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

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