Single Molecule Force Spectroscopy Reveals Distinctions in Key Biophysical Parameters of αβ T-Cell Receptors Compared with Chimeric Antigen Receptors Directed at the Same Ligand

Abstract

Chimeric antigen receptor (CAR) T-cell therapies exploit facile antibodymediated targeting to elicit useful immune responses in patients. This work directly compares binding profiles of CAR and αβ T-cell receptors (TCR) with single cell and single molecule optical trap measurements against a shared ligand. DNA-tethered measurements of peptide-major histocompatibility complex (pMHC) ligand interaction in both CAR and TCR exhibit catch bonds with specific peptide agonist peaking at 25 and 14 pN, respectively. While a conformational transition is regularly seen in TCR−pMHC systems, that of CAR− pMHC systems is dissimilar, being infrequent, of lower magnitude, and irreversible. Slip bonds are observed with CD19-specific CAR T-cells and with a monoclonal antibody mapping to the MHC α2 helix but indifferent to the bound peptide. Collectively, these findings suggest that the CAR−pMHC interface underpins the CAR catch bond response to pMHC ligands in contradistinction to slip bonds for CARs targeting canonical ligands.

Catch bond models may explain how force amplifies TCR signaling and antigen discrimination

“…1b). Since the TCRβ subunit has also been proposed to undergo FG-loop-regulated conformational change 6,24,25 , we assume that disruption of the α 1 α 2 – β 2m joint would also result in tilting of the TCR–pMHC bonding interface and shifting of the mechanical load from the TCR Vβ–Cβ joint to the Vα–Cα joint, leading to partial unfolding of the Vα–Cα joint (Fig. 1b).…”

“…Besides binding properties of the TCR–pMHC complex, its structural features and conformational changes have been suggested to be important for TCR triggering. For example, TCR–pMHC docking orientation has been correlated to its ability to trigger T cell signaling 15,22,23 .Partial unfolding or allosteric regulation of either the TCR and/or MHC molecules has been inferred from mechanical experiments and steered molecular dynamics (SMD) simulations of pulling single TCR–pMHC bonds 6,8,24,25 . Whereas the extent of these conformational changes has been correlated to the strength of TCR–pMHC catch bonds 6,8,24 , the two phenomena have not been integrated into a mathematical model to explore their potential connection.…”

“…Partial unfolding or allosteric regulation of either the TCR and/or MHC molecules has been inferred from mechanical experiments and steered molecular dynamics (SMD) simulations of pulling single TCR–pMHC bonds 6,9,25,26 . Whereas the extent of these conformational changes has been correlated to the strength of TCR–pMHC catch bonds 6,9,25 , the two phenomena have not been integrated into a mathematical model to explore their potential connection.…”

CD28-CAR-T cell activation through FYN kinase signaling rather than LCK enhances therapeutic performance

“… 12 , 13 We used CHO cells expressing single-chain peptide MHC (pMHC) as antigen-presenting cells (APCs) ( Figures S1 B and S1C). 14 We found that anti-pMHC CARs have similar biophysical properties to TCR 15 but that the CD8 co-receptor is dispensable for CAR signaling. 14 CD8 is typically considered to be important for bringing LCK into the IS.…”

TCR-mimic bispecific antibodies to target the HIV-1 reservoir

“…Whether scDb are able to activate CTLs better than natural recognition by αβTCRs remains to be seen. Contrary to conventional receptor–ligand interactions exemplified by antigen–antibody binding, bioforces are essential for nonthermal equilibrium, mechanosensor-based αβT cell activation ( 81 – 90 ). αβT cell motility during immune surveillance and the local cytoskeletal machinery place physical load on individual αβTCR-pMHC bonds, tuning the sensitivity and specificity of αβTCR recognition ( 82 ).…”