Computational Design of the Affinity and Specificity of a Therapeutic T Cell Receptor

Abstract

T cell receptors (TCRs) are key to antigen-specific immunity and are increasingly being explored as therapeutics, most visibly in cancer immunotherapy. As TCRs typically possess only low-to-moderate affinity for their peptide/MHC (pMHC) ligands, there is a recognized need to develop affinity-enhanced TCR variants. Previous in vitro engineering efforts have yielded remarkable improvements in TCR affinity, yet concerns exist about the maintenance of peptide specificity and the biological impacts of ultra-high affinity. As opposed to in vitro engineering, computational design can directly address these issues, in theory permitting the rational control of peptide specificity together with relatively controlled increments in affinity. Here we explored the efficacy of computational design with the clinically relevant TCR DMF5, which recognizes nonameric and decameric epitopes from the melanoma-associated Melan-A/MART-1 protein presented by the class I MHC HLA-A2. We tested multiple mutations selected by flexible and rigid modeling protocols, assessed impacts on affinity and specificity, and utilized the data to examine and improve algorithmic performance. We identified multiple mutations that improved binding affinity, and characterized the structure, affinity, and binding kinetics of a previously reported double mutant that exhibits an impressive 400-fold affinity improvement for the decameric pMHC ligand without detectable binding to non-cognate ligands. The structure of this high affinity mutant indicated very little conformational consequences and emphasized the high fidelity of our modeling procedure. Overall, our work showcases the capability of computational design to generate TCRs with improved pMHC affinities while explicitly accounting for peptide specificity, as well as its potential for generating TCRs with customized antigen targeting capabilities.

How structural adaptability exists alongside HLA-A2 bias in the human αβ TCR repertoire

“…For analysis of D26α-Arg65 pair, the L98βW single mutant was used. We note that, excluding the sites of the two mutations, the structures of the high-affinity and wild-type DMF5 TCR complexes are essentially identical (13,65). The utilization of affinity-enhancing mutations is unlikely to impact the double-mutant cycle experiments, owing to the structural similarities and the robust nature of double-mutant cycle analysis (40,41).…”

“…Errors were propagated using standard statistical error propagation methods (63). High-affinity variants of the DMF5 TCR were used to facilitate the analysis (65). For analysis of Tyr48β-Arg65 pair, the D26αY/L98βW double mutant was used.…”

A generalized framework for computational design and mutational scanning of T-cell receptor binding interfaces

“…The A6 and B7 TCRs also share the same germlinederived Vβ chain, although crystallographic structures and biophysical studies of A6 and B7 with Tax 11-19 /HLA-A2 showed structural and thermodynamic differences in binding (Davis-Harrison et al, 2005). We modeled 740 point mutations in the B7-Tax 11-19 /HLA-A2 interface using Rosetta (Das and Baker, 2008;Kaufmann et al, 2010) and the scheme described in Pierce et al (2014). As performed previously, effects were determined by scoring the complex, then separating the components and separately scoring the TCR and pMHC in order to calculate a 'binding score' (Kortemme and Baker, 2002).…”

“…1b). Utilizing the ZAFFI score function first developed for the A6 TCR and refined with the DMF5 TCR (Haidar et al, 2009;Pierce et al, 2014) led to an improved but still weak correlation (R = 0.47; Fig. 1c).…”

“…Not only can interactions be manipulated in a way that more appropriately addresses peptide specificity, affinity increments can in principle be more tightly controlled. Towards these goals, structureguided design has been used to modify a small number of TCRs (Haidar et al, 2009;Malecek et al, 2014;Pierce et al, 2014;Zoete et al, 2013). Recently, we used structure-guided design to engineer variants of the DMF5 TCR, which has been used clinically in immunotherapy for melanoma and continues to serve as a model TCR for improving cancer immunotherapy (Johnson et al, 2009).…”