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 ?/? T-cell receptors (TCR?/?). However, potential mispairing of introduced TCR?/?-chains with endogenous ?/?-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the V?-Linker-V?-fragment to the TCR C?-domain and coexpression of the TCR C?-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 TCR?. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCR?-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCR?/?-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 V?-Li-V?-fragment through the design of a novel disulfide bond between a V?- and a linker-resident residue close to V?. Multimer-stainings, and cytotoxicity-, IFN?-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCR?-formation without impairing avidity of scTCR/C? in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/C? inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCR?/?-positive T-cells.

SUBMITTER: Knies D 

PROVIDER: S-EPMC5008279 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature