I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Mathieu
Ferrari
,
Matteo
Righi
,
Vania
Baldan
,
Patrycja
Wawrzyniecka
,
Anna
Bulek
,
Alexander
Kinna
,
Biao
Ma
,
Reyisa
Bughda
,
Zulaikha
Akbar
,
Saket
Srivastava
,
Isaac
Gannon
,
Mathew
Robson
,
James
Sillibourne
,
Ram
Jha
,
Mohamed
El-Kholy
,
Oliver Muhammad
Amin
,
Evangelia
Kokalaki
,
Mohammed Amin
Banani
,
Rehan
Hussain
,
William
Day
,
Wen Chean
Lim
,
Priyanka
Ghongane
,
Jade R.
Hopkins
,
Dennis
Jungherz
,
Marco
Herling
,
Martin
Welin
,
Sachin
Surade
,
Michael
Dyson
,
John
Mccafferty
,
Derek
Logan
,
Shaun
Cordoba
,
Simon
Thomas
,
Andrew
Sewell
,
Paul
Maciocia
,
Shimobi
Onuoha
,
Martin
Pule
Open Access
Abstract: Peripheral T cell lymphomas are typically aggressive with a poor prognosis. Unlike other hematologic malignancies, the lack of target antigens to discriminate healthy from malignant cells limits the efficacy of immunotherapeutic approaches. The T cell receptor expresses one of two highly homologous chains [T cell receptor β-chain constant (TRBC) domains 1 and 2] in a mutually exclusive manner, making it a promising target. Here we demonstrate specificity redirection by rational design using structure-guided computational biology to generate a TRBC2-specific antibody (KFN), complementing the antibody previously described by our laboratory with unique TRBC1 specificity (Jovi-1) in targeting broader spectrum of T cell malignancies clonally expressing either of the two chains. This permits generation of paired reagents (chimeric antigen receptor-T cells) specific for TRBC1 and TRBC2, with preclinical evidence to support their efficacy in T cell malignancies.
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Feb 2024
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Yuan
Chen
,
Georgina H.
Mason
,
D. Oliver
Scourfield
,
Alexander
Greenshields-Watson
,
Tracey A.
Haigh
,
Andrew K.
Sewell
,
Heather M.
Long
,
Awen M.
Gallimore
,
Pierre
Rizkallah
,
Bruce J.
Maclachlan
,
Andrew
Godkin
Diamond Proposal Number(s):
[20147, 29502, 29990]
Open Access
Abstract: CD4+ T cells recognize a broad range of peptide epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which contribute to immune memory and limit COVID-19 disease. We demonstrate that the immunogenicity of SARS-CoV-2 peptides, in the context of the model allotype HLA-DR1, does not correlate with their binding affinity to the HLA heterodimer. Analyzing six epitopes, some with very low binding affinity, we solve X-ray crystallographic structures of each bound to HLA-DR1. Further structural definitions reveal the precise molecular impact of viral variant mutations on epitope presentation. Omicron escaped ancestral SARS-CoV-2 immunity to two epitopes through two distinct mechanisms: (1) mutations to TCR-facing epitope positions and (2) a mechanism whereby a single amino acid substitution caused a register shift within the HLA binding groove, completely altering the peptide-HLA structure. This HLA-II-specific paradigm of immune escape highlights how CD4+ T cell memory is finely poised at the level of peptide-HLA-II presentation.
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Aug 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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I04-1-Macromolecular Crystallography (fixed wavelength)
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Garry
Dolton
,
Cristina
Rius
,
Md Samiul
Hasan
,
Aaron
Wall
,
Barbara
Szomolay
,
Enas
Behiry
,
Thomas
Whalley
,
Joel
Southgate
,
Anna
Fuller
,
Théo
Morin
,
Katie
Topley
,
Li Rong
Tan
,
Philip J. R.
Goulder
,
Owen B.
Spiller
,
Pierre J.
Rizkallah
,
Lucy C.
Jones
,
Thomas R.
Connor
,
Andrew K.
Sewell
Diamond Proposal Number(s):
[29502]
Open Access
Abstract: We studied the prevalent cytotoxic CD8 T-cell response mounted against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike glycoprotein269-277 epitope (sequence YLQPRTFLL) via the most frequent Human Leukocyte Antigen (HLA) class I worldwide, HLA A∗02. The Spike P272L mutation that has arisen in at least 112 different SARS-CoV-2 lineages to date, including in lineages classified as ‘variants of concern’, was not recognised by the large CD8 T-cell response seen across cohorts of HLA A∗02+ convalescent patients and individuals vaccinated against SARS-CoV-2, despite these responses comprising of over 175 different individual T-cell receptors. Viral escape at prevalent T-cell epitopes restricted by high frequency HLAs may be particularly problematic when vaccine immunity is focussed on a single protein such as SARS-CoV-2 Spike providing a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlighting the need for monitoring T-cell escape in new SARS-CoV-2 variants.
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Jul 2022
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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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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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I02-Macromolecular Crystallography
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Bruce J.
Maclachlan
,
Garry
Dolton
,
Athanasios
Papakyriakou
,
Alexander
Greenshields-Watson
,
Georgina H.
Mason
,
Andrea
Schauenburg
,
Matthieu
Besneux
,
Barbara
Szomolay
,
Tim
Elliott
,
Andrew K.
Sewell
,
Awen
Gallimore
,
Pierre
Rizkallah
,
David K.
Cole
,
Andrew
Godkin
Diamond Proposal Number(s):
[10462]
Abstract: CD4+ T-cells recognize peptide antigens, in the context of human leukocyte antigen (HLA) class II molecules (HLA-II), which through peptide flanking residues (PFRs) can extend beyond the limits of the HLA-binding. The role of the PFRs during antigen recognition is not fully understood; however, recent studies have indicated that these regions can influence TCR affinity and pHLA-II stability. Here, using various biochemical approaches including peptide sensitivity ELISA and ELISpot assays, peptide binding assays and HLA-II tetramer staining, we focused on CD4+ T-cell responses against a tumor antigen, 5T4 oncofetal trophoblast glycoprotein (5T4), which have been associated with improved control of colorectal cancer. Despite their weak T-cell receptor (TCR) binding affinity, we found that anti-5T4 CD4+ T-cells are polyfunctional and that their PFRs are essential for TCR recognition of the core bound nonamer. The high-resolution (1.95 Å) crystal structure of HLA-DR1 presenting the immunodominant 20-mer peptide 5T4111-130, combined with molecular dynamic simulations, revealed how PFRs explore the HLA-proximal space to contribute to antigen reactivity. These findings advance our understanding of what constitutes an HLA-II epitope and indicate that PFRs can tune weak-affinity TCR-pHLA-II interactions.
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Oct 2019
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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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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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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Katie
Tungatt
,
Garry
Dolton
,
Sophie B.
Morgan
,
Meriem
Attaf
,
Anna
Fuller
,
Thomas
Whalley
,
Johanneke D.
Hemmink
,
Emily
Porter
,
Barbara
Szomolay
,
Maria
Montoya
,
John A.
Hammond
,
John J.
Miles
,
David K.
Cole
,
Alain
Townsend
,
Mick
Bailey
,
Pierre
Rizkallah
,
Bryan
Charleston
,
Elma
Tchilian
,
Andrew K.
Sewell
Diamond Proposal Number(s):
[10462, 14843, 20147]
Open Access
Abstract: There is increasing evidence that induction of local immune responses is a key component of effective vaccines. For respiratory pathogens, for example tuberculosis and influenza, aerosol delivery is being actively explored as a method to administer vaccine antigens. Current animal models used to study respiratory pathogens suffer from anatomical disparity with humans. The pig is a natural and important host of influenza viruses and is physiologically more comparable to humans than other animal models in terms of size, respiratory tract biology and volume. It may also be an important vector in the birds to human infection cycle. A major drawback of the current pig model is the inability to analyze antigen-specific CD8+ T-cell responses, which are critical to respiratory immunity. Here we address this knowledge gap using an established in-bred pig model with a high degree of genetic identity between individuals, including the MHC (Swine Leukocyte Antigen (SLA)) locus. We developed a toolset that included long-term in vitro pig T-cell culture and cloning and identification of novel immunodominant influenza-derived T-cell epitopes. We also generated structures of the two SLA class I molecules found in these animals presenting the immunodominant epitopes. These structures allowed definition of the primary anchor points for epitopes in the SLA binding groove and established SLA binding motifs that were used to successfully predict other influenza-derived peptide sequences capable of stimulating T-cells. Peptide-SLA tetramers were constructed and used to track influenza-specific T-cells ex vivo in blood, the lungs and draining lymph nodes. Aerosol immunization with attenuated single cycle influenza viruses (S-FLU) induced large numbers of CD8+ T-cells specific for conserved NP peptides in the respiratory tract. Collectively, these data substantially increase the utility of pigs as an effective model for studying protective local cellular immunity against respiratory pathogens.
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May 2018
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