Final optimized junction for dsTCRcp is usually shown in Supplemental Figure 2

Final optimized junction for dsTCRcp is usually shown in Supplemental Figure 2. DOI: Figure 2figure product 2. Open in a separate window Optimization of domain-swapped junctions for dsTCRV and dsTCRcp.(A) Amino acid sequences of F5 TCR V-C and V-C domain boundaries are shown. trials (Bendle et al., 2010; Bunse, 2014). Although no severe adverse events have been attributed to TCR mispairing in designed T cell trials (Rosenberg, 2010), autoreactive off-target and off-tumor designed T cell responses have caused deaths (Linette et al., 2013; Morgan et al., 2013, 2010). These underscore the need to safeguard against TCR mispairing-related autoreactivity, particularly as more potent immunotherapy regimes are employed. Efforts to prevent TCR mispairing can be broadly categorized as either engineering the transduced TCR (adding interchain disulfide bonds, murinizing portions of the TCR, expressing TCR as a single chain) (Uckert and Schumacher, 2009) or reducing expression of the endogenous TCR (shRNA knockdown (Bunse, 2014; Okamoto et al., 2009) or genomic knockout [Provasi et al., 2012]). Although several engineering strategies improve pairing between the transduced chains, total prevention of mispairing has not been achieved (Thomas et al., 2007) BIO-1211 and murine TCRs are immunogenic (Davis, 2010). Endogenous TCR knockout prevents mispairing, but the considerable processing currently required to generate these cells is usually incompatible with clinical protocols. The ideal answer will prevent mispairing entirely, eliminating the risk of autoimmunity. Additionally, modifications made to the launched TCR chains should avoid foreign sequences to minimize immunogenicity. Finally, these modifications must be restricted to the constant TCR domains, such that they can be applied without further optimization to any TCR of therapeutic Rabbit polyclonal to ADCYAP1R1 interest. We describe a novel approach for preventing TCR mispairing that meets these criteria. We show that this approach is usually further improved by combining it with the complementary strategy of endogenous TCR knockdown. Results Domain-swapped BIO-1211 TCR (dsTCR) design Our approach to prevent TCR mispairing exploits the molecular requirements for TCR biogenesis and function. The TCR and chains each contain a membrane-distal variable immunoglobulin domain name (V), which imparts specificity, and several constant domains including a membrane-proximal constant immunoglobulin domain name (C), a connecting BIO-1211 peptide (cp), a transmembrane helix (TM), and a short cytoplasmic tail (cyto) (Physique 1A). To achieve functional form, the TCR heterodimer must assemble with six additional chains (CD3 dimers , , and 2), which facilitate export of the TCR complex to the cell surface and mediate signal transduction upon antigen binding (Call and Wucherpfennig, 2005). If the TCR/CD3 complex is not put together properly prior to export, it is degraded (Bonifacino, 1989). Assembly with CD3 requires contacts within the constant domains of both the TCR and chains (Call et al., 2002; Kuhns and Davis, 2007; Xu and Call, 2006), most critically the basic residues within the transmembrane domains (Call et al., 2002)(Physique 1B). We designed interchain domain-swapped (ds) TCRs in which select constant domains of the TCR and chains are exchanged in a reciprocal manner (Physique 1C). Correctly paired / dsTCRs maintain all domains necessary to assemble with CD3 and to enact tumor-targeted immunity. By contrast, mispaired heterodimers comprising one dsTCR chain and one wild-type (wt) TCR chain lack domains necessary to assemble with CD3 or to enact autoimmune responses (Physique 1d). Open in a separate window Physique 1. Schematic outlining the domain-swapped TCR strategy.(A) The TCR/CD3 complex comprises the antigen-specific variable (V) Ig domain name and constant domains (constant Ig, C; connecting peptide, cp; transmembrane helix, TM; and cytoplasmic tail, cyto), which assemble with BIO-1211 CD3 chains. CD3 chains are required for export of the TCR/CD3 complex to the cell surface and for signaling. Parallel horizontal lines represent the cell membrane. (B) Schematic showing key interactions.