I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Rita
Szoke-Kovacs
,
Sophie
Khakoo
,
Victor
Lopes Rangel
,
Pietro
Della Cristina
,
Johanne
Pentier
,
Rahul
Khanolkar
,
Sam
El-Ajouz
,
Robert
Simmons
,
David K.
Cole
,
Peter
Gogolak
,
Mariolina
Salio
,
Vijaykumar
Karuppiah
Diamond Proposal Number(s):
[22870, 33930]
Open Access
Abstract: Introduction: Immunotherapy is emerging as an efficacious treatment for some cancers, complementing traditional chemo-radiation therapies. Specific markers at the cell surface of cancer cells can be used as immunotherapy targets. However, many of these markers are defined by a patient’s genetic background, limiting their use across the human population.
Methods: Here, we investigated the non-polymorphic antigen presenting molecule, CD1c, that is only expressed on subsets of mature hematopoietic cells, as a potential immunotherapy target with reduced risk of off-tumor on-target toxicity in healthy tissues.
Results and discussion: We identified a T cell receptor (TCR) which recognises CD1c in a lipid independent manner and determined the crystal structure of the TCR-CD1c complex which revealed flexibility around the lipid binding region, and a new binding mechanism of auto-antigen recognition. We generated affinity enhanced variants of the TCR and fused them to an anti-CD3 antibody for T cell redirection. Lipidomic analysis revealed promiscuous lipid recognition of CD1c by the affinity enhanced TCR variants, with preference for larger lipid head group, a finding which is supported by the crystal structure. The bispecific molecule induced potent re-directed T cell killing of CD1c positive cell lines. These proof-of-concept findings demonstrate that CD1c targeting TCR bispecific engagers might be good candidates for the development of non-MHC restricted, universal therapeutics for the treatment of CD1c+ leukemias.
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Jul 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[17077, 28224]
Open Access
Abstract: T cell receptors (TCRs) recognize specific peptides presented by human leukocyte antigens (HLAs) on the surface of antigen presenting cells and are involved in fighting pathogens and cancer surveillance. Canonical docking orientation of TCRs to their target peptide-HLAs (pHLAs) is essential for T cell activation, with reverse binding TCRs lacking functionality. TCR binding geometry and molecular interaction footprint with pHLAs is typically obtained by determining the crystal structure. Here, we describe the use of a cross-linking tandem mass spectrometry (XL-MS/MS) method to decipher the binding orientation of several TCRs to their target pHLAs. Cross-linking sites were localized to specific residues and their molecular interactions showed differentiation between TCRs binding in canonical or reverse orientations. Structural prediction and crystal structure determination of two TCR-pHLA complexes validated these findings. The XL-MS/MS method described herein offers a faster and simpler approach for elucidating TCR-pHLA binding orientation and interactions.
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Mar 2025
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I04-Macromolecular Crystallography
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Richard J.
Suckling
,
Cevriye
Pamukcu
,
Robert A.
Simmons
,
Daniel
Fonseca
,
Emma
Grant
,
Rory
Harrison
,
Saher A.
Shaikh
,
Rahul C.
Khanolkar
,
Hemza
Ghadbane
,
Andrew
Creese
,
Miriam
Hock
,
Thomas G.
Gligoris
,
Marco
Lepore
,
Vijaykumar
Karuppiah
,
Mariolina
Salio
Diamond Proposal Number(s):
[28224]
Open Access
Abstract: Introduction: The MHC-class-I-related molecule MR1 presents small metabolites of microbial and self-origin to T cells bearing semi-invariant or variant T cell receptors. One such T cell receptor, MC.7.G5, was previously shown to confer broad MR1-restricted reactivity to tumor cells but not normal cells, sparking interest in the development of non-MHC-restricted immunotherapy approaches.
Methods/Results: Here we provide cellular, biophysical, and crystallographic evidence that the MC.7.G5 TCR does not have pan-cancer specificity but is restricted to a rare allomorph of MR1, bearing the R9H mutation.
Discussion: Our results underscore the importance of in-depth characterization of MR1-reactive TCRs against targets expressing the full repertoire of MR1 allomorphs.
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Mar 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Vijaykumar
Karuppiah
,
Dhaval
Sangani
,
Lorraine
Whaley
,
Robert
Pengelly
,
Pelin
Uluocak
,
Ricardo J.
Carreira
,
Miriam
Hock
,
Pietro Della
Cristina
,
Paulina
Bartasun
,
Paula
Dobrinic
,
Nicola
Smith
,
Keir
Barnbrook
,
Ross A.
Robinson
,
Stephen
Harper
Diamond Proposal Number(s):
[22870, 28224]
Open Access
Abstract: Specificity of a T cell receptor (TCR) is determined by the combination of its interactions to the peptide and human leukocyte antigen (HLA). TCR-based therapeutic molecules have to date targeted a single peptide in the context of a single HLA allele. Some peptides are presented on multiple HLA alleles, and by engineering TCRs for specific recognition of more than one allele, there is potential to expand the targetable patient population. Here, as a proof of concept, we studied two TCRs, S2 and S8, binding to the PRAME peptide antigen (ELFSYLIEK) presented by HLA alleles HLA-A*03:01 and HLA-A*11:01. By structure-guided affinity maturation targeting a specific residue on the HLA surface, we show that the affinity of the TCR can be modulated for different alleles. Using a combination of affinity maturation and functional T cell assay, we demonstrate that an engineered TCR can target the same peptide on two different HLA alleles with similar affinity and potency. This work highlights the importance of engineering alloselectivity for designing TCR based therapeutics suitable for differing global populations.
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Nov 2024
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I04-Macromolecular Crystallography
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Gavuthami
Murugesan
,
Rachel L.
Paterson
,
Rakesh
Kulkarni
,
Veronica
Ilkow
,
Richard J.
Suckling
,
Mary M.
Connolly
,
Vijaykumar
Karuppiah
,
Robert
Pengelly
,
Archana
Jadhav
,
Jose
Donoso
,
Tiaan
Heunis
,
Wilawan
Bunjobpol
,
Gwilym
Philips
,
Kafayat
Ololade
,
Daniel
Kay
,
Anshuk
Sarkar
,
Claire
Barber
,
Ritu
Raj
,
Carole
Perot
,
Tressan
Grant
,
Agatha
Treveil
,
Andrew
Walker
,
Marcin
Dembek
,
Dawn
Gibbs-Howe
,
Miriam
Hock
,
Ricardo J.
Carreira
,
Kate E.
Atkin
,
Lucy
Dorrell
,
Andrew
Knox
,
Sarah
Leonard
,
Mariolina
Salio
,
Luis F.
Godinho
Diamond Proposal Number(s):
[28224]
Open Access
Abstract: The non-polymorphic HLA-E molecule offers opportunities for new universal immunotherapeutic approaches to chronic infectious diseases. Chronic Hepatitis B virus (HBV) infection is driven in part by T cell dysfunction due to elevated levels of the HBV envelope (Env) protein hepatitis B surface antigen (HBsAg). Here we report the characterization of three genotypic variants of an HLA-E-binding HBsAg peptide, Env371-379, identified through bioinformatic predictions and verified by biochemical and cellular assays. Using a soluble affinity-enhanced T cell receptor (TCR) (a09b08)-anti-CD3 bispecific molecule to probe HLA-E presentation of the Env371-379 peptides, we demonstrate that only the most stable Env371-379 variant, L6I, elicits functional responses to a09b08-anti-CD3-redirected polyclonal T cells co-cultured with targets expressing endogenous HBsAg. Furthermore, HLA-E-Env371-379 L6I-specific CD8+ T cells are detectable in HBV-naïve donors and people with chronic HBV after in vitro priming. In conclusion, we provide evidence for HLA-E-mediated HBV Env peptide presentation, and highlight the effect of viral mutations on the stability and targetability of pHLA-E molecules.
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Nov 2024
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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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Andrew
Poole
,
Vijaykumar
Karuppiah
,
Annabelle
Hartt
,
Jaafar N.
Haidar
,
Sylvie
Moureau
,
Tomasz
Dobrzycki
,
Conor
Hayes
,
Christopher
Rowley
,
Jorge
Dias
,
Stephen
Harper
,
Keir
Barnbrook
,
Miriam
Hock
,
Charlotte
Coles
,
Wei
Yang
,
Milos
Aleksic
,
Aimee Bence
Lin
,
Ross
Robinson
,
Joe D.
Dukes
,
Nathaniel
Liddy
,
Marc
Van Der Kamp
,
Gregory D.
Plowman
,
Annelise
Vuidepot
,
David K.
Cole
,
Andrew D.
Whale
,
Chandramouli
Chillakuri
Diamond Proposal Number(s):
[22870]
Open Access
Abstract: Neoantigens derived from somatic mutations are specific to cancer cells and are ideal targets for cancer immunotherapy. KRAS is the most frequently mutated oncogene and drives the pathogenesis of several cancers. Here we show the identification and development of an affinity-enhanced T cell receptor (TCR) that recognizes a peptide derived from the most common KRAS mutant, KRASG12D, presented in the context of HLA-A*11:01. The affinity of the engineered TCR is increased by over one million-fold yet fully able to distinguish KRASG12D over KRASWT. While crystal structures reveal few discernible differences in TCR interactions with KRASWT versus KRASG12D, thermodynamic analysis and molecular dynamics simulations reveal that TCR specificity is driven by differences in indirect electrostatic interactions. The affinity enhanced TCR, fused to a humanized anti-CD3 scFv, enables selective killing of cancer cells expressing KRASG12D. Our work thus reveals a molecular mechanism that drives TCR selectivity and describes a soluble bispecific molecule with therapeutic potential against cancers harboring a common shared neoantigen.
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Sep 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
Open Access
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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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12788]
Open Access
Abstract: In the native pathway to therapeutic cannabinoid biosynthesis in Cannabis sativa, the three‐step production of a key intermediate, olivetolic acid, is catalysed by the enzymes tetraketide synthase (TKS; linear tetraketide intermediate production in two stages) and olivetolic acid cyclase (OAC; final C2 → C7 aldol condensation). In the absence of OAC, a nonenzymatic C2 → C7 decarboxylative aldol condensation of the tetraketide intermediate occurs forming olivetol. TKS is a type III polyketide synthase, and the question arises why it is unable to form olivetolic acid directly, but instead forms this unwanted side product. We determined the TKS, CoA complex structure, and performed structurally guided mutagenesis studies to identify potential residues responsible for cyclization pathway discrimination in type III polyketide synthases. Prior studies suggested an ‘aldol switch’ is necessary to allow linear tetraketide intermediate release prior to cyclization, thereby enabling subsequent olivetolic acid production by OAC. However, our studies do not support the presence of a universal or predictable ‘aldol switch’ consensus sequence. Instead, we propose the mode of ordered active site water activation between type III polyketide synthases catalysing different cyclization mechanisms is subtle and homologue‐specific. Our work indicates that subtle structural variations between homologous enzymes can have a major mechanistic impact on the catalytic outcome. This highlights the importance of embedding high‐resolution structural analysis of multiple enzyme homologues with classical site‐directed mutagenesis studies when investigating highly similar enzymes with different mechanistic pathway outcomes.
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Oct 2019
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[8997]
Open Access
Abstract: Natural competence is the term used to describe the uptake of “naked” extracellular DNA by bacteria; it plays a significant role in horizontal genetic exchange. It is associated with type IV pili, and specialized competence pili mediate DNA uptake. Here, we show that the crystal structure of a competence-associated protein from Thermus thermophilus, ComZ, consists of a type II secretion pseudopilin-like domain, with a large β-solenoid domain inserted into the β-sheet of the pilin-like fold. ComZ binds with high affinity to another competence-associated pilin, PilA2, which lies adjacent to the comZ gene in the genome. The crystal structure of PilA2 revealed a similar type II secretion pseudopilin-like fold, with a small subdomain; docking simulations predicted that PilA2 binds between the pseudopilin-like and β-solenoid domains of ComZ. Electrophoretic shift analysis and DNase protection studies were used to show that ComZ alone and the ComZ/PilA2 complex are able to bind DNA. Protection against reductive dimethylation was used in combination with mass spectrometry and site-directed mutagenesis to identify two lysine residues in ComZ which are involved in DNA binding. They are located between the two domains in ComZ, on the opposite side from the predicted PilA2 binding site. These results suggest a model in which PilA2 assists ComZ in forming the competence pilus tip and DNA binds to the side of the fiber. The results demonstrate how a type IV pilin can be adapted to a specific function by domain insertion and provide the first structural insights into a tip-located competence pilin.
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Jun 2019
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