Secretion of 4-1BB Ligand Crosslinked to PD-1 Checkpoint Inhibitor Potentiates Chimeric Antigen Receptor T Cell Solid Tumor Efficacy.

Abstract

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of hematological malignancies but has yet to achieve similar success in solid tumors due to a lack of persistence and function in the tumor microenvironment. We previously reported the augmentation of CAR T cell therapy in an engineered solid tumor model through the secretion of anti-PD-1 single-chain fragment variable region (scFv), as shown by enhanced CAR T cell antitumor efficacy, expansion, and vitality. We have since improved the platform to create a superior cellular product-CAR T cells secreting single-chain trimeric 4-1BB ligand fused to anti-PD-1 scFv (αPD1-41BBL). 4-1BB signaling promotes cytotoxic T lymphocyte proliferation and survival but targeting 4-1BB with agonist antibodies in the clinic has been hindered by low antitumor activity and high toxicity. CAR T cells using 4-1BB endodomain for costimulatory signals have demonstrated milder antitumor response and longer persistence compared to CAR T cells costimulated by CD28 endodomain. We have, for the first time, engineered CD28-costimulated CAR T cells to secrete a fusion protein containing the soluble trimeric 4-1BB ligand. In vitro and in vivo, CAR19.αPD1-41BBL T cells exhibited reduced inhibitory receptor upregulation, enhanced persistence and proliferation, and a less differentiated memory status compared to CAR T cells without additional 4-1BB:4-1BBL costimulation. Accordingly, CAR19.αPD1-41BBL T cell-treated mice displayed significantly improved tumor growth control and overall survival. Spurred on by our preclinical success targeting CD19 as a model antigen, we produced mesothelin-targeting CAR T cells and confirmed the enhanced solid tumor efficacy of αPD1-41BBL-secreting CAR T cells.