Targeting CAR to the Peptide-MHC Complex Reveals Distinct Signaling Compared to That of TCR in a Jurkat T Cell Model

Abstract

Chimeric antigen receptor T cells (CAR-T) utilize T cell receptor (TCR) signaling cascades and the recognition functions of antibodies. This allows T cells, normally restricted by the major histocompatibility complex (MHC), to be redirected to target cells by their surface antigens, such as tumor associated antigens (TAAs). CAR-T technology has achieved significant successes in treatment of certain cancers, primarily liquid cancers. Nonetheless, many challenges hinder development of this therapy, such as cytokine release syndrome (CRS) and the efficacy of CAR-T treatments for solid tumors. These challenges show our inadequate understanding of this technology, particularly regarding CAR signaling, which has been less studied. To dissect CAR signaling, we designed a CAR that targets an epitope from latent membrane protein 2 A (LMP2 A) of the Epstein–Barr virus (EBV) presented on HLA*A02:01. Because of this, CAR and TCR signaling can be compared directly, allowing us to study the involvement of other signaling molecules, such as coreceptors. This comparison revealed that CAR was sufficient to bind monomeric antigens due to its high affinity but required oligomeric antigens for its activation. CAR sustained the transduced signal significantly longer, but at a lower magnitude, than did TCR. CD8 coreceptor was recruited to the CAR synapse but played a negligible role in signaling, unlike for TCR signaling. The distinct CAR signaling processes could provide explanations for clinical behavior of CAR-T therapy and suggest ways to improve the technology.

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

“…It has been reported that CAR T-cells form nonclassical immunological synapses, unlike the typical bulls-eye signature of central, peripheral, and distal supramolecular activation complexes that are observed between αβ T cells and their targets . Ligand-induced receptor dimerization was reported for CAR T-cells targeting soluble ligands. , CAR signaling was found to be unique and different from that of TCRs and even distinct among various CAR constructs. , Wu and co-workers compared bulk activation of TCR T-cells and CAR T-cells targeting LMP2/A*0201, revealing that although CAR binds the monomeric antigen due to its high affinity, it requires multimeric antigen for activation. CAR calcium flux reached and sustained a plateau, whereas for TCR it decreased after reaching a peak.…”

“…40,41 CAR signaling was found to be unique and different from that of TCRs and even distinct among various CAR constructs. 9,42 Wu and coworkers compared bulk activation of TCR T-cells and CAR Tcells targeting LMP2/A*0201, 19 revealing that although CAR binds the monomeric antigen due to its high affinity, it requires multimeric antigen for activation. CAR calcium flux reached and sustained a plateau, whereas for TCR it decreased after reaching a peak.…”

“…Although CD8 coreceptor was recruited to the CAR synapse, its role in calcium flux was not significant, unlike for the TCR. 19 Switchable dual receptor CAR-T cells (sdCAR-T) were found to be more effective than conventional CAR T-cells against solid tumors. 43 Redirection and regulation of CAR Tcell activity were shown by using a bifunctional small molecule "switch".…”

“…We sought to characterize the nature of the CAR–pMHC bonds with direct comparison to the TCR–pMHC bond at the single molecule level. TCR and CARs were produced and displayed on Jurkat76 cells as described (Figures S1–S4). The SMSC assay permits direct lifetime measurement of a single TCR/CAR-pMHC bond as a function of force by using optical tweezers to pull directly on a bead tethered via DNA linker to pMHC bound to a single receptor decorating the surface of a coverslip-affixed cell (Figure A). , Note that pMHC is linked to the DNA strand by a half-antibody to ensure an individual interaction.…”

Application and Design of Switches Used in CAR

“…Another key factor was the observation of MHC-restricted antigen recognition, a phenomenon that makes T cell receptors (TCRs) require not only an antigen but also an MHC molecule to activate them (Doherty and Zinkernagel, 1996) [ 8 ]. The chimeric antigen receptor is designed to recognize antigens independently of the MHC, which greatly increases its effectiveness in killing, especially when the tumor cell lacks an MHC [ 9 ]. The MHC is also important in distinguishing between self and foreign cells.…”