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

Abstract

BackgroundAntibody–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.MethodsADC-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.ResultsThe 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.ConclusionsWe elucidated the immunoregulatory properties of a GPC2-targeted ADC and showed robust efficacy of combination immunotherapies in diverse neuroblastoma preclinical models.

Targeted Drug Delivery by MMAE Farnesyl-Bioconjugated Multivalent Chemically Self-Assembled Nanorings Induces Potent Receptor-Dependent Immunogenic Cell Death

“…The potential for cytotoxic drugs to enhance their antitumor response through immunologic cell death (ICD) is a growing area of therapeutic interest since it combines a cytotoxic response with immunological activation toward the tumor cells . A number of cytotoxic drugs including ADCs have been found to induce ICD and thus in combination with immunomodulatory agents, such as anti-PD-1 antibodies, result in an enhanced and durable level of tumor elimination at lower doses of ADCs. − Similar to ADCs bearing MMAE, E1-DHFR 2 -MMAE CSANs were shown to induce the hallmarks of ICD . A431 cells cocultured with T-cells and treated with E1-DHFR 2 -MMAE CSANs resulted in enhanced expression of CD25 by the T-cells, which is a marker of T-cell activation.…”

Blocking MIF secretion enhances CAR T-cell efficacy against neuroblastoma

“…CD19.CAR was designed using a CD8α leader, followed by a scFv derived from the FMC63 antibody, a CD8 hinge and transmembrane domain, and a 4-1BB and CD3-ζ co-stimulatory domains. DNA transfections, lentivirus production using second and third-generation lentiviral systems and virus transductions were performed as previously described 61 .…”

“…3b, Supplement Fig 3b). We next used CAR T-cells targeting GPC2, which have shown robust safety and efficacy in diverse preclinical models of neuroblastoma and are currently being tested clinically (NCT05650749), to further investigate the roles of MIF and MDK in CAR T-cell modulation 44,60,61 . To study this interaction, we generated neuroblastoma cell lines with genetic depletion of MDK (SK-N-AS-shMDK) and MIF (SKNBE2C-shMIF) using shRNAmediated silencing (Supplement Fig.…”

Clinical application of immunogenic cell death inducers in cancer immunotherapy: turning cold tumors hot

“…Similarly, the immunogenicity-inducing effects of another ADC drug targeting aberrantly expressed oncoproteins GPC2 (D3-GPC2-PBD) were explored. When applied to GPC2-expressing murine neuroblastomas, the drug caused changes in ICD markers such as CRT and HSP70/90, modulated the microenvironment, and improved macrophage phagocytosis, which was enhanced when CD47 was blocked by the combination ( Pascual-Pasto et al, 2022 ). These examples suggest that ADCs are powerful ICD inducers with a promising future in combination therapies.…”