Route of 41BB/41BBL Costimulation Determines Effector Function of B7-H3-CAR.CD28ζ T Cells

Abstract

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. In this study, we initially performed a detailed analysis of T cells expressing B7-H3-CARs with different hinge/transmembrane (CD8α versus 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 whether 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 one of four 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.

A Novel Orthotopic Implantation Technique for Osteosarcoma Produces Spontaneous Metastases and Illustrates Dose-Dependent Efficacy of B7-H3-CAR T Cells

“…We show that collagen-tumor cell scaffolds surgically implanted into the tibia of mice reliably produced local and systemic metastatic OS. Likewise, we demonstrate that CAR T cells targeting B7-H3, a tumor antigen that is expressed in a high percent of OS (15)(16)(17), have antitumor activity in a dose dependent fashion against primary and metastatic OS. Thus, the described model should be highly relevant for the preclinical evaluation and optimization of cell-based immunotherapies.…”

“…The OS cell line LM7, a derivative of SaOS-2, was kindly provided by Dr. Eugenie Kleinerman (MD Anderson Cancer Center, Houston, TX, USA) ( 18 ). LM7 cells expressing green fluorescent protein (GFP) and firefly luciferase (ffluc) (LM7.ffluc) previously generated in our laboratory were used for all experiments ( 15 ). LM7 was grown in DMEM (GE Healthcare, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (GE Healthcare) and 1% Glutamax (Thermo Fisher Scientific, Waltham, MA, USA).…”

B7-H3 Chimeric Antigen Receptor Redirected T Cells Target Anaplastic Lymphoma Kinase-Positive Anaplastic Large Cell Lymphoma

“…The internal signal domain chosen in this study was a 4-1BB signaling domain. Although it is has been shown that B7-H3.CAR-28ζ-Ts and B7-H3.CAR-BBζ-Ts showed equivalent antitumor properties in tumor models [ 17 ], incorporating 4-1BB signaling into the CAR molecular cellular domain could enhance CAR-T cell expansion in vivo in certain models [ 27 ]. This might account for the persistence of B7-H3 CAR-T cells upon repeated antigen stimulation.…”

CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality?

“…T cells express 41BB upon activation, and studies have demonstrated that expression of the 41BB ligand (41BBL) on the cell surface of CD28.z-CAR T cells endows CAR T cells with superior effector function in comparison to incorporating the 41BB signaling domain directly into the CAR. 55,190 Other ligands of the TNF superfamily have also been expressed on the cell surface of CAR T cells, including CD40L. 165 Lastly, investigators are actively exploring the activation of TLR pathways through inducible co-stimulatory molecules containing signaling domains of MyD88, 69,191 While common g-cytokines signal through JAK1/JAK2, and predominantly STAT5, IL-12 and IL-23 signal through JAK2, TYK2, and STAT3 and/or STAT4.…”