Route of 41BB/41BBL costimulation determines effector function of B7-H3-CAR.CD28ζ T cells

B7-H3 is actively being explored as an immunotherapy target for pediatric patients with solid tumors using monoclonal antibodies or T cells expressing chimeric antigen receptors (CARs). B7-H3-CARs containing a 41BB costimulatory domain are currently favored by several groups based on preclinical studies. Here we initially performed a detailed analysis of T cells expressing B7-H3-CARs with different hinge/transmembrane (CD8α vs CD28) and CD28 or 41BB costimulatory domains (CD8α/CD28, CD8α/41BB, CD28/CD28, CD28/41BB). Only subtle differences in effector function were observed between CAR T-cell populations in vitro. However, CD8α/CD28-CAR T cells consistently outperformed other CAR T-cell populations in three animal models, resulting in a significant survival advantage. We next explored if adding 41BB signaling to CD8α/CD28-CAR T cells would further enhance effector function. Surprisingly, incorporating 41BB signaling into the CAR endodomain had detrimental effects, while expressing 41BBL on the surface of CD8α/CD28-CAR T cells enhanced their ability to kill tumor cells in repeat stimulation assays. Furthermore, 41BBL expression enhanced CD8α/CD28-CAR T-cell expansion in vivo and improved antitumor activity in 1 of 4 evaluated models. Thus, our study highlights the intricate interplay between CAR hinge/transmembrane and costimulatory domains. Based on our study, we selected CD8α/CD28-CAR T cells expressing 41BBL for early phase clinical testing.

Learning Objectives:

1. Understand B7-H3 as a promising immunotherapy target found at high levels on many tumor types

2. Identify Optimal CAR design as a determined empirically for each new construct