Immune Checkpoint Blockade via PD-L1 Potentiates More CD28-Based than 4-1BB-Based Anti-Carbonic Anhydrase IX Chimeric Antigen Receptor T Cells

Abstract

Introduction: Chimeric antigen receptor (CAR) T cells are capable to be activated directly when in contact with a specific tumor antigen, becoming a new potent strategy against cancer. The complete regression of clear cell renal cell carcinoma (ccRCC) obtained pre-clinically with anti-carbonic anhydrase IX (CAIX) G36 CAR T cells in doses equivalent to ≅10 8 CAR T cells/kg renewed the potential of this target to treat ccRCC and other tumors in hypoxia. The immune checkpoint blockade (ICB) brought durable clinical responses for ccRCC in adjuvant settings and metastatic scenarios, becoming an important pillar treatment.Objectives: This project tested CD8α/4-1BB compared to CD28-based anti-CAIX CAR T cells releasing anti-programmed cell death ligand-1 (PD-L1) IgG4 for human ccRCC treatment in vitro and in an orthotopic NSG mice model in vivo. Methodology:Lentiviruses containing the different CAR constructions to be tested were produced, concentrated, and the transduction efficiency was determined by flow cytometry and IgG secretion. The cytotoxic effects of anti-CAIX CAR T were analyzed by flow cytometry and lactate dehydrogenase activity. The secretion of IL-2 and IFNγ was determined by ELISA. The exhaustion status was determined by flow cytometry. Alanine (ALT) and aspartate (AST) transaminases activity was determined by spectrophotometry.