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.

Diana Knies,Wolfgang Herr,Hans Stauss,Matthias Theobald,Tana Omokoko,Oezlem Yildiz,Pedro Romero,Matthias Birtel,Shao-An Xue,Ugur Sahin,Ralf-Holger Voss,Sebastian Klobuch,Philippe Guillaume,Simone Thomas,Hakim Echchannaoui

doi:10.18632/oncotarget.8385

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.

Highlights

T-cell receptors (TCR) are immunglobulin-like folded membrane proteins expressed on CD4+ / CD8+ T-cells with the ability to recognize complexes of processed peptides associated with MHC-molecules on antigen presenting cells (APC)
By taking advantage of a silencing mutation in TCR CDR3α our results clearly demonstrated the incidence of TCR alpha-chain (TCRα)-mispairing with a 3-domain single chain TCR for a human and a mouse scTCR on a molecular basis in the absence of potentially interfering endogenous TCRα/β-chains
We assess the extent of hybrid TCR-formation for a single chain TCR format and identify specific modifications to those TCRs that reduce mispairing to negligible levels

Summary

Introduction

T-cell receptors (TCR) are immunglobulin-like folded membrane proteins expressed on CD4+ / CD8+ T-cells with the ability to recognize complexes of processed peptides associated with MHC-molecules on antigen presenting cells (APC). Current strategies in immunotherapy of cancer aim at equipping patients’ T-cells with tumor reactive TCRs by e.g. retroviral gene transfer ex vivo and to reinfuse them systemically. The heterologous overexpression of the TCR in T-cells necessitates optimization of the TCR framework to accomplish biochemical inertness against endogenous TCRs. since firstly, the TCR is a heterodimer comprising TCRα- and TCRβ-chain and secondly, the retroviral introduction of an exogenous TCR does not override expression of the endogenous TCR, the formation of mixed TCR chain pairing with unpredictable consequences on self-antigen recognition is a distinct possibility [2]. Neoreactivities which end up with autoimmunity [3, 4] may impose severe adverse reactions in adoptive TCR gene transfer-based clinical trials. Endogenous TCRs have been targeted in T-cells via sequence-specific siRNA-technology [10], or genomic editing by zinc finger nucleases [11], or TALENs [12]

Methods

Results

Conclusion