I02-Macromolecular Crystallography
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Rory M.
Crean
,
Bruce J.
Maclachlan
,
Florian
Madura
,
Thomas
Whalley
,
Pierre J.
Rizkallah
,
Christopher J.
Holland
,
Catriona
Mcmurran
,
Stephen
Harper
,
Andrew
Godkin
,
Andrew K.
Sewell
,
Christopher R.
Pudney
,
Marc W.
Van Der Kamp
,
David K.
Cole
Diamond Proposal Number(s):
[6232]
Open Access
Abstract: Immuno-oncology approaches that utilise T cell receptors (TCRs) are becoming highly attractive because of their potential to target virtually all cellular proteins, including cancer specific epitopes, via the recognition of peptide-human leukocyte antigen complexes (pHLA) presented at the cell surface. However, because natural TCRs generally recognise cancer derived pHLAs with very weak affinities, efforts have been made to enhance their binding strength, in some cases by several million-fold. Here, we investigated the mechanisms underpinning human TCR affinity enhancement by comparing the crystal structures of engineered enhanced affinity TCRs with that of their wildtype progenitors. Additionally, we performed molecular dynamics simulations to better understand the energetic mechanisms driving the affinity enhancements. These data demonstrate that supra-physiological binding affinities can be achieved without altering native TCR-pHLA binding modes via relatively subtle modifications to the interface contacts, often driven through the addition of buried hydrophobic residues. Individual energetic components of the TCR-pHLA interaction governing affinity enhancements were distinct and highly variable for each TCR, often resulting from additive, or knock-on, effects beyond the mutated residues. This comprehensive analysis of affinity enhanced TCRs has important implications for the future rational design of engineered TCRs as efficacious and safe drugs for cancer treatment.We demonstrate that the native TCR-pHLA conformation is compatible with supra-physiological binding affinities via subtle modifications to the interface contacts, often driven through the addition of buried hydrophobic residues. This comprehensive analysis of affinity enhanced TCRs has important implications for the future rational design of engineered TCRs for cancer therapy.
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Jul 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Florian
Madura
,
Pierre J.
Rizkallah
,
Mateusz
Legut
,
Christopher J.
Holland
,
Anna
Fuller
,
Anna
Bulek
,
Andrea J.
Schauenburg
,
Andrew
Trimby
,
Jade R.
Hopkins
,
Stephen
Wells
,
Andrew
Godkin
,
John J.
Miles
,
Malkit
Sami
,
Yi
Li
,
Nathaniel
Liddy
,
Bent K.
Jakobsen
,
E. Joel
Loveridge
,
David K.
Cole
,
Andrew K.
Sewell
Diamond Proposal Number(s):
[4352, 6232]
Open Access
Abstract: 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 nonapeptides 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.
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May 2019
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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David K.
Cole
,
Anna
Fuller
,
Garry
Dolton
,
Efthalia
Zervoudi
,
Mateusz
Legut
,
Kim
Miles
,
Lori
Blanchfield
,
Florian
Madura
,
Christopher J.
Holland
,
Anna M.
Bulek
,
John S.
Bridgeman
,
John J.
Miles
,
Andrea J. A.
Schauenburg
,
Konrad
Beck
,
Brian D.
Evavold
,
Pierre
Rizkallah
,
Andrew K.
Sewell
Diamond Proposal Number(s):
[4532, 6232]
Abstract: Serial accumulation of mutations to fixation in the SLYNTVATL (SL9) immunodominant, HIV p17 Gag-derived, HLA A2-restricted cytotoxic T lymphocyte 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 destabilise 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 re-evaluation of this exemplar model of HIV TCR escape.
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Nov 2017
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: 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.
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Dec 2014
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I04-Macromolecular Crystallography
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David K.
Cole
,
Kim
Miles
,
Florian
Madura
,
Chris
Holland
,
Andrea
Schauenburg
,
Andrew J.
Godkin
,
Anna
Bulek
,
Anna
Fuller
,
Hephzibah
Akpovwa
,
Philip G
Pymm
,
Nathaniel
Liddy
,
Malkit
Sami
,
Yi
Li
,
Pierre
Rizkallah
,
Bent K.
Jakobsen
,
Andrew K.
Sewell
Open Access
Abstract: This work was supported by the Medical Research Council (to A. K. S., T. J. G., and I. B.), Central Laboratory of the Research Councils (CLRC) Daresbury Laboratory, the Diamond Light Source (Midlands BAG MX310), the Danish Medical Research Council (to U. H.), the NOVO Nordic Foundation (to U. H.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. ), the Lundbeck Foundation (U. H.), and Fonden til Lægevidenskabens Fremme (to U. H.).
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Jan 2014
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Florian
Madura
,
Pierre J.
Rizkallah
,
Kim M.
Miles
,
Christopher J.
Holland
,
Anna M.
Bulek
,
Anna
Fuller
,
Andrea J. A.
Schauenburg
,
John
Miles
,
Nathaniel
Liddy
,
Malkit
Sami
,
Yi
Li
,
Moushumi
Hossain
,
Brian M.
Baker
,
Bent K.
Jakobsen
,
Andrew K.
Sewell
,
David K.
Cole
Open Access
Abstract: The T-cell receptor (TCR) recognizes peptides bound to major histocompatibility molecules (MHC) and allows T-cells to interrogate the cellular proteome for internal anomalies from the cell surface. The TCR contacts both MHC and peptide in an interaction characterized by weak affinity (KD = 100 nm to 270 μm). We used phage-display to produce a melanoma-specific TCR (α24β17) with a 30,000-fold enhanced binding affinity (KD = 0.6 nm) to aid our exploration of the molecular mechanisms utilized to maintain peptide specificity. Remarkably, although the enhanced affinity was mediated primarily through new TCR-MHC contacts, α24β17 remained acutely sensitive to modifications at every position along the peptide backbone, mimicking the specificity of the wild type TCR. Thermodynamic analyses revealed an important role for solvation in directing peptide specificity. These findings advance our understanding of the molecular mechanisms that can govern the exquisite peptide specificity characteristic of TCR recognition.
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May 2013
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I03-Macromolecular Crystallography
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Julia
Ekeruche-Makinde
,
Mathew
Clement
,
David K.
Cole
,
Emily S. J.
Edwards
,
Kristin
Ladell
,
John J.
Miles
,
Katherine K.
Matthews
,
Anna
Fuller
,
Katy A.
Lloyd
,
Florian
Madura
,
Garry M.
Dolton
,
Johanne
Pentier
,
Anna
Lissina
,
Emma
Gostick
,
Tiffany K.
Baxter
,
Brian M.
Baker
,
Pierre
Rizkallah
,
David A.
Price
,
Linda
Wooldridge
,
Andrew K.
Sewell
Open Access
Abstract: Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8+ T-cell clone (MEL5) with an enhanced ability to recognize the HLA A*0201-Melan A27–35 (HLA A*0201-AAGIGILTV) antigen expressed on the surface of malignant melanoma cells. We used combinatorial peptide library screening to design an optimal peptide sequence that enhanced functional activation of the MEL5 clone, but not other CD8+ T-cell clones that recognized HLA A*0201-AAGIGILTV poorly. Structural analysis revealed the potential for new contacts between the MEL5 T-cell receptor and the optimized peptide. Furthermore, the optimized peptide was able to prime CD8+ T-cell populations in peripheral blood mononuclear cell isolates from multiple HLA A*0201+ individuals that were capable of efficient HLA A*0201+ melanoma cell destruction. This proof-of-concept study demonstrates that it is possible to design altered peptide antigens for the selection of superior T-cell clonotypes with enhanced antigen recognition properties.
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Oct 2012
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: Successful immunity requires that a limited pool of 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.
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Sep 2012
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: 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.
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Aug 2012
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Anna
Bulek
,
David
Cole
,
Ania
Skowera
,
Garry
Dolton
,
Stephanie
Gras
,
Florian
Madura
,
Anna
Fuller
,
John
Miles
,
Emma
Gostick
,
David A.
Price
,
Jan W.
Drijfhout
,
Robin R.
Knight
,
Guo C.
Huang
,
Nikolai
Lissin
,
Peter E.
Molloy
,
Linda
Fothergill-Gilmore
,
Bent K.
Jakobsen
,
Jamie
Rossjohn
,
Mark
Peakman
,
Pierre J.
Rizkallah
,
Andrew K.
Sewell
Open Access
Abstract: The structural characteristics of the engagement of major histocompatibility complex (MHC) class II–restricted self antigens by autoreactive T cell antigen receptors (TCRs) is established, but how autoimmune TCRs interact with complexes of self peptide and MHC class I has been unclear. Here we examined how CD8+ T cells kill human islet beta cells in type 1 diabetes via recognition of a human leukocyte antigen HLA-A*0201–restricted glucose-sensitive preproinsulin peptide by the autoreactive TCR 1E6. Rigid 'lock-and-key' binding underpinned the 1E6–HLA-A*0201–peptide interaction, whereby 1E6 docked similarly to most MHC class I–restricted TCRs. However, this interaction was extraordinarily weak because of limited contacts with MHC class I. TCR binding was highly peptide centric, dominated by two residues of the complementarity-determining region 3 (CDR3) loops that acted as an 'aromatic-cap' over the complex of peptide and MHC class I (pMHCI). Thus, highly focused peptide-centric interactions associated with suboptimal TCR-pMHCI binding affinities might lead to thymic escape and potential CD8+ T cell–mediated autoreactivity.
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Jan 2012
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