# Multi-parametric profiling of IL-7-augmented GD2.CART products in a phase 1 clinical trial

## TL;DR

This study uses advanced flow cytometry to profile GD2-directed CAR T cells in a clinical trial, finding that adding an IL-7 receptor improves their function and effectiveness in treating brain tumors.

## Contribution

The study introduces a 33-color spectral cytometry approach to profile CAR T cell products and demonstrates how co-expressing C7R improves GD2.CART performance.

## Key findings

- C7R co-expression enhances GD2.CART activation, cytotoxicity, and infiltration.
- CD8+ T cell clusters associated with clinical responses were identified through unsupervised clustering.
- Spectral cytometry profiling reveals key determinants of CAR T cell therapy efficacy.

## Abstract

CAR T cell (CART) therapy holds promise for cancer treatment, but heterogeneity among products limits clinical effectiveness, making systematic profiling essential to identify predictors of success. Recently, a phase 1 clinical trial investigated whether a constitutively active IL-7 receptor (C7R) could safely improve the function and persistence of GD2-directed CARTs (GD2.CARTs) in pediatric patients with high-grade CNS tumors. We analyzed infusion products from trial participants using a custom-designed 33-color full spectrum flow cytometry (FSFC) panel combined with an image-based tumor killing assay to characterize CART products and evaluate the impact of C7R on GD2.CART performance. Patient-specific variations in T cell composition were linked to therapeutic success, with C7R co-expression enhancing the functional phenotype of GD2.CARTs compared to CAR-only products. Unsupervised clustering identified CD8+ T cells associated with clinical responses, marked by activation, infiltration, resilience, and cytotoxicity. Our FSFC-based profiling approach reveals determinants of CART efficacy and supports strategies to optimize adoptive immunotherapy.

•33-marker spectral cytometry enables high-dimensional CART product profiling•C7R co-expression enhances GD2.CART activation, cytotoxicity, and infiltration•Distinct CD8+ T cell clusters are enriched in products linked to clinical response•Spectral cytometry-based profiling approach reveals determinants of CART efficacy

33-marker spectral cytometry enables high-dimensional CART product profiling

C7R co-expression enhances GD2.CART activation, cytotoxicity, and infiltration

Distinct CD8+ T cell clusters are enriched in products linked to clinical response

Spectral cytometry-based profiling approach reveals determinants of CART efficacy

Health sciences; Immunology; Laboratory medicine; Medicine; Oncology