An optimized single chain TCR scaffold relying on the assembly with the native CD3-complex prevents residual mispairing with endogenous TCRs in human T-cells

Abstract

Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/β T-cell receptors (TCRα/β). However, potential mispairing of introduced TCRα/β-chains with endogenous β/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vβ-fragment to the TCR Cβ-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/β-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vβ-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vβ. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/β-positive T-cells.

Persistent donor derived Vδ4 T cell clones may improve survival for recurrent T cell acute lymphoblastic leukemia after HSCT and DLI

“…These results may support that the donor derived Vδ4 T cell clone we identified here may be specific to any T-ALL antigen and work efficiently for lysing T-ALL blasts. For confirming the function of Vδ4 T cells, TCR γδ gene could be cloned from this Vδ4 T cells and transferred to healthy T cells to detect the specific cytotoxicity [28, 29]. If the anti-leukemia effect could be confirmed, such clonally expanded Vδ4 T cells in this case may be considered as specific DLI to avoid GvHD development.…”

Engineering antigen‐specific NK cell lines against the melanoma‐associated antigen tyrosinase via TCR gene transfer

“…Tyrosinase (368)(369)(370)(371)(372)(373)(374)(375)(376)(377) and Melan-A/Mart-1 (27)(28)(29)(30)(31)(32)(33)(34)(35) peptides as well as APC conjugated Tyrosinase (368-377) /HLA-A2 pentamers were purchased from ProImmune Ltd. For Pentamer labeling, 0.5 × 10 6 NK-92 cells were washed once with PBS and incubated with 10 μL of Tyrosinase (368-377) /HLA-A2 pentamers at 4°C for 30 min. The labeled cells were then washed twice with PBS and analyzed by flow cytometry.…”

Clustering of CD3ζ is sufficient to initiate T cell receptor signaling

“…To verify if LIC-CD3z was signaling competent, we first investigated whether CD3ζ clustering was sufficient to trigger downstream signaling events, measured here as Ca 2+ fluxes. We transfected LIC-CD3z into αβTCR-deficient T cells, Jurkat 76 cells, that have essentially no endogenous CD3 expression on the cell surface (Heemskerk et al, 2003, Knies et al, 2016. Thus, any signaling exhibited in these cells would be restricted to LIC-CD3z and would not involve other components of the TCR complex.…”

“…The exact mechanisms of how ligand binding to the extracellular domain of the αβTCR triggers CD3 phosphorylation of the intracellular motif remains unclear. Ligand binding may induce conformational changes (Gil et al, 2002, Lee et al, 2015 to facilitate coordinated re-arrangements of the TCR-CD3 subunits. However, conformational changes cannot explain TCR signaling induced by antibodies or triggering of simpler versions of the TCR such as chimeric antigen receptors (CARs).…”