Single-chain VαVβ T-cell receptors function without mispairing with endogenous TCR chains

Abstract

Transduction of exogenous T cell receptor (TCR) genes into patients’ activated peripheral blood T cells is a potent strategy to generate large numbers of specific T cells for adoptive therapy of cancer and viral diseases. However, the remarkable clinical promise of this powerful approach is still being overshadowed by a serious potential consequence: mispairing of the exogenous TCR chains with endogenous TCR chains. These “mixed” heterodimers can generate new specificities that result in graft-versus-host reactions. Engineering TCR constant regions of the exogenous chains with a cysteine promotes proper pairing and reduces the mispairing, but, as we show here, does not eliminate the formation of mixed heterodimers. By contrast, deletion of the constant regions, through use of a stabilized Vα/Vβ single-chain TCR (scTv), avoided mispairing completely. By linking a high-affinity scTv to intracellular signaling domains, such as Lck and CD28, the scTv was capable of activating functional T cell responses in the absence of either the CD3 subunits or the co-receptors, and circumvented mispairing with endogenous TCRs. Such transduced T cells can respond to the targeted antigen independent of CD3 subunits via the introduced scTv, without the transduced T cells acquiring any new undefined and potentially dangerous specificities.

T-cells “à la CAR-T(e)” – Genetically engineering T-cell response against cancer

“…One can also swap residues [49] or entire domains [50] between the α and β chains to promote exquisite pairing. To overcome the competition for the CD3ζ chains between endogenous and exogenous TCRs, other strategies were elaborated such as the generation of chimeric TCR fused to the intra cellular region of CD3ζ [51] or of a single-chain TCR in which the variable α and β portions are linked (similarly to scFv) [52]. Alternatively, one can also knock down the expression of the endogenous TCR using shRNA or RNAi [53,54] zing fingers [55],TALEN [56] or more recently CRISPR/Cas9 systems [57,58] ( Fig.…”

A chimeric antigen receptor for TRAIL-receptor 1 induces apoptosis in various types of tumor cells

“…The TR1-scFv-CAR-expression vectors were constructed as previously described [11][12][13]. To construct the cytoplasmic domain of CAR, CD137 (4-1BB) cDNA was first isolated from human PBLs; human CD3f, CD8a and CD28 cDNAs were purchased from OriGene Technologies (Rockville, MD, USA).…”

Clinical translation and regulatory aspects of CAR/TCR-based adoptive cell therapies—the German Cancer Consortium approach

“…However, in humans this has not been observed so far. Moreover, a number of approaches have been developed to reduce the risk for mispairing, including the introduction of additional cysteine bonds, murinization, also encompassing potentially less immunogenic minimal murine sequences of constant regions, and the construction of single-chain TCRs by fusing the introduced alpha-and beta-chains [46][47][48][49][50]. Another special feature of TCRs is the binding affinity which is much lower as compared to antibodies [51].…”