I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Andrew
Chancellor
,
Robert A.
Simmons
,
Rahul C.
Khanolkar
,
Vladimir
Nosi
,
Aisha
Beshirova
,
Giuliano
Berloffa
,
Rodrigo
Colombo
,
Vijaykumar
Karuppiah
,
Johanne M.
Pentier
,
Vanessa
Tubb
,
Hemza
Ghadbane
,
Richard J.
Suckling
,
Keith
Page
,
Rory M.
Crean
,
Alessandro
Vacchini
,
Corinne
De Gregorio
,
Verena
Schaefer
,
Daniel
Constantin
,
Thomas
Gligoris
,
Angharad
Lloyd
,
Miriam
Hock
,
Velupillai
Srikannathasan
,
Ross A.
Robinson
,
Gurdyal S.
Besra
,
Marc W.
Van Der Kamp
,
Lucia
Mori
,
Raffaele
Calogero
,
David K.
Cole
,
Gennaro
De Libero
,
Marco
Lepore
Diamond Proposal Number(s):
[22870, 28224]
Abstract: Mucosal-associated invariant T (MAIT) cells use canonical semi-invariant T cell receptors (TCR) to recognize microbial riboflavin precursors displayed by the antigen-presenting molecule MR1. The extent of MAIT TCR crossreactivity toward physiological, microbially unrelated antigens remains underexplored. We describe MAIT TCRs endowed with MR1-dependent reactivity to tumor and healthy cells in the absence of microbial metabolites. MAIT cells bearing TCRs crossreactive toward self are rare but commonly found within healthy donors and display T-helper-like functions in vitro. Experiments with MR1-tetramers loaded with distinct ligands revealed significant crossreactivity among MAIT TCRs both ex vivo and upon in vitro expansion. A canonical MAIT TCR was selected on the basis of extremely promiscuous MR1 recognition. Structural and molecular dynamic analyses associated promiscuity to unique TCRβ-chain features that were enriched within self-reactive MAIT cells of healthy individuals. Thus, self-reactive recognition of MR1 represents a functionally relevant indication of MAIT TCR crossreactivity, suggesting a potentially broader role of MAIT cells in immune homeostasis and diseases, beyond microbial immunosurveillance.
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Sep 2023
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Garry
Dolton
,
Cristina
Rius
,
Aaron
Wall
,
Barbara
Szomolay
,
Valentina
Bianchi
,
Sarah A. E.
Galloway
,
Md Samiul
Hasan
,
Théo
Morin
,
Marine E.
Caillaud
,
Hannah L.
Thomas
,
Sarah
Theaker
,
Li Rong
Tan
,
Anna
Fuller
,
Katie
Topley
,
Mateusz
Legut
,
Meriem
Attaf
,
Jade R.
Hopkins
,
Enas
Behiry
,
Joanna
Zabkiewicz
,
Caroline
Alvares
,
Angharad
Lloyd
,
Amber
Rogers
,
Peter
Henley
,
Christopher
Fegan
,
Oliver
Ottmann
,
Stephen
Man
,
Michael D.
Crowther
,
Marco
Donia
,
Inge Marie
Svane
,
David K.
Cole
,
Paul E.
Brown
,
Pierre
Rizkallah
,
Andrew K.
Sewell
Open Access
Abstract: The T cells of the immune system can target tumors and clear solid cancers following tumor-infiltrating lymphocyte (TIL) therapy. We used combinatorial peptide libraries and a proteomic database to reveal the antigen specificities of persistent cancer-specific T cell receptors (TCRs) following successful TIL therapy for stage IV malignant melanoma. Remarkably, individual TCRs could target multiple different tumor types via the HLA A∗02:01-restricted epitopes EAAGIGILTV, LLLGIGILVL, and NLSALGIFST from Melan A, BST2, and IMP2, respectively. Atomic structures of a TCR bound to all three antigens revealed the importance of the shared x-x-x-A/G-I/L-G-I-x-x-x recognition motif. Multi-epitope targeting allows individual T cells to attack cancer in several ways simultaneously. Such “multipronged” T cells exhibited superior recognition of cancer cells compared with conventional T cell recognition of individual epitopes, making them attractive candidates for the development of future immunotherapies.
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Jul 2023
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Alexander
Greenshields-Watson
,
Meriem
Attaf
,
Bruce J.
Maclachlan
,
Thomas
Whalley
,
Cristina
Rius
,
Aaron
Wall
,
Angharad
Lloyd
,
Hywel
Hughes
,
Kathryn E.
Strange
,
Georgina H.
Mason
,
Andrea J.
Schauenburg
,
Sarah L.
Hulin-Curtis
,
James
Geary
,
Yuan
Chen
,
Sarah N.
Lauder
,
Kathryn
Smart
,
Dhanasekaran
Vijaykrishna
,
Miguel L.
Grau
,
Mikhail
Shugay
,
Robert
Andrews
,
Garry
Dolton
,
Pierre J.
Rizkallah
,
Awen M.
Gallimore
,
Andrew K.
Sewell
,
Andrew J.
Godkin
,
David K.
Cole
Diamond Proposal Number(s):
[10462, 14843]
Open Access
Abstract: T cell recognition of peptides presented by human leukocyte antigens (HLAs) is mediated by the highly variable T cell receptor (TCR). Despite this built-in TCR variability, individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD8+ T cells. Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA. However, few examples exist for CD4+ T cells. Here, we investigate CD4+ T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity. We identify five internal epitopes that are commonly recognized by CD4+ T cells in five HLA-DR1+ subjects and show conservation across viral strains and zoonotic reservoirs. TCR repertoire analysis demonstrates several shared gene usage biases underpinned by complementary biochemical features evident in a structural comparison. These epitopes are attractive targets for vaccination and other T cell therapies.
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Jul 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[17077]
Open Access
Abstract: T cell-mediated immunity is governed primarily by T cell receptor (TCR) recognition of peptide human leukocyte antigen complexes (pHLA) and is essential for immunosurveillance and disease control. This interaction is generally stabilised by interactions between the HLA surface and TCR germline encoded complementarity determining region (CDR) loops 1 and 2, whereas peptide selectivity is guided by direct interactions with the TCR CDR3 loops. Here, we solved the structure of a newly identified TCR in complex with a clinically relevant peptide derived from the cancer testis antigen melanoma antiGEn-A4 (MAGE-A4). The TCR bound pHLA in a position shifted toward the peptide’s N-terminus. This enabled the TCR to achieve peptide selectivity via an indirect mechanism, whereby the TCR sensed the first residue of the peptide through HLA residue Trp167, which acted as a tuneable gateway. Amino acid substitutions at peptide position 1 predicted to alter the HLA Trp167 sidechain conformation abrogated TCR binding, indicating that this indirect binding mechanism is essential for peptide recognition. These findings extend our understanding of the molecular rules that underpin antigen recognition by TCRs and have important implications for the development of TCR-based therapies.
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Jun 2020
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Charlotte
Coles
,
Rachel M.
Mulvaney
,
Sunir
Malla
,
Andrew
Walker
,
Kathrine J.
Smith
,
Angharad
Lloyd
,
Kate L.
Lowe
,
Michelle L.
Mccully
,
Ruth
Martinez Hague
,
Milos
Aleksic
,
Jane
Harper
,
Samantha J.
Paston
,
Zoe
Donnellan
,
Fiona
Chester
,
Katrin
Wiederhold
,
Ross A.
Robinson
,
Andrew
Knox
,
Andrew R.
Stacey
,
Joseph
Dukes
,
Emma
Baston
,
Sue
Griffin
,
Bent K.
Jakobsen
,
Annelise
Vuidepot
,
Stephen
Harper
Open Access
Abstract: The molecular rules driving TCR cross-reactivity are poorly understood and, consequently, it is unclear the extent to which TCRs targeting the same Ag recognize the same off-target peptides. We determined TCR–peptide–HLA crystal structures and, using a single-chain peptide–HLA phage library, we generated peptide specificity profiles for three newly identified human TCRs specific for the cancer testis Ag NY-ESO-1157–165–HLA-A2. Two TCRs engaged the same central peptide feature, although were more permissive at peripheral peptide positions and, accordingly, possessed partially overlapping peptide specificity profiles. The third TCR engaged a flipped peptide conformation, leading to the recognition of off-target peptides sharing little similarity with the cognate peptide. These data show that TCRs specific for a cognate peptide recognize discrete peptide repertoires and reconciles how an individual’s limited TCR repertoire following negative selection in the thymus is able to recognize a vastly larger antigenic pool.
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Apr 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Christopher J.
Holland
,
Rory M.
Crean
,
Johanne M.
Pentier
,
Ben
De Wet
,
Angharad
Lloyd
,
Velupillai
Srikannathasan
,
Nikolai
Lissin
,
Katy A.
Lloyd
,
Thomas H.
Blicher
,
Paul J.
Conroy
,
Miriam
Hock
,
Robert J.
Pengelly
,
Thomas E.
Spinner
,
Brian
Cameron
,
Elizabeth A.
Potter
,
Anitha
Jeyanthan
,
Peter E.
Molloy
,
Malkit
Sami
,
Milos
Aleksic
,
Nathaniel
Liddy
,
Ross A.
Robinson
,
Stephen
Harper
,
Marco
Lepore
,
Chris R.
Pudney
,
Marc W.
Van Der Kamp
,
Pierre J.
Rizkallah
,
Bent K.
Jakobsen
,
Annelise
Vuidepot
,
David K.
Cole
Diamond Proposal Number(s):
[17077, 14843]
Abstract: Tumor-associated peptide–human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface–expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents.
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Apr 2020
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I24-Microfocus Macromolecular Crystallography
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Sarah A. E.
Galloway
,
Garry
Dolton
,
Meriem
Attaf
,
Aaron
Wall
,
Anna
Fuller
,
Cristina
Rius
,
Valentina
Bianchi
,
Sarah
Theaker
,
Angharad
Lloyd
,
Marine E.
Caillaud
,
Inge Marie
Svane
,
Marco
Donia
,
David K.
Cole
,
Barbara
Szomolay
,
Pierre
Rizkallah
,
Andrew K.
Sewell
Diamond Proposal Number(s):
[14843]
Open Access
Abstract: Recent immunotherapeutic approaches using adoptive cell therapy, or checkpoint blockade, have demonstrated the powerful anti-cancer potential of CD8 cytotoxic T-lymphocytes (CTL). While these approaches have shown great promise, they are only effective in some patients with some cancers. The potential power, and relative ease, of therapeutic vaccination against tumour associated antigens (TAA) present in different cancers has been a long sought-after approach for harnessing the discriminating sensitivity of CTL to treat cancer and has seen recent renewed interest following cancer vaccination successes using unique tumour neoantigens. Unfortunately, results with TAA-targeted “universal” cancer vaccines (UCV) have been largely disappointing. Infectious disease models have demonstrated that T-cell clonotypes that recognise the same antigen should not be viewed as being equally effective. Extrapolation of this notion to UCV would suggest that the quality of response in terms of the T-cell receptor (TCR) clonotypes induced might be more important than the quantity of the response. Unfortunately, there is little opportunity to assess the effectiveness of individual T-cell clonotypes in vivo. Here, we identified effective, persistent T-cell clonotypes in an HLA A2+ patient following successful tumour infiltrating lymphocyte (TIL) therapy. One such T-cell clone was used to generate super-agonist altered peptide ligands (APLs). Further refinement produced an APL that was capable of inducing T-cells in greater magnitude, and with improved effectiveness, from the blood of all 14 healthy donors tested. Importantly, this APL also induced T-cells from melanoma patient blood that exhibited superior recognition of the patient's own tumour compared to those induced by the natural antigen sequence. These results suggest that use of APL to skew the clonotypic quality of T-cells induced by cancer vaccination could provide a promising avenue in the hunt for the UCV “magic bullet.”
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Mar 2019
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[12332]
Open Access
Abstract: T cell receptor (TCR) recognition of foreign peptide fragments, presented by peptide major histocompatibility complex (pMHC), governs T-cell mediated protection against pathogens and cancer. Many factors govern T-cell sensitivity, including the affinity of the TCR-pMHC interaction and the stability of pMHC on the surface of antigen presenting cells. These factors are particularly relevant for the peptide vaccination field, in which more stable pMHC interactions could enable more effective protection against disease. Here, we discuss a method for the determination of pMHC stability that we have used to investigate HIV immune escape, T-cell sensitivity to cancer antigens and mechanisms leading to autoimmunity.
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Jul 2017
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B23-Circular Dichroism
I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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David K.
Cole
,
Hugo A.
Van Den Berg
,
Angharad
Lloyd
,
Michael D.
Crowther
,
Konrad
Beck
,
Julia
Ekeruche-Makinde
,
John J.
Miles
,
Anna M.
Bulek
,
Garry
Dolton
,
Andrea J.
Schauenburg
,
Aaron
Wall
,
Anna
Fuller
,
Mathew
Clement
,
Bruno
Laugel
,
Pierre J.
Rizkallah
,
Linda
Wooldridge
,
Andrew K.
Sewell
Diamond Proposal Number(s):
[6232, 8096, 10462, 10049, 9308, 12332]
Open Access
Abstract: T-cell cross-reactivity is essential for effective immune surveillance but has also been implicated as a pathway to autoimmunity. Previous studies have demonstrated that T-cell receptors (TCRs) that focus on a minimal motif within the peptide are able to facilitate a high level of T-cell cross-reactivity. However, the structural database shows that most TCRs exhibit less focused antigen binding involving contact with more peptide residues. To further explore the structural features that allow the clonally expressed TCR to functionally engage with multiple peptide-major histocompatibility complexes (pMHCs), we examined the ILA1 CD8 T-cell clone that responds to a peptide sequence derived from human telomerase reverse transcriptase. The ILA1 TCR contacted its pMHC with a broad peptide binding footprint encompassing spatially distant peptide residues. Despite the lack of focused TCR-peptide binding, the ILA1 T-cell clone was still cross-reactive. Overall, the TCR-peptide contacts apparent in the structure correlated well with the level of degeneracy at different peptide positions. Thus, the ILA1 TCR was less tolerant of changes at peptide residues that were at, or adjacent to, key contact sites. This study provides new insights into the molecular mechanisms that control T-cell cross-reactivity with important implications for pathogen surveillance, autoimmunity, and transplant rejection.
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Jan 2017
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8096, 10462]
Open Access
Abstract: Human CD8+ cytotoxic T lymphocytes (CTLs) can mediate tumour regression in melanoma through the specific recognition of human leukocyte antigen (HLA)-restricted peptides. Because of the relatively weak affinity of most anti-cancer T-cell receptors (TCR), there is growing emphasis on immunizing melanoma patients with altered peptide ligands in order to induce strong anti-tumour immunity capable of breaking tolerance towards these self-antigens. Previous studies have shown that these immunogenic designer peptides are not always effective and detailed structural analyses will be required in order to improve future success rates. The melanocyte differentiation protein, glycoprotein (gp)100, encodes a naturally processed epitope that is an attractive target for melanoma immunotherapies, in particular peptide-based vaccines. Previous studies have shown that substitutions at peptide residue Glu3 have a broad negative impact on polyclonal T-cell responses. Here, we describe the first atomic structure of a natural cognate TCR in complex with this gp100 epitope, and highlight the relatively high affinity of the interaction. Alanine scan mutagenesis performed across the gp100280-288 peptide showed that Glu3 was critically important for TCR binding. Unexpectedly, structural analysis demonstrated that the Glu3>Ala substitution resulted in a molecular switch that was transmitted to adjacent residues, abrogating TCR binding and T-cell recognition through knock-on effects. These findings help to clarify the mechanism of T-cell recognition of gp100 during melanoma responses and could direct the development of altered peptides for vaccination.
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Feb 2016
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