Structural and biophysical insights from targeting melanoma using genetically modified T-cell receptors

CD8+ T-cells recognise pathogens and cancer through a specific interaction between the T-cell receptor (TCR) and a 8-14 amino-acid residue peptide presented by class I major histocompatibility complex (pMHCI) molecules expressed on the target cell surface. The first structures of murine and human TCR/pMHC complexes, published in 1996, revealed a number of important features of the TCR/pMHC interface. Currently, <25 unique human TCR/pMHC complexes are reported in the literature. This is a relatively low number compared with the number of antibody or unligated pMHC structures. The lack of structural information regarding human TCR/pMHC complexes has compromised the determination of a comprehensive and accepted set of rules that govern T-cell antigen recognition. Difficulties in generating TCR/pMHC complex crystals partly explain the low number of these structures. The first part of this thesis reports the development of a new crystallization screen (TOPS) designed specifically for the generation of such protein crystals. I also had access to MART-1-specific TCRs, the MART-1 protein being expressed by virtually all fresh melanoma tumour specimens. Different human leukocyte antigen (HLA)- A*0201-restricted peptides from this protein are presented at the melanoma cell surface. As TCRs are known to bind to cancer-derived “self” peptides with weak affinity, there is considerable interest in designing enhanced affinity TCRs for the recognition of HLA-A*0201-MART-1. My work concentrated on the MART-1- specific TCR MEL5 and its affinity-enhanced variant selected by phage display, α24β17. I analysed the biophysical properties of α24β17 and determined that it bound HLA-A*0201-MART-1 with >30,000-fold enhanced affinity and distinct thermodynamics. Comparison of TCR/HLA-A*0201-MART-1 complex structures solved with TOPS and binding biophysics showed that: (i) TCR affinity can be enhanced by increasing interactions between the TCR and the MHC surface; (ii) soluble α24β17 retains the peptide specificity by a novel mechanism involving interactions with solvent molecules; and, (iii) MEL5 interaction with the physiologically relevant MART-127-35 nonameric antigen led to a peptide anchor residue switch, a TCR-induced modification that has never been observed before. I also initiated a preliminary study on the generation of genetically modified Jurkat cells and CD8+ T-cells expressing a range of affinity-enhanced TCRs directed against melanoma for adoptive cell therapy. These results suggested that melanoma specificity is retained after MEL5 transduction and that there is no need to optimize beyond a TCR affinity threshold to obtain optimal T-cell activation. Collectively, these data shed light on the complex and unpredictable nature of T-cell antigen recognition

Dual molecular mechanisms govern escape at immunodominant HLA A2-restricted HIV epitope

Serial accumulation of mutations to fixation in the SLYNTVATL (SL9) immunodominant, HIV p17 Gag-derived, HLA A2-restricted CTL epitope produce the SLFNTIAVL triple mutant ‘ultimate’ escape variant. These mutations in solvent-exposed residues are believed to interfere with TCR recognition, although confirmation has awaited structural verification. Here, we solved a TCR co-complex structure with SL9 and the triple escape mutant to determine the mechanism of immune escape in this eminent system. We show that, in contrast to prevailing hypotheses, the main TCR contact residue is 4N and the dominant mechanism of escape is not via lack of TCR engagement. Instead, mutation of solvent exposed residues in the peptide destabilize the peptide-HLA and reduce peptide density at the cell surface. These results highlight the extraordinary lengths that HIV employs to evade detection by high-affinity TCRs with a broad peptide-binding footprint and necessitate reevaluation of this exemplar model of HIV TCR escape

Structural basis for ineffective T-cell responses to MHC anchor residue-improved 'heteroclitic' peptides

MHC anchor residue-modified “heteroclitic” peptides have been used in many cancer vaccine trials and often induce greater immune responses than the wild-type peptide. The best-studied system to date is the decamer MART-1/Melan-A26–35 peptide, EAAGIGILTV, where the natural alanine at position 2 has been modified to leucine to improve human leukocyte antigen (HLA)-A*0201 anchoring. The resulting ELAGIGILTV peptide has been used in many studies. We recently showed that T cells primed with the ELAGIGILTV peptide can fail to recognize the natural tumor-expressed peptide efficiently, thereby providing a potential molecular reason for why clinical trials of this peptide have been unsuccessful. Here, we solved the structure of a TCR in complex with HLA-A*0201-EAAGIGILTV peptide and compared it with its heteroclitic counterpart , HLA-A*0201-ELAGIGILTV. The data demonstrate that a suboptimal anchor residue at position 2 enables the TCR to “pull” the peptide away from the MHC binding groove, facilitating extra contacts with both the peptide and MHC surface. These data explain how a TCR can distinguish between two epitopes that differ by only a single MHC anchor residue and demonstrate how weak MHC anchoring can enable an induced-fit interaction with the TCR. Our findings constitute a novel demonstration of the extreme sensitivity of the TCR to minor alterations in peptide conformation

Enhanced detection of antigen-specific CD4+ T cells using altered peptide flanking residue peptide-MHC class II multimers

Fluorochrome-conjugated peptide–MHC (pMHC) class I multimers are staple components of the immunologist’s toolbox, enabling reliable quantification and analysis of Ag-specific CD8+ T cells irrespective of functional outputs. In contrast, widespread use of the equivalent pMHC class II (pMHC-II) reagents has been hindered by intrinsically weaker TCR affinities for pMHC-II, a lack of cooperative binding between the TCR and CD4 coreceptor, and a low frequency of Ag-specific CD4+ T cell populations in the peripheral blood. In this study, we show that peptide flanking regions, extending beyond the central nonamer core of MHC-II–bound peptides, can enhance TCR–pMHC-II binding and T cell activation without loss of specificity. Consistent with these findings, pMHC-II multimers incorporating peptide flanking residue modifications proved superior for the ex vivo detection, characterization, and manipulation of Ag-specific CD4+ T cells, highlighting an unappreciated feature of TCR–pMHC-II interactions

T-cell Receptor (TCR)-Peptide Specificity Overrides Affinity-enhancing TCR-Major Histocompatibility Complex Interactions

αβ T-cell receptors (TCRs) engage antigens using complementarity-determining region (CDR) loops that are either germ line-encoded (CDR1 and CDR2) or somatically rearranged (CDR3). TCR ligands compose a presentation platform (major histocompatibility complex (MHC)) and a variable antigenic component consisting of a short “foreign” peptide. The sequence of events when the TCR engages its peptide-MHC (pMHC) ligand remains unclear. Some studies suggest that the germ line elements of the TCR engage the MHC prior to peptide scanning, but this order of binding is difficult to reconcile with some TCR-pMHC structures. Here, we used TCRs that exhibited enhanced pMHC binding as a result of mutations in either CDR2 and/or CDR3 loops, that bound to the MHC or peptide, respectively, to dissect the roles of these loops in stabilizing TCR-pMHC interactions. Our data show that TCR-peptide interactions play a strongly dominant energetic role providing a binding mode that is both temporally and energetically complementary with a system requiring positive selection by self-pMHC in the thymus and rapid recognition of non-self-pMHC in the periphery

TCR‐induced alteration of primary MHC peptide anchor residue

The HLA‐A*02:01‐restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T‐cells‐1 (MART‐1) protein, represents one of the best‐studied tumor associated T‐cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA‐A*02:01 and TCRs from clinically relevant T‐cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5‐HLA‐A*02:01‐AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide–MHC anchoring. This “flexing” at the TCR–peptide–MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well‐studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells

TCR/pMHC optimized protein crystallization screen

The interaction between the clonotypic αβ T cell receptor (TCR), expressed on the T cell surface, and peptide-major histocompatibility complex (pMHC) molecules, expressed on the target cell surface, governs T cell mediated autoimmunity and immunity against pathogens and cancer. Structural investigations of this interaction have been limited because of the challenges inherent in the production of good quality TCR/pMHC protein crystals. Here, we report the development of an ‘intelligently designed’ crystallization screen that reproducibly generates high quality TCR/pMHC complex crystals suitable for X-ray crystallographic studies, thereby reducing protein consumption. Over the last 2 years, we have implemented this screen to produce 32 T cell related protein structures at high resolution, substantially contributing to the current immune protein database. Protein crystallography, used to study this interaction, has already extended our understanding of the molecular rules that govern T cell immunity. Subsequently, these data may help to guide the intelligent design of T cell based therapies that target human diseases, underlining the importance of developing optimized approaches for crystallizing novel TCR/pMHC complexes

Minimal conformational plasticity enables TCR cross-reactivity to different MHC class II heterodimers

Successful immunity requires that a limited pool of αβ T-cell receptors (TCRs) provide cover for a vast number of potential foreign peptide antigens presented by ‘self’ major histocompatibility complex (pMHC) molecules. Structures of unligated and ligated MHC class-I-restricted TCRs with different ligands, supplemented with biophysical analyses, have revealed a number of important mechanisms that govern TCR mediated antigen recognition. HA1.7 TCR binding to the influenza hemagglutinin antigen (HA306–318) presented by HLA-DR1 or HLA-DR4 represents an ideal system for interrogating pMHC-II antigen recognition. Accordingly, we solved the structure of the unligated HA1.7 TCR and compared it to both complex structures. Despite a relatively rigid binding mode, HA1.7 T-cells could tolerate mutations in key contact residues within the peptide epitope. Thermodynamic analysis revealed that limited plasticity and extreme favorable entropy underpinned the ability of the HA1.7 T-cell clone to cross-react with HA306–318 presented by multiple MHC-II alleles