B7-H3 Expression Research Articles

Comprehensive characterization of B7 family members in breast cancer: B7-H5 switch reverses breast cancer from “immuno-cold” into “immuno-hot” status

The members of the classic B7 family regulate the immune microenvironment of several malignant tumors. However, the potential relationship between the B7 family and the breast cancer (BrCa) tumor immune microenvironment has remained elusive. In the present study, we provide a comprehensive explanation of the expression, clinical significance, mutation, and immune cell infiltration of B7 family molecules in BrCa. First, we recruited 10 patients with BrCa surgery from the Wuxi Maternal and Child Health Hospital and performed single-cell RNA sequencing (scRNA-seq) analysis to investigate the distribution of B7 family members in multiple immune cell subsets. We focused on B7-2, B7-H3, and B7-H5 molecules of the B7 family and constructed tumor microarrays by self-recruiting patients to perform multiple immunohistochemical (mIHC) analyses and study tumor expression of B7-2, B7-H3, B7-H5 and CD8+ immune cell infiltration. B7-H5 displayed a strong correlation with CD8+ immune cell infiltration. In summary, B7-H5 provides a new perspective for the identification of immunothermal subtypes of BrCa and could function as a switch to reverse BrCa from an “immunologically cold” state to an “immunologically hot” state.Graphical abstract

The deubiquitinase USP2a promotes tumor immunosuppression by stabilizing immune checkpoint B7–H4 in lung adenocarcinoma harboring EGFR-activating mutants

B7–H4 is an immune checkpoint crucial for inhibiting CD8+ T-cell activity. A clinical trial is underway to investigate B7–H4 as a potential immunotherapeutic agent. However, the regulatory mechanism of B7–H4 degradation via the ubiquitin-proteasome pathway (UPP) remains poorly understood. In this study, we discovered that proteasome inhibitors effectively increased B7–H4 expression, while EGFR-activating mutants promoted B7–H4 expression through the UPP. We screened B7–H4 binding proteins by co-immunoprecipitation and mass spectrometry and found that USP2a acted as a deubiquitinase of B7–H4 by removing K48- and K63-linked ubiquitin chains from B7–H4, leading to a reduction in B7–H4 degradation. EGFR mutants enhanced B7–H4 stability by upregulating USP2a expression. We further investigated the role of USP2a in tumor growth in vivo. Depletion of USP2a in L858R/LLC cells inhibited tumor cell proliferation, consequently suppressing tumor growth in immune-deficient nude mice by destabilizing downstream molecules such as Cyclin D1. In an immune-competent C57BL/6 mouse tumor model, USP2a abrogation facilitated infiltration of CD95+CD8+ effector T cells and hindered infiltration of Tim-3+CD8+ and LAG-3+CD8+ exhausted T cells by destabilizing B7–H4. Clinical lung adenocarcinoma samples showed a significant correlation between B7–H4 abundance and USP2a expression, indicating the contribution of the EGFR/USP2a/B7–H4 axis to tumor immunosuppression. In summary, this study elucidates the dual effects of USP2a in tumor growth by stabilizing Cyclin D1, promoting tumor cell proliferation, and stabilizing B7–H4, contributing to tumor immunosuppression. Therefore, USP2a represents a potential target for tumor therapy.

Pancreatic Epithelial IL17/IL17RA Signaling Drives B7-H4 Expression to Promote Tumorigenesis.

IL17 is required for the initiation and progression of pancreatic cancer, particularly in the context of inflammation, as previously shown by genetic and pharmacological approaches. However, the cellular compartment and downstream molecular mediators of IL17-mediated pancreatic tumorigenesis have not been fully identified. This study examined the cellular compartment required by generating transgenic animals with IL17 receptor A (IL17RA), which was genetically deleted from either the pancreatic epithelial compartment or the hematopoietic compartment via generation of IL17RA-deficient (IL17-RA-/-) bone marrow chimeras, in the context of embryonically activated or inducible Kras. Deletion of IL17RA from the pancreatic epithelial compartment, but not from hematopoietic compartment, resulted in delayed initiation and progression of premalignant lesions and increased infiltration of CD8+ cytotoxic T cells to the tumor microenvironment. Absence of IL17RA in the pancreatic compartment affected transcriptional profiles of epithelial cells, modulating stemness, and immunological pathways. B7-H4, a known inhibitor of T-cell activation encoded by the gene Vtcn1, was the checkpoint molecule most upregulated via IL17 early during pancreatic tumorigenesis, and its genetic deletion delayed the development of pancreatic premalignant lesions and reduced immunosuppression. Thus, our data reveal that pancreatic epithelial IL17RA promotes pancreatic tumorigenesis by reprogramming the immune pancreatic landscape, which is partially orchestrated by regulation of B7-H4. Our findings provide the foundation of the mechanisms triggered by IL17 to mediate pancreatic tumorigenesis and reveal the avenues for early pancreatic cancer immune interception. See related Spotlight by Lee and Pasca di Magliano, p. 1130.

The Comprehensive Characterization of B7-H3 Expression in the Tumor Microenvironment of Lung Squamous Cell Carcinoma: A Retrospective Study.

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

IDeate-Lung02: A phase 3, randomized, open-label study of ifinatamab deruxtecan (I-DXd) vs treatment of physician’s choice (TPC) in relapsed small cell lung cancer (SCLC).

TPS8126 Background: SCLC is an aggressive malignancy, and patients (pts) with extensive stage (ES)-SCLC have a relatively short median overall survival (OS), with limited treatment options beyond first-line (1L) therapy. B7 homolog 3 (B7-H3 [CD276]) is a transmembrane protein expressed in many solid tumors but with low/no expression in normal tissue. In SCLC, B7-H3 is highly expressed and associated with poor prognosis and lack of response to standard therapy. I-DXd is a B7-H3-directed antibody–drug conjugate comprising an anti-B7-H3 mAb linked to a topoisomerase I inhibitor payload (DXd) via a stable cleavable linker, designed to enhance selective tumor cell death and reduce systemic exposure. I-DXd demonstrated durable efficacy (objective response rate [ORR] 52.4%; median duration of response [DoR] 5.9 mo; median OS 12.2 mo) irrespective of B7-H3 status, with a manageable safety profile in 21 ES-SCLC pts with a median of 2 prior lines of therapy in a Phase 1/2 trial (NCT04145622). I-DXd is being investigated in an ongoing Phase 2 trial in pretreated ES-SCLC, IDeate-Lung01 (NCT05280470). We describe a Phase 3 trial comparing the efficacy and safety of I-DXd vs TPC in relapsed SCLC. Methods: IDeate-Lung02 (NCT06203210) is a global, multicenter, randomized, open-label, Phase 3 trial in adult pts with relapsed SCLC who have received 1 prior line of platinum-based therapy and have an ECOG PS of ≤1; pts with asymptomatic brain metastases are eligible. Pts are randomized 1:1 to receive I-DXd 12 mg/kg iv every 3 wks (n = 234) or TPC (n = 234; topotecan, amrubicin, or lurbinectedin). Randomization is stratified by chemotherapy-free interval after 1L therapy (< 90 vs ≥90 days); TPC (topotecan vs amrubicin vs lurbinectedin); prior treatment with PD-(L)1 (yes vs no); and presence/history of asymptomatic brain metastases (yes vs no). Study treatment continues until disease progression, unacceptable toxicity, withdrawal of consent, death, loss to follow-up, or other reasons per protocol. Radiographic tumor assessments occur every 6 wks for 48 wks, then every 12 wks. Dual primary efficacy endpoints are ORR per RECIST v1.1 by blinded independent central review (BICR) and OS. Secondary efficacy endpoints are ORR per investigator; progression-free survival, DoR, disease control rate and time to response by BICR and investigator; and patient-reported outcomes. Safety endpoints include incidence of treatment-emergent adverse events (TEAEs), deaths, serious TEAEs, and TEAEs leading to dose modification or discontinuation. Immunogenicity of I-DXd and relationship between B7-H3 expression and clinical outcome are also secondary objectives. ORR will be analyzed using a Cochran-Mantel-Haenszel test at a 2-sided 1% alpha level, and OS will be analyzed using a log-rank test at a 2-sided 4% significance level, under a 2-look group sequential design. Clinical trial information: NCT06203210 .

ARTEMIS-001: Data from a phase 1a/b study of HS-20093 in patients with relapsed small cell lung cancer (SCLC).

8093 Background: SCLC is characterized by a high rate of proliferation and poor prognosis, and new therapies are urgently needed. B7-H3 is highly expressed in SCLC. HS-20093, a B7-H3-targeted antibody-drug conjugate (ADC), demonstrated antitumor activity in advanced solid tumors in the dose escalation part of ARTEMIS-001 study (Jie Wang et al., JCO, 2023) (NCT05276609). Here, we present results on the efficacy and safety of the expansion doses in patients (pts) with SCLC from phase 1a/b. Methods: ARTEMIS-001 study consisted of dose escalation (1a) and expansion (1b) part. Pts were treated with doses of 1.0 to 16.0 mg/kg HS-20093 every 3 weeks in dose escalation, and were treated with doses at 8.0 mg/kg and 10.0 mg/kg randomly in dose expansion. Pts with SCLC were required to have received prior platinum-based standard therapy. B7-H3 expression was retrospectively evaluated by IHC. Results: As of data cutoff November 30th 2023, a total of 56 pts with extensive stage SCLC (ES-SCLC) were enrolled and received ≥1 dose of HS-20093 with doses at 8.0 mg/kg (n=31) or 10.0 mg/kg (n=25). Median prior lines of therapy was 2.0 (range: 1-6). All pts received platinum plus etoposide and 73.2% (41/56) received immunotherapy. Safety profile was consistent with previous reports. The most common grade≥3 treatment-related adverse events (≧10%) were neutropenia, leukopenia, lymphopenia, thrombocytopenia and anemia. Out of 56 pts, 52 pts were efficacy evaluable (8.0 mg/kg: 31 pts; 10.0 mg/kg: 21 pts). HS-20093 showed encouraging efficacy in relapsed ES-SCLC (Table). Tumor shrinkage in target lesions occurred in 96.2% (50/52) pts with a post-baseline scan. Deep response defined as tumor shrinkage ≥50% was obtained in 44.2% (23/52) pts. Median overall survival has not yet reached. Responses were observed regardless of B7-H3 expression. Pharmacokinetic (PK) showed approximately dose-proportional increase in exposure with a half-life of 3-7 days. PK profiles of total antibody and ADC were similar and exposure to payload was considerably low. Conclusions: HS-20093 demonstrated promising antitumor activity and manageable safety in pts with previously-treated SCLC. Phase 3 study is planned to compare the efficacy and safety of HS-20093 with standard-of-care chemotherapy in relapsed SCLC. Clinical trial information: NCT05276609 . [Table: see text]

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 .

A phase 1/2, first-in-human, open-label, dose-escalation study of TAK-280, an investigational B7-H3 x CD3ε conditional bispecific redirected activation (COBRA) T-cell engager, in adult patients with unresectable, locally advanced, or metastatic solid tumors.

TPS2684 Background: TAK-280 is a novel COBRA T-cell engager that targets the B7 homolog 3 protein (B7-H3), an antigen highly expressed in a range of solid tumors including metastatic castration-resistant prostate cancer (mCRPC) and non–small cell lung cancer. In its prodrug form, TAK-280 binds to B7-H3, but not to CD3ε. Once in the protease-rich tumor microenvironment, TAK-280 undergoes protease-mediated activation through formation of CD3ε-binding active dimers, which stimulate CD3 T-cell activation and cytotoxic antitumor response against co-engaged B7-H3–expressing cells. In preclinical studies, TAK-280 demonstrated cleavage-dependent conditionality, engagement of tumor target antigen, and induction of T-cell mediated tumor killing. TAK-280 has the potential to target solid tumors that are not amenable to treatment with conventional first- or second-generation T-cell engagers, while limiting toxicity to normal tissues. Methods: This phase 1/2 study is enrolling adult patients with unresectable, locally advanced, or metastatic solid tumors described in the literature to have enhanced B7-H3 expression, who are ineligible or intolerant to standard therapies. Other eligibility criteria include Eastern Cooperative Oncology Group performance status ≤1 and measurable disease per RECIST v1.1. Key exclusion criteria include history of autoimmune disease, major surgery ≤8 weeks before first dose of TAK-280, and history of clinically significant cardiac or gastrointestinal disorders. During phase 1 dose escalation, TAK-280 is administered as an intravenous infusion, once weekly, in 28-day cycles at a pre-defined dose level range. Patients who do not experience dose-limiting or other unacceptable toxicities may receive up to 14 treatment cycles depending on response; patients are treated until disease progression, unacceptable toxicity, or withdrawal from study. The dose-escalation phase will determine two recommended doses for expansion to be assessed during the cohort-expansion phase. The primary endpoint is incidence of dose-limiting or other toxicities during dose escalation; secondary endpoints include pharmacokinetic parameters, response assessment per RECIST v1.1, overall response rate, duration of response, progression-free survival, overall survival, disease control rate, and prostate-specific antigen response in patients with mCRPC. Cytokine release syndrome is closely managed and reported per American Society for Transplantation and Cellular Therapy consensus grading. Approximately 182 patients are planned to be enrolled (42 in dose escalation and 100–142 in cohort-expansion); as of the data cutoff date of Jan 10, 2024, 18 patients have received the study drug. Clinical trial information: NCT05220098 .

Safety and efficacy of B7-H3 targeting CAR-T cell therapy for patients with recurrent GBM.

2062 Background: Glioblastoma Multiforme (GBM) is one of the most deadly cancers, and nearly all patients experience recurrence upon the current standard of care for newly diagnosed GBM. The recurrent GBM (rGBM) exhibits a median survival of less than 8 months with limited therapeutic options. We have previously cloned the B7-H3 gene, characterized its function, demonstrated altered B7-H3 expression in human solid tumors, and identified B7-H3 as a promising therapeutic target. We present herein the preliminary findings of an investigator-initiated trial (NCT05241392), where patients with rGBM were treated with autologous chimeric antigen receptor-T (CAR-T) cells targeting B7-H3 (TX103). Methods: This is an open, single-arm, "3+3" dose-escalation and multiple-dose study. The primary objective was to evaluate the safety and the maximal-tolerated dose. Secondary objectives included survival outcomes, tumor responses, immunological responses, and pharmacokinetic parameters. Patients aged 18 to 75 were enrolled with confirmed recurrence by image scan and B7-H3 expression >= 30% by immunohistochemistry analysis. TX103 was administered intracavity and/or intraventricularly to patients via an Ommaya reservoir. TX103 was given biweekly to patients each cycle at dose levels 1, 2, and 3 (20, 60, 150 million cells, respectively), with four cycles as one course. Results: Between March 2022 and January 2024, 13 patients received at least one intracranial infusion of TX103, with 3, 4, and 6 patients in dose levels 1, 2, and 3, respectively. The median number of infusion cycles was 4 (Min, Max 1,9). All patients were included in the safety analysis, revealing no dose-limiting toxicity or CAR-T treatment-related death. All patients experienced at least one adverse event (AE), and the treatment-related AEs (TRAEs) included cytokine release syndrome, increased intracranial pressure (ICP), headache, epilepsy, decreased level of consciousness, vomiting, and pyrexia. Most TRAEs were grade 1-2, while three grade-3 TRAEs were observed: one case of increased ICP in dose level 2, one case of epilepsy, and another case of decreased level of consciousness in dose level 3. As of January 2024, six patients from dose levels 1 and 2 were eligible for a 12-month survival assessment; the 12-month overall survival (OS) rate was 83.3% (95% CI: 58.3%~100%), and the median OS was 20.3 mo (95% CI: 20.3~not reach). Two of the three patients from dose level 2 achieved partial and complete responses, respectively. Following CAR-T administration, significant increases in cytokines, including IL-6 and IFN-γ, and elevated copy numbers of the CAR gene were observed in the cerebrospinal fluid, while only minimal elevations were noted in the peripheral blood. Conclusions: In this trial, treating rGBM with TX103 is deemed safe and tolerated. These interim results support further study using TX103 to enhance the survival outcomes in patients with rGBM.

STRIvE-02 Arm C: Phase 1 study of concurrent PD-1 inhibition with B7-H3 CAR T cell immunotherapy for recurrent/refractory pediatric solid tumors.

e14626 Background: B7-H3 expression by a broad range of pediatric solid tumors has led to development of B7-H3-directed chimeric antigen receptor (B7-H3-CAR) T cells. However, early phase clinical trials of B7-H3-CAR T cells in recurrent/refractory solid tumors demonstrated inconsistent CAR T cell expansion and limited anti-tumor activity. We subsequently developed a dual transduced CAR product expressing B7-H3-CAR and CD19-CAR (CARB7H3x19) to enhance CAR T cell expansion with antigenic stimulation by normal B cells (STRIvE-02 Arm B). Augmented expansion was observed but CAR T cell persistence was variable, leading to a hypothesis that concurrent use of pembrolizumab (pembro), a PD1-inhibitor, would help prolong persistence, leading to improved anti-tumor activity. Methods: Patients up to age 26 years with recurrent/refractory solid tumors (excluding primary CNS) enrolled onto Arm C of STRIvE-02 to examine safety and feasibility of CARB7H3x19 at a dose of 0.5 x 106 CD19-CAR T/kg followed by pembro 2 mg/kg (max 200 mg) either 7- (Dose regimen (DR) 1) or 21-days (DR 2) post-CAR T cell using a 3+3 statistical design. All subjects received lymphodepletion with fludarabine and cyclophosphamide prior to CAR T cells. CTCAEv5 was used for toxicity grading. Results: Of six patients (pts) enrolled (age range 11–25 yrs, median 20 yrs, four received CAR T cell+pembro; three received DR1 pembro (day (D)+7), and one received DR2 (D+21). A pt on DR2 with nonseminomatous germ cell tumor developed grade (gr) 5 immune effector cell-associated hyperinflammatory syndrome (IEC-HS) on D+17 prior to pembro, resulting in trial suspension. One pt did not receive CAR T cells and is unevaluable. Adverse effects occurring in pts who received pembro included gr 3 fever in setting of neutropenia (n=1), and gr 2 hyperthyroidism (n=1), with no dose limiting toxicity attributed to CAR T cell+pembro. Maximum circulating CAR T expansion for Arm C was 192.18 cells/uL (range 6.86 – 192.18 cells/uL) with median persistence of 28 days (range 28 - 84), compared to a maximum circulating CAR T expansion of 740.95 cells/uL (range 8.29 – 740.95 cells/uL) and median persistence of 65 days (range 21 - 274) in pts treated on Arm B receiving the same CAR dose level (n=8). D+28 disease restaging in the four Arm C pts who received CAR T cells+pembro demonstrated stable disease (n=1) and disease progression (n=3). Conclusions: Although we observed tolerable toxicity among patients who received CAR T cell+pembro, the small number of patients limit full toxicity assessment. T cell expansion and persistence was observed, but the effects of pembro on CAR T cell fitness are unclear. Phenotypic characterization of circulating CAR T cells is underway to further assess impact of pembro. Of note, this is the first reported death attributed to IEC-HS in a patient after receiving B7-H3-CAR T cells. Investigations into etiology are ongoing. Clinical trial information: NCT04483778 .

A phase Ia/Ib, non-randomized, open-label, dose escalation and expansion trial of the B7-H6/CD3 T-cell engager BI 765049 with or without ezabenlimab in Asian patients with advanced solid tumors.

TPS2669 Background: Bispecific T-cell engagers represent a promising therapeutic approach for patients with solid tumors; however, these agents require an appropriate target antigen. B7-H6 is an encouraging target as it is expressed on multiple solid tumors but shows only limited expression on normal tissue (1). BI 765049 is a novel immunoglobulin G (IgG)-like T-cell engager designed to simultaneously bind B7-H6-expressing tumor cells and CD3 on T-cells resulting in the formation of a cytolytic synapse that triggers apoptosis of the tumor cells. Methods: NCT06091930 is a Phase I, non-randomized, open-label, multi-center, dose escalation and expansion trial that aims to assess the safety and tolerability of BI 765049 alone or in combination with programmed cell death protein 1 inhibitor ezabenlimab, in patients with advanced or unresectable gastric cancer, colorectal cancer (CRC), pancreatic ductal adenocarcinoma, hepatocellular cancer, head and neck squamous cell carcinoma, or non-small cell lung cancer. Patients must have progressed on, or be ineligible for, standard therapies. Key inclusion criteria include confirmed B7-H6 expression (central pathology review) except in CRC; ≥1 evaluable target lesion outside of the central nervous system (Response Evaluation Criteria in Solid Tumors [RECIST] v1.1); and Eastern Cooperative Oncology Group performance status 0/1. Patients with prior B7-H6-targeted treatment are ineligible. The study is divided into four parts: dose escalation and expansion of BI 765049 monotherapy (Parts I and II, respectively), and in combination with ezabenlimab (Parts III and IV, respectively). BI 765049 dose escalation will be guided by a Bayesian logistic regression model with overdose control. Treatment will continue until progressive disease or discontinuation for other reasons, or for up to 36 months. The primary endpoint for Parts I and III is the occurrence of dose-limiting toxicities; and for Parts II and IV it is objective response (OR; determined by the investigator; RECIST v1.1). The secondary endpoints for Parts I and III are pharmacokinetic (PK) parameters and OR (RECIST v1.1); and for Parts II and IV secondary endpoints are progression-free survival, duration of response, disease control, PK measurements, and OR (immunotherapy RECIST [iRECIST]). 1. Zhang W, et al. Clin Cancer Res 2022;28(23):5190–5201. Clinical trial information: NCT06091930 .

Targeting both GD2 and B7-H3 using bispecific antibody improves tumor selectivity for GD2-positive tumors.

Disialoganglioside 2 (GD2), overexpressed by cancers such as melanoma and neuroblastoma, is a tumor antigen for targeted therapy. The delivery of conventional IgG antibody technologies targeting GD2 is limited clinically by its co-expression on nerves that contributes to toxicity presenting as severe neuropathic pain. To improve the tumor selectivity of current GD2-targeting approaches, a next-generation bispecific antibody targeting GD2 and B7-H3 (CD276) was generated. Differential expression of human B7-H3 (hB7-H3) was transduced into GD2+ B78 murine melanoma cells and confirmed by flow cytometry. We assessed the avidity and selectivity of our GD2-B7-H3 targeting bispecific antibodies (INV34-6, INV33-2, and INV36-6) towards GD2+/hB7-H3- B78 cells relative to GD2+/hB7-H3+ B78 cells using flow cytometry and competition binding assays, comparing results an anti-GD2 antibody (dinutuximab, DINU). The bispecific antibodies, DINU, and a non-targeted bispecific control (bsAb CTRL) were conjugated with deferoxamine for radiolabeling with Zr-89 (t1/2 = 78.4 h). Using positron emission tomography (PET) studies, we evaluated the in vivo avidity and selectivity of the GD2-B7-H3 targeting bispecific compared to bsAb CTRL and DINU using GD2+/hB7-H3+ and GD2+/hB7-H3- B78 tumor models. Flow cytometry and competition binding assays showed that INV34-6 bound with high avidity to GD2+/hB7-H3+ B78 cells with high avidity but not GD2+/hB7-H3+ B78 cells. In comparison, no selectivity between cell types was observed for DINU. PET in mice bearing the GD2+/hB7-H3- and GD2+/hB7-H3+ B78 murine tumor showed similar biodistribution in normal tissues for [89Zr]Zr-Df-INV34-6, [89Zr]Zr-Df-bsAb CTRL, and [89Zr]Zr-Df-DINU. Importantly, [89Zr]Zr-Df-INV34-6 tumor uptake was selective to GD2+/hB7-H3+ B78 over GD2+/hB7-H3- B78 tumors, and substantially higher to GD2+/hB7-H3+ B78 than the non-targeted [89Zr]Zr-Df-bsAb CTRL control. [89Zr]Zr-Df-DINU displayed similar uptake in both GD2+ tumor models, with uptake comparable to [89Zr]Zr-Df-INV34-6 in the GD2+/hB7-H3+ B78 model. The GD2-B7-H3 targeting bispecific antibodies successfully improved selectivity to cells expressing both antigens. This approach should address the severe toxicities associated with GD2-targeting therapies by reducing off-tumor GD2 binding in nerves. Continued improvements in bispecific antibody technologies will continue to transform the therapeutic biologics landscape.

Chimeric Antigen Receptor T Cell with an Inducible Caspase-9 Suicide Gene Eradicates Uveal Melanoma Liver Metastases via B7-H3 Targeting.

Uveal melanoma (UM) is the most common intraocular malignant tumor. Despite successful treatment of the primary tumor, about 50% of patients will recur with systemic diseases for which there are no effective treatment strategies. Here we investigated the preclinical efficacy of a chimeric antigen receptor (CAR) T-cell-based immunotherapy targeting B7-H3. B7-H3 expression on primary and metastatic human UM samples and cell lines was assessed by RNA sequencing, flow cytometry, and immunohistochemistry. Antitumor activity of CAR T cells targeting B7-H3 was tested in vitro with UM cell lines, patient-derived organotypic tumor spheroids from patients with metastatic UM, and in immunodeficient and humanized murine models. B7-H3 is expressed at high levels in >95% UM tumor cells in vitro and in vivo. We generated a B7-H3 CAR with an inducible caspase-9 (iCas9) suicide gene controlled by the chemical inducer of dimerization AP1903, which effectively kills UM cells in vitro and eradicates UM liver metastases in murine models. Delivery of iCas9.B7-H3 CAR T cells in experimental models of UM liver metastases demonstrates a durable antitumor response, even upon tumor rechallenge or in the presence of a significant metastatic disease burden. We demonstrate effective iCas9.B7-H3 CAR T-cell elimination in vitro and in vivo in response to AP1903. Our studies demonstrate more effective tumor suppression with iCas9.B7-H3 CAR T cells as compared to a B7-H3-targeted humanized monoclonal antibody. These studies support a phase I clinical trial with iCas9.B7-H3 CAR T cells to treat patients with metastatic UM.

Prognostic Value of B7H4 Expression in Patients with Solid Cancers: A Systematic Review and Meta-Analysis.

V-set domain-containing T-cell activation inhibitor 1 (aliases VTCN1, B7H4) participates in tumour immune escape by delivering inhibitory signals to T cells. The purpose of this article was to assess the B7H4 prognostic value in solid cancers. Three databases were searched for relevant articles. The main endpoints were overall survival (OS), disease-specific survival (DSS), progression-free survival (PFS), recurrence-free survival (RFS), and disease-free survival (DFS). Appropriate hazard ratios (HRs) were pooled. The R studio software (version 4.0.3) was used for data analysis. Thirty-one studies met the inclusion criteria. High expression of B7H4 was associated with worse OS (HR = 1.52, 95% CI: 1.37-1.68) but not with DSS (HR = 1.14, 95% CI: 0.49-2.63), RFS (HR = 1.77, 95% CI: 0.75-4.18), DFS (HR = 1.29, 95% CI: 0.8-2.09), or PFS (HR = 1.71, 95% CI: 0.91-3.2) in patients with solid cancers. High expression of B7H4 is associated with a poorer prognosis in patients with solid cancers. B7H4 is a promising prognostic biomarker and immunotherapeutic target for various solid cancers because of its activity in cancer immunity and tumourigenesis.

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

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

Abstract PO4-03-13: High B7-H3 expression with low PD-L1 expression identifies armored-cold tumors suggesting potential anti-B7-H3 therapy in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is generally regarded as the most aggressive subtype among breast cancer. B7 homolog 3 protein (B7-H3), also known as CD276, is an emerging immune checkpoint molecule, which plays a dual role in the immune system. Multiple anti-B7-H3 therapies has been raised, such as DS-7300, MGA271, MGD009, and B7-H3 chimeric antigen receptor T-cell immunotherapy (CAR-T). However, the expression of B7-H3 in TNBC and its value as a biomarker identifying therapeutic options has not been elucidated Methods: A total of 165 samples (83 non-TNBC and 82 TNBC) were included in this study, and 6 samples included paired para-tumor samples. In addition, 30 TNBC patients receiving standardized neoadjuvant chemotherapy (NAT) were recruited. After 8 cycles of NAT, the response to NAT was assessed by RECIST1.1 criterion before surgical operation. Moreover, multiple public cohorts of breast cancer and TNBC were obtained, including the TCGA dataset, the METABRIC dataset, the GSE176307 (durvalumab-based therapy) dataset, the PRJNA558949 (durvalumab-based therapy) dataset, the GSE194040 (paclitaxel-based therapy) dataset, and the GSE34138 (anthracycline-based therapy) dataset. Based on multiple in-house and public cohorts, we investigated the expression features of B7-H3 in breast cancer and checked the anti-tumor effect of the B7-H3 monoclonal antibody in mouse model. We also developed a novel classifier combined B7-H3 and PD-L1 expression in TNBC. Results: B7-H3 was highly expressed in tumor tissues in the in-house and the TCGA cohorts. B7-H3 expression did not differ significantly between non-TNBC and TNBC samples in the in-house and the TCGA cohorts, excepting for the METABIRC cohort. There was no notable difference in CD8+ cells infiltration between B7-H3 low and high expressed non-TNBC samples, but CD8+ cells was notably higher in the B7-H3 low expressed TNBC samples. The similar findings were also observed in the TCGA and the METABIRC cohorts. In addition, enrichment analysis of transcriptome data and analysis of in-house samples showed that B7-H3 was positively related to most collagens. Moreover, anti-B7-H3 therapy significantly inhibited tumor growth in mouse TNBC model, notably increased immune cells infiltration, and also reduced collagen deposition within the tumor. Given the notable value of PD-L1 in predicting immunotherapy responses, we also combined B7-H3 and PD-L1 expression to establish a novel subtyping strategy. TNBC patients with B7-H3highPDL1low feature exhibited the lowest anti-tumor immune infiltration and higher collagen deposition level. In the durvalumab-dependent cohort, paclitaxel-dependent cohort, anthracycline-dependent cohort, and our recruited NAT cohort, the B7-H3highPDL1low subgroup exhibited the lowest therapeutic response. Conclusions: Overall, this research provides a novel subtyping strategy based on the combination of B7-H3/PD-L1 expression that could potentially be applied to predict the therapeutic responses in TNBC, and suggests a potential biomarker-guided anti-B7-H3 therapy. Based on the classifier, we can select potential beneficiaries to deliver personalized medical services. Citation Format: Jie Mei, Yun Cai, Ziyi Fu, Yongmei Yin. High B7-H3 expression with low PD-L1 expression identifies armored-cold tumors suggesting potential anti-B7-H3 therapy in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-03-13.

Expression of B7-H4 in endometrial cancer and its impact on patients' prognosis.

The aim of the study was to evaluate the B7-H4 expression in endometrial cancer cells and to investigate its relationship with patient prognosis and clinicopathological features of the disease. We performed a single-center, retrospective cohort study that included endometrial cancer patients treated between 2012 and 2019. B7-H4 expression in specimens obtained from 63 patients was examined by immunohistochemical staining. The evaluation of B7H4 immunoreactivity was assessed using Immunoreactivity Scoring (IRS) system. B7-H4 reactivity was observed in all, except one, endometrial cancer patients (98%). Staining intensity: no reaction in one case, weak in 16 (24%) patients, moderate in 25 (37%), and strong in 22 (35%). Twenty-nine (46%) patients showed B7-H4 immunoreactivity in more than 60% of cells, while, in 18 (29%) cases and 16 (25%) patients, the percentages were 30-60% and < 30% respectively. Median IRS was 2 (range 0-6). We found a significantly worse overall survival (OS) rate for patients with high versus low B7-H4 IRS (p = 0.03), however, in multivariate analysis, the difference in patient survival was close to the significance level (p = 0.052). B7-H4 expression was not related to histopathological type of the tumor, tumor grade, lymph node metastases, or the FIGO stage of the disease. Our result suggests that B7-H4 expression might be a useful prognostic factor in endometrial cancer.

Epithelial Expressed B7-H4 Drives Differential Immunotherapy Response in Murine and Human Breast Cancer.

This translational study confirms the association of B7-H4 expression with a cold immune microenvironment in breast cancer and offers preclinical studies demonstrating a potential role for B7-H4 in suppressing response to checkpoint therapy. However, analysis of two clinical trials with checkpoint inhibitors in the early and metastatic settings argue against B7-H4 as being a mechanism of clinical resistance to checkpoints, with clear implications for its candidacy as a therapeutic target.

Enhancing surgical outcomes: accurate identification and removal of prostate cancer with B7-H3-targeted NIR-II molecular imaging.

One of the main reasons for prostate cancer (PCa) recurrence is the difficulty in identifying and removing cancerous lesions during surgery. Accurately localizing and excising cancerous tissue remains a significant challenge. The second near-infrared window (NIR-II, 1000-1700nm) fluorescence offers enhanced resolution, a high signal-to-noise ratio, and the potential for deeper tissue penetration. However, this technology is not currently employed for intraoperative imaging of PCa. This study aims to construct a new NIR-II probe targeting B7-H3 (AbB7-H3-800CW) for accurate intraoperative identification and resection of PCa. Based on the differential expression of B7-H3 in PCa, we designed a novel imaging probe to accurately identify and guide the resection of preclinical PCa models and ex vivo human PCa tissues using NIR-II fluorescence imaging technology. Analyzing tissue samples from 60 clinical cases of PCa, along with benign prostatic hyperplasia and normal prostate tissue from 22 cases, we observed a significant difference in B7-H3 protein expression levels (P < 0.001). Subcutaneous and orthotopic mouse models of PCa were imaged using NIR-II fluorescence after AbB7-H3-800CW injection, showing promising results with successful tumor targeting and high-contrast images achieved within 24-48h post-injection. The imaging also enabled the detection of occult PCa lesions approximately 1mm in diameter. In addition, imaging analysis of human PCa and adjacent tissues using AbB7-H3-800CW incubation revealed that cancer tissues exhibited a significantly higher fluorescence intensity than adjacent tissues (P < 0.05), which was conducive to the evaluation of tumor resection margin in vitro. The findings revealed that B7-H3 was a compelling imaging target for PCa. The AbB7-H3-800CW molecular imaging probe is capable of accurately identifying PCa lesions and guiding their removal. This approach can potentially reduce the rate of surgical margins under NIR-II fluorescence guidance.

Abstract P66: Off-The-Shelf Epstein Barr Virus Specific T Cells Armored with B7-H3-Targeting Chimeric Antigen Receptors Is an Effective and Safe Tumor- Agnostic Therapy For B7-H3 Positive Solid Malignancies

Abstract In contrast to its limited expression in normal tissues, the B7 homolog 3 protein (B7-H3) is over-expressed in many human tumors, making it an attractive target for chimeric antigen receptor (CAR) T cell therapy. To address the technical challenges limiting broader patient access to autologous CAR T cell therapy, we armoured Epstein-Barr Virus Specific T Cells (EBVSTs) with CARs as an off-the-shelf therapy against solid tumors. We identified and utilised a nanobody as the domain specifically targeting B7-H3 in our CAR construct to reduce the stability issues associated with scFv-based domains. Our B7H3.CAR armored EBVSTs demonstrated potent in-vitro and in-vivo activity against multiple B7-H3 positive tumor and patient-derived xenografts models. Analysis of human healthy tissues and tumors microarrays indicated that B7-H3 expression, while elevated in patient tumors, was differentially low in healthy tissues. Consistent with this, mouse T cells expressing a murine equivalent of our B7H3.CAR displayed good on-target off-tumor safety in immunocompetent mice bearing syngeneic tumors. Further in-vitro evaluation revealed that while human T, B and NK cells were unaffected by B7H3.CAR EBVSTs targeting, monocytes were readily targeted due to the upregulation of B7-H3. Consequent to this targeting and depletion of B7-H3 positive human myeloid cells which are key mediators of CRS, a low incidence of cytokine release syndrome was observed in humanized mice after B7H3.CAR EBVST treatment. Lastly, we showed that B7H3.CAR EBVSTs can target myeloid-derived suppressor cells (MDSCs) that express B7-H3 and in doing so, alleviate MDSC-driven immune suppression. Our findings demonstrate that our nanobody-based B7H3.CAR EBVSTs present not only an effective and safe off-the-shelf approach for targeting any B7-H3-positive solid tumor while also addressing challenges that hindered the clinical development of CAR T cells in such indications. Citation Format: Siok Ping Yeo, Lindsay Kua, Jin Wei Tan, Joanna Kristyn Lim, Fiona Wong, May Delos Santos, Chek Meng Poh, Angeline Goh XH, Xin Yu Koh, Xiao Hua Zhou, Rajarethinam R, Qing Feng Chen, Zhisheng Her, Ivan D Horak, Lionel Low, Kar Wai Tan. Off-The-Shelf Epstein Barr Virus Specific T Cells Armored with B7-H3-Targeting Chimeric Antigen Receptors Is an Effective and Safe Tumor- Agnostic Therapy For B7-H3 Positive Solid Malignancies [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P66.