CD28 Co-Stimulus Achieves Superior CAR T Cell Effector Function against Solid Tumors Than 4-1BB Co-Stimulus.

Ana Textor,Annika Winkler,Angelika Eggert,Jana Rolff,Kathleen Anders,Maria Stecklum,Annette Künkele,Anton G Henssen,Uta E Höpken,Thomas Blankenstein,Anika Klaus,Johannes H Schulte,Michael C Jensen,Laura Grunewald,Silke Schwiebert

doi:10.3390/cancers13051050

Abstract

Simple SummaryEfficient trafficking and survival of CAR T cells within the hostile tumor microenvironment are important prerequisites for potent solid tumor attack that have not yet been achieved. We deployed monospecific murine instead of polyclonal human T cells for CAR T cell generation to evaluate second generation L1CAM- and HER2-specific CARs with different spacer length and either the CD28 or 4-1BB co-stimulatory domain in mouse models of neuroblastoma and ovarian carcinoma. This mouse-in-mouse approach ensured CAR T cell trafficking unhindered by species-specific discrepancies and demonstrated superior solid tumor attack by CAR T cells harboring the CD28 compared to 4-1BB co-stimulatory domain. Our approach has the potential to improve prediction and selection of promising clinical CAR candidates against solid tumors in the future.Spacer or co-stimulatory components in chimeric antigen receptor (CAR) design influence CAR T cell effector function. Few preclinical mouse models optimally support CAR candidate pre-selection for clinical development. Here we use a model in which murine CAR T cells can be exploited with human tumor xenografts. This mouse-in-mouse approach avoids limitations caused by species-specific factors crucial for CAR T cell survival, trafficking and function. We compared trafficking, expansion and tumor control for T cells expressing different CAR construct designs targeting two antigens (L1CAM or HER2), structurally identical except for spacer (long or short) or co-stimulatory (4-1BB or CD28) domains to be evaluated. Using monoclonal, murine-derived L1CAM-specific CAR T cells in Rag-/- mice harboring established xenografted tumors from a human neuroblastoma cell line revealed a clear superiority in CAR T cell trafficking using CD28 co-stimulation. L1CAM-targeting short spacer-CD28/ζ CAR T cells expanded the most at the tumor site and induced initial tumor regression. Treating patient-derived neuroblastoma xenografts with human L1CAM-targeting CAR T cells confirmed the superiority of CD28 co-stimulus. CD28 superiority was also demonstrated with HER2-specific CAR T cells (targeting ovarian carcinoma xenografts). Our findings encourage incorporating CD28 signaling into CAR design for adoptive T cell treatment of solid tumors.

Highlights

Chimeric antigen receptor (CAR) T cell therapy is a promising clinical approach in cancer treatment where patients’ own T cells are engineered to express a synthetic receptor that redirects specificity towards recognizing and killing cancer cells
After co-culture of murine L1CAM-specific chimeric antigen receptor (CAR) T cells with titrated numbers of L1CAM+ SK-N-BE(2) neuroblastoma cells (Supplementary Figure S2A), Ifng and Il2 cytokine release were quantified by enzyme-linked immunosorbent assay (ELISA) (Figure 1B)
The anti-cancer activity of HER2-specific CAR T cells with long spacers differed, with HER2-LS-28/ζ CAR T cells achieving slightly delayed tumor eradication in all mice and HER2-LS-4-1BB/ζ CAR T cells failing to induce any significant tumor regression. These findings demonstrate that HER2-specific CAR T cells with a short spacer element performed well, irrespective of co-stimulatory domain used, and that CAR designs benefit from CD28 co-stimulation in the ovarian tumor model

Summary

Introduction

Chimeric antigen receptor (CAR) T cell therapy is a promising clinical approach in cancer treatment where patients’ own T cells are engineered to express a synthetic receptor that redirects specificity towards recognizing and killing cancer cells. Due to limited efficacy in clinical trials, one (second generation) or two (third generation) co-stimulatory signaling domains (most often CD28 or/and 4-1BB) were added to improve CAR T cell persistence and, effector functions [1,2,3]. We use CAR T cells targeting the glycosylated CE7 epitope of the L1 cell adhesion molecule, L1CAM (formerly CD171), which is expressed on tumor cells and a promising target for neuroblastoma and ovarian cancer [5,6,7]. Children diagnosed with refractory neuroblastoma were treated with L1CAM-targeting CAR T cells harboring the 4-1BB co-stimulatory domain in a clinical phase I trial (NCT02311621, Available online: https:clinicaltrials.gov (accessed on 1 March 2021))

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