Expression Levels Of B7-H4 Research Articles

B7-H3 and ICAM-1 are potentially therapeutic targets for thyroid carcinoma

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

ARTEMIS-002: Phase 2 study of HS-20093 in patients with relapsed or refractory osteosarcoma.

11507 Background: Patients (pts) with relapsed and refractory (R/R) osteosarcoma have a poor prognosis with limited therapeutic options. HS-20093 is a novel antibody-drug conjugate (ADC) targeting B7-H3, which showed preliminary anti-tumor activity in phase 1 ARTEMIS-001 study (NCT05276609). Here we report results in R/R osteosarcoma pts treated with HS-20093 from ARTEMIS-002 study (NCT05830123). Methods: ARTEMIS-002 trial is an open label, two-arm phase 2 trial in pts with R/R osteosarcoma or other sarcomas progressed upon standard systemic treatment. Based on the results of the phase 1 trial that the maximum tolerated dose was 12 mg/kg once every 3 weeks (Q3W) intravenous infusion, the osteosarcoma pts in the phase 2 trial were randomized to receiving HS-20093 either at 8 mg/kg or 12 mg/kg Q3W at a ratio of 1:1. The primary endpoint was objective response rate (ORR) according to RECIST1.1. B7-H3 expression was retrospectively evaluated by IHC in osteosarcoma FFPE tissue. Results: A total of 34 pts with R/R osteosarcoma were enrolled from June to December in 2023, receiving HS-20093 at the dose of either 8.0 mg/kg (N = 15) or 12.0 mg/kg (N = 19). Median age was 21.5 years (range: 18~65). At baseline, most pts were evaluated with clinical stage IV disease (32/34, 94.1%) and pulmonary metastasis (28/34, 82.4%). Twenty-two pts (64.7%) had received ≥3 prior lines therapy. Twenty-six pts (76.5%) had received 4 types of standard chemotherapies consisting of platinum, anthracyclines, ifosfamide and methotrexate. Treatment emergent adverse event (TEAEs) occurred in 33 pts (97.1%). The common grade 3/4 TEAEs (≥5%) were: neutropenia, leukopenia, thrombocytopenia, lymphopenia and anemia. The incidences of discontinuations, dose withhold and dose reductions were 2.9%, 11.8% and 23.5%, respectively. There was no TEAE leading to death. As of cut-off date (25 December, 2023), the median follow-up time was 4.1 months (95% CI: 1.4~5.5) among 21 response-evaluable pts (11 treated with 8 mg/kg and 10 with 12.0 mg/kg). The ORR of 12.0 mg/kg HS-20093 was 20.0%. Two confirmed partial responses were observed in pts with 12.0 mg/kg and remained on response until last follow-up, of which the longest duration of response was 4.0 months. The disease control rate was 81.8% (9/11) and 100% (10/10) in pts with 8 mg/kg and 12.0 mg/kg. The median progression-free survival of all 21 pts was not mature. B7-H3 is highly expressed in osteosarcoma with median H-score 185 (0~260). No correlation was observed between tumor response and B7-H3 expression level. The PK exposure of HS-20093 ADC, total Ab and payload increased with dose, approximately proportional to dose, with a half-life of 4 to 6 days, and no or minor accumulation after multiple doses of Q3W. Conclusions: The data has demonstrated that HS-20093 exhibited promising antitumor activity with acceptable toxicity in pts of heavily-pretreated R/R osteosarcoma. The enrollment of ARTEMIS-002 is continuing. Clinical trial information: NCT05830123 .

IMMU-25. ANALYSIS OF IMMUNOSUPPRESSIVE MYELOID CELLS IN THE GLIOBLASTOMA TUMOR MICROENVIRONMENT REVEALS THERAPEUTICALLY TARGETABLE IMMUNOMODULATORY SIGNALS ACROSS POPULATIONS

Abstract Immunosuppression in glioblastoma (GBM) remains a critical and poorly understood barrier to the development of effective novel therapies. Increasing evidence points to the critical role of immunosuppressive myeloid cells in the immunosuppressive tumor microenvironment and an intriguing target for ameliorating tumor-mediated immune suppression. Efforts to characterize the myeloid milieu in the tumor microenvironment have revealed a highly heterogenous population of cells with the capacity to exert immunosuppressive effects. In order to maximize the efficacy of potential therapeutic targets, we sought to identify immune checkpoint markers expressed across a range of immunomodulatory myeloid cell populations in the tumor microenvironment of patients undergoing resection for GBM. Freshly collected ultrasonic aspirate obtained during the primary surgery for GBM resection (based on preoperative imaging and intraoperative frozen section, later confirmed on final pathology) was prepared for high dimensional flow cytometry to quantify the immune checkpoint marker expression profile of a number of described immunosuppressive myeloid cell populations, including both monocytic and granulocytic myeloid-derived suppressor cells (mMDSC and gMDSC), and CD16+ non-classical monocytes (NCM). mMDSCs expressed particularly high levels of TIGIT (53.9±19.27%), which was also highly expressed in the NCM population (25.03±5.44%). NCMs also demonstrated high levels of B7H3 expression (39.85±18.05%), which has recently been implicated as an important immune checkpoint protein in GBM. gMDSCs demonstrated a rather different expression profile, with significantly higher levels of VISTA expression than other immunomodulatory markers (p< 0.05). These findings underscore the diversity of immunomodulatory signaling in the tumor-associated myeloid cell compartment, emphasizing the challenges in developing single-target immune checkpoint inhibitor therapy in GBM. Nevertheless, this data points to some common patterns of expression across major immunomodulatory cell populations that remain understudied in GBM and could herald more promising avenues of immunotherapy development.

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.

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.

Preconditioning of radiotherapy enhances efficacy of B7-H3-CAR-T in treating solid tumor models

AimsLimited efficacy of chimeric antigen receptor T (CAR-T) cells in treating solid tumors is largely due to the antigen heterogeneity and immunosuppressive tumor microenvironment (TME). B7-H3 is over-expressed in most kind of solid tumors, making it a promising target for cancer treatment. This study aims to explore the effect of B7-H3-CAR-T therapy combined with radiotherapy in treating solid tumor models. MethodsIrradiated tumor cell lines were prepared and tested. A humanized B7-H3-CAR-T was constructed, and it was evaluated that B7-H3-CAR-T cytotoxicity against solid tumor models with preconditioning of radiotherapy in vitro and vivo. ResultsIrradiation was found to increase expression level of B7-H3 in pancreatic cancer (PANC-1), colorectal cancer (HCT-15, SW620), acute myelocytic leukemia (AML-5), epidermoid carcinoma (KB) and glioma (U87-MG) human cell lines significantly. 6Gy irradiation was also found to up-regulate tumor-infiltration molecule like intracellular adhesion molecule-1 ICAM-1 or FAS in HCT-15 cells, supporting a possible synergistic enhancement effect of radiotherapy. In vitro and in vivo experiments demonstrated that irradiation indeed significantly enhanced the ability of B7-H3-CAR-T to infiltrate and kill tumors. Interestingly in dual-tumor mouse model study, not only tumor cells on irradiation side were eradicated completely, irradiation also enhanced CAR-T tumor-killing ability on non-irradiated side, confirming the abscopal effect of irradiation existed with CAR-T therapy. ConclusionsOur results suggest that B7-H3-CAR-T therapy combined with radiotherapy may be a promising modality in treating solid tumors.

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]

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 .

Spatial Immunoprofiling of Adenoid Cystic Carcinoma Reveals B7-H4 Is a Therapeutic Target for Aggressive Tumors.

Adenoid cystic carcinoma (ACC) is a heterogeneous malignancy, and no effective systemic therapy exists for metastatic disease. We previously described two prognostic ACC molecular subtypes with distinct therapeutic vulnerabilities, ACC-I and ACC-II. In this study, we explored the ACC tumor microenvironment (TME) using RNA-sequencing and spatial biology to identify potential therapeutic targets. Tumor samples from 62 ACC patients with available RNA-sequencing data that had been collected as part of previous studies were stained with a panel of 28 validated metal-tagged antibodies. Imaging mass cytometry (IMC) was performed using the Fluidigm Helios CyTOF instrument and analyzed with Visiopharm software. The B7-H4 antibody-drug conjugate AZD8205 was tested in ACC patient-derived xenografts (PDX). RNA deconvolution revealed that most ACCs are immunologically "cold," with approximately 30% being "hot." ACC-I tumors with a poor prognosis harbored a higher density of immune cells; however, spatial analysis by IMC revealed that ACC-I immune cells were significantly restricted to the stroma, characterizing an immune-excluded TME. ACC-I tumors overexpressed the immune checkpoint B7-H4, and the degree of immune exclusion was directly correlated with B7-H4 expression levels, an independent predictor of poor survival. Two ACC-I/B7-H4-high PDXs obtained 90% complete responses to a single dose of AZD8205, but none were observed with isotype-conjugated payload or in an ACC-II/B7-H4 low PDX. Spatial analysis revealed that ACC subtypes have distinct TMEs, with enrichment of ACC-I immune cells that are restricted to the stroma. B7-H4 is highly expressed in poor-prognosis ACC-I subtype and is a potential therapeutic target.

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&E images from serial sections and performed Oncotopix® Discovery software, which considered pathology inputs and machine learning to analyze H&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.

Abstract 4891: Ifinatamab deruxtecan (I-DXd), a novel B7-H3-targeting antibody-drug conjugate, demonstrates efficient payload delivery into tumor through target-dependent internalization

Abstract Ifinatamab deruxtecan (I-DXd; DS-7300) is a novel ADC which consists of an anti-B7-H3 antibody linked with a DNA topoisomerase I inhibiting anti-tumor agent, DXd. I-DXd has demonstrated potent anti-tumor activity in B7-H3-positive preclinical cancer models and showed early signs of promising clinical efficacy in patients with heavily pretreated, advanced solid tumors. ADCs are postulated to exert cytotoxicity specifically in tumors that express the target antigen via target-specific uptake of the compound. Accordingly, effective action of an ADC depends on multiple dynamic steps including antigen-specific binding, internalization, trafficking to lysosomes, linker cleavage and payload release, as well as payload potency. The goal of this research was to characterize I-DXd molecular dynamics in preclinical tumor cell models by investigating the relationship among target expression and dynamics, its translocation to lysosomes, lysosomal enzyme-mediated linker cleavage, and payload release after I-DXd treatment. As a result, I-DXd demonstrated target dependent internalization, efficient trafficking to lysosomes, and subsequent payload release in the studied tumor cell lines. Payload release levels generally correlated with target expression in vitro using a panel of cell lines with varying levels of B7-H3 expression. Also, in cell-line derived xenograft models, tumor tissue payload concentration corresponded to I-DXd binding at the cell membrane. These results suggest the importance of cell surface B7-H3 expression and ADC dynamics that lead to payload release in target expressing tumors to exert anti-tumor activity in non-clinical model. In addition to target-dependent uptake in tumor cells, it was observed in viable human lung tumor slices that B7-H3 was also expressed in several non-tumor cells including cancer-associated fibroblasts, endothelial cells, and macrophages in the tumor microenvironment and I-DXd was shown to be internalized in these B7-H3-expressing non-tumor cells. In summary, this pre-clinical study characterizes the relationship between B7-H3 expression in the tumor microenvironment and I-DXd molecular dynamics. Our results support developing I-DXd as a targeted therapy against advanced solid tumors known to overexpress B7-H3. Citation Format: Len Katsumata, Tsuneo Deguchi, Jun Hasegawa, Michiko Yamato, Yumi Nishiya, Akiko Watanabe, Tomoyo Honda, Megumi Minami, Naomi Kasanuki, Takanori Maejima, Sumie Muramatsu, Monika Herrmann, Nina Schulte, Gernot Polier, Xuya Wang, Takanori Yoshida, Nanae Izumi, Xiaozhong Qian, Toshinori Agatsuma, Kenji Nakamaru. Ifinatamab deruxtecan (I-DXd), a novel B7-H3-targeting antibody-drug conjugate, demonstrates efficient payload delivery into tumor through target-dependent internalization. [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 4891.

MALAT-1: Immunomodulatory lncRNA hampering the innate and the adaptive immune arms in triple negative breast cancer

BackgroundTriple negative breast cancer (TNBC) is known as hot immunogenic tumor. Yet, it is one of the most aggressive BC subtypes. TNBC evolve several tactics to evade the immune surveillance phenomena, one of which is shedding of natural killer (NK) cells activating immune ligands such as MICA/B and/or by inducing the expression of the immune checkpoints such as PD-L1 and B7-H4. MALAT-1 is an oncogenic lncRNA. MALAT-1 immunogenic profile is not well investigated. AimThe study aims at exploring the immunogenic role of MALAT-1 in TNBC patients and cell lines and to identify its molecular mechanism in altering both innate and adaptive immune cells present at the tumor microenvironment of TNBC MethodsBC patients (n = 35) were recruited. Primary NK cells and cytotoxic T lymphocytes were isolated from normal individuals using the negative selection method. MDA-MB-231 cells were cultured and transfected by several oligonucleotides by lipofection technique. Screening of ncRNAs was performed using q-RT-PCR. Immunological functional analysis experiments were performed upon co-culturing primary natural killer cells and cytotoxic T lymphocytes using LDH assay. Bioinformatics analysis was performed to identify potential microRNAs targeted by MALAT-1. ResultsMALAT-1 expression was significantly upregulated in BC patinets with a profound expression in TNBC patients compared to their normal counterparts. Correlation analysis revealed a positive correlation between MALAT-1, tumor size and lymph node metastasis. Knocking down of MALAT-1 in MDA-MB-231 cells resulted in a significant induction of MICA/B, repression of PD-L1 and B7-H4 expression levels. Enhancement of cytotoxic activity of co-cultured NK and CD8+ cells with MALAT-1 siRNAs transfected MDA-MB-231 cells. In silico analysis revealed that miR-34a and miR-17–5p are potential targets to MALAT-1; accordingly, they were found to be downregulated in BC patients. Forcing the expression of miR-34a in MDA-MB-231 cells resulted in a significant induction in MICA/B levels. Ectopic expression of miR-17–5p in MDA-MB-231 cells significantly repressed the expression of PD-L1 and B7-H4 checkpoints. Validations of MALAT-1/miR-34a" and "MALAT-1/miR-17–5p axes were performed by a series of co-transfections and functional assessment of cytotoxic profile of primary immune cells. ConclusionThis study proposes a novel epigenetic alteration exerted by TNBC cells mainly by inducing the expression of MALAT-1 lncRNA. MALAT-1 mediates innate and adaptive immune suppression events partially via targeting miR-34a/MICA/B and miR-175p/PD-L1/B7-H4 axes in TNBC patients and cell lines.

Abstract P2-20-06: Anti-B7-H3 Antibody (T-1A5) Blocks Immunomodulatory Function of B7-H3 and Enhances NK and T Cell–Mediated Cytotoxicity against Breast Cancer Cells

Abstract One of the main factors contributing to breast cancer (BC) initiation and metastasis is immune suppression by tumor cells. Immune checkpoint blockade has recently been shown to overcome tumor-induced immune suppression, but a significant proportion of patients do not respond, implying that more effective cancer immunotherapies are required. B7-H3 belongs to B7 family of immune checkpoint proteins that is overexpressed in various human malignancies. Here, we hypothesize that blocking B7-H3 using monoclonal antibodies (mAbs) enhances immune cell proliferation and function. To test our hypothesis, B7-H3 expression was determined using RNA-seq data from primary and metastatic breast tumors, and adjacent normal tissues, from the TCGA and METABRIC databases. To assess B7-H3 protein expression in BC, we performed immunohistochemistry (IHC) on frozen primary tumor tissues (n=50) and adjacent normal tissues (n=23) from TNBC patients. In addition, to investigate the immunomodulatory effect of B7-H3 in BC, we performed IHC for T-cell markers in subsets of patient tissues with high and low levels of B7-H3 expression. Next, we assessed B7-H3 expression in over 13 BC cell lines, including TNBC and ER+, PR+, and HER2+ cell lines, as well as TNBC PDX-derived cells. Moreover, to determine the effect of B7-H3 knockdown on NK- and T-cell activity, we co-cultured control and B7H3–KD BC cells in the presence and absence of NK and T cells and measured the induction of apoptosis in BC cells through IncuCyte live-cell imaging system. The effect of a novel B7H3-blocking mAb (clone T-1A5, isotype IgG1) on NK-cell and T-cell-mediated cytotoxicity in BC cell lines was examined using live-cell imaging. In the TCGA and METABRIC databases, B7-H3 was found to be significantly overexpressed (P< 0.0001) in tumor tissues than in adjacent normal tissues of BC patients. A survival analysis by the log-rank test indicated that patients with B7-H3high tumors had significantly lower progression-free (P=0.01) and relapse-free (P=0.0026) survival than patients with B7-H3low tumors. Moreover, B7-H3 is significantly upregulated in all the BC subtypes including basal, luminal A, luminal B and Her2-enriched with highest expression in basal type BC. Relative mRNA quantification and flow cytometry analysis demonstrated strong B7-H3 expression in most of the BC cell lines including TNBC and ER+, PR+, and HER2+ cell lines, as well as TNBC PDX-derived cells. Furthermore, IHC analysis revealed that compared to the matched normal tissue, B7-H3 expression was substantially higher in tumor tissue (N=16, P< 0.001). Also, patients with high B7-H3 expression had significantly lower numbers of CD3+, CD4+, and CD8+ T-cell, compared to patients with low B7-H3 expression (P< 0.001), indicating immunosuppressive role of B7-H3. Furthermore, NK cell and T cell mediated killing was significantly higher in B7H3-KD BC cell lines compared to control cells. We observed a significant increase in the killing of BC cells by NK cells and T cells in the presence of anti-B7H3 mAb T-1A5 in a concentration dependent manner (P < 0.001), suggesting that anti-B7H3 antibodies suppress the immunomodulatory function of B7-H3 and enhance NK and T cell–mediated ADCC in BC. To determine the antibody-dependent cell-mediated cytotoxicity, we developed a human-mouse chimera of T-1A5 (chT-1A5) and tested in combination with NK cells. Interestingly, we found a concentration and time dependent increase in ADCC in BC cells in the presence of chT-1A5 antibody. Our data suggests that B7-H3 is overexpressed in primary BC and inhibits immune-cell infiltration. Moreover, blocking the immunomodulatory functions of B7-H3 using anti-B7H3 antibody T-1A5 enhances NK and T cell–mediated killing of BC cells. Citation Format: Vivek Anand, Anudishi Tyagi, Venkata Lokesh Battula. Anti-B7-H3 Antibody (T-1A5) Blocks Immunomodulatory Function of B7-H3 and Enhances NK and T Cell–Mediated Cytotoxicity against Breast Cancer Cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-06.

Clinical Significance of the Expression of Co-Stimulatory Molecule B7-H3 in Papillary Thyroid Carcinoma.

Background: B7-H3, also known as CD276, an important immune checkpoint member of the B7-CD28 family, is confirmed as a promising target after PD-L1 in clinical trials. Although the overexpression of B7-H3 has been associated with invasive metastatic potential and poor prognosis in multiple types of cancer, nothing is known regarding the expression profiles of B7-H3 in papillary thyroid carcinoma (PTC). In this study, we carried out a large-scale analysis of B7-H3 expression in PTC patients and evaluated the potential clinical significance of B7-H3. Methods: In total, data from 1,210 samples, including 867 cases from TCGA and four GEO datasets, were collected for B7-H3–related transcriptome analyses, and 343 postoperative, whole-tumor sections were collected from patients with PTC at our institute for B7-H3–specific immunohistochemistry (IHC) staining. The statistical analysis was primarily accomplished using the R project for statistical computing. Results: B7-H3 positivity was found in 84.8% of PTC patients (291/343), and the mRNA and protein expression levels of B7-H3 in PTC were markedly higher than those of para-tumor tissues (p < 0.001), demonstrating that B7-H3 can serve as a potential diagnostic biomarker for PTC. The significant upregulation of B7-H3 in PTC is caused by distinct patterns of CNVs and CpG DNA methylation. Functional enrichment analysis confirmed that high B7-H3 expression was significantly associated with specific immune features and angiogenesis. High B7-H3 protein expression was associated with tumor size (p = 0.022), extrathyroidal extension (ETE) (p = 0.003), and lymph node metastasis (LNM) (p < 0.001). More importantly, multivariate analysis confirmed that B7-H3 was an independent predictor of relapse-free survival (RFS) (p < 0.05). In the subgroup analysis, positive B7-H3 staining was associated with worse RFS in patients with primary tumor size ≥2 cm (p < 0.05), age ≥55 years (p < 0.05), LNM (p = 0.07), multifocality (p < 0.05), and ETE (p < 0.05). In addition, Circos plots indicated that B7-H3 was significantly associated with other immune checkpoints in the B7-CD28 family. Conclusion: This is the first comprehensive study to elucidate the expression profile of B7-H3 in PTC. Our observations revealed that B7-H3 is a novel independent biomarker for predicting LNM and disease recurrence for PTC patients, and it thus may serve as an indicator that could be used to improve risk-adapted therapeutic strategies and a novel target for immunotherapy strategies for patients who undergo an aggressive disease course.

Overexpression of B7-H3 Is Associated With Poor Prognosis in Laryngeal Cancer.

The immune checkpoint molecule, B7-H3, which belongs to the B7 family, has been shown to be overexpressed in various cancers. Its role in tumors is not well defined, and many studies suggest that it is associated with poor clinical outcomes. The effect of B7-H3 on laryngeal cancer has not been reported. This study investigated the expression of B7-H3 in laryngeal squamous cell carcinoma (LSCC), and its relationship with clinicopathological factors and prognosis of LSCC patients. The gene expression quantification data and clinical data of LSCC retrieved from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database were analyzed to determine the diagnostic and prognostic roles of B7-H3. Quantitative real-time polymerase chain reaction (qRT-PCR) was then performed to determine the gene expression level of B7-H3 between LSCC tissues and paired normal adjacent tissues. In addition, TCGA RNA-seq data was analyzed to evaluate the expression level of B7 family genes. Next, the protein expression of B7-H3 and CD8 in LSCC was determined using immunohistochemistry and immunofluorescence. qRT-PCR results showed that the expression level of B7-H3 mRNA was significantly higher in LSCC tissues than in adjacent normal tissues. Similar results were obtained from the TCGA analysis. The expression of B7-H3 was significantly associated with T stage, lymph node metastasis, and pathological tumor node metastasis (TNM) stage, and it was also an independent factor influencing the overall survival time (OS) of patients with LSCC. In addition, B7-H3 was negatively correlated with CD8+T cells. These results show that B7-H3 is upregulated in LSCC. Therefore, B7-H3 may serve as a biomarker of poor prognosis and a promising therapeutic target in LSCC.

Intracerebral administration of CTLA-4 and PD-1 immune checkpoint blocking monoclonal antibodies in patients with recurrent glioblastoma: a phase I clinical trial

BackgroundPatients with recurrent glioblastoma (rGB) have a poor prognosis with a median overall survival (OS) of 30–39 weeks in prospective clinical trials. Intravenous administration of programmed cell death protein 1...

Cancer Cell B7-H3 Expression Is More Prevalent in the Pancreato-Biliary Subtype of Ampullary Cancer Than in Pancreatic Cancer

B7-H3 is an immunomodulatory member of the B7-superfamily with limited expression in normal tissues, but overexpression in several types of cancer. Therefore it is currently being explored as a potential target for cancer immunotherapy. The biological relevance of B7-H3 expression in pancreatic cancer is unclear, while there are no data on B7-H3 expression in ampullary cancer. We aimed to compare intra-tumoral B7-H3 expression between these two closely related cancer types and analyze its association with post-surgical disease course. B7-H3 expression levels were determined by immunohistochemistry in tissue microarrays of resected tumors of 137 pancreatic cancer patients and 83 patients with ampullary cancer of the pancreato-biliary subtype. B7-H3 was more frequently expressed in cancer cells of ampullary cancer patients compared to pancreatic cancer patients (51% versus 21%; p< 0.001). In ampullary cancer patients, but not in pancreatic cancer patients, B7-H3 cancer cell expression was associated with longer disease-free survival and patient survival. However, the prognostic value of B7-H3 was lost upon adjustment for CA19-9 levels. The frequencies of B7-H3 expression in tumor stroma did not differ between the two types of cancer (66% versus 63%). In both cancer types, stromal B7-H3 expression was not associated with post-surgical disease course. Compared to pancreatic cancer, B7-H3 is more frequently expressed in cancer cells of patients with the pancreato-biliary subtype of ampullary cancer. These data suggest that B7-H3 may represent an interesting potential target for immunotherapy in ampullary cancer rather than in pancreatic cancer.

Tumor B7-H3 expression in diagnostic biopsy specimens and survival in patients with metastatic prostate cancer.

Prostate cancer spans a broad spectrum from indolent to deadly disease. In the management of prostate cancer, diagnostic biopsy specimens are important sources of data that inform the selection of treatment. B7-H3 (CD276), an immune checkpoint molecule, has emerged as a promising immunotherapy target. B7-H3 expression is related to adverse clinical outcomes in various types of cancer; however, little is known concerning the association between tumor B7-H3 expression in diagnostic biopsy specimens and clinical outcome in patients with metastatic prostate cancer. We evaluated tumor B7-H3 expression levels in diagnostic biopsy specimens from 135 patients with metastatic prostate cancer and 113 patients with localized prostate cancer. High B7-H3 expression was more frequently observed in patients with metastatic cancer than in those with localized cancer (31 vs. 12%; p = 0.0003). In patients with localized cancer, the B7-H3 expression status was not associated with biochemical recurrence-free survival. However, among patients with metastatic cancer, high B7-H3 expression was independently associated with high disease-specific mortality (multivariable hazard ratio [HR] = 2.72; p = 0.047) and overall mortality rates (multivariable HR = 2.04; p = 0.025). Tumor B7-H3 expression in diagnostic biopsy specimens may be a useful biomarker for identifying highly aggressive metastatic prostate cancer. Given the potential utility of anti-B7-H3 immunotherapy, this information may aid in stratifying prostate cancer based on its responsiveness to B7-H3-targeted treatment.

The expression and clinical significance of B7-H3 and miR-145 in lung cancer patients with malignant pleural effusion.

To investigate the expression and clinical significance of costimulatory molecule B7-H3 and microRNA-145 (miR-145) in lung cancer patients with malignant pleural effusion. A total of 100 cases of patients with lung cancer who admitted to our hospital for treatment from March 2017 to September 2018 were selected. Forty-nine cases of patients diagnosed with malignant pleural effusion were included in the study group, and 51 cases with benign pleural effusion in the control group. The content of B7-H3 in pleural effusion of the two groups was detected by enzyme-linked immunosorbent assay (ELISA), and the expression of miR-145 in pleural effusion of the two groups was analyzed by Real-time quantitative PCR (qRT-PCR). The relationships between the expressions of B7-H3 and miR-145 and the clinicopathological characteristics were analyzed. The diagnostic value of B7-H3 and miR-145 in lung cancer was analyzed. The expression level of B7-H3 in the study group was significantly higher than that in the control group (p<0.050), while the expression level of miR-145 was significantly lower than that in the control group (p<0.050). The expression levels of B7-H3 and miR-145 in the study group were correlated with lymph node metastasis, differentiation degree of lung cancer and TNM stage (p<0.001). The sensitivity and specificity of miR-145 in single diagnosis of lung cancer were 64.71% and 79.59%, respectively. The sensitivity and specificity of B7-H3 in single diagnosis of lung cancer were 80.39% and 61.22%, respectively. B7-H3 and miR-145 are abnormally expressed in lung cancer, and are closely related to the lymphatic metastasis, differentiation degree and TNM stage of lung cancer. They may be potential markers for the diagnosis of malignant pleural effusion in lung cancer in the future.

B7-H3-Targeted CAR-T Cells Exhibit Potent Antitumor Effects on Hematologic and Solid Tumors

Recently, B7-H3 was frequently reported to be overexpressed in various cancer types and has been suggested to be a promising target for cancer immunotherapy. In the present study, we analyzed the mRNA expression of B7-H3 in The Cancer Genome Atlas (TCGA) database and validated its expression across multiple cancer types. We then generated a novel B7-H3-targeted chimeric antigen receptor (CAR) and tested its antitumor activity both in vitro and in vivo. The B7-H3 expression heterogeneity and variation were frequent. Moderate or even high expression levels of B7-H3 were also observed in some tumor-adjacent tissues, but the staining intensity was weaker than that in tumor tissues. B7-H3 expression was absent or very low in normal tissues and organs. Flow cytometry indicated that the mean expression level of B7-H3 in eight bone marrow specimens from patients with acute myeloid leukemia (AML) was 57.2% (range 38.8–80.4). Furthermore, we showed that the B7-H3-targeted CAR-T cells exhibited significant antitumor activity against AML and melanoma in vitro and in xenograft mouse models. In conclusion, although B7-H3 represents a promising pan-cancer target, and B7-H3-redirected CAR-T cells can effectively control tumor growth, the expression heterogeneity and variation have to be carefully considered in translating B7-H3-targeted CAR-T cell therapy into clinical practice.