Targeting B7-H3 Research Articles

Targeting B7-H3-A Novel Strategy for the Design of Anticancer Agents for Extracranial Pediatric Solid Tumors Treatment.

Recent scientific data recognize the B7-H3 checkpoint molecule as a potential target for immunotherapy of pediatric solid tumors (PSTs). B7-H3 is highly expressed in extracranial PSTs such as neuroblastoma, rhabdomyosarcoma, nephroblastoma, osteosarcoma, and Ewing sarcoma, whereas its expression is absent or very low in normal tissues and organs. The influence of B7-H3 on the biological behavior of malignant solid neoplasms of childhood is expressed through different molecular mechanisms, including stimulation of immune evasion and tumor invasion, and cell-cycle disruption. It has been shown that B7-H3 knockdown decreased tumor cell proliferation and migration, suppressed tumor growth, and enhanced anti-tumor immune response in some pediatric solid cancers. Antibody-drug conjugates targeting B7-H3 exhibited profound anti-tumor effects against preclinical models of pediatric solid malignancies. Moreover, B7-H3-targeting chimeric antigen receptor (CAR)-T cells demonstrated significant in vivo activity against different xenograft models of neuroblastoma, Ewing sarcoma, and osteosarcoma. Finally, clinical studies demonstrated the potent anti-tumor activity of B7-H3-targeting antibody-radioimmunoconjugates in metastatic neuroblastoma. This review summarizes the established data from various PST-related studies, including in vitro, in vivo, and clinical research, and explains all the benefits and potential obstacles of targeting B7-H3 by novel immunotherapeutic agents designed to treat malignant extracranial solid tumors of childhood.

Abstract 6304: Preclinical development of a next generation antibody drug conjugate (ADC) targeting B7-H3 for treatment of solid tumors

Abstract B7-H3 (CD276), an immune checkpoint member of the B7/CD28 family, is widely expressed in multiple cancers and is associated with poor prognosis. YL201 is a novel B7-H3-targeting antibody-drug conjugate (ADC) and built on MediLink’s tumor microenvironment activable linker-payload platform (TMALIN platform). This ADC is comprised of an anti-B7-H3 human monoclonal antibody conjugated to a novel topoisomerase 1 inhibitor via a protease-cleavable linker. The novel linker-payload and site-specific conjugation of YL201 results in a homogeneous and more hydrophilic ADC. In addition, the binding specificity, affinity and avidity of antibodies show no impact after conjugation. In vitro, upon binding to B7-H3, the antigen-ADC complexes are internalized to lysosomal vesicles releasing the toxic payload, whose cytotoxic features drive cell cycle arrest, apoptosis, elevation of PARP/caspase 7 expression, as well as cell death and bystander effect. In preclinical studies, YL201 is effective in causing tumor regressions in both cell line- and patient-derived xenograft (CDX and PDX) mouse models at doses that are well tolerated. The in vivo pharmacokinetics in monkeys showed that YL201 is deemed as ‘highly stable’ in circulation. Safety evaluations demonstrated acceptable safety profile and no off-target toxicity in monkeys. These non-clinical data suggest the stability of linker in circulation but efficiently release the payload in tumors, and eventually, expanded the therapeutic window. Taken together, preclinical data suggest that YL201 could be a promising treatment strategy for B7-H3 positive cancer patients. Citation Format: Jian Xu, Qing Zong, Wei Wang, Qigang Liu, Sasha Stann, Jiaqiang Cai. Preclinical development of a next generation antibody drug conjugate (ADC) targeting B7-H3 for treatment of solid tumors. [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 6304.

372 IPSC-Derived NK cells Exhibit Potent In Vitro and In Vivo Tumorcidal Activity Against Patient-Derived Glioblastoma Stem Cells (GSCs)

INTRODUCTION: Natural Killer (NK) cells play pivotal roles in cancer immune surveillance and harbor potent anti-tumor activities. We have engineered an induced pluripotent stem cells (iPSCs) derived, NK cell (termed FT538) that express 1) a high-affinity non-cleavable CD16 Fc receptor to facilitate antibody engagement 2) a membrane-bound IL-15/IL-15 receptor fusion to enhance NK activity and persistence and 3) a knock-out of CD38 to enhance NK metabolic fitness. FT538 is produced in a GMP-compliant manner as an off-the-shelf immunotherapy. Clinical experiences with FT538 have shown evidence of durable anti-tumor immunity after single administration in select lymphoma patients. METHODS: Laboratory characterization and murine modeling. RESULTS: Without specific antibody to engage CD16, FT538 exhibited potent in vitro activity against ∼80% of a panel of isocitrate dehydrogenase wild-type patient derived xenograft (PDX) glioblastoma lines, including MGG8. When directly injected into the murine brain, 50% of the FT538 persisted for >2 weeks without addition of exogenous cytokine. Moreover, the mice exhibited no evidence of neurologic compromise, and brains harvested after injection show no signs of FT538 activation. In vivo intratumoral injection of FT538 into an orthotopically established MGG8 glioblastoma caused an ∼20 fold increase in granzyme B release by FT538, with a comparable increase of cleaved Caspase 3 in the tumor proper. In independent experiments, >90% of mice orthotopically implanted MGG8 survived beyond 100 days after intra-tumoral FT538 while all mice treated with mock injection died by 60 days. For glioblastoma PDX refractory to FT538, such as MGG4, tumoricidal activity of FT583 can be induced in vitro and in vivo by the addition of a tri-specific engager targeting B7H3, a surface protein highly expressed in glioblastomas. CONCLUSIONS: Our results suggest that FT538 great promise as a glioblastoma therapy, with plans for translation into a first-in-human clinical trial.

Radioimmunotherapy Targeting B7-H3 in situ glioma models enhanced antitumor efficacy by Reconstructing the tumor microenvironment.

Radionuclide drug conjugates (RDCs) with antibodies serve as a novel approach for the treatment of malignant tumors including glioblastoma. However, RDCs require optimal antibodies to work efficiently. Hu4G4, a novel B7-H3-targeting humanized monoclonal IgG1 antibody, is highly specific for the human B7-H3 protein (a marker of tumor cells, including glioblastoma cells). Herein, we established 131I-labeled hu4G4 (131I-hu4G4) and showed that it specifically bound to B7-H3 with high affinity (Kd = 0.99 ± 0.07 nM) and inhibited the growth of U87 cells in vitro. 131I-hu4G4 displayed potent in situ antitumor activity in a mouse model of glioma based on GL261 Red-Fluc-B7-H3 cells. More importantly, 131I-hu4G4 remodeled the tumor microenvironment and promoted the transformation of glioma from "cold" to "hot" tumors by promoting CD4+ and CD8+ T cell infiltration and the polarization of M2 to M1. Therefore, the antitumor activity observed with 131I-hu4G4, together with its ability to enhance antitumor immune responses, makes it a novel candidate for radioimmunotherapy of glioblastoma.

EXTH-64. IPSC-DERIVED NK CELLS EXHIBIT POTENT IN VITRO AND IN VIVO TUMORCIDAL ACTIVITY AGAINST PATIENT-DERIVED GLIOBLASTOMA STEM CELLS (GSCS)

Abstract INTRODUCTION Natural Killer (NK) cells play pivotal roles in cancer immune surveillance and harbor potent anti-tumor activities. We have engineered an induced pluripotent stem cells (iPSCs) derived, NK cell (termed FT538) that express 1) a high-affinity non-cleavable CD16 Fc receptor to facilitate antibody engagement, 2) a membrane-bound IL-15/IL-15 receptor fusion to enhance NK activity and persistence and 3) a knock-out of CD38 to enhance NK metabolic fitness. FT538 is produced in a GMP-compliant manner as an off-the-shelf immunotherapy. Clinical experiences with FT538 have shown evidence of durable anti-tumor immunity after single administration in select lymphoma patients. METHODS We performed pre-clinical studies to characterize the tumoricidal activity of FT573 against glioblastomas. RESULTS Without specific antibody to engage CD16, FT538 exhibited potent in vitro activity against ~80% of a panel of isocitrate dehydrogenase wild-type patient-derived xenograft (PDX) glioblastoma lines, including MGG8. When directly injected into the murine brain, 50% of the FT538 persisted for > 2 weeks without addition of exogenous cytokine. Moreover, the mice exhibited no evidence of neurologic compromise, and brains harvested after injection show no signs of FT538 activation. In vivo intratumoral injection of FT538 into an orthotopically established MGG8 glioblastoma caused a ~20 fold increase in granzyme B release by FT538, with a comparable increase of cleaved-caspase 3 in the tumor. In independent experiments, > 90% of mice orthotopically implanted MGG8 survived beyond 100 days after intra-tumoral FT538 while all mice treated with mock injection died by 60 days. For glioblastoma PDX refractory to FT538, such as MGG4, tumoricidal activity of FT583 can be induced in vitro and in vivo by the addition of a tri-specific engager targeting B7H3, a surface protein highly expressed in glioblastomas. CONCLUSION Our results demonstrate great promise of FT538 as a glioblastoma therapy, with plans for translation into a first-in-human clinical trial.

Hopes on immunotherapy targeting B7-H3 in neuroblastoma

Neuroblastoma is one of the most aggressive cancer forms in children, with highly heterogenous clinical manifestations ranging from spontaneous regression to high metastatic capacity. High-risk neuroblastoma has the highest mortality rates of all pediatric cancers, highlighting the urgent need for effective novel therapeutic interventions. B7-H3 immune checkpoint protein is highly expressed in neuroblastoma, and it is involved in oncogenic signaling, tumor cell plasticity, and drug resistance. Immunotherapies based on immune checkpoint inhibition have improved patient survival in several human cancers, and recent reports provide preclinical evidence on the benefits of targeting B7-H3 in neuroblastoma, with emphasis on novel CAR T/NK-cell approaches. Here, we summarize the current status of neuroblastoma targeted therapies, with a focus on B7-H3 as a promising novel immunoregulatory therapeutic target for high-risk neuroblastoma.

Clinical significance of B7-H3 and HER2 co-expression and therapeutic value of combination treatment in gastric cancer

BackgroundGastric cancer (GC) is a digestive system malignancy. Trastuzumab (a HER2-targeted monoclonal antibody) is an important targeted drug for GC. However, the drug resistance limits its clinical efficacy. B7-H3 was suggested to be a promising target for cancer immunotherapy. This study aimed to investigate the clinical significance of B7-H3 and HER2 co-expression and the therapeutic value of combination treatment in GC. MethodsWe examined the expression of B7-H3 and HER2 in 268 GC patients by immunohistochemistry. Pearson test was used to analyze the correlation between categorical variables. Overall survival was assessed by Kaplan–Meier analysis. All in vitro experiments using HER2-positive GC cells were treated with small interfering RNA targeting B7-H3/HER2 or B7-H3 blocking antibody 3E8/trastuzumab to verify the antitumor efficacy of the combination therapy. GC xenograft mouse models were established to evaluate the in vivo anti-tumor effect of combined therapy. ResultsThere was a significant correlation between B7-H3 and HER2 expression in GC tissues. High co-expression of B7-H3 and HER2 was associated with poor prognosis (P = 0.007) and could be an independent risk factor for survival. In addition, knockdown or targeted therapies of B7-H3/HER2 significantly suppressed cell proliferation, migration, invasion and adhesion in vitro. Trastuzumab combined with 3E8 was significantly effective at reducing mice tumor growth than monotherapy. ConclusionHigh co-expression of B7-H3 and HER2 indicates a poor prognosis, and combination therapy targeting B7-H3 and HER2 could be an immunotherapeutic strategy for GC.

Abstract 330: Targeting B7-H3 in prostate cancer: Preclinical proof of concept with MGC018, an investigational anti-B7-H3 antibody-drug conjugate

Abstract Introduction: Prostate cancer is the second most common cancer among men worldwide. In 2021, it is estimated that 248,530 men in the United States will be diagnosed with prostate cancer, and 34,130 will die from the disease. Although current treatments have success initially, development of resistance commonly leads to recurrence of an incurable castrate-resistant form of the disease. Thus, significant need for novel therapies to improve the outcome of castrate-resistant prostate cancer remains. B7-H3 (CD276), a member of the B7 family of immunomodulatory molecules, is overexpressed in primary and metastatic prostate cancer, and correlates with disease severity and poor clinical outcome. MGC018, a duocarmycin-based B7-H3 antibody-drug conjugate, is currently being evaluated in clinical studies. Here, MGC018 was explored preclinically to assess the potential for targeting B7-H3 in prostate cancer. Methods: Immunohistochemistry studies were performed to define the expression of B7-H3 in prostate cancer tissue microarrays (TMA). In vivo efficacy studies were conducted with human prostate cancer cell line-derived xenograft (CDX) models to explore the antitumor activity of MGC018 as a single agent and in combination with Poly (ADP-ribose) polymerase (PARP) and androgen receptor (AR) inhibitors. Based on the results in the CDX studies, in vivo efficacy studies were extended to a panel of metastatic prostate cancer patient-derived xenograft (PDX) models, which exhibit heterogenous expression of B7-H3 and more closely mimic the biological characteristics of patient tumors. Results: Staining of prostate tumor TMAs revealed high expression of B7-H3 in primary and metastatic prostate cancer. Of the prostate samples evaluated, 95% (38/40) of the tumor samples were positive for B7-H3 (H-score ≥ 20): 65% had H-scores greater than 200, while 20% and 10% had H-scores between 101-200 and 1-100, respectively. MGC018 demonstrated in vitro cytotoxicity toward B7-H3-positive human prostate cancer cell lines. The in vitro cytotoxicity translated to potent antitumor activity in vivo toward prostate cancer CDX models, and the antitumor activity of MGC018 was enhanced when combined with inhibitors of PARP or AR. In PDX models of metastatic prostate cancer, MGC018 was active as a single agent toward heterogeneous B7-H3-expressing tumors, and combining MGC018 with inhibitors of PARP or AR led to a greater response in some models. Conclusion: B7-H3 is frequently overexpressed in prostate cancer. MGC018 demonstrated potent antitumor activity in vivo toward CDX and PDX models of prostate cancer, and enhanced antitumor activity when combined with inhibitors of PARP or AR. These results support prostate cancer as an indication that may be responsive to ADC-based treatments directed toward B7-H3. MGC018 is being investigated in metastatic prostate cancer in a Phase 1/2 clinical study. Citation Format: Juniper A. Scribner, Francine Z. Chen, Anushka De Costa, Ying Li, Michael Chiechi, Thomas Son, Jeff Hooley, Jonathan Li, Scott Koenig, Chet Bohac, Ezio Bonvini, Paul A. Moore, Deryk Loo. Targeting B7-H3 in prostate cancer: Preclinical proof of concept with MGC018, an investigational anti-B7-H3 antibody-drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 330.

Abstract 2019: Tumor-targeting and efficacy of B7H3/GD2 bispecific SNIPER antibodies

Abstract Introduction: GD2 is expressed on neuroblastoma, melanomas, small cell lung cancers and sarcomas. While it is expressed minimally on normal tissues, it is expressed on nerve cells and, anti-GD2 (dinutuximab) treatment can cause neuropathic pain. To increase tumor specificity, we developed a bispecific SNIPER antibody to simultaneously target two tumor antigens (GD2 and B7H3). B7H3 is overexpressed on multiple tumor types, with minimal expression on most normal cells and is absent on nerve cells. The goal of this SNIPER is to enhance tumor-specificity, while reducing toxicity, ultimately improving efficacy for cancers expressing both targets. Experimental Procedures: SNIPER specificity was tested by flow cytometry for binding to GD2 +/- B7H3 expressing cells. Using an Incucyte S3, we tested antibody internalization properties [compared to anti-B7H3 and dinutuximab monoclonal antibodies (mAbs)] and antibody dependent cellular-cytotoxicity (ADCC) capabilities. Mice bearing variants of GD2/B7H3-expressing tumors were intravenously injected with 89Zr- radiolabeled SNIPER and its longitudinal in vivo biodistribution was monitored via positron emission tomography imaging. In vivo efficacy studies of SNIPER were tested against mice bearing either melanoma or neuroblastoma tumors that express both GD2 and B7H3. Summary of Data: In vitro and in vivo tumor specificity testing confirmed that SNIPER specifically targets B7H3+/GD2+ cells, but it does not bind to GD2+/B7H3- cells (which simulate nerve cells). We observed high internalization of anti-GD2 mAbs; we did not observe antibody internalization of either the SNIPER or anti-B7H3 mAbs. SNIPER was as effective at ADCC as the dinutuximab, but we saw minimal ADCC with the anti-B7H3 mAbs. An afucosylated version of SNIPER showed significantly enhanced ADCC compared to dinutuximab. Our in vivo efficacy studies found that SNIPER was as as dinutuximab when given at the same dose. Conclusions: The Fab arms of SNIPER targeting GD2 and B7H3 each have low-to-moderate affinity. SNIPER binds with strong avidity when both arms bind to their antigens on the same cell. Stronger avidity through both arms binding results in high-tumor specificity. Because SNIPER should not bind to nerves, it may be possible to administer increased doses of SNIPER beyond the tolerable dose of dinutuximab, which could further improve efficacy. Ongoing studies include antitumor efficacy testing, nerve binding assays and assessing reduction of pain in vivo with SNIPER. Citation Format: Amy K. Erbe, Arika Feils, Zack Rosenkrans, Jessica Wiwczar, Daniel Gerhardt, Bonnie Hammer, Mildred Felder, Mark Bercher, Alina Hampton, Lizzie Frankel, Dan Spiegelman, Noah Tsarovsky, Alexander Rakhmilevich, Jacquelyn Hank, Bryan Glaser, Reinier Hernandez, Roland Green, Paul M. Sondel. Tumor-targeting and efficacy of B7H3/GD2 bispecific SNIPER antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2019.

DIPG-53. Long-term survival from a Phase 1 dose-escalation trial using convection-enhanced delivery (CED) of radioimmunotherapeutic124I-omburtamab for treatment of diffuse intrinsic pontine glioma (DIPG).

BACKGROUND: Median survival from DIPG is less than one year. In a phase 1 dose escalation study (clinicaltrials.gov NCT01502917) 124I-omburtamab targeting B7-H3 was administered intratumorally using CED. METHODS: CED was performed between 4-14 weeks post radiation therapy. Using a 3 + 3 design, 124I-omburtamab was escalated from 0.25-10.0 mCi and infusion volumes (Vi) from 250-10,000 µl with serial 124I PET/CT performed up to ~1 week post-administration. Toxicities were assessed for 30 days. Dose escalation safety was evaluated. Survival was calculated using Kaplan-Meier statistics. RESULTS: 46 children were treated and evaluable for toxicity and survival;4 patients received partial doses and were evaluable for toxicity only. Three patients experienced dose limiting toxicities. Eleven patients had transient treatment related grade 3 toxicities with no grade 4 or 5 toxicities. Grade 3 nervous system toxicities included: muscular weakness(n=8), dysarthria(n=4), ataxia(n=3), dysphagia(n=3), and gait disturbance(n=1). Lesion absorbed doses ranged from 1,000-1,500cGy/mCi, with lesion-to-whole body radiation absorbed-dose ratios of ~900. A dose of 8mCi and infusion volume of 8,000 µl is safe and may provide a distribution volume up to 20cm3. Median survival was 15.3 months (n =46, 95% CI 12.7, 17.3). Survival rate estimates (95% CI) at 1, 2, 3 and 5 years were 0.67 (0.55;0.82); 0.18 (0.09;0.35); 0.10 (0.04;0.26); and 0.05 (0.01;0.20). Four patients survived >3 years; two remain alive (61+ and 106+ months);two have died (44 and 53 month) with distant CNS disease and one with extra-CNS metastasis. CONCLUSION: Administration of escalating doses and volumes of 124I-omburtamab via CED was a viable option for this patient subgroup. The median overall survival was increased 3-4 months compared to historical controls. Anecdotal long-term survival if validated with a planned phase 2 trial would support the concept of whole neuroaxis treatment in combination with CED in a subset of DIPG patients.

Targeting B7-H3 in prostate cancer: Phase 2 trial in localized prostate cancer using the anti-B7-H3 antibody enoblituzumab, with biomarker correlatives.

5015 Background: B7-H3/CD276, a member of the B7 superfamily, is highly expressed in prostate cancer (PCa) and is associated with rapid biochemical recurrence and early metastases. B7-H3 is the only checkpoint candidate to have a presumptive androgen receptor binding site, suggesting interaction with the androgen axis. Enoblituzumab (MacroGenics) is an investigational humanized Fc-optimized B7-H3–targeting antibody that induces antibody dependent cellular cytotoxicity (ADCC). Methods: In this phase 2 single-arm biomarker-rich neoadjuvant trial, men with operable intermediate- and high-risk localized prostate cancer (Grade Groups 3-5) were enrolled to evaluate the safety, anti-tumor efficacy, and immunogenicity of enoblituzumab when given prior to prostatectomy. Patients received enoblituzumab (15 mg/kg IV weekly x 6) prior to surgery. Prostate glands were harvested 2 weeks after the last dose, and were examined for pathologic and immunologic endpoints. The co-primary outcomes were safety and PSA0 at 1 year post-op. Pre-planned secondary outcomes were PSA and Gleason grade group change from biopsy to prostatectomy. Results: 32 men were enrolled. Grade 3/4 adverse events occurred in 12% of patients. One patient developed a grade-3 infusion reaction, and one had immune myocarditis that improved with steroids. Pre-prostatectomy PSA declines of >10% were observed in 31% of patients (95% CI: 16-50%). PSA0 at 1 year post-op was seen in 66% of men (95% CI: 47-81%). Median time to PSA recurrence was not reached, with a median follow-up of 30 months. Gleason group upgrade, no change, and downgrade was observed in 13%, 37%, and 50% of patients. Gleason grade group changes were significantly associated with enoblituzumab treatment compared to 1:1 matched historical controls (p=0.023). Tumor microenvironment profiling by NanoString GeoMx spatial proteomics and PanCancer IO 360 mRNA expression analysis revealed post-treatment upregulation of CD8+ T cells, PD-1/PD-L1 expression, and immune activation (granzyme B, IFN signaling, myeloid inflammation). There was a significant association between CD8+ T-cell increases and Gleason grade group declines. First-in-human antigen spread profiling revealed no safety concerns. TCR sequencing showed focused peripheral expansion of tumor associated T-cell clones that correlated with PSA0 at 1 year. Whole exome and RNAseq data, and clinical correlations, will be presented. Conclusions: In this neoadjuvant trial, inhibition of B7-H3 with enoblituzumab demonstrated favorable safety and encouraging activity in localized PCa patients. Data suggest robust intratumoral induction (adaptive upregulation) of immune checkpoints, T-cell activation, and myeloid inflammation. Enoblituzumab-induced peripheral expansion of tumor associated T-cell clones may be associated with tumor control. Clinical trial information: NCT02923180.

Antitumor Response of Anti-B7-H3 CAR-T Cells with Humanized scFv in Solid Tumors

We prepared a humanized alternative B7-H3 CAR (B7-H3-haCAR-T) targeting B7-H3 which contained the humanized singl-chain varinlde fragment (scFv) from humanized 8H9 Monoclonal Antibody (hu8H9mAb). The antitumor effects of B7-H3-haCAR-T cells were evaluated in B7-H3 overexpressed tumor cells (in vitro) and B7-H3 xenograft models (in vivo). The specific tumour killing ability of B7-H3-haCAR-T in overexpressing B7-H3 tumour cells was verified by cytotoxicity and ELISA tests. In addition, B7-H3 haCAR-T cells were also noted to suppress the tumour growth remarkably well in the xenograft murine models, and the survival time in the haCAR-T cell treatment group was appreciably longer than that in the control group. The specific recognition and highly efficient tumoricidal behavior of B7-H3 haCAR-T provide a basis for future clinical studies with humanized scFv-transduced CAR-T cells targeting solid tumors.

DS-7300a, a DNA Topoisomerase I Inhibitor, DXd-Based Antibody-Drug Conjugate Targeting B7-H3, Exerts Potent Antitumor Activities in Preclinical Models.

B7-H3 is overexpressed in various solid tumors and has been considered as an attractive target for cancer therapy. Here, we report the development of DS-7300a, a novel B7-H3-targeting antibody-drug conjugate with a potent DNA topoisomerase I inhibitor, and its in vitro profile, pharmacokinetic profiles, safety profiles, and in vivo antitumor activities in nonclinical species. The target specificity and species cross-reactivity of DS-7300a were assessed. Its pharmacologic activities were evaluated in several human cancer cell lines in vitro and xenograft mouse models, including patient-derived xenograft (PDX) mouse models in vivo. Pharmacokinetics was investigated in cynomolgus monkeys. Safety profiles in rats and cynomolgus monkeys were also assessed. DS-7300a specifically bound to B7-H3 and inhibited the growth of B7-H3-expressing cancer cells, but not that of B7-H3-negative cancer cells, in vitro. Additionally, treatment with DS-7300a and DXd induced phosphorylated checkpoint kinase 1, a DNA damage marker, and cleaved PARP, an apoptosis marker, in cancer cells. Moreover, DS-7300a demonstrated potent in vivo antitumor activities in high-B7-H3 tumor xenograft models, including various tumor types of high-B7-H3 PDX models. Furthermore, DS-7300a was stable in circulation with acceptable pharmacokinetic profiles in monkeys, and well tolerated in rats and monkeys. DS-7300a exerted potent antitumor activities against B7-H3-expressing tumors in in vitro and in vivo models, including PDX mouse models, and showed acceptable pharmacokinetic and safety profiles in nonclinical species. Therefore, DS-7300a may be effective in treating patients with B7-H3-expressing solid tumors in a clinical setting.

126P Novel B7-H3 targeting dual nanobody NK cell engagers display robust activity against a broad spectrum of solid and hematologic malignancies

B7-H3 is a checkpoint molecule under intense investigation as an immune therapy target. We previously showed that dual camelid nanobody tri-specific killer engager (TriKE) (GTB-5550) specifically bound B7-H3 on PC3/C4-2 prostate cancer (PCa) cells and activated peripheral blood (PB) NK cells. We have since developed a dual camelid bispecific killer engager (BiKE) targeting B7-H3 and show that both GTB-5550, which harbors wild-type IL-15, and BiKE display broad activity against B7-H3-expressing tumors.

Anti-B7-H3 Antibody (T-1A5) Blocks Immunomodulatory Function of B7-H3 and Enhances NK Cell-Mediated Cytotoxicity Against Acute Myeloid Leukemia Cells

Background: The immune checkpoint molecule B7-H3 (CD276) is overexpressed in various solid tumors and hematological malignancies; however, its expression is limited in normal tissue, which makes it an attractive therapeutic target in cancer. Several monoclonal antibodies (mAbs) targeting B7-H3 have shown promising results against solid tumors. However, B7-H3's role in acute myeloid leukemia (AML) remains unexplored. Here, we hypothesized that targeting B7-H3 using mAbs alters the immunomodulatory function of B7-H3 and enhances NK cell-mediated cytotoxicity against AML cells.Methods: B7-H3 protein expression was analyzed in the peripheral blood (PB) and bone marrow of 100 patients with AML and 20 healthy donors by flow cytometry and tested for associations with multiple clinical parameters and disease outcomes. To investigate B7-H3's role in immunomodulation, we stably knocked down B7-H3 in AML cell lines including OCI-AML3, MV4-11, and U937 and co-cultured them with activated human NK cells. NK cell-induced apoptosis was measured by annexin-v binding assay using an IncuCyte live-cell imaging system. B7-H3-blocking mAbs (clones T-1A5, HEK5-1B3, and 58B1) were tested for their effect on NK-cell-mediated cytotoxicity in AML cell lines using live-cell imaging. AML xenograft (OCI-AML3) or patient-derived xenograft (PDX) models were used to determine the effect of anti-B7-H3 antibodies on AML growth. In vivo AML growth was monitored by measuring human CD45 positive cells with flow cytometry. A human-mouse chimeric (ch) antibody was generated based on sequences from the T-1A5 antibody, and its binding site on the B7-H3 protein was characterized by epitope mapping. Further, we evaluated the effect of chT-1A5 on NK cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) in primary AML cells and healthy donor PB-derived mononuclear cells (PBMCs).Results: Expression of B7-H3 was significantly higher in AML patients than in healthy donors (p < 0.01) and was higher in CD34 positive than in CD34 negative AML cells (p < 0.01). High B7-H3 expression was associated with poor overall survival (p = 0.04) and prognostic risk scores (p = 0.05). NK cell-mediated apoptosis was 3-fold higher in all 3 B7-H3-knockdown AML cell lines than in scrambled control cells, suggesting that B7-H3 is an important immunomodulator of NK cells. Moreover, we observed a significant increase in NK cell-mediated killing of AML cells in the presence of anti-B7-H3 mAbs (p < 0.01). In vivo, anti-B7-H3 antibodies significantly inhibited AML growth and extended survival in PDX models compared to IgG control treatment. Among PDX-bearing mice treated with the three anti-B7-H3 antibodies, the T-1A5 antibody-treated group survived for longer than the other groups. In combination with NK cells, T-1A5 treatment also significantly increased the survival of AML xenograft-bearing mice compared to NK + IgG control treatment. These data suggest that the T-1A5 antibody blocks B7-H3 and enhances NK cell-mediated cytotoxicity in AML cells in vivo. Next, we found that a chT-1A5 antibody induced NK cell-mediated ADCC in primary AML cells and cell lines in a dose-dependent manner. In contrast, it did not induce any ADCC activity in healthy donor-derived PBMCs, suggesting that chT-1A5 is not toxic to healthy cells. Moreover, the chT-1A5 antibody combined with human NK cells dramatically inhibited leukemia growth and extended survival in B7-H3 positive AML PDX models (p < 0.001) compared to the control (rituximab-treated) group. Finally, epitope mapping using peptides derived from the B7-H3 protein identified the FG loop region of B7-H3 as the binding site for the T-1A5 antibody, which may be involved in the immunomodulatory function of B7-H3.Conclusion: B7-H3 is overexpressed in AML cells, and its expression is associated with poor overall survival. Anti-B7-H3 antibodies block B7-H3's immunomodulatory function and inhibit AML growth in vivo. A chT-1A5 antibody in combination with NK cells induced ADCC in primary AML cells in vitro and in vivo but had no effect on PBMCs from healthy donors. Therefore, targeting B7-H3 could benefit AML patients, specifically those with a poor clinical prognosis. DisclosuresBattula: Tolero Pharmaceuticals: Research Funding.

Abstract 950: Targeting B7-H3 in squamous cell carcinoma of the head and neck: Preclinical proof-of-concept with the investigational anti-B7-H3 antibody-drug conjugate, MGC018

Abstract Introduction: Squamous cell carcinoma of the head and neck (SCCHN) is the seventh most common cancer worldwide, accounting for ~ 4% of all cancers in the United States. Although PD-1-directed therapy has efficacy in SCCHN, 85-95% of patients progress following initial response. B7-H3 is a member of the B7 family of immunomodulatory molecules, is overexpressed in SCCHN, and correlates with disease severity and poor clinical outcome. Furthermore, consistent with its putative coinhibitory function, B7-H3 expression in SCCHN is inversely correlated with the number of tumor infiltrating CD8+ T-cells. We are developing therapeutics targeting B7-H3, including enoblituzumab, an Fc-engineered anti-B7-H3 monoclonal antibody, and MGC018, a duocarmycin-based B7-H3 ADC, both of which are currently being evaluated in clinical studies. Here the potential of MGC018 was explored in preclinical models as a proof of concept for targeting B7-H3 in SCCHN. Methods: Immunohistochemistry studies were performed to define the expression of B7-H3 in SCCHN tissue microarrays (TMA). Single and repeat-dose in vivo efficacy studies were conducted in CD-1 nude mice with cell line-derived SCCHN human tumor xenografts to explore the relationship between Cmax, exposure and antitumor activity, and to define the minimal efficacious dose in these models. Based on results in these cell-derived xenograft (CDX) studies, in vivo efficacy studies were extended to a panel of SCCHN patient-derived xenograft (PDX) models, which more closely mimic the biological characteristics of the patient tumor and exhibit heterogenous expression of B7-H3. Results: Analysis of B7-H3 expression on a SCCHN TMA confirmed and extended previously reported expression of B7-H3 in SCCHN. Of the SCCHN samples evaluated, 90% (36/40) of the tumor samples were positive for B7-H3: 35% (14/40) had H-scores greater than 200, with the remaining 22 samples equally distributed between the H-score range of 101-200 and 1-100 (~ 28% each). MGC018 demonstrated specific, dose-dependent in vitro cytotoxicity toward SCCHN human tumor cell lines. The in vitro cytotoxicity translated to potent antitumor activity in vivo against SCCHN CDX models, with a single administration of 3 mg/kg resulting in complete responses in 7/7 mice in the FaDu model. In the PDX setting (H-scores 120-283), repeat dose administration every week or two weeks with MGC018 at 3 mg/kg/dose, led to regressions and/or stable disease in 10/18 models, and a delay in tumor growth in 5 additional models. Conclusion: B7-H3 is frequently overexpressed in SCCHN. At clinically relevant dose levels, MGC018 demonstrated potent antitumor activity in vivo toward SCCHN CDX mouse models and the majority of SCCHN PDX mouse models examined. These results support SCCHN as a potential indication that may be responsive to ADC-based treatments directed toward B7-H3. Citation Format: Juniper A. Scribner, Francine Z. Chen, Ying Li, Michael Chiechi, Thomas Son, Jeff Hooley, Scott Koenig, Paul A. Moore, Ezio Bonvini, Chet Bohac, Deryk Loo. Targeting B7-H3 in squamous cell carcinoma of the head and neck: Preclinical proof-of-concept with the investigational anti-B7-H3 antibody-drug conjugate, MGC018 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 950.

Testing of B7-H3 targeting antibody-drug conjugate (ADC) MGC018 in models of pediatric solid tumors by the Pediatric Preclinical Testing Consortium (PPTC).

10037 Background: B7-H3 (encoded by the CD276 gene) is an immunoregulatory molecule that is widely expressed in pediatric embryonal tumors and sarcomas, while expression is limited for normal tissues. Among PPTC models, osteosarcoma models showed highest CD276 transcript levels followed by Wilms tumor, neuroblastoma, rhabdomyosarcoma, and Ewing sarcoma. Protein expression by IHC generally followed expression at the RNA level, with high expression by IHC observed across a range of models. MGC018 is a duocarmycin-based humanized ADC targeting B7-H3 that shows selective cytotoxicity for B7-H3 expressing cancer cells, robust in vivo activity against a range of adult cancer preclinical models, a favorable pharmacokinetic and safety profile in cynomolgus monkeys, and has entered clinical testing for adults with cancer. Herein we report the in vivo antitumor activity of MGC018 against preclinical models of pediatric solid tumors. Methods: MGC018 was tested at 6 mg/kg administered intraperitoneally (IP) on Day 1. SYD988, an anti-CD20 ADC with the same linker and payload as MGC018, was used as a control arm at 6 mg/kg IP on Day 1. Osteosarcoma models, due to their slower growth kinetics and lower rates of tumor regression, were tested with n = 10 mice per arm, while the sarcoma and neuroblastoma models were tested with n of 1 or n of 2 designs, respectively. Activity was evaluated using response criteria mirroring clinical criteria (progressive disease (PD), stable disease (SD), partial response (PR), complete response (CR), and maintained CR (MCR)], by minimum relative tumor volume across all models, and by event-free survival (EFS) comparisons between treatment groups. Results: For neuroblastoma models (n = 9), 3 and 2 neuroblastoma models showed objective responses (PR/CR/MCR) or SD, respectively, to MGC018, while none showed an objective response or SD to SYD988. For osteosarcoma, results are mature for 1 of 5 models, with this model showing an MCR response to MGC018 and a PD response to SYD988. For Ewing sarcoma (n = 5), a PR and MCR were observed to MGC018 with the remaining models showing PD; for SYD988 a single CR was observed with the remaining 4 models showing PD. For rhabdomyosarcoma (n = 3) 2 MCR and 1 PD were noted for MGC018, while only PD was noted for SYD988. EFS duration exceeding 100 days to a single dose of MGC018 was observed for 2 rhabdomyosarcoma, 1 Ewing sarcoma, and 1 neuroblastoma model. Evaluation of the relationship between B7-H3 expression and response to MGC018 will be performed when results from all studies are completed. Conclusions: B7-H3 is an important target for immuno-oncology agents for childhood cancers. Our results provide proof-of-principle for the ability of MGC018 to produce profound antitumor activity in select pediatric solid tumor models in a B7-H3 specific manner.

Targeting B7-H3 via chimeric antigen receptor T cells and bispecific killer cell engagers augments antitumor response of cytotoxic lymphocytes

BackgroundB7-H3, an immune-checkpoint molecule and a transmembrane protein, is overexpressed in non-small cell lung cancer (NSCLC), making it an attractive therapeutic target. Here, we aimed to systematically evaluate the value of B7-H3 as a target in NSCLC via T cells expressing B7-H3-specific chimeric antigen receptors (CARs) and bispecific killer cell engager (BiKE)-redirected natural killer (NK) cells.MethodsWe generated B7-H3 CAR and B7-H3/CD16 BiKE derived from an anti-B7-H3 antibody omburtamab that has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. Antitumor efficacy and induced-immune response of CAR and BiKE were evaluated in vitro and in vivo. The effects of B7-H3 on aerobic glycolysis in NSCLC cells were further investigated.ResultsB7-H3 CAR-T cells effectively inhibited NSCLC tumorigenesis in vitro and in vivo. B7-H3 redirection promoted highly specific T-cell infiltration into tumors. Additionally, NK cell activity could be specially triggered by B7-H3/CD16 BiKE through direct CD16 signaling, resulting in significant increase in NK cell activation and target cell death. BiKE improved antitumor efficacy mediated by NK cells in vitro and in vivo, regardless of the cell surface target antigen density on tumor tissues. Furthermore, we found that anti-B7-H3 blockade might alter tumor glucose metabolism via the reactive oxygen species-mediated pathway.ConclusionsTogether, our results suggest that B7-H3 may serve as a target for NSCLC therapy and support the further development of two therapeutic agents in the preclinical and clinical studies.

NURS-02. CLINICAL MANAGEMENT OF PATIENTS RECEIVING CAR T CELL THERAPY FOR CNS TUMORS

Chimeric antigen receptor (CAR) T cells are an innovative new therapy with proven efficacy in some pediatric cancers such as leukemia and lymphoma, but much less experience in solid tumors, especially tumors of the central nervous system (CNS). Seattle Children’s has three open Phase 1 CAR T cell studies (BrainChild-01, -02, and -03 targeting HER2, EGFR, and B7-H3, respectively) for recurrent/refractory CNS tumors and DIPG (BrainChild-03 only). As of December 2019, four patients have been treated at Seattle Children’s Hospital with CAR T cells infused on a weekly schedule through indwelling catheters into the tumor resection cavity or ventricular system. Given the scrutiny of clinical care needed for Phase 1 studies, we are now able to report detailed clinical information that we have learned during the treatment of these patients. Clinical care includes the judicious use of steroids, the clinical support of patient’s symptoms pre- and post-infusion, and the management of peritumoral edema. We will also discuss the psychosocial support needed for families who travel long distances to receive this therapy compounded by the many emotional components of being enrolled on any Phase 1 trial. Case studies and experience from a Nurse Practitioner role will be provided and discussed.

Selected Articles from This Issue

B7-H3, a member of the B7 family of immune regulators, is often overexpressed in cancers. In this manuscript, Scribner and colleagues disclose MGC018, an antibody-drug conjugate (ADC) targeting B7-H3. MGC018 carries a duocarmycin payload linked to the humanized IgG1/kapp. monoclonal antibody. Their ADC showed antitumor activity in a range of patient-derived xenografts, including triple-negative breast cancer, prostate cancer, and head and neck squamous cell carcinoma. Preclinical results in this First Disclosure support the clinical development of MGC018 in solid cancers.There are many clinical and pathological similarities between human and canine mucosal melanomas. Two axes common to both species are the Ras/MAPK and PI3K/Akt/mTOR pathways. Wei and colleagues investigated the use of an MEK inhibitor (trametinib) previously investigated in canine models combined with the PI3K/mTOR inhibitor sapanisertib. The combined therapy reduced pS6/pERK, modified Bim/Bcl-xL expression, and limited tumor metastasis. Optimizing the dosage strategy required daily dose of trametinib and staggering the dose of sapanisertib. Taken together, the data indicates careful dose refinement is required for this combined approach and shows cytotoxicity in mucosal melanoma.Resistance to tyrosine kinase inhibitors and immunotherap. continues to be a concern for lung cancer patients. In this manuscript, Jin and colleagues exploit and observed overexpression in CD73 using a humanized anti-CD73 monoclonal antibody and antibody-drug conjugate (ADC). Treatment with the MMAE-bearing CD73-ADC generated anti-tumor responses against CD73-high tumors and led to accumulation of pro-inflammatory macrophages and dendritic cells. Their results underline the potential for the clinical development of CD73-ADC for CD73-dysregulated lung cancer patients.There is still a high need for solutions to recurrent, persistent, or metastatic cervical cancer. Anti-angiogenic therapies have shown promise, and continued translation requires predictive biomarkers of response. One cost-efficient biomarker that is well-suited to guide second-line therapy after anti-VEG therapy are circulating tumor cells (CTCs). Therefore, Tewari and colleagues investigated their association with survival and ability to predict anti-VEGF therapy. Patients above and below the median CTCs were stratified as those with and without bevacizumab. Bevacizumab therapy reduced the hazard of death and progression for patients with high pre-treatment CTCs. Therefore, CTCs may have prognostic significance in cervical cancer.