B7-H3 Levels Research Articles

B7-H3 is widely expressed in soft tissue sarcomas

PurposeTargeted 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 methodsThis 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.ResultsSpecimens 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.ConclusionThese 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.

B7H3 Immune Checkpoint Overexpression Is Associated with Decreased Complete Response Rates to Neoadjuvant Therapy in Locally Advanced Rectal Cancer.

Rectal cancer accounts for approximately one-third of colorectal cancers, with over 340,000 deaths globally in 2022. Despite advancements in treatment, the five-year overall survival for locally advanced rectal cancer (LARC) remains at 74%, with significant morbidity. B7H3 (CD276), an immune checkpoint protein, plays a role in tumor progression and resistance to therapy, and correlates with poor prognosis in various cancers, including colorectal cancer. This study aims to evaluate the expression of B7H3 in LARC and its impact on overall complete response (oCR) rates to neoadjuvant therapy. A retrospective study was conducted on 60 patients with LARC who received neoadjuvant chemoradiation (nCRT) followed by total mesorectal excision (TME). B7H3 expression was assessed using immunohistochemistry on surgical specimens. Expression levels were categorized as high or low based on a composite score, and their association with oCR rates was analyzed. High B7H3 expression was observed in 60% of patients, with 73.5% showing expression in more than 50% of tumor cells. Patients who achieved oCR had significantly lower B7H3 expression compared to those with residual disease (p < 0.001). No nuclear expression of B7H3 was detected. No significant correlation was found between B7H3 expression and other clinicopathological variables, except for a higher likelihood of non-restorative surgery in patients with elevated B7H3 levels (p = 0.049). Mucinous adenocarcinoma had high expression of B7H3. Elevated B7H3 expression is associated with reduced oCR rates in LARC, highlighting its potential role as a prognostic biomarker. Further studies with larger cohorts are warranted to validate these findings and explore B7H3-targeted therapies as a treatment strategy for LARC.

DIPG-11. PRECLINICAL EFFICACY OF B7-H3 CAR T CELLS IN COMBINATION WITH ONC206

Abstract Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric tumor of the pons affecting over 300 children in the U.S. each year. Chimeric antigen receptor (CAR) T cells are a targeted immunotherapy with remarkable clinical success against hematological malignancies and now are being investigated against central nervous system (CNS) tumors. B7-H3 is a surface antigen specifically expressed on nearly all DIPG but not on normal brain providing an attractive therapeutic window. Therefore, Seattle Children’s opened BrainChild-03 (NCT04185038), a first-in-human phase 1 trial of repeatedly dosed intracranial B7-H3 CAR T cells for children with DIPG. We found the highest planned dose (10x107 cells) to be tolerable and observed preliminary signals of efficacy. ONC206 is a small molecule ClpP-agonist and DRD2-antagonist derivative of ONC201 that is currently being evaluated on PNOC-023 (NCT04732065). Here, we set out to evaluate the combinatorial potential of these modalities. ONC206 effectively reduced DIPG tumor cell viability (IC50 = ~300 nM compared to ONC201 ~1400 nM) against four treatment-naïve patient-deriving DIPG models representing a range of molecular subtypes. Using multiple orthogonal systems, apoptosis of DIPG cells was confirmed. We found that ONC206 drives tumor cell mitochondrial dysfunction and upregulation of DR5, a cell-death receptor involved in the TRAIL pathway, whereas levels of B7-H3, DRD2, and ClpP remain constant. We previously published the efficacy of B7-H3 CAR T cells against DIPG, but here - using low effector:tumor (E:T) ratios (e.g. 0.5:1) of activated B7-H3 CAR T cells and low-dose ONC206 (e.g. 200 nM) - we found a significant in vitro combinatorial benefit against DIPG (p&amp;lt;0.01). Supportive biologic studies suggest this could be an additive benefit and in vivo studies are currently ongoing. Based on the clinical translation of B7-H3 CAR T cells and ONC206, we aim to further validate this combinatorial benefit to help future patients.

PD-L1 and alternative immune checkpoints (AICs) in non-small cell lung cancer (NSCLC): Insights into the tumor immune microenvironment (TIME) and immunotherapy (IO) outcomes.

8531 Background: PD-L1 and recently identified AICs (B7x, B7-H3, and HHLA2) may function as key immune evasion mechanisms in NSCLC and influence IO efficacy. This study explores the expression patterns of PD-L1 and AICs within the TIME and their impact on IO outcomes in NSCLC, including key molecular subtypes. Methods: NSCLC samples (n=27,629) were analyzed (Caris Life Sciences) with NextGen Sequencing on DNA (592 genes or whole exome)/RNA (whole transcriptome). Tumor cell PD-L1 expression (22c3) was assessed by IHC. Gene expression profiles were analyzed for IFN-γ score and QuantiSEQ was used to assess immune cell infiltration in the TIME. Real-world overall survival (rwOS: from time of biopsy to last contact), time on treatment [TOT: from the first pembrolizumab (pembro) treatment to the last], and OS on pembro (OSpem: from pembro start to last contact) were calculated. Mann-Whitney U, X2/Fisher-Exact, and log-rank tests were applied where appropriate. Results: PD-L1 IHC+ NSCLC and cases with high PD-L1 RNA expression had higher IFN-γ score and immune cell fractions (p&lt;0.0001 for both) vs PD-L1 IHC- and low expression, respectively. Additionally, PD-L1 IHC+ NSCLC was associated with (a/w) higher median transcript levels (TPM) of PD-L1 and B7-H3, while PD-L1 IHC- NSCLC was a/w higher TPM of HHLA2 and B7x (p&lt;0.0001 for all). AICs expression varied by molecular subtype, e.g., higher HHLA2/B7x RNA in EGFR mutant vs wild-type (WT) NSCLC. Moreover, PD-L1 IHC+ NSCLC and cases with high PD-L1 RNA and low B7H3 RNA were a/w longer rwOS, OSpem, and TOT; high HHLA2 RNA was a/w longer rwOS and OSpem with similar TOT; and low B7x RNA expression was a/w longer OSpem and TOT with similar rwOS (Table). Conclusions: Both PD-L1 IHC+ NSCLC and cases with high PD-L1 RNA expression are a/w a more inflammatory TIME, and superior survival and IO outcomes. Moreover, consistent with our prior HHLA2 IHC work (PMIDs: 27553831, 29374053), HHLA2 RNA expression was significantly higher in PD-L1 IHC- and EGFR mutant cases. These findings support the potential utility of transcriptomics along with protein expression for assessing novel predictive biomarkers for IO. Furthermore, the distinct expression profiles of AICs identified in our study may represent key subtypes mediating IO response in WT and EGFR mutant NSCLC. [Table: see text]

Abstract 2453: Soluble B7H3 has a potential of novel serum biomaker for colorectal cancer

Abstract B7H3 (also known as CD276, an immune checkpoint molecule) is aberrantly over expressed in many types of cancer. Recently, several reports indicate a crucial role for B7H3 in promoting carcinogenesis and metastasis. We investigated whether concentration of the soluble B7H3 could be used as a new prognostic predictor of colorectal cancer by measuring serum level of B7H3. Recently we established a new mAb to B7H3 and developed sandwich ELISA-system detecting human serum B7H3 with high sensitivity and specificity. Measuring the levels of B7H3 in the serum specimens of colorectal cancer donors, we examined the usefulness of B7H3 as a novel biomarker for colorectal cancers. We tested the blood from the 82 donors of colorectal cancers and 9 healthy donors who agreed in writing to this research, just before the operation. The mean value + 2SD in 9 healthy donors was set as the cutoff value. We classified cancer patients into 2 groups with high value of B7H3 and low value of B7H3. We also analyzed the relationships between serum B7H3 levels and disease-free survival (DFS) and overall survival (OS). Serum B7H3 levels were also compared with diagnostic biomarkers such as CEA and CA19-9. B7H3 immunostaining was also performed on tissue samples. The serum levels of B7H3 in patients with colorectal cancer were significantly higher than those of healthy donors. However, there were no correlation between B7H3 and other diagnostic markers CEA and CA19-9. Regarding DFS, the higher value group of serum B7H3 showed a tendency for DFS to decrease in Stage II and III. Regarding OS, there was no significant difference of serum levels of B7H3 in Stage I and IV cases, however, in Stage II and III, OS decreased significantly in the high B7H3 group. In B7H3 immunostaining, the tissue was not sufficiently stained. These data indicate that serum B7-H3 should be useful for novel biomarker for a rationale of disease monitoring and prognosis of colorectal cancer. Citation Format: Akihiro Koizumi, Tomoaki Ito, Kazunori Kato, Takumi Iwasawa, Tomoyuki Kushida, Koichi Sato, Chikako Okamoto. Soluble B7H3 has a potential of novel serum biomaker for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2453.

Abstract 6391: Spatial proteomic profiling unveils pre-existing anti-tumor immunity as a hallmark of exceptional benefit from immunotherapy in NSCLC

Abstract Background: Immunotherapy is firmly established as a treatment regimen in various solid tumors due to its exceptional benefits observed in a select group of patients. Despite widespread use of immune checkpoint blockade (ICB) across diverse solid tumors, including non-small cell lung cancer (NSCLC), the quest for a clinically informative biomarker for long-term benefit remains unmet. Here we investigate the potential utility of clinicopathological and spatial proteomic profiling of tumor specimens from NSCLC patients to identify predictive biomarkers of long-term benefit to ICB. Methods: Forty-nine patients diagnosed with advanced NSCLC who received ICB at Hospital del Mar, Barcelona between 2017-2021 were included. Long-term responders (LTR, n=21) were defined as patients achieving a maintained radiologic response for more than 2 years, and short-term responders (STR, n=28) as patients presenting disease progression within the first six months of ICB initiation. Clinicopathological information, incidence of immune related adverse (irAES) and PD-L1 tumor proportion score assessed by IHC was available for all of them. DSP GeoMx was performed in subset of patients (LTR n=14 and STR n=14), to assess 49 immune biomarkers. Pancytokeratin (panCK; epithelial), CD3, CD20 and SYTO 13 (nuclear) were utilized as morphology biomarkers. Regions of interest were placed in tissue areas containing tumor and segmented in Tumor (PanCK+) and tumor microenvironment (TME) (PanCK-) that was used on downstream analysis. Digital counts were normalized using the protein expression of housekeeper proteins. Statistical analysis was performed using linear mixed models. A P value less than 0.05 was considered significant. Results: Our analysis revealed specific characteristics of LTR patients compared with STR, namely LTR population was enriched for PD-L1 positive in tumors (p=0.005) and a higher incidence of immune-related adverse events (irAEs) (p=0.001). Within the Tumor compartment, LTR patients displayed significantly higher levels of IDO1, CD8, PD-L1, CD45, HLA-DR and STING, while STR patients exhibit higher levels of B7-H3, CD56 and OX40L (p&amp;lt;0.05 for all proteins). Comparison analysis of the TME compartment between LTR and STR revealed augmented levels of IDO1, CD8, CD45, CD11c, CD27 and CD3 in LTR patients, and lower levels of B7H3 and OX40L compared with STR patients (p&amp;lt;0.05 for all proteins). Conclusions: Our comprehensive analysis of metastatic NSCLC patients treated with ICB has unveiled distinct clinicopathological and immunological features associated with long-term benefit of ICB, highlighting the presence of pre-existing antitumor immunity as a stronger predictor of long-term benefit. These findings offer insights into potential biomarkers and therapeutic strategies for enhancing ICB outcomes in metastatic NSCLC. Citation Format: Sharia Hernandez, Rafael Bach, Mario Giner, Wei Lu, Larisa Kostousov, Sean Barnes, Khaja Khan, Laura Masfarré, Xavier Villanueva, Ignacio Sanchéz, Nil Navarro, Álvaro Taus, Miguel Galindo, Max Hardy, Raúl Del Rey-Vergara, Albert Iñañez, Beatriz Sanchez-Espiridion, Laura Moliner, Sergi Clavé, Beatriz Bellosillo, Ana Rovira, Júlia Perera-Bel, Ignacio Wistuba, Edurne Arriola, Luisa M. Solis, Pedro Rocha. Spatial proteomic profiling unveils pre-existing anti-tumor immunity as a hallmark of exceptional benefit from immunotherapy in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6391.

The Impact of Laparoscopic Appendectomy and Open Appendectomy on B7-H3-Mediated Intrinsic Immune Response in Children with Acute Suppurative Appendicitis.

Surgery impairs immune function and increases postoperative complications. B7H3, a co-stimulatory molecule, plays a crucial role in immune regulation. The present study examined the impact of B7H3 on the postoperative immune response in children with acute suppurative appendicitis (ASA) by comparing preoperative and postoperative B7H3 levels in laparoscopic surgery (LA) and open appendectomy (OA). 198 pediatric ASA patients were enrolled. The researcher group performed LA, while the control group performed OA. Perioperative time, recovery time of gastrointestinal function, time to pass gas, length of incision, and length of hospitalization were compared in the perioperative period. Additionally, an ELISA assay was conducted to examine the levels of inflammatory factors and B7H3 and CD28. Short-term postoperative complications were also evaluated. Compared with the control group, the research group had a short operative time, gastrointestinal function recovery time, gas time, and hospitalization time. The short-term complication rate was significantly lower in the research group. More importantly, B7H3 and CD28 were insignificantly different preoperatively, but they were all reduced postoperatively. Moreover, the reduction was more pronounced in the research group. The same results were noted in inflammatory factors and immune markers, which were non-significant different preoperatively and were typically reduced postoperatively, particularly in the research group. Finally, postoperative B7H3 was positively correlated with both inflammatory factors and immune cell levels. B7H3 was reduced in both postoperative periods, and the reduction was more pronounced in the LA group. B7H3 may be involved in postoperative recovery by modulating postoperative inflammation and immune responses.

Tumor-derived biomarkers predict efficacy of B7H3 antibody-drug conjugate treatment in metastatic prostate cancer models.

Antibody-drug conjugates (ADCs) are a promising targeted cancer therapy; however, patient selection based solely on target antigen expression without consideration for cytotoxic payload vulnerabilities has plateaued clinical benefits. Biomarkers to capture patients who might benefit from specific ADCs have not been systematically determined for any cancer. We present a comprehensive therapeutic and biomarker analysis of a B7H3-ADC with pyrrolobenzodiazepine(PBD) payload in 26 treatment-resistant, metastatic prostate cancer (mPC) models. B7H3 is a tumor-specific surface protein widely expressed in mPC, and PBD is a DNA cross-linking agent. B7H3 expression was necessary but not sufficient for B7H3-PBD-ADC responsiveness. RB1 deficiency and/or replication stress, characteristics of poor prognosis, and conferred sensitivity were associated with complete tumor regression in both neuroendocrine (NEPC) and androgen receptor positive (ARPC) prostate cancer models, even with low B7H3 levels. Non-ARPC models, which are currently lacking efficacious treatment, demonstrated the highest replication stress and were most sensitive to treatment. In RB1 WT ARPC tumors, SLFN11 expression or select DNA repair mutations in SLFN11 nonexpressors governed response. Importantly, WT TP53 predicted nonresponsiveness (7 of 8 models). Overall, biomarker-focused selection of models led to high efficacy of in vivo treatment. These data enable a paradigm shift to biomarker-driven trial designs for maximizing clinical benefit of ADC therapies.

B7-H3 and CD47 co-expression in gastric cancer is a predictor of poor prognosis and potential targets for future dual-targeting immunotherapy.

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. 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. 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. 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.

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 Associates with IMPDH2 and Regulates Cancer Cell Survival

Lung cancer is one of the most common cancers worldwide, and despite improvements in treatment regimens, patient prognosis remains poor. Lung adenocarcinomas develop from the lung epithelia and understanding how specific genetic and environmental factors lead to oncogenic transformation in these cells is of great importance to define the pathways that contribute to tumorigenesis. The recent rise in the use of immunotherapy to treat different cancers has prompted the exploration of immune modulators in tumour cells that may provide new targets to manipulate this process. Of these, the B7 family of cell surface receptors, which includes PD-1, is of particular interest due to its role in modulating immune cell responses within the tumour microenvironment. B7-H3 (CD276) is one family member that is upregulated in many cancer types and suggested to contribute to tumour–immune interactions. However, the function and ligand(s) for this receptor in normal lung epithelia and the mechanisms through which the overexpression of B7-H3 regulate cancer progression in the absence of immune cell interactions remain unclear. Here, we present evidence that B7-H3 is associated with one of the key rate-limiting metabolic enzymes IMPDH2, and the localisation of this complex is altered in human lung cancer cells that express high levels of B7-H3. Mechanistically, the IMPDH2:B7-H3 complex provides a protective role in cancer cells to escape oxidative stress triggered by chemotherapy, thus leading to cell survival. We further demonstrate that the loss of B7-H3 in cancer cells has no effect on growth or migration in 2D but promotes the expansion of 3D spheroids in an IMPDH2-dependent manner. These findings provide new insights into the B7-H3 function in the metabolic homeostasis of normal and transformed lung cancer cells, and whilst this molecule remains an interesting target for immunotherapy, these findings caution against the use of anti-B7-H3 therapies in certain clinical settings.

Effect of antiviral and immunomodulatory treatment on a cytokine profile in patients with COVID-19.

The severity of COVID-19 is associated with an elevated level of a variety of inflammatory mediators. Increasing evidence suggests that the Th17 response contributes to the severity of COVID-19 pneumonia, whereas Th22 response plays a regulatory role in SARS-CoV-2 infection. Two main types of available COVID-19 treatments are antivirals and immunomodulatory drugs; however, their effect on a cytokine profile is yet to be determined. This study aim to analyse a cytokine profile in peripheral blood from patients with COVID-19 (n=44) undergoing antiviral or/and immunomodulatory treatment and healthy controls (n=20). Circulating CD4+ and CD8+ T cells and their intracellular expression of IL-17A and IL-22 were assessed by flow cytometry. Initial results showed an overexpression of IL-17F, IL-17A, CCL5/RANTES, GM-CSF, IL-4, IL-10, CXCL-10/IP-10 and IL-6 in COVID-19 patients compared to healthy controls. Treatment with remdesivir resulted in a significant decline in concentrations of IL-6, IL-10, IFN-alpha and CXCL10/IP-10. Immunomodulatory treatment contributed to a significant downregulation of IL-10, IFN-alpha, CXCL10/IP-10 and B7-H3 as well as upregulation of IL-22 and IL-1 beta. A combination of an antiviral and immunomodulatory treatment resulted in a significant decrease in IL-17F, IL-10, IFN-alpha, CXCL10/IP-10 and B7-H3 levels as well as an increase in IL-17A and IL-1 beta. We found significantly higher percentage of both CD4+ and CD8+ T cells producing IL-17A and CD4+ T cells producing IL-22 in patients with COVID-19. Administration of antiviral or/and immunomodulatory treatment resulted in a significant downregulation of pro-inflammatory cytokine expression and an upregulation of T cell absolute counts in most cases, thus showing effectiveness of treatment in COVID-19. SARS-CoV-2 infection induced cytokine overexpression in hospitalized patients with COVID-19 as well as lymphopenia, particularly a decrease in CD4+ and CD8+ T cell counts. Moreover, despite the reduced counts of CD4+ and CD8+ T cells, both subsets showed overactivation and increased expression of IL-17A and IL-22, thus targeting Th17 response might alleviate inflammatory response in severe disease.

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.

Canine osteosarcoma as a solid tumor model for evaluation of dual valent B7H3-CXCR2 CAR T cell therapy

Abstract To develop more effective solid tumor CAR T cell therapies, we utilized the spontaneous canine metastatic osteosarcoma (OS) animal model. Our studies evaluated the relative activity of single B7H3 and dual-valent B7H3-CXCR2 CAR constructs. Studies were also designed to determine if modifying the tumor microenvironment (TME) with oral immunotherapy improved CAR activity, based on recent studies of losartan and propranolol in dogs with OS and glioma. We evaluated B7H3 expression on canine OS cell lines using flow cytometry and OS biopsies using IHC. High levels of B7-H3 were observed on OS cell lines and OS biopsies, whereas normal canine tissues were negative or low. To assess the utility of the dual CAR we determined that canine OS cell lines secreted high IL8 spontaneously. To assess the functionality of canine B7-H3 CAR T cells, we generated canine CAR T cells from whole blood from dogs with cancer. We next determined that canine dual CAR T cells exerted specific killing and cytokine activity against B7H3+ canine OS cell lines. To further establish functionality, we evaluated the two CAR constructs in a mouse xenograft model of canine OS. We found that the dual CAR construct was significantly more active than the single CAR construct in slowing tumor growth. We found that losartan and propranolol blocked migration of mouse inflammatory monocytes to xenografted canine OS tumors. Future steps will be to evaluate the effectiveness of dual CAR T cells with TME modification in dogs with metastatic OS, to investigate the translational relevance in human patients. Results from these studies in dogs will likely give more clinically relevant results for evaluating new CAR T cell therapies and bridging results between mouse models and human clinical trials. The V Foundation, The Shipley Family Foundation, and NIH T32 training grant

Abstract LB102: Hexyl-(cuban-1-yl-methyl)-biguanide (HCB) suppresses N-glycosylation of immune checkpoint proteins B7-H3 and B7-H4, reverses tumor hypoxia, decreases intratumoral regulatory T cells, and increases intratumoral CD8+ T cells in the ovarian dependent ER+HER2- SSM2ucd mammary cancer allograft model

Abstract Introduction: Immune checkpoint blockade (ICB) has clinical activity in triple negative breast cancer (TNBC) but is less effective in the ER+HER2- signature, where there is a cold immune microenvironment (IM) and regulatory T cells (Tregs) may suppress effector T cells. Agents that activate the IM by turning cold tumors hot may support ICB. The biguanides hexyl-benzyl-biguanide (HBB) and its bioisostere hexyl-(cuban-1-yl-methyl)-biguanide (HCB) are candidate agents to activate the IM because they potently inhibit biosynthesis of immunosuppressive epoxyeicosatrienoic acids (EETs) and EET-driven oxidative phosphorylation (OXPHOS), while blocking N-glycosylation of immune checkpoint (IC) proteins. We hypothesized that reversal of hypoxia by biguanides in the ovarian dependent ER+HER2- STAT1 KO SSM2ucd mouse mammary carcinoma (MC) model would suppress Tregs and promote effector T cells in the tumor IM. While the SSM2ucd model did not express immune checkpoint protein PD-L1 (B7-H1), it did express related IC proteins B7-H3 and B7-H4. We hypothesized that by inhibiting OXPHOS and reducing N-glycosylation of immune checkpoint proteins, HBB and HCB may promote efficacy of ICB. We chose the SSM2ucd model to test impact of HCB on the ER+ MC IM. Results: SSM2ucd cells exhibited longer tumor latency (60 days) than the basal 4T1 (10 days) and 67NR (20 days) mouse MC models. SSM2ucd tumor reimplantation shortened latency by more than half, to 20 days. Immunohistochemistry showed that B7-H3 and B7-H4 protein levels were 1.2 (P=0.001) and 1.3-fold (P=0.04) higher in reimplanted tumors vs. control. In SSM2ucd cells, HCB inhibited N-glycosylation of B7-H3 (P=0.01) by 35% and B7-H4 (P=0.02) by 45% and suppressed TGFβ induction of B7-H3 by 21% (P=0.02) and B7-H4 by 79% (P=0.001) at 24 hours, while 14,15-EET promoted N-glycosylation of B7-H3 (1.2-fold; P=0.03) and B7-H4 (1.3-fold; P=0.04) at 4 hours. Effects of HBB and HCB on anti-CD3 and anti-CD28 stimulated mouse splenocytes were assayed. The proliferative effects of HBB on CD4+ and CD8+ cells peaked at 12 uM (p&amp;lt;0.001) and for HCB at 6.25 uM (p&amp;lt;0.001). Tregs decreased with HCB at a threshold of 12 uM (p &amp;lt;0.001) and with HBB at 25 uM (P &amp;lt; 0.001) while 14,15-EET increased Tregs (1.2 fold; P=0.02) and suppressed the CD8+:Treg ratio (0.79; P=0.02). In the SSM2ucd mammary allograft model, although HCB 12mg/kg daily did not inhibit tumor growth, it reduced intratumoral hypoxia by 20% (P=0.01), increased CD8+ TIL by 4.4-fold (P=0.04), decreased Treg:CD4+ TIL ratio by 76% (P=0.02), and decreased Treg:CD8+ TIL ratio by 88% (P=0.01) relative to control. Conclusion: B7-H3 and B7-H4 expression inversely correlated with latency of ER+ MC and may represent targets for immune checkpoint antibodies and their drug conjugates. HCB, an inhibitor of OXPHOS and EET biosynthesis, reduced intratumoral hypoxia, increased CD8+ TIL and reduced the Treg:CD8+ ratio, potentially supporting ICB therapy of ER+ MC by turning cold tumors hot. Supported by CDMRP BCRP Grant BC180596, Award Number W81XWH-19-1-0099 Citation Format: Zhijun Guo, Jianxun Lei, Hrishi Venkatesh, David Owen, Adam Bass, Christine Cannon, Joshua McCarra, Brenda Koniar, Craig Flory, Beverly Norris, Robert J. Schumacher, Swaathi Jayaraman, John Hawse, Emmanuel S. Antonarakis, Emanuel F. Petricoin, Julia Wulfkuhle, Robert D. Cardiff, Elizabeth A. Ambrose, Gunda I. Georg, Kaylee L. Schwertfeger, Michael A. Farrar, Brad St. Croix, Matthew P. Goetz, David A. Potter. Hexyl-(cuban-1-yl-methyl)-biguanide (HCB) suppresses N-glycosylation of immune checkpoint proteins B7-H3 and B7-H4, reverses tumor hypoxia, decreases intratumoral regulatory T cells, and increases intratumoral CD8+ T cells in the ovarian dependent ER+HER2- SSM2ucd mammary cancer allograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB102.

Abstract 4626: Spatial profiling of the tumor microenvironment in head and neck cancer identifies immune checkpoints and proteins involved in cell death signaling biomarkers of immunotherapy response

Abstract Immune checkpoint inhibitors (ICIs) Pembrolizumab and Nivolumab have shown promising results in patients with recurrent or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC). Nonetheless, only up to 20% of patients may benefit from them. Next generation predictive biomarkers of response to ICI therapies are currently needed. This retrospective study profiled pre-treatment tissue samples collected from R/M HNSCC patients (n=30) treated with Pembrolizumab or Nivolumab. Targeted spatial proteomics profiling was performed using NanoString’s GeoMx® Digital Spatial Profiler (GeoMx). To develop an informed region-of-interest (ROI) selection strategy for the GeoMx, we analyzed H&amp;E images from serial sections and performed Oncotopix® Discovery software, which considered pathology inputs and machine learning to analyze H&amp;E imagery and 4-channel GeoMx visualization inputs. We profiled 80 proteins simultaneously using the GeoMx across modules in cell death, immune activation, immune cell typing, immuno-oncology drug target, immune profiling, PI3K/AKT signaling, and pan-tumor markers.. Next, we performed differential expression (DE) analysis of the proteins localized in the tumor and stromal compartments against response to therapy parameters of complete, partial, stable and progressive disease (according to RECIST criteria). Four-channel GeoMx immunofluorescence images provided per-ROI cell counts (total and phenotyped based on 3 markers), and these data were used to aid in area and cell count normalization within the tumor and stromal compartments. We found that patients responsive to immunotherapy had higher expression levels of PD-L1, Bcl-2, BCLX, and BIM in the tumor, whereas VISTA, FOXP3, and CD66b were downregulated. In the stromal compartment, it was found that responders had higher expression levels of B7-H3, CD40, and SMA, but lower expression of PARP, S100B, and NY-ESO-1 in comparison to non-responders. In terms of best response analysis, patients with PR (n=5) had higher levels of PD-L1, ER-alpha, and CD68 expression, but lower levels of VISTA, CD27, and CD95/Fas in tumor regions than patients with PD (n=8). In the stroma, PD-L1, CD68, and HLA-DR were upregulated, while VISTA, BIM, and BAD were downregulated in patients with PR versus those with PD. We found that informed ROI selection strategy aided in defining key features in the tumor microenvironment for comparative analysis across samples, and for data normalization methods. Moreover, we found that immune checkpoints and proteins involved in cell death signaling play important roles in the immune responsive tumor microenvironment of HNSCC based on response to immunotherapy. Citation Format: Habib Sadeghirad, Ning Liu, James Monkman, Chin Wee Tan, Caroline Cooper, Jeni Caldera, Sarah Church, Fabian Schneider, James Robert Mansfield, Ken O'Byrne, Melissa Davis, Brett Hughes, Arutha Kulasinghe. Spatial profiling of the tumor microenvironment in head and neck cancer identifies immune checkpoints and proteins involved in cell death signaling biomarkers of immunotherapy response. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4626.

Serum and bronchoalveolar lavage fluid levels of soluble B7H3 in patients with interstitial lung diseases

BackgroundThe B7 family member B7H3/CD276 was recently reported to be involved in the pathophysiology of idiopathic pulmonary fibrosis (IPF). However, the association of B7H3 with prognosis in patients with fibrosing interstitial lung diseases (ILDs), including IPF, remains unclear. This study was investigated to determine the potential of soluble B7H3 (sB7H3) as a biomarker to predict prognosis in patients with fibrosing ILDs. MethodsPatients with ILDs from various categories who underwent bronchoalveolar lavage (BAL) were included in the study. The relationship between sB7H3 levels in serum or BAL fluid (BALF) and clinical variables at the time of ILD diagnosis was studied retrospectively. All patients who met the fibrosing ILD criteria were followed for 5 years. ResultsWe found that coexisting malignancy affected the serum, but not the BALF, sB7H3 levels. There was no significant correlation between serum and BALF levels of sB7H3 in 49 ILD patients without malignancy (11 with sarcoidosis, 5 with drug-induced ILD, 22 with IPF, and 11 with ILD associated with systemic sclerosis). We also found that the BALF levels, but not serum levels, of sB7H3 at the time of ILD diagnosis had independent prognostic potential on 5−year survival in patients with fibrosing ILDs. Of note, patients with a higher level of BALF sB7H3 at diagnosis (≥0.100 ng/mL) showed significantly shorter survival than those with lower levels. ConclusionsThis study suggests that BALF sB7H3 could be a novel prognostic biomarker in a broad range of fibrosing ILD patients.

B7-H3 chimeric antigen receptor-modified T cell shows potential for targeted treatment of acute myeloid leukaemia

Background and aimsChimeric antigen receptor (CAR)-T cell therapy is a novel type of immunotherapy. However, the use of CAR-T cells to treat acute myeloid leukaemia (AML) has limitations. B7-H3 is expressed in several malignancies, including some types of AML cells. However, its expression in normal tissues is low. Therefore, B7-H3 is ideal for targeted AML therapy.Materials and methodsFirst, we constructed B7-H3 CAR that can target B7-H3, and then constructed B7-H3-CAR-T cells in vitro, which were co-incubated with six AML cell lines expressing different levels of B7-H3, respectively. The toxicity and cytokines were detected by flow cytometry. In vivo, AML model was established in B-NSG mice to study the toxicity of B7-H3-CAR T on AML cells.ResultsIn vitro functional tests showed that B7-H3-CAR-T cells were cytotoxic to B7-H3-positive AML tumor cells and had good scavenging effect on B7-H3-expressing AML cell lines, and the cytokine results were consistent. In vivo, B7-H3-CAR-T cells significantly inhibited tumor cell growth in a mouse model of AML, prolonging mouse survival compared with controls.ConclusionB7-H3-CAR-T cells may serve as a novel therapeutic method for the targeted treatment of AML.

Construction of a pyroptosis-related lncRNAs signature for predicting prognosis and immunotherapy response in glioma.

Recent studies have proved that pyroptosis-related long non-coding RNAs (PRlncRNAs) are closely linked to tumor progression, prognosis, and immunity. Here, we systematically evaluated the correlation of PRlncRNAs with glioma prognosis. This study included 3 glioma cohorts (The Cancer Genome Atlas, Chinese Glioma Genome Atlas, and Gravendeel). Through Pearson correlation analysis, PRlncRNAs were screened from these 3 cohorts. Univariate Cox regression analysis was then carried out to determine the prognostic PRlncRNAs. A pyroptosis-related lncRNAs signature (PRLS) was then built by least absolute shrinkage and selection operator and multivariate Cox analyses. We systematically evaluated the correlation of the PRLS with the prognosis, immune features, and tumor mutation burden in glioma. A total of 14 prognostic PRlncRNAs overlapped in all cohorts and were selected as candidate lncRNAs. Based on The Cancer Genome Atlas cohort, a PRLS containing 7 PRlncRNAs was built. In all cohorts, the PRLS was proved to be a good predictor of glioma prognosis, with a higher risk score related to a poorer prognosis. We observed obvious differences in the immune microenvironment, immune cell infiltration level, and immune checkpoint expression in low- and high-risk subgroups. Compared with low-risk cases, high-risk cases had lower Tumor Immune Dysfunction and Exclusion scores and greater tumor mutation burden, indicating that high-risk cases can be more sensitive to immunotherapy. A nomogram combining PRLS and clinical parameters was constructed, which showed more robust and accurate predictive power. In conclusion, the PRLS is a potentially useful indicator for predicting prognosis and response to immunotherapy in glioma. Our findings may provide a useful insight into clinically individualized treatment strategies for patients.

B7-H3: A Useful Emerging Diagnostic Marker for Colon Cancer.

Colon cancer is the second most common malignancy causing the majority of cancer-related deaths. B7-H3 concentrations have drawn major interest as possible diagnostic biomarkers of cancer. The aim of this study was to measure the preoperative serum B7-H3 levels and to determine those that are replaced in colon cancer. We measured preoperative serum B7-H3 concentrations of 90 patients aged 57-69 years diagnosed with colon cancer and 50 age-matched healthy individuals. B7-H3 levels were determined using the sandwich enzyme-linked immunosorbent assay (ELISA). Patients were categorized by stage based on the TNM staging system, and the serum levels of B7-H3 were compared between patients with different TNM stages. No statistically significant difference was found between the patient and control groups in terms of age and gender. Preoperative serum B7-H3 levels were statistically significantly higher in patients with colon cancer than in the healthy group (p < 0.001). Preoperative serum B7-H3 concentration of the stage IV patients was significantly higher than those of the patients with stage I and stage II disease. In addition, higher serum B7-H3 levels were associated with lymph node status and distant metastasis in colon cancer. We showed that B7-H3 is highly expressed in colon cancer and can be used as a candidate diagnostic biomarker and a potential target in colon cancer in future.