GPC2 antibody-drug conjugate reprograms the neuroblastoma immune milieu to enhance macrophage-driven therapies.

BACKGROUND: Antibody-drug conjugates (ADCs) that deliver cytotoxic drugs to tumor cells have emerged as an effective and safe anticancer therapy. ADCs may induce immunogenic cell death (ICD) to promote additional endogenous antitumor immune responses. Here, we characterized the immunomodulatory properties of D3-GPC2-PBD, a pyrrolobenzodiazepine (PBD) dimer-bearing ADC that targets glypican 2 (GPC2), a cell surface oncoprotein highly differentially expressed in neuroblastoma. METHODS: ADC-mediated induction of ICD was studied in GPC2-expressing murine neuroblastomas in vitro and in vivo. ADC reprogramming of the neuroblastoma tumor microenvironment was profiled by RNA sequencing, cytokine arrays, cytometry by time of flight and flow cytometry. ADC efficacy was tested in combination with macrophage-driven immunoregulators in neuroblastoma syngeneic allografts and human patient-derived xenografts. RESULTS: The D3-GPC2-PBD ADC induced biomarkers of ICD, including neuroblastoma cell membrane translocation of calreticulin and heat shock proteins (HSP70/90) and release of high-mobility group box 1 and ATP. Vaccination of immunocompetent mice with ADC-treated murine neuroblastoma cells promoted T cell-mediated immune responses that protected animals against tumor rechallenge. ADC treatment also reprogrammed the tumor immune microenvironment to a proinflammatory state in these syngeneic neuroblastoma models, with increased tumor trafficking of activated macrophages and T cells. In turn, macrophage or T-cell inhibition impaired ADC efficacy in vivo, which was alternatively enhanced by both CD40 agonist and CD47 antagonist antibodies. In human neuroblastomas, the D3-GPC2-PBD ADC also induced ICD and promoted tumor phagocytosis by macrophages, which was further enhanced when blocking CD47 signaling in vitro and in vivo. CONCLUSIONS: We elucidated the immunoregulatory properties of a GPC2-targeted ADC and showed robust efficacy of combination immunotherapies in diverse neuroblastoma preclinical models.

Development of GPC2-directed chimeric antigen receptors using mRNA for pediatric brain tumors.

BACKGROUND: Pediatric brain tumors are the leading cause of cancer death in children with an urgent need for innovative therapies. Glypican 2 (GPC2) is a cell surface oncoprotein expressed in neuroblastoma for which targeted immunotherapies have been developed. This work aimed to characterize GPC2 expression in pediatric brain tumors and develop an mRNA CAR T cell approach against this target. METHODS: We investigated GPC2 expression across a cohort of primary pediatric brain tumor samples and cell lines using RNA sequencing, immunohistochemistry, and flow cytometry. To target GPC2 in the brain with adoptive cellular therapies and mitigate potential inflammatory neurotoxicity, we used optimized mRNA to create transient chimeric antigen receptor (CAR) T cells. We developed four mRNA CAR T cell constructs using the highly GPC2-specific fully human D3 single chain variable fragment for preclinical testing. RESULTS: We identified high GPC2 expression across multiple pediatric brain tumor types including medulloblastomas, embryonal tumors with multilayered rosettes, other central nervous system embryonal tumors, as well as definable subsets of highly malignant gliomas. We next validated and prioritized CAR configurations using in vitro cytotoxicity assays with GPC2-expressing neuroblastoma cells, where the light-to-heavy single chain variable fragment configurations proved to be superior. We expanded the testing of the two most potent GPC2-directed CAR constructs to GPC2-expressing medulloblastoma and high-grade glioma cell lines, showing significant GPC2-specific cell death in multiple models. Finally, biweekly locoregional delivery of 2-4 million GPC2-directed mRNA CAR T cells induced significant tumor regression in an orthotopic medulloblastoma model and significantly prolonged survival in an aggressive orthotopic thalamic diffuse midline glioma xenograft model. No GPC2-directed CAR T cell related neurologic or systemic toxicity was observed. CONCLUSION: Taken together, these data show that GPC2 is a highly differentially expressed cell surface protein on multiple malignant pediatric brain tumors that can be targeted safely with local delivery of mRNA CAR T cells, laying the framework for the clinical translation of GPC2-directed immunotherapies for pediatric brain tumors.

Serial Profiling of Circulating Tumor DNA Identifies Dynamic Evolution of Clinically Actionable Genomic Alterations in High-Risk Neuroblastoma.

Neuroblastoma evolution, heterogeneity, and resistance remain inadequately defined, suggesting a role for circulating tumor DNA (ctDNA) sequencing. To define the utility of ctDNA profiling in neuroblastoma, 167 blood samples from 48 high-risk patients were evaluated for ctDNA using comprehensive genomic profiling. At least one pathogenic genomic alteration was identified in 56% of samples and 73% of evaluable patients, including clinically actionable ALK and RAS-MAPK pathway variants. Fifteen patients received ALK inhibition (ALKi), and ctDNA data revealed dynamic genomic evolution under ALKi therapeutic pressure. Serial ctDNA profiling detected disease evolution in 15 of 16 patients with a recurrently identified variant-in some cases confirming disease progression prior to standard surveillance methods. Finally, ctDNA-defined ERRFI1 loss-of-function variants were validated in neuroblastoma cellular models, with the mutant proteins exhibiting loss of wild-type ERRFI1's tumor-suppressive functions. Taken together, ctDNA is prevalent in children with high-risk neuroblastoma and should be followed throughout neuroblastoma treatment. SIGNIFICANCE: ctDNA is prevalent in children with neuroblastoma. Serial ctDNA profiling in patients with neuroblastoma improves the detection of potentially clinically actionable and functionally relevant variants in cancer driver genes and delineates dynamic tumor evolution and disease progression beyond that of standard tumor sequencing and clinical surveillance practices. See related commentary by Deubzer et al., p. 2727. This article is highlighted in the In This Issue feature, p. 2711.

A GPC2 antibody-drug conjugate is efficacious against neuroblastoma and small-cell lung cancer via binding a conformational epitope.

Glypican 2 (GPC2) is a MYCN-regulated, differentially expressed cell-surface oncoprotein and target for immune-based therapies in neuroblastoma. Here, we build on GPC2's immunotherapeutic attributes by finding that it is also a highly expressed, MYCN-driven oncoprotein on small-cell lung cancers (SCLCs), with significantly enriched expression in both the SCLC and neuroblastoma stem cell compartment.By solving the crystal structure of the D3-GPC2-Fab/GPC2 complex at 3.3 Å resolution, we further illustrate that the GPC2-directed antibody-drug conjugate (ADC; D3-GPC2-PBD), that links a human GPC2 antibody (D3) to DNA-damaging pyrrolobenzodiazepine (PBD) dimers, binds a tumor-specific, conformation-dependent epitope of the core GPC2 extracellular domain. We then show that this ADC induces durable neuroblastoma and SCLC tumor regression via induction of DNA damage, apoptosis, and bystander cell killing, notably with no signs of ADC-induced in vivo toxicity. These studies provide preclinical data to support the clinical translation of ADCs targeting GPC2.