Specific Increase in Potency via Structure-Based Design of a TCR

Abstract

Adoptive immunotherapy with antigen-specific T lymphocytes is a powerful strategy for cancer treatment. However, most tumor antigens are non-reactive “self” proteins, which presents an immunotherapy design challenge. Recent studies have shown that tumor-specific T cell receptors (TCRs) can be transduced into normal peripheral blood lymphocytes, which persist after transfer in about 30% of patients and effectively destroy tumor cells in vivo. Although encouraging, the limited clinical responses underscore the need for enrichment of T cells with desirable anti-tumor capabilities prior to patient transfer. In this study, we used structure-based design to predict point mutations of a TCR (DMF5) that enhance its binding affinity for an agonist tumor antigen-major histocompatibility complex (pMHC), Mart-1(27L)-HLA-A2, which elicits full T cell activation to trigger immune responses. We analyzed the effects of selected TCR point mutations on T cell activation potency and analyzed cross-reactivity with related antigens. Our results showed that the mutated TCRs had improved T cell activation potency, while retaining a high degree of specificity. Such affinity-optimized TCRs have demonstrated to be very specific for Mart-1 (27L), the epitope for which they were structurally designed. And even though of limited clinical relevance, these studies open the possibility for future structural-based studies that could potentially be used in adoptive immunotherapy to treat melanoma while avoiding adverse autoimmunity-derived effects.

Structural Model of the Extracellular Assembly of the TCR-CD3 Complex

“…T-cells expressing wild type 2B4 TCR and mutated TCRs were activated by Chinese Hamster Ovary (CHO) cells expressing pMHC (I-E k /K5) (Krogsgaard et al, 2005), and IL-2 production was quantified by ELISA (Malecek et al, 2013) (Figure S5). The area under the curve for IL-2 production, an overall measure for cumulative response (Hioe et al, 2010; Malecek et al, 2014), was determined and compared to the wild type to identify the change in IL-2 production.…”

“…The mutant mouse 2B4 TCR construct were generated by PCR using overlapping primers containing the mutant sequences and cloned into retroviral pMX vector (Kitamura et al, 2003). Retroviral transductions of the hybridoma cells were done as previously described (Malecek et al, 2014; Malecek et al, 2013; Zhong et al, 2013; Zhong et al, 2010). The transduced cells were stained with PE anti-CD3ε and APC anti-TCRβ (clone H57-597) antibodies.…”

ATLAS: A database linking binding affinities with structures for wild-type and mutant TCR-pMHC complexes

“…Precise prediction and manipulation of both TCR affinity and specificity is therefore essential for designing effective T‐cell‐based therapeutics. A number of methods have been developed for altering TCR‐pMHC interactions, including in vitro molecular evolution and structure‐guided protein design . Structure‐guided design algorithms can alter affinity and specificity directly and efficiently, but are limited by the accuracy of their scoring functions.…”

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

“…Structure-guided design has been used to generate a small number of high affinity TCRs, as well as manipulate binding specificity (Haidar et al, 2009;Malecek et al, 2014;Pierce et al, 2014;Zoete et al, 2013). However, although the TCR-pMHC structural database has grown significantly in recent years, wide-scale application of structure-guided TCR design is hindered by several complexities.…”

“…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).…”