Synergistic T cell signaling by 41BB and CD28 is optimally achieved by membrane proximal positioning within parallel chimeric antigen receptors

Abstract

Summary Second generation (2G) chimeric antigen receptors (CARs) contain a CD28 or 41BB co-stimulatory endodomain and elicit remarkable efficacy in hematological malignancies. Third generation (3G) CARs extend this linear blueprint by fusing both co-stimulatory units in series. However, clinical impact has been muted despite compelling evidence that co-signaling by CD28 and 41BB can powerfully amplify natural immune responses. We postulate that effective dual co-stimulation requires juxta-membrane positioning of endodomain components within separate synthetic receptors. Consequently, we designed parallel (p)CARs in which a 2G (CD28+CD3ζ) CAR is co-expressed with a 41BB-containing chimeric co-stimulatory receptor. We demonstrate that the pCAR platform optimally harnesses synergistic and tumor-dependent co-stimulation to resist T cell exhaustion and senescence, sustaining proliferation, cytokine release, cytokine signaling, and metabolic fitness upon repeated stimulation. When engineered using targeting moieties of diverse composition, affinity, and specificity, pCAR T cells consistently elicit superior anti-tumor activity compared with T cells that express traditional linear CARs.

Engineering of an Avidity-Optimized CD19-Specific Parallel Chimeric Antigen Receptor That Delivers Dual CD28 and 4-1BB Co-Stimulation

“…We have recently demonstrated superior anti-tumor function of pCAR T-cells, in which a CD28-containing 2G CAR is co-expressed with a 4-1BB-containing CCR ( 10 ). To test the applicability of the pCAR platform to targeting of CD19, we co-expressed each of the mutant scFv-based 2G CARs described above with a 4-1BB CCR that contains an unmodified FMC63 scFv.…”

“…Cytolytic activity of all pCAR T-cell populations was similar or superior compared to the corresponding mutant scFv-based 2G CAR, or the F-2 CAR ( Figure 4B ). We have previously shown that a docking effect of the CCR can potentiate cytolytic activity of some, but not all pCARs, when compared to the parental CAR ( 10 ). Analysis of immune synapse formation by these T-cell populations could help to uncover mechanisms that may explain this variable response of pCAR T-cells.…”

“…Moreover, we also observed that pCAR T-cells outperformed the combination of a 1G CAR and a dual CD28 + 4-1BB CCR ( 12 ), potentially consistent with the importance of membrane proximity in effective co-stimulation. In mice in which pCAR T-cells achieved complete tumor rejection, enhanced functional persistence of these cells was confirmed by successful rejection of a delayed tumor rechallenge ( 10 ).…”

“…We have previously shown that CAR T-cell co-stimulation both by CD28 and 4-1BB is optimally delivered by two separate fusion receptors. To leverage this, we engineered parallel (p)CARs in which a CD28-containing 2G CAR is co-expressed with a 4-1BB containing chimeric co-stimulatory receptor (CCR) ( 10 ). We observed that pCAR T-cells demonstrate more durable activity in several tumor models, indicated by enhanced proliferation, cytokine release and cytolytic function and lowered expression of exhaustion and senescence markers.…”

Cancer-Homing CAR-T Cells and Endogenous Immune Population Dynamics

“…Dual CAR design can also be exploited to enhance signaling by 4-1BB and CD28 domains, which is best achieved by the membrane proximal positioning of both signaling units, within separate, parallel CAR [ 88 ].…”