-H3 expression was negatively regulated by AR during the early phase of ADT treatment, but positively associated with PCa proliferation during the remainder of disease progression. Our findings suggest its use as a biomarker for diagnosis, prognosis, and ADT treatment response, and the potential of combining ADT and B7-H3 targeting immunotherapy for hormone-naïve PCa treatment to prevent fatal CRPC relapse.

Clinical significance of B7-H3 expression in circulating CD4+CD25high T cells, CD14+ monocytes, and plasma for the progression of HIV infection

BackgroundB7-H3 is an important immune checkpoint molecule that plays a negative role in immune regulation. This study was aimed to explore B7-H3 expression in HIV-infected patients and its clinical significance.MethodsTo explore the expression and clinical significance of B7-H3 in HIV-infected patients, we investigated the B7-H3 expression pattern and the correlation of B7-H3 expression with clinical parameters of HIV-infected patients with different levels of CD4+ T cells. To assess the role of B7-H3 in regulating the function of T cells in HIV infection, we performed a proliferation assay and T cell function test in vitro.ResultsB7-H3 expression in HIV-infected patients was significantly higher than that in healthy controls. mB7-H3 expression on CD4+CD25high T cells and CD14+ monocytes increased with disease progression. mB7-H3 expression on CD4+CD25high T cells and monocytes was negatively correlated with lymphocyte count, CD4+T cell count, and positively correlated with HIV viral load in HIV-infected patients. when the number of CD4+ T cells in HIV-infected patients was ≥ 200/µL, sB7-H3 and mB7-H3 expression levels on CD4+CD25high T cells and monocytes were negatively correlated with lymphocyte count, CD4+T cell count. sB7-H3 and mB7-H3 expression on monocytes were positively correlated with HIV viral load. B7-H3 inhibited the proliferation of lymphocytes and the secretion of IFN-γ in vitro, especially the ability of CD8+ T cells to secrete IFN-γ.ConclusionsB7-H3 played an important negative regulatory role in anti-HIV infection immunity. It could be used as a potential biomarker for the progression of HIV infection and a novel target for the treatment of HIV infection.

Heterogeneous Expression of PD-L1, B7x, B7-H3, and HHLA2 in Pulmonary Sarcomatoid Carcinoma and the Related Regulatory Signaling Pathways.

Immunotherapy has transformed lung cancer management, but PSC remains an aggressive subtype with a poor prognosis. This study investigates the differential expression of PD-L1 and alternative immune checkpoints (ICs; B7x, B7-H3, and HHLA2), and genetic alterations in PSCs. Tumor specimens of 41 PSC patients were evaluated. PD-L1, B7x, B7-H3, and HHLA2 were positive in 75.0%, 67.6%, 73.0%, and 91.9% of tumors, respectively. PD-L1 expression was significantly higher in the epithelial compared to the sarcomatoid component (median TPS: 50% vs. 0%, p = 0.010). Expression of PD-L1 in both components was only seen in 32.1% of patients. However, at least one IC was expressed in 92.9% of epithelial and 100% of sarcomatoid components. Furthermore, METex14 was detected in 19.5% of patients and was associated with a higher sarcomatoid percentage. Our preclinical studies revealed that METex14 induced PD-L1 expression via MAPK or PI3K/Akt pathways, and MET inhibitors decreased PD-L1 expression. Our findings demonstrate distinct expressions of ICs in PSC subcomponents. Thus, combination IC inhibition as a therapeutic strategy in PSC warrants further exploration. A high percentage of METex14 in PSC and its role in regulating PD-L1 expression reveal different therapeutic targets in this aggressive NSCLC subtype.

A clinicopathological analysis of supratentorial ependymoma, ZFTA fusion-positive: utility of immunohistochemical detection of CDKN2A alterations and characteristics of the immune microenvironment

EPN-ZFTA is a rare brain tumor where prognostic factors remain unclear and no effective immunotherapy or chemotherapy is currently available. Therefore, this study investigated its clinicopathological features, evaluated the utility of MTAP and p16 IHC as surrogate markers of CDKN2A alterations, and characterized the immune microenvironment of EPN-ZFTA. Thirty surgically removed brain tumors, including 10 EPN-ZFTA, were subjected to IHC. MLPA was performed for CDKN2A HD in 20 ependymal tumors, including EPN-ZFTA. The 5-years OS and PFS of EPN-ZFTA were 90% and 60%, respectively. CDKN2A HD was detected in two cases of EPN-ZFTA; these cases were immunohistochemically negative for both MTAP and p16 and recurred earlier after surgery. As for the immune microenvironment of EPN-ZFTA, B7-H3, but not PD-L1, was positive in all cases of EPN-ZFTA; Iba-1-positive or CD204-positive macrophages were large, while infiltrating lymphocytes were small, in number in EPN-ZFTA. Collectively, these results indicate the potential of MTAP and p16 IHC as useful surrogate markers of CDKN2A HD in EPN-ZFTA, and tumor-associated macrophages, including the M2 type, may contribute to its immune microenvironment. Furthermore, the expression of B7-H3 in EPN-ZFTA may indicate the usefulness of B7-H3 as a target of immune checkpoint chemotherapy for EPN-ZFTA via B7-H3 pathway.

IMG-04. POTENTIAL TARGETS FOR THERANOSTICS IN PEDIATRIC HIGH-GRADE CENTRAL NERVOUS SYSTEM (CNS) TUMORS

Abstract Theranostics refers to the combination of a radioactive drug to identify (diagnose) and another radioactive drug to deliver therapy to cancer. In adult cancer several radiopharmaceuticals with specificity for receptors like cx-chemokine-receptor type 4 (CXCR-4), prostate-membrane specific antigen (PSMA), and somatostatine receptor (SSTR2a) are used. Especially PSMA for prostate cancer and SSTR2a ligands for neuro-endocrine tumors have proven their value. Moreover, novel promising targets like B7-H3 are upcoming. In children the potential of such theranostic drugs have been minimally explored. AIM of this study is to explore the expression of theranostic markers CXCR-4, PSMA, SSTR2a and B7-H3 in a broad cohort of high-grade pediatric CNS tumors to determine the potential of these imaging ligands in this cohort. METHODS The expression of CXCR-4, PSMA, SSTR2a and B7-H3 were examined both by RNA (by bulk RNAsequencing) and protein level (by immunohistochemistry (IHC)) (RNA n=161/IHC n=52) in pediatric high-grade CNS tumors: ependymomas (n=33/11), atypical teratoid rhabdoid tumors (ATRT) (n=9/7), medulloblastomas (n=51/11), diffuse midline gliomas H3K27-altered (n=27/9) and other high-grade gliomas (n=41/15). RESULTS mRNA expression of CXCR-4 was present but low in most tumor types, with the highest expression in medulloblastoma. Immunohistochemistry showed variable CXCR-4 expression, with the highest expression in some medulloblastomas, ependymomas and ATRTs. PSMA (FOLH1) mRNA and protein expression was generally very low. As expected SSTR2a showed a very high mRNA and protein expression only in medulloblastoma samples. Remarkably, B7-H3 (CD276) showed a relatively high expression in all high-grade pediatric CNS tumor types, both on RNA and protein level. CONCLUSION Of the four potential theranostic targets tested, SSTR2a is an interesting antigen in medulloblastoma. CXCR-4 could be of interest for individual patients with medulloblastoma, ATRT or ependymoma. Finally, B7-H3 is widely but variably expressed on all different tumor types, and seems a promising novel target for theranostics in pediatric CNS tumors.

MDB-45. SELECTIVE B7-H3 INHIBITORS NI1 AND NI4 REDUCE AGGRESSIVENESS OF GROUP 3 MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is a primary childhood malignancy of the central nervous system (CNS). Clinically heterogenous MB are sub-grouped into four molecular subgroups, i.e. Wingless (WNT), Sonic Hedgehog (SHH), Group 3, and Group 4. Among all, Group 3 MB (G3MB) is known to be the most aggressive form, with less than 50% five-year survival. G3MB are associated with large-cell/anaplastic (LCA) histology, distinctive methylation profile, amplification of MYC, and isochromosome 17q (i17q). Targeting these high-risk molecular features unique to G3MB has been challenging, necessitating alternative approaches to therapy for G3-MB. One innovative approach is immunotherapy, which is largely impeded by inhibitory immune checkpoints. B7 homolog 3 (B7-H3/CD276), an immunosuppressive immune checkpoint protein and member of B7 superfamily, is significantly enriched in pediatric malignancies of CNS including G3MB. The enhanced expression of B7-H3/CD276 has been associated with aggressive and metastatic potential. B7-H3/CD276 is also a primary target of miR-1253, a novel tumor suppressor gene silenced in G3MB. Using a structure-based drug discovery pipeline, we screened the ChemBridge library of compounds to discover selective B7-H3 small-molecule inhibitors. Selective B7-H3 inhibitors Ni1 and Ni4 exhibited strong binding scores, CNS penetrance, and oral bioavailability. In vitro, both compounds had appreciably low IC50 values of 3µM (Ni1) and 0.75µM (Ni4). Further, both compounds inhibited wound healing and colony formation. Ni1 shifted the thermostability of B7-H3, suggesting in vitro biding. Ni1 has also undergone PK/PD and MTD studies in vivo, showing stability in serum, good blood-brain barrier permeability, and a half-life of 8 hours. We are now elucidating B7-H3-activated downstream signaling through JAK2/STAT3 signaling and testing the in vivo efficacy for Ni1 against G3MB tumors in a murine model.

IMMU-20. EVALUATION OF B7-H3 NANOBODY CAR-T CELLS IN PEDIATRIC GLIOBLASTOMA

Abstract Chimeric antigen receptor (CAR)-T cell therapies are an undoubted success in childhood acute lymphoblastic leukemia; however, this success has not translated clinically to any childhood solid tumor. B7-H3 has been found to be highly expressed in pediatric brain tumors and correlates with tumor progression and poor prognosis. We utilized a novel nanobody based CAR-T cell which can bind tumor antigens with high affinity. We hypothesize that anti-B7-H3 nanobody CAR-T cells mediate more potent antitumor effects both in vitro and in vivo compared to existing antibody-based B7-H3 CAR-T cells. High expression of B7-H3 was confirmed on the surface of two pediatric glioblastoma cell lines by flow cytometry and Quantibrite staining. B7-H3 nanobodies isolated from camel phage libraries were cloned into lentiviral CAR-T cell constructs. We manufactured CAR-T cells by lentiviral transduction of human T cells. Glioblastoma tumor cells were co-cultured with B7-H3 CAR-T cells vs. control T cells. We confirmed B7-H3 specific cytokine release by CAR-T cells via ELISA and effective tumor cell killing via two different cytotoxicity assays (impedance or luciferase based). Nanobody based B7-H3 CAR-T cells exhibited statistically significant higher cytokine release and cytotoxicity compared to antibody based B7-H3 CAR-T cells. In vivo studies are currently evaluating the efficacy of intracerebroventricular delivery of B7-H3 CAR-T cells against two orthotopic glioblastoma models utilizing multiple human donor T cells in immunocompromised mice. We are simultaneously developing a syngeneic orthotopic glioblastoma model to evaluate the nanobody based B7-H3 CAR-T cell cross-reactive in mice utilizing an immunocompetent model. Careful investigation of CAR-T cell therapy in appropriate immunocompetent in vivo models is needed to develop effective immunotherapy for clinical application.

B7H3 Role in Reshaping Immunosuppressive Landscape in MSI and MSS Colorectal Cancer Tumours.

The study aimed to assess the expression of B7H3 concerning clinicopathological and histological parameters, including MSI/MSS status, CD-8 cells, tumour-infiltrating lymphocytes (TILs), budding, TNM scale and grading. Moreover, we analyzed the B7H3-related pathways using available online datasets and the immunological context of B7H3 expression, through the 48-cytokine screening panel of cancer tissues homogenates, immunogenic features and immune composition. The study included 158 patients diagnosed with CRC. To assess B7H3 levels, we performed an immunohistochemistry method (IHC) and enzyme-linked immunosorbent assay (ELISA). To elucidate the immune composition of colorectal cancer, we performed the Bio-Plex Pro Human 48-cytokine panel. To study biological characteristics of B7H3, we used online databases. Expression of B7H3 was upregulated in CRC tumour tissues in comparison to adjacent noncancerous margin tissues. The concentrations of B7H3 in tumours were positively associated with T parameter of patients and negatively with tumour-infiltrating lymphocytes score. Additionally, Principal Component Analysis showed that B7H3 expression in tumours correlated positively with cytokines associated with M2-macrophages and protumour growth factors. The expression of B7H3 in tumours was independent of MSI/MSS status. These findings will improve our understanding of B7H3 role in colorectal cancer immunity. Our study suggests that B7-H3 is a promising potential target for cancer therapy. Further studies must clarify the mechanisms of B7H3 overexpression and its therapeutic importance in colorectal cancer.

Abstract B040: Novel immunotherapy strategies targeting PTEN and TP53 defects in advanced prostate cancer

Abstract Prostate cancer is the most diagnosed cancer in men worldwide and the leading cause of cancer death in men worldwide. Genetic inactivation of PTEN and TP53 are common in advanced prostate cancers. Checkpoint immunotherapy has yielded meaningful responses across many cancers but shown modest activity in advanced prostate cancer. Prior studies showed that overexpression of immune checkpoint B7-H3 (CD276) correlates with the increased risks of clinical recurrence, disease spread, and poor outcomes in various cancer types, including prostate cancer. However, the roles of B7-H3 in prostate cancer development and its tumor microenvironment remain unclear, partially due to the lack of tissue-specific deletion mouse models. This gap in knowledge hinders the application of immunotherapy targeting B7-H3 in prostate cancers. To identify PTEN- and p53-associated immune checkpoints, we performed multi-omics analyses of expression patterns of 51 checkpoint molecules in human prostate cancer samples and found that B7-H3 is one of the most significantly overexpressed immune checkpoints in prostate tumors containing PTEN and TP53 genetic inactivation. Mechanistically, we found that the PTEN-AKT pathway co-operates with p53 pathway in modulating B7-H3 expression in cancer cells. In Pten/Trp53 genetically engineered mouse (GEM) models, prostate-specific deletion of Cd276 resulted in markedly delayed tumor progression and reversed immunosuppression in the tumor microenvironment. Furthermore, we developed and evaluated novel combinatorial immunotherapy strategies targeting B7-H3 in advanced prostate cancer with PTEN and TP53 deficiencies. Citation Format: Wei Shi, Yin Wang, Yuehui Zhao, Di Zhao. Novel immunotherapy strategies targeting PTEN and TP53 defects in advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B040.

Correlation of low B7-H6 expression with efficacy of immunotherapy inpatients with melanoma.

e21556 Background: B7-H6 (NCR3LG1) can bind to NKp30 to trigger antitumor NK cell activation and cytokine secretion. Our previous studies have shown that in gastric cancer, B7-H6 high expression was associated with better prognosis and the low expression of B7-H6 group was correlated with better immune microenvironment. However, the effect of B7-H6 expression on immunotherapy is unknown. Herein, we used the data from a clinically annotated cohort of melanoma patients treated with ICI to analyze the influence of B7-H6 expression on immunotherapy. Methods: The gene expression profile, gene mutation information and clinical data were extracted from the supplementary data in melanoma cohort (PMID: 31792460). Maximally selected rank statistics was used to select the optimum threshold for B7-H6 expression, and the patients was divided into two groups according to the expression of B7-H6, high group and low group. Kaplan-Meier survival analysis was used to evaluate the influence of B7-H6 expression on OS prognosis. Associations between variables and OS survival were tested using univariate and multivariate Cox and displayed by forest (R package). Results: We selected 3.14 as the optimum threshold of B7-H6 expression, and the patients was divided into high group (B7-H6 expression＞3.14) and low group (B7-H6 expression≤3.14). The results showed that low group was significantly associated with longer overall survival (p value = 0.048) in cohort of melanoma patients treated with anti-PD1 ICI, which was consistent with our previous results that low expression of B7-H6 group was correlated with better immune microenvironment. We selected primary lesion type, TMB and the expression of B7-H6 factors for univariate and multivariate analysis. TMB-H (top 25%) (p value = 0.051) and B7-H6 (p value = 0.051) low expression were associated with a better OS. Then the patients were divided into four groups according to the TMB and B7-H6 expression and analyzed the difference in the four groups. The results showed that in patients with TMB-L, B7-H6 low group had significant better OS than the B7-H6 high group (p value = 0.032). Conclusions: The low B7-H6 expression was correlated with better efficacy immunotherapy. The effect of B7-H6 expression on immunotherapy needs to be further prospective trial validation.

B7H3 expression in sarcoma.

e23517 Background: Targeted therapy development in sarcoma has been burdened by the heterogeneity of this rare group of tumors. B7H3 is an immune checkpoint molecule in the same family as PD-L1 (also known as B7H1). Its limited expression in non-cancerous tissues and over-expression in numerous tumor types makes it an attractive target for cancer immunotherapy. Available data suggests high expression of B7H3 in certain sarcoma subtypes, including rhabdomyosarcoma, osteosarcoma, liposarcoma, Ewing sarcoma, synovial sarcoma and chondrosarcoma. This study was conducted to further explore B7H3 expression patterns across sarcoma subtypes and the relationship, if any, with PD-1 and PD-L1 expression. Methods: This single-institution, retrospective analysis evaluated soft tissue sarcoma Formalin-Fixed Paraffin-Embedded tumor specimens in patients with a variety of different subtypes. Specimens were evaluated for expression of B7H3, vessel positivity for B7H3, B7H3 staining pattern, and expression of PD-L1 and PD-1. The association between B7H3 expression and baseline and tumor factors was tested using the Mantel Haenszel test or two-sample T-test. Overall survival was estimated using the Kaplan-Meier method where survival time was calculated from diagnosis to death (or last follow-up for those last known to be alive). Results: 156 sarcoma specimens encompassing 16 subtypes were included. B7H3 was broadly expressed across sarcoma subtypes, with some degree of expression found in 91% of samples and 61% demonstrating high levels of B7H3 expression. All tumor samples expressing B7H3 exhibited the biomarker in at least 80% of tumor cells per sample (with a mean of 92.34% of cells testing positive for B7H3). All samples positive for B7H3 showed both cytoplasmic and membranous expression, and B7H3 vessel positivity was found in 90% (137/152) of samples. There were no significant differences in expression or level of expression when stratified by sarcoma subtype, prior treatment(s), tumor size, tumor grade or patient age. No significant difference in overall survival was seen regardless of B7H3 expression or expression levels across sarcoma subtypes. There was no correlation between B7H3 expression and expression of PD-L1 or PD-1 in tumor cells. Conclusions: This is the most comprehensive analysis of B7H3 expression in sarcoma to date. This data shows high levels of B7H3 expression across sarcoma subtypes, suggesting its feasibility as a therapeutic target for future sarcoma treatments. Future clinical trials are needed to evaluate its numerous actions and if manipulation of B7H3 can be employed to improve outcomes in sarcoma. [Table: see text]

XMT-1660: A phase 1b trial of a B7-H4 targeted antibody drug conjugate (ADC) in breast, endometrial, and ovarian cancers.

TPS3154 Background: Breast cancer (BC) is the most commonly diagnosed cancer and one of the leading causes of cancer death in women. Despite significant therapeutic advances, the majority of patients with unresectable or recurrent/metastatic disease eventually develop resistance to available standard of care (SOC) therapies. High B7-H4 expression has been observed in several cancers including breast, endometrial, and ovarian, with limited expression in normal tissue. As a member of the CD28/B7 family of cell surface proteins, it promotes tumorigenesis by suppressing anti-tumor immunity. XMT-1660 is a B7-H4-directed Dolasynthen antibody drug conjugate designed with a precise, optimized drug-to-antibody ratio and a DolaLock microtubule inhibitor payload with controlled bystander effect. In the preclinical setting, XMT-1660 has demonstrated anti-tumor activity in TNBC and ER+/HER2- patient-derived xenograft mouse models, which included tumors from heavily pre-treated patients (Collins et al, AACR 2022). Increased anti-tumor activity tended to be more frequent in preclinical models with higher B7-H4 expression, providing rationale for a Ph1 clinical trial. Methods: The Ph1 trial includes a first-in-human open-label dose escalation (DES) portion followed by dose expansion (EXP) evaluating XMT-1660 in patients with BC, EC, and OC following progression on SOC as applicable (i.e., CDK4/6i + ET; platinum-based chemotherapy). In the DES, Bayesian Optimal Interval (BOIN) design will be used to determine the MTD. Patients will receive XMT-1660 IV Q3 weeks. Primary endpoints in DES are to assess safety and determine a recommended phase 2 dose (RP2D) and assessment of preliminary efficacy as a secondary endpoint. In parallel with DES, optional backfill cohorts will enroll patients into tumor-type specific cohorts (TNBC, HR+/HER2- BC, EC, or OC) at a selected dose level from DES and can be initiated at multiple dose levels simultaneously. In the EXP portion, cohorts enrolling TNBC, ER+/HER2- BC, EC/OC are planned and additional patients may be enrolled based on emerging data. The primary endpoint of EXP is to assess safety and tolerability, overall response rate, disease control rate, and duration of response. Secondary endpoints include pharmacokinetic analysis and assessment of antidrug antibodies. Patients are not selected by B7-H4 status, but baseline tumors samples are collected for retrospective tumor tissue evaluation. The trial is currently enrolling patients. NCT05377996. Clinical trial information: NCT05377996 .

COORDINATED CHANGES IN THE TUMOR MICROENVIRONMENT (TME) ARE ASSOCIATED WITH INCREASED RISK OF THERAPEUTIC FAILURE IN NEWLY DIAGNOSED DIFFUSE LARGE B‐CELL LYMPHOMA (DLBCL)

Introduction: Despite advances, 20%–40% of patients with DLBCL do not achieve remission or relapse after an initial response. We aimed to identify immunological factors associated with early clinical failure in DLBCL. Methods: Patients with DLBCL treated with rituximab and anthracycline-based chemotherapy were prospectively enrolled in the University of Iowa/Mayo Clinic Lymphoma Specialized Program of Research Excellence (SPORE). Tissue microarrays (TMAs) containing specimens from diagnosis were analyzed using the Digital Spatial Profiling (DSP) system quantifying expression of 58 proteins (Figure 1A). Event-free survival (EFS) was defined as time from diagnosis to relapse, progression, or death from any cause. Patients with EFS of at least 24 months were classified as achieving EFS24. Following normalization, logistic regression was performed on DSP data to adjust for IPI and cell of origin. Multiplex immunofluorescence (MxIF) using the CODEX system was performed on a biopsy specimen from a representative patient. Machine learning models were used for cell segmentation and classification (Figure 1B). Results: Specimens from 446 DLBCL patients were available on TMAs, of which 321 (72%) achieved EFS24. DSP data was acquired in 395 unique DLBCL patients and showed that increased expression of CD163 (Log2FC = 2.9; p = 0.0009), BCL2 (Log2FC = 1.4; p = 0.047), and PDL1 (Log2FC = 1.3; p = 0.05) were significantly associated with increased risk of EFS24 failure (Figure 1A, red arrows). Conversely, increased expression of CD11c (Log2FC = 0.5; p = 0.003), HLA-DR (Log2FC = 0.4; p = 0.01), CD357/GITR (Log2FC = 0.8; p = 0.01), CD137 (Log2FC = 0.7; p = 0.02), and B7H3 (Log2FC = 0.9; p = 0.02) were significantly associated with decreased risk of EFS24 failure (Figure 1A, green arrows). A total of 3004 single cell events were acquired using MxIF, of which 55% were classified as B cells, 17% as T cells, 8% as macrophages, and 20% as other cells. Within B cells, 81% were classified as HLA-DR+, 73% as CD11c+, 54% as BCL2+, while; 45% were positive for all 3 antigens (Figure C1–4). GITR expression was confirmed in 3% of T cells and showed a memory (CD45RO+), activated (CD38+) phenotype (Figure D1–4). The research was funded by: Department of Defense (DOD), Lymphoma Research Foundation (LRF), National Cancer Institute (NCI) Keywords: aggressive B-cell non-Hodgkin lymphoma, genomics, epigenomics, and other -omics, microenvironment No conflicts of interests pertinent to the abstract.

Exploring the potential of combination immune checkpoint strategies in non-small cell lung cancer (NSCLC).

9037 Background: The clinical outcomes for patients with NSCLC remain suboptimal despite recent advancements in anti-PD-1/PD-L1 immunotherapies. Thus, identification of rational combinations of novel immune checkpoint (IC) strategies is imperative. This study aimed to comprehensively characterize the expression of IC targets to inform drug development and design effective combinatorial strategies, to advance the search for optimized therapies for NSCLC. Methods: We analyzed the differential gene expression of IC targets in 537 primary lung adenocarcinoma (LUAD) samples and 59 normal lung tissue samples pre-immunotherapy receipt using RNA-sequencing data from The Cancer Genome Atlas. Gene expression of IC-targets including PDCD1 (PD-1), CD274 (PD-L1), CTLA4, TIGIT, LAG3, HAVCR2 (TIM-3), VSIR (VISTA), ICOS, CD276 (B7-H3), BTLA, IDO1, KLRD1 (CD94/NKG2A), NT5E (CD73), TNFRSF4 (OX-40), TNFRSF18 (GITR), and TNFRSF9 (4-1BB) were obtained. Differential gene expression was determined intra-tumorally by comparison of transcripts per million and inter-tumorally using the DESeq2 algorithm. Log2 fold change >0.5 and false discovery rate adjusted P<0.05 were used as criteria for significant differential expression. Hierarchical clustering was used to identify subsets of tumors with similar profiles of IC-targetable gene expression. Results: Tumors showed significantly increased expression of TNFRSF18 (GITR), NT5E (CD73), TNFRSF9 (4-1BB), LAG3, TIGIT, CTLA4, PD-1, BTLA, B7-H3, and OX-40 (Table 1). On the other hand, tumors exhibited significant downregulation of CD274 (PD-L1), HAVCR2 (TIM-3), ICOS, KLRD1 (CD94/NKG2A), and VSIR (VISTA). Hierarchical clustering revealed recurrent patterns of IC gene overexpression which were characterized by: 1) IDO1-hi, 2) CD276-mid + TNFRSF18-mid, 3) VSIR-hi, 4) CD276-hi, 5) NT5E-hi, and 6) CD276-mid + NT5E-mid. Interestingly, patterns of IC gene expression distinguish tumors from controls. Of note, high VSIR expression was specific to normal tissue rather than tumors. Conclusions: Our study identified many IC targetable genes that were significantly overexpressed in tumors. However, subsets of tumors showed distinct and segregable combinations of overexpressed IC genes. These results suggest heterogeneous mechanisms of immune evasion in NSCLC, which may explain suboptimal outcomes with anti-PD-1/PD-L1 based strategies in this patient population. Our findings emphasize the importance of biomarker-guided approaches for the optimal selection of ICs and the development of rational combinatorial immunotherapies in NSCLC.

Pan-cancer associations of B7-H3 (CD276) transcriptional expression across human malignancies.

2624 Background: B7-H3 ( CD276) is a transmembrane glycoprotein of the B7 superfamily that includes PD-L1 and CTLA-4. B7-H3 targeted therapeutics have demonstrated promising results in both solid tumors and hematologic cancers. To understand the applicability of B7-H3 targeted therapies, we characterized the landscape of clinical outcomes, genomic, transcriptomic, and immunological features associated with B7-H3 mRNA expression in > 93,000 samples across > 45 cancer types from a large real-world clinico-genomics database. Methods: DNA (592-gene/whole exome) and RNA (whole transcriptome) sequencing was performed for samples submitted to Caris Life Sciences (Phoenix, AZ). Differential expression of individual genes was analyzed, with pathway enrichment assessed by Gene Set Enrichment Analyses (GSEA). RNA deconvolution was used to estimate immune cell infiltration of tumor microenvironments using quanTIseq (Finotello, 2019). Real-world overall survival (OS) was determined from insurance claims data and Kaplan-Meier estimates were calculated. Statistical significance was determined using X2 and Mann-Whitney U tests with Benjamini-Hochberg corrections for multiple hypothesis testing where appropriate. Results: B7-H3 mRNA displayed robust expression in many prevalent cancer types, including prostate, pancreatic, breast, colorectal, ovarian, and lung cancers (n = 3407, 4624, 8639, 12,862, 8004, and 13,502, respectively). High B7-H3 expression predicted worse OS in ovarian cancer (1.38 HR, 95% CI 1.23-1.53, p < 0.001) and lung adenocarcinoma (1.45 HR, 95% CI 1.34-1.57, p < 0.001), and better OS in prostate (0.84 HR, 95% CI 0.71-0.99, p < 0.05). Notably, high B7-H3 was detected in tumors with known driver mutations (e.g. EGFR, ESR1, KRAS, SPOP) or undruggable therapeutic targets ( TP53, APC). When examining Hallmark gene signatures, we found that B7-H3-high samples demonstrated consistent enrichment for pathways involved in epithelial to mesenchymal transition (EMT), WNT, TGF-Beta, and Notch signaling (FDR < 0.05) across cancer types. Additionally, high B7-H3 expression was correlated with greater proportions of pro-inflammatory M1 macrophages but lower proportions of effector CD8+ T cells and NK cells across multiple cancer types. Conclusions: We demonstrate B7-H3 to be a promising pan-tumor target, with most solid tumors showing robust expression which is often associated with worse OS. In several major cancers, high B7-H3 expression was associated with mutational and transcriptomic repertoires not easily targeted by current precision therapies. We find B7-H3-high tumors to have increased potential for myeloid-driven immune induction, making them potentially more amenable to immunomodulatory therapies, in particular, those targeting B7-H3. Further work is warranted to elucidate different mechanistic interactions of B7-H3 with specific oncogenic pathways in each cancer type.

ARTEMIS-001: Phase 1 study of HS-20093, a B7-H3–targeting antibody-drug conjugate, in patients with advanced solid tumor.

3017 Background: B7-H3, a member of the B7 superfamily, is highly expressed in various solid tumors, but has limited expression at low level in normal tissues. HS-20093 is a B7-H3-targeted antibody-drug conjugate. It could be an attractive therapeutic option for advanced solid tumors, especially the patients (pts) who have failed in multi-line therapy requiring novel and more effective treatment. Methods: This is a first-in-human, multicenter, open-label phase 1 study of HS-20093 in advanced solid tumors (NCT05276609). Dose escalation part assessed safety and tolerability of intravenous HS-20093 with doses ranging from 1.0 to 16.0 mg/kg. The dose escalation schedule utilized an accelerated titration and interval 3+3 design. Pts with advanced solid tumor were required to have received prior standard therapy. HS-20093 is administrated intravenously every 3 weeks. Results: The study completed all planned dose cohorts (1.0 to 16.0 mg/kg) as of cutoff date (20 December 2022). In dose escalation study, 53 pts of multiple tumor types were enrolled and received ≥1 dose of HS-20093, which included 29 pts with non-small cell lung cancer, 11 pts with small cell lung cancer (SCLC), 9 pts with sarcoma and 4 pts with other advanced solid tumors. At baseline, 25 pts (47.2%) had received ≥3 prior lines therapy with a mean of 3.2 (range, 1-12) prior lines of therapy. Three pts experienced dose-limiting toxicities (1 pt at 12.0 mg/kg; 2 pts at 16.0 mg/kg). The maximum tolerated dose was determined to be 12.0 mg/kg. Treatment-emerged adverse events (TEAEs) occurred in 53 pts (100%). The most common TEAEs overall in ≥30% of pts were leukopenia, neutropenia, anemia, pyrexia, nausea, thrombocytopenia, hypoalbuminemia, vomiting, lymphopenia, infusion-related reaction and fatigue. No interstitial lung disease was reported. Of 40 response-evaluable pts, regardless of baseline B7-H3 expression level, 14 partial responses (PRs) were observed in pts treated with HS-20093 from 4.0 mg/kg to 12.0 mg/kg doses (response rate: 35.0%), including 9 confirmed PRs and 5 PRs awaiting confirmation. The disease control rate was 85.0% (34/40, 95% CI: 70.2-94.3). The patient with the longest treatment duration (349 days) remains on treatment. In the subset of 9 SCLC pts, 7 PRs were observed (response rate: 77.8%) with the median depth of response of 50.5%, including 3 confirmed PRs and 4 PRs awaiting confirmation. All responses of SCLC pts occurred at the first disease assessment; the median time to first response was 6 weeks. HS-20093 displayed anti-tumor activity in SCLC pts who have progressed on prior derivative of camptothecin treatment. Conclusions: The safety profile of HS-20093 was acceptable. HS-20093 demonstrated promising antitumor activity in several tumor types, especially in SCLC. The further dose expansion study on efficacy and safety of HS-20093 in selected tumor types is ongoing. Clinical trial information: NCT05276609 .