I04-1-Macromolecular Crystallography (fixed wavelength)
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Emily A.
Bates
,
James A.
Davies
,
Jana
Váňová
,
Davor
Nestić
,
Valerie S.
Meniel
,
Sarah
Koushyar
,
Tabitha G.
Cunliffe
,
Rosie M.
Mundy
,
Elise
Moses
,
Hanni K.
Uusi-Kerttula
,
Alexander T.
Baker
,
David K.
Cole
,
Dragomira
Majhen
,
Pierre J.
Rizkallah
,
Toby
Phesse
,
John D.
Chester
,
Alan L.
Parker
Diamond Proposal Number(s):
[18812]
Open Access
Abstract: Oncolytic virotherapies (OV) hold immense clinical potential. OV based on human adenoviruses (HAdV) derived from HAdV with naturally low rates of pre-existing immunity will be beneficial for future clinical translation. We generated a low seroprevalence HAdV-D10 serotype vector incorporating an αvβ6 integrin selective peptide, A20, to target αvβ6 positive tumour cell types. HAdV-D10 has limited natural tropism. Structural and biological studies of HAdV-D10 knob protein highlighted low affinity engagement with native adenoviral receptors CAR and sialic acid. HAdV-D10 fails to engage blood coagulation Factor X, potentially eliminating “off-target” hepatic sequestration in vivo. We engineered A20 peptide that selectively binds αvβ6 integrin into the DG loop of HAdV-D10 fiber knob. Assays in αvβ6+ cancer cell lines, demonstrated significantly increased transduction mediated by αvβ6 targeted variants compared to controls, confirmed microscopically. HAdV-D10.A20 resisted neutralization by neutralizing HAdV-C5 sera. Systemic delivery of HAdV-D10.A20 resulted in significantly increased GFP expression in BT20 tumours. Replication competent HAdV-D10.A20 demonstrated αvβ6 integrin selective cell killing in vitro and in vivo. HAdV-D10 possesses characteristics of a promising virotherapy, combining low seroprevalence, weak receptor interactions and reduced off-target uptake. Incorporation of an αvβ6 integrin selective peptide resulted in HAdV-D10.A20, with significant potential for clinical translation.
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Mar 2022
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Claire
Barber
,
Victoria Arena
De Souza
,
Rachel L.
Paterson
,
Magdalena
Martin-Urdiroz
,
Nitha Charles
Mulakkal
,
Velupillai
Srikannathasan
,
Mary
Connolly
,
Gwilym
Phillips
,
Tein
Foong-Leong
,
Robert
Pengelly
,
Vijaykumar
Karuppiah
,
Tressan
Grant
,
Marcin
Dembek
,
Anil
Verma
,
Dawn
Gibbs-Howe
,
Thomas H.
Blicher
,
Andrew
Knox
,
Ross A.
Robinson
,
David K.
Cole
,
Sarah
Leonard
Abstract: The non-polymorphic class Ib molecule, human leukocyte antigen (HLA)-E, primarily presents peptides from HLA class Ia leader peptides, providing an inhibitory signal to NK cells via CD94/NKG2 interactions.
Although peptides of pathogenic origin can also be presented by HLA-E to T cells, the molecular basis underpinning their role in antigen surveillance is largely unknown. Here, we solved a co-complex crystal structure of a T cell receptor (TCR) with an HLA-E presented peptide (pHLA-E) from bacterial (Mycobacterium tuberculosis) origin, and the first TCR-pHLA-E complex with a non-canonically presented peptide from viral (human immuno-deficiency virus; HIV) origin. The structures provided a molecular foundation to develop a novel method to introduce cysteine traps using non-natural amino acid chemistry that stabilized pHLA-E complexes whilst maintaining native interface contacts between the TCRs and different pHLA-E complexes. These pHLA-E monomers could be used to isolate pHLA-E specific T cells, with obvious utility for studying pHLA-E restricted T cells, and for the identification of putative therapeutic TCRs.
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Feb 2022
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Stephen
Man
,
James E.
Redman
,
Deborah L.
Cross
,
David K.
Cole
,
Ilona
Can
,
Bethan
Davies
,
Shaikh Shimaz
Hashimdeen
,
Reiss
Reid
,
Sian
Llewellyn-Lacey
,
Kelly L.
Miners
,
Kristin
Ladell
,
Anya
Lissina
,
Paul E.
Brown
,
Linda
Wooldridge
,
David A.
Price
,
Pierre J.
Rizkallah
Diamond Proposal Number(s):
[10462, 14843]
Abstract: The human CD8+ T cell clone 6C5 has previously been shown to recognize the tert-butyl-modified Bax161–170 peptide LLSY(3-tBu)FGTPT presented by HLA-A*02:01. This nonnatural epitope was likely created as a by-product of fluorenylmethoxycarbonyl protecting group peptide synthesis and bound poorly to HLA-A*02:01. In this study, we used a systematic approach to identify and characterize natural ligands for the 6C5 TCR. Functional analyses revealed that 6C5 T cells only recognized the LLSYFGTPT peptide when tBu was added to the tyrosine residue and did not recognize the LLSYFGTPT peptide modified with larger (di-tBu) or smaller chemical groups (Me). Combinatorial peptide library screening further showed that 6C5 T cells recognized a series of self-derived peptides with dissimilar amino acid sequences to LLSY(3-tBu)FGTPT. Structural studies of LLSY(3-tBu)FGTPT and two other activating nonamers (IIGWMWIPV and LLGWVFAQV) in complex with HLA-A*02:01 demonstrated similar overall peptide conformations and highlighted the importance of the position (P) 4 residue for T cell recognition, particularly the capacity of the bulky amino acid tryptophan to substitute for the tBu-modified tyrosine residue in conjunction with other changes at P5 and P6. Collectively, these results indicated that chemical modifications directly altered the immunogenicity of a synthetic peptide via molecular mimicry, leading to the inadvertent activation of a T cell clone with unexpected and potentially autoreactive specificities
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Jul 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18812]
Open Access
Abstract: The human adenovirus (HAdV) phylogenetic tree is diverse, divided across seven species and comprising over 100 individual types. Species D HAdV are rarely isolated with low rates of pre-existing immunity, making them appealing for therapeutic applications. Several species D vectors have been developed as vaccines against infectious diseases where they induce robust immunity in pre-clinical models and early phase clinical trials. However, many aspects of the basic virology of species D HAdV, including their basic receptor usage and means of cell entry, remain understudied.
Here, we investigated HAdV-D49, which previously has been studied for vaccine and vascular gene transfer applications. We generated a pseudotyped HAdV-C5 presenting the HAdV-D49 fiber knob protein (HAdV-C5/D49K). This pseudotyped vector was efficient at infecting cells devoid of all known HAdV receptors, indicating HAdV-D49 uses an unidentified cellular receptor. Conversely, a pseudotyped vector presenting the fiber knob protein of the closely related HAdV-D30 (HAdV-C5/D30K), differing in four amino acids to HAdV-D49, failed to demonstrate the same tropism. These four amino acid changes resulted in a change in isoelectric point of the knob protein, with HAdV-D49K possessing a basic apical region compared to a more acidic region in HAdV-D30K. Structurally and biologically we demonstrate that HAdV-D49 knob protein is unable to engage CD46, while potential interaction with CAR is extremely limited by extension of the DG loop. HAdV-C5/49K efficiently transduced cancer cell lines of pancreatic, breast, lung, oesophageal and ovarian origin, indicating it may have potential for oncolytic virotherapy applications, especially for difficult to transduce tumor types.
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Dec 2020
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I03-Macromolecular Crystallography
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Open Access
Abstract: The CD8 T cell response to the HLA-A2-restricted epitope LLWNGPMAV (LLW) of the non-structural protein 4b of Yellow Fever Virus (YFV) is remarkably immunodominant, highly prevalent and powerful in YFV-vaccinated humans. Here we used a combinatorial peptide library screening in the context of an A2/LLW-specific CD8 T cell clone to identify a superagonist that features a methionine to isoleucine substitution at position 7. Based on in silico modeling, the functional enhancement of this LLW-7I mutation was associated with alterations in the structural dynamics of the peptide in the major histocompatibility complex (pMHC) binding with the T cell receptor (TCR). While the TCR off-rate of LLW-7I pMHC is comparable to the wild type peptide, the rigidity of the 7I peptide seems to confer less entropy loss upon TCR binding. This LLW-7I superagonist is an example of improved functionality in human CD8 T cells associated with optimized ligand rigidity for TCR binding and not with changes in TCR:pMHC off-rate kinetics.
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Sep 2020
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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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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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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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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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