TCRs Genetically Linked to CD28 and CD3ε Do Not Mispair with Endogenous TCR Chains and Mediate Enhanced T Cell Persistence and Anti-Melanoma Activity

Abstract

Adoptive transfer of T cells that are gene engineered to express a defined TCR represents a feasible and promising therapy for patients with tumors. However, TCR gene therapy is hindered by the transient presence and effectiveness of transferred T cells, which are anticipated to be improved by adequate T cell costimulation. In this article, we report the identification and characterization of a novel two-chain TCR linked to CD28 and CD3ε (i.e., TCR:28ε). This modified TCR demonstrates enhanced binding of peptide–MHC and mediates enhanced T cell function following stimulation with peptide compared with wild-type TCR. Surface expression of TCR:28ε depends on the transmembrane domain of CD28, whereas T cell functions depend on the intracellular domains of both CD28 and CD3ε, with IL-2 production showing dependency on CD28:LCK binding. TCR:28ε, but not wild-type TCR, induces detectable immune synapses in primary human T cells, and such immune synapses show significantly enhanced accumulation of TCR transgenes and markers of early TCR signaling, such as phosphorylated LCK and ERK. Importantly, TCR:28ε does not show signs of off-target recognition, as evidenced by lack of TCR mispairing, as well as preserved specificity. Notably, when testing TCR:28ε in immune-competent mice, we observed a drastic increase in T cell survival, which was accompanied by regression of large melanomas with limited recurrence. Our data argue that TCR transgenes that contain CD28, and, thereby, may provide T cell costimulation in an immune-suppressive environment, represent candidate receptors to treat patients with tumors.

Engineering Chimeric Antigen Receptor T-Cells for Racing in Solid Tumors: Don’t Forget the Fuel

“…Interestingly, similar to TCRs, increasing receptor-binding strength can augment T-cell function, but there is an affinity threshold beyond which there is no further gain in activity, and target density impacts CAR T-cell activation (79–81). Along with scFv, other tumor-binding moieties including an anti-integrin peptide α5β6 (82), heregulin (83), interleukin 13-zetakine (84), NKG2D (85), vasculature endothelial growth factor (VEGF) (86), and TCRs (87) have been incorporated into functional CARs. Finally, universal CARs, including an avidin ectodomain (88) and an anti-FITC scFv (89) for recognizing targets bound by biotinylated and FITC-labeled mAbs, respectively, have been developed.…”

Adoptive T-cell therapy: a need for standard immune monitoring

“…CAR T cells did not persist in the circulation for more than 30 days following infusion due to an immune response directed against the CAR construct [35][36][37]. Also another CAR T-cell trial in ovarian cancer patients showed a very short in vivo CAR T-cell persistence, and in addition this study revealed a lack of localization of 111 In-labeled CAR T cells to the tumor sites, except in one patient [38]. In contrast to the above two studies, Brenner et al showed prolonged in vivo persistence of a CAR that was introduced in Epstein-Barr virus (EBV)-specific T cells and emphasized the importance of costimulatory molecules that were more present on these EBV specific T cells.…”

“…However, progress in receptor design, such as costimulatory receptors [111] in combination with T-cell preparation toward optimally fit T cells [112], may eliminate the need for NMA and IL-2 and may make treatments more simplified and less of a burden to patients. Such developments would also allow monitoring of immune cells in the peripheral blood prior to and during T-cell treatment in search for parameters that are related to therapy outcome.…”

Antigen receptor-redirected T cells derived from hematopoietic precursor cells lack expression of the endogenous TCR/CD3 receptor and exhibit specific antitumor capacities

“…Cross-pairing may induce unexpected autoreactivity and GvHD as well as reduce the expression levels of the transgenic TCR 13,16 . In the approach described here, additional measures were taken to prevent interference with the tumor-specific TCR expression: The linked CD3ζ and CD28 signaling sequences force both transgenic chains to preferentially pair with each other and not with endogenous chains 17,20 . In addition, as these TCRs are surface-expressed independent of CD3, there is no competition for endogenous CD3 as the cytoplasm brightly stains for superfluous CD3 that cannot incorporate into the membrane due to lack of a TCR.…”