Abstract 5118: Neuroblastoma extracellular vesicles present GPC2 and activate GPC2 CAR T cells in an antigen-dependent manner
Authors: Giudice Anna M. , Matlaga Stephanie , Pascual-Pasto Guillem , Schuerch Patrick M. , Rouin Geoffrey , McIntyre Brendan , Zecchino Vincent P. , Bosse Kristopher R. 
Published: March 2024

Glypican 2 (GPC2) chimeric antigen receptor (CAR) T cells are safe and efficacious in neuroblastoma and are currently being tested in a first-in-human Phase 1 clinical trial at the Children’s Hospital of Philadelphia. Tumor-derived extracellular vesicles (TEVs) are nanosized vesicles secreted by cancer cells, often enriched with glypicans. However, the presence of GPC2 on neuroblastoma TEVs and any interaction with GPC2 CAR T cells has not been explored.

High-levels of GPC2 were found on TEVs isolated from neuroblastoma preclinical models [7 cell lines, circulating EVs from 6 patient-derived xenograft (PDX)-bearing mice] and from the peripheral blood of 16 neuroblastoma patients, but not on circulating EVs from 7 non-tumor bearing mice or 12 healthy human donors. The level of circulating GPC2+ EVs isolated from the peripheral blood of PDX-bearing mice positively correlated with PDX size (R=0.96; P&lt;0.0001) and parent PDX GPC2 expression. To evaluate how GPC2 on EVs modulates CAR T cell functionality, we generated EVs with varied levels of GPC2 (Hi/Lo/Neg) and co-incubated each EV subset with GPC2 CAR T cells. Here using immunofluorescence and flow assays we found that EVs bind GPC2 CAR T cells proportional to the amount of GPC2 on their surface. Further, GPC2Hi EV-CAR T cell synapses induced potent T cell activation shown by CD69/Granzyme B expression and release of IL-2/IFN-γ. GPC2Hi EV pre-incubated CAR T cells also displayed enhanced target neuroblastoma cell cytotoxicity, inducing 4-fold more specific cytotoxicity than CAR T cells incubated with control GPC2Neg EVs. In vivo we observed that injection of GPC2Hi EVs into GPC2Lo neuroblastoma SK-N-AS xenografts genetically altered to have significantly decreased endogenous EV production (via RAB27A knock-out) greatly enhanced the efficacy of co-infused GPC2 CAR T cells (p&lt;0.05). Similarly, in an isogenic SK-N-AS-GPC2 xenograft murine model treated with a limiting number of GPC2 CAR T cells concurrently with the EV-inhibiting drug GW4869, GPC2 CAR efficacy was dependent on the presence of GPC2Hi EVs. Here, pharmacologic EV inhibition in mice decreased GPC2 CAR efficacy which could be rescued by intratumoral injection of GPC2Hi EVs. In both in vivo models, the presence of intratumoral GPC2Hi EVs resulted in a significantly enhanced tumor infiltration of activated (CD69+/CD25+) GPC2 CAR T cells (p&lt;0.05).

Taken together, GPC2Hi EVs are selectively secreted from neuroblastomas and bind and activate GPC2 CAR T cells enhancing their anti-tumor cytotoxicity. To further capitalize on these findings, we engineered GPC2Hi EVs to have an extended circulation half-life through albumin binding or tumor targeting via GD2 binding that are currently being tested in vivo and results will be reported. EVs offer a versatile platform for CAR antigen presentation and should be further validated as a strategy to enhance CAR T cell efficacy for solid tumors.

Citation Format: Anna M. Giudice, Stephanie Matlaga, Guillem Pascual-Pasto, Patrick M. Schuerch, Geoffrey Rouin, Brendan McIntyre, Vincent P. Zecchino, Kristopher R. Bosse. Neuroblastoma extracellular vesicles present GPC2 and activate GPC2 CAR T cells in an antigen-dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5118.