I03-Macromolecular Crystallography
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Gareth C. G.
Davies
,
Neesha
Dedi
,
Paul S.
Jones
,
Lara
Kevorkian
,
David
Mcmillan
,
Cristina
Ottone
,
Monika-Sarah E. D.
Schulze
,
Anthony
Scott-Tucker
,
Roohi
Tewari
,
Shauna
West
,
Michael
Wright
,
Tania F.
Rowley
Diamond Proposal Number(s):
[5006]
Open Access
Abstract: Gremlin-1, a high-affinity antagonist of bone morphogenetic proteins (BMP)-2, −4, and −7, is implicated in tumor initiation and progression. Increased gremlin-1 expression, and therefore suppressed BMP signaling, correlates with poor prognosis in a range of cancer types. A lack of published work using therapeutic modalities has precluded the testing of the hypothesis that blocking the gremlin-1/BMP interaction will provide benefits to patients. To address this shortfall, we developed ginisortamab (UCB6114), a first-in-class clinical anti-human gremlin-1 antibody, currently in clinical development for the treatment of cancer, along with its murine analog antibody Ab7326 mouse immunoglobulin G1 (mIgG1). Surface plasmon resonance assays revealed that ginisortamab and Ab7326 mIgG1 had similar affinities for human and mouse gremlin-1, with mean equilibrium dissociation constants of 87 pM and 61 pM, respectively. The gremlin-1/Ab7326 antigen-binding fragment (Fab) crystal structure revealed a gremlin-1 dimer with a Fab molecule bound to each monomer that blocked BMP binding. In cell culture experiments, ginisortamab fully blocked the activity of recombinant human gremlin-1, and restored BMP signaling pathways in human colorectal cancer (CRC) cell lines. Furthermore, in a human CRC – fibroblast co-culture system where gremlin-1 is produced by the fibroblasts, ginisortamab restored BMP signaling in both the CRC cells and fibroblasts, demonstrating its activity in a relevant human tumor microenvironment model. The safety and efficacy of ginisortamab are currently being evaluated in a Phase 1/2 clinical trial in patients with advanced solid tumors (NCT04393298).
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Dec 2023
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B21-High Throughput SAXS
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Emma
Davé
,
Oliver
Durrant
,
Neha
Dhami
,
Joanne
Compson
,
Janice
Broadbridge
,
Sophie
Archer
,
Asher
Maroof
,
Kevin
Whale
,
Karelle
Menochet
,
Pierre
Bonnaillie
,
Emily
Barry
,
Gavin
Wild
,
Claude
Peerboom
,
Pallavi
Bhatta
,
Mark
Ellis
,
Matthew
Hinchliffe
,
David P.
Humphreys
,
Sam P.
Heywood
Open Access
Abstract: TrYbe® is an Fc-free therapeutic antibody format, capable of engaging up to three targets simultaneously, with long in vivo half-life conferred by albumin binding. This format is shown by small-angle X-ray scattering to be conformationally flexible with favorable ‘reach’ properties. We demonstrate the format’s broad functionality by co-targeting of soluble and cell surface antigens. The benefit of monovalent target binding is illustrated by the lack of formation of large immune complexes when co-targeting multivalent antigens. TrYbes® are manufactured using standard mammalian cell culture and protein A affinity capture processes. TrYbes® have been formulated at high concentrations and have favorable drug-like properties, including stability, solubility, and low viscosity. The unique functionality and inherent developability of the TrYbe® makes it a promising multi-specific antibody fragment format for antibody therapy.
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Feb 2023
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[25110]
Open Access
Abstract: Monoclonal antibody (mAb) aggregation can present major challenges for the development of biotherapeutics. An understanding of the molecular mechanisms of mAb aggregation is highly desirable both because it allows the performance of informed risk assessments regarding the criticality of mAb aggregates and because it may facilitate rational stabilization of aggregation prone regions. Here, we report the generation and isolation of dimer species of an IgG4 mAb (mAb1) that were present in stressed material under differing levels of temperature stress. We demonstrate the power of combining established higher order techniques with non-routine analysis, such as small-angle X-ray scattering, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and protein conformational array enzyme-linked immunosorbent assay (PCA ELISA), and show that dimer species formed under temperature stress are structurally distinct from those present in unstressed mAb1. Specifically, stress-induced dimers are shown to adopt a more elongated conformation with a greater degree of unfolding when compared to native dimers. Analysis by HDX-MS and PCA ELISA, supported by in silico shape and charge molecular docking, enabled the identification of residues in both the variable and constant domains that appear to play a significant role in the dimerization of mAb1. Furthermore, we show that dimers formed under temperature stress are significantly more long-lived than those present in unstressed mAb1. We also present evidence that mAb1 dimers can behave as aggregation nuclei, and that dimers produced under high-temperature stress do so to a greater extent. This work presents an advancement in our understanding of the molecular mechanisms of mAb aggregation and highlights the importance of structural characterization of dimer species during the development of mAb biotherapeutics.
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Oct 2022
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I04-Macromolecular Crystallography
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Carolina T.
Orozco
,
Manuela
Bersellini
,
Lorraine M.
Irving
,
Wesley W.
Howard
,
David
Hargreaves
,
Paul W. A.
Devine
,
Elise
Siouve
,
Gareth J.
Browne
,
Nicholas J.
Bond
,
Jonathan J.
Phillips
,
Peter
Ravn
,
Sophie E.
Jackson
Open Access
Abstract: Although monoclonal antibodies have greatly improved cancer therapy, they can trigger side effects due to on-target, off-tumor toxicity. Over the past decade, strategies have emerged to successfully mask the antigen-binding site of antibodies, such that they are only activated at the relevant site, for example, after proteolytic cleavage. However, the methods for designing an ideal affinity-based mask and what parameters are important are not yet well understood. Here, we undertook mechanistic studies using three masks with different properties and identified four critical factors: binding site and affinity, as well as association and dissociation rate constants, which also played an important role. HDX-MS was used to identify the location of binding sites on the antibody, which were subsequently validated by obtaining a high-resolution crystal structure for one of the mask-antibody complexes. These findings will inform future designs of optimal affinity-based masks for antibodies and other therapeutic proteins.
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Jul 2022
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[12346]
Open Access
Abstract: The success of therapeutic antibodies is largely attributed for their exquisite specificity, homogeneity, and functionality. There is, however, a need to engineer antibodies to extend and enhance their potency. One parameter is functional affinity augmentation, since antibodies matured in vivo have a natural affinity threshold. Generation of multivalent antibodies is one option capable of surpassing this affinity threshold through increased avidity. In this study, we present a novel platform consisting of an array of multivalent antibody formats, termed Quads, generated using the self-assembling tetramerization domain from p53. We demonstrate the versatility of this tetramerization domain by engineering anti-tumor necrosis factor (TNF) Quads that exhibit major increases in binding potency and in neutralizing TNF-mediated cytotoxicity compared to parental anti-TNF molecules. Further, Quads are amenable to fusion with different binding domains, allowing generation of novel multivalent monospecific and bispecific formats. Quads are thus a novel group of molecules that can be engineered to yield potential therapeutics with novel modalities and potencies.
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Jan 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Mark
Austin
,
Daniel
Burschowsky
,
Denice T. Y.
Chan
,
Lesley
Jenkinson
,
Stuart
Haynes
,
Agata
Diamandakis
,
Chitra
Seewooruthun
,
Alexandra
Addyman
,
Sebastian
Fiedler
,
Stephanie
Ryman
,
Jessica
Whitehouse
,
Louise H.
Slater
,
Andreas V.
Hadjinicolaou
,
Uzi
Gileadi
,
Ellen
Gowans
,
Yoko
Shibata
,
Michelle
Barnard
,
Teresa
Kaserer
,
Pooja
Sharma
,
Nadia M.
Luheshi
,
Robert W.
Wilkinson
,
Tristan J.
Vaughan
,
Sarah V.
Holt
,
Vincenzo
Cerundolo
,
Mark D.
Carr
,
Maria A. T.
Groves
Diamond Proposal Number(s):
[19880]
Open Access
Abstract: Arginase 2 (ARG2) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine. The dysregulated expression of ARG2 within specific tumor microenvironments generates an immunosuppressive niche that effectively renders the tumor ‘invisible’ to the host’s immune system. Increased ARG2 expression leads to a concomitant depletion of local L-arginine levels, which in turn leads to suppression of anti-tumor T-cell-mediated immune responses. Here we describe the isolation and characterization of a high affinity antibody (C0021158) that inhibits ARG2 enzymatic function completely, effectively restoring T-cell proliferation in vitro. Enzyme kinetic studies confirmed that C0021158 exhibits a noncompetitive mechanism of action, inhibiting ARG2 independently of L-arginine concentrations. To elucidate C0021158’s inhibitory mechanism at a structural level, the co-crystal structure of the Fab in complex with trimeric ARG2 was solved. C0021158’s epitope was consequently mapped to an area some distance from the enzyme’s substrate binding cleft, indicating an allosteric mechanism was being employed. Following C0021158 binding, distinct regions of ARG2 undergo major conformational changes. Notably, the backbone structure of a surface-exposed loop is completely rearranged, leading to the formation of a new short helix structure at the Fab-ARG2 interface. Moreover, this large-scale structural remodeling at ARG2’s epitope translates into more subtle changes within the enzyme’s active site. An arginine residue at position 39 is reoriented inwards, sterically impeding the binding of L-arginine. Arg39 is also predicted to alter the pK A of a key catalytic histidine residue at position 160, further attenuating ARG2’s enzymatic function. In silico molecular docking simulations predict that L-arginine is unable to bind effectively when antibody is bound, a prediction supported by isothermal calorimetry experiments using an L-arginine mimetic. Specifically, targeting ARG2 in the tumor microenvironment through the application of C0021158, potentially in combination with standard chemotherapy regimens or alternate immunotherapies, represents a potential new strategy to target immune cold tumors.
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Jan 2020
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I04-Macromolecular Crystallography
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Janet
Sim
,
Jonathan T.
Sockolosky
,
Emma
Sangalang
,
Shelley
Izquierdo
,
Darlene
Pedersen
,
William
Harriman
,
Ardian S.
Wibowo
,
Josh
Carter
,
Anup
Madan
,
Laura
Doyle
,
Ons
Harrabi
,
Steven E.
Kauder
,
Amy
Chen
,
Tracy C.
Kuo
,
Hong
Wan
,
Jaume
Pons
Open Access
Abstract: Targeting the CD47-signal-regulatory protein α (SIRPα) pathway represents a novel therapeutic approach to enhance anti-cancer immunity by promoting both innate and adaptive immune responses. Unlike CD47, which is expressed ubiquitously, SIRPα expression is mainly restricted to myeloid cells and neurons. Therefore, compared to CD47-targeted therapies, targeting SIRPα may result in differential safety and efficacy profiles, potentially enabling lower effective doses and improved pharmacokinetics and pharmacodynamics. The development of effective SIRPα antagonists is restricted by polymorphisms within the CD47-binding domain of SIRPα, necessitating pan-allele reactive anti-SIRPα antibodies for therapeutic intervention in diverse patient populations. We immunized wild-type and human antibody transgenic chickens with a multi-allele and multi-species SIRPα regimen in order to discover pan-allelic and pan-mammalian reactive anti-SIRPα antibodies suitable for clinical translation. A total of 200 antibodies were isolated and screened for SIRPα reactivity from which approximately 70 antibodies with diverse SIRPα binding profiles, sequence families, and epitopes were selected for further characterization. A subset of anti-SIRPα antibodies bound to both human SIRPα v1 and v2 alleles with high affinity ranging from low nanomolar to picomolar, potently antagonized the CD47/SIRPα interaction, and potentiated macrophage-mediated antibody-dependent cellular phagocytosis in vitro. X-ray crystal structures of five anti-SIRPα antigen-binding fragments, each with unique epitopes, in complex with SIRPα (PDB codes 6NMV, 6NMU, 6NMT, 6NMS, and 6NMR) are reported. Furthermore, some of the anti-SIRPα antibodies cross-react with cynomolgus SIRPα and various mouse SIRPα alleles (BALB/c, NOD, BL/6), which can facilitate preclinical to clinical development. These properties provide an attractive rationale to advance the development of these anti-SIRPα antibodies as a novel therapy for advanced malignancies.
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Jun 2019
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I04-Macromolecular Crystallography
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Bryan
Smith
,
Andrea
Kiessling
,
Rocio
Lledo-Garcia
,
Kate L.
Dixon
,
Matthew C.
Catley
,
Paul
Atherfold
,
Lena E.
D’hooghe
,
Helene
Finney
,
Kevin
Greenslade
,
Hanna
Hailu
,
Lara
Kevorkian
,
Daniel
Lightwood
,
Christoph
Meier
,
Rebecca
Munro
,
Omar
Qureshi
,
Kaushik
Sarkar
,
Sophie P.
Shaw
,
Roohi
Tewari
,
Alison
Turner
,
Kerry
Tyson
,
Shauna
West
,
Stevan
Shaw
,
Frank R.
Brennan
Open Access
Abstract: Rozanolixizumab (UCB7665), a humanized high-affinity anti-human neonatal Fc receptor (FcRn) monoclonal antibody (IgG4P), has been developed to reduce pathogenic IgG in autoimmune and alloimmune diseases. We document the antibody isolation and compare rozanolixizumab with the same variable region expressed in various mono-, bi- and trivalent formats. We report activity data for rozanolixizumab and the different molecular formats in human cells, FcRn-transgenic mice, and cynomolgus monkeys. Rozanolixizumab, considered the most effective molecular format, dose-dependently and selectively reduced plasma IgG concentrations in an FcRn-transgenic mouse model (no effect on albumin). Intravenous (IV) rozanolixizumab dosing in cynomolgus monkeys demonstrated non-linear pharmacokinetics indicative of target-mediated drug disposition; single IV rozanolixizumab doses (30 mg/kg) in cynomolgus monkeys reduced plasma IgG concentration by 69% by Day 7 post-administration. Daily IV administration of rozanolixizumab (initial 30 mg/kg loading dose; 5 mg/kg daily thereafter) reduced plasma IgG concentrations in all cynomolgus monkeys, with low concentrations maintained throughout the treatment period (42 days). In a 13-week toxicology study in cynomolgus monkeys, supra-pharmacological subcutaneous and IV doses of rozanolixizumab (≤ 150 mg/kg every 3 days) were well tolerated, inducing sustained (but reversible) reductions in IgG concentrations by up to 85%, with no adverse events observed. We have demonstrated accelerated natural catabolism of IgG through inhibition of IgG:FcRn interactions in mice and cynomolgus monkeys. Inhibition of FcRn with rozanolixizumab may provide a novel therapeutic approach to reduce pathogenic IgG in human autoimmune disease. Rozanolixizumab is being investigated in patients with immune thrombocytopenia (NCT02718716) and myasthenia gravis (NCT03052751).
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Aug 2018
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Ralph
Adams
,
Laura
Griffin
,
Joanne E.
Compson
,
Mark
Jairaj
,
Terry
Baker
,
Tom
Ceska
,
Shauna
West
,
Oliver
Zaccheo
,
Emma
Davé
,
Alastair Dg.
Lawson
,
David P.
Humphreys
,
Sam
Heywood
Open Access
Abstract: We generated an anti-albumin antibody, CA645, to link its Fv domain to an antigen-binding fragment (Fab), thereby extending the serum half-life of the Fab. CA645 was demonstrated to bind human, cynomolgus, and mouse serum albumin with similar affinity (1–7 nM), and to bind human serum albumin (HSA) when it is in complex with common known ligands. Importantly for half-life extension, CA645 binds HSA with similar affinity within the physiologically relevant range of pH 5.0 – pH 7.4, and does not have a deleterious effect on the binding of HSA to neonatal Fc receptor (FcRn). A crystal structure of humanized CA645 Fab in complex with HSA was solved and showed that CA645 Fab binds to domain II of HSA. Superimposition with the crystal structure of FcRn bound to HSA confirmed that CA645 does not block HSA binding to FcRn. In mice, the serum half-life of humanized CA645 Fab is 84.2 h. This is a significant extension in comparison with < 1 h for a non-HSA binding CA645 Fab variant. The Fab-HSA structure was used to design a series of mutants with reduced affinity to investigate the correlation between the affinity for albumin and serum half-life. Reduction in the affinity for MSA by 144-fold from 2.2 nM to 316 nM had no effect on serum half-life. Strikingly, despite a reduction in affinity to 62 µM, an extension in serum half-life of 26.4 h was still obtained. CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, 5FUZ and 5FUO, respectively.
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Jun 2016
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I02-Macromolecular Crystallography
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Stevan
Shaw
,
Tim
Bourne
,
Chris
Meier
,
Bruce
Carrington
,
Rich
Gelinas
,
Alistair
Henry
,
Andrew
Popplewell
,
Ralph
Adams
,
Terry
Baker
,
Steve
Rapecki
,
Diane
Marshall
,
Adrian
Moore
,
Helen
Neale
,
Alastair
Lawson
Open Access
Abstract: Interleukin-6 (IL-6) is a critical regulator of the immune system and has been widely implicated in autoimmune disease. Here, we describe the discovery and characterization of olokizumab, a humanized antibody to IL-6. Data from structural biology, cell biology and primate pharmacology demonstrate the therapeutic potential of targeting IL-6 at “Site 3”, blocking the interaction with the signaling co-receptor gp130.
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Apr 2014
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