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- PDB-8blq: Cryo-EM structure of the CODV-IL13-RefAb triple complex -

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Basic information

Entry
Database: PDB / ID: 8blq
TitleCryo-EM structure of the CODV-IL13-RefAb triple complex
Components
  • CODV-Fab, heavy chain
  • CODV-Fab, light chain
  • Interleukin-13, human
  • RefAb, heavy chain
  • RefAb, light chain
KeywordsIMMUNE SYSTEM / Cytosolic / bispecific / Fab / therapeutical
Function / homology
Function and homology information


negative regulation of complement-dependent cytotoxicity / cytokine receptor binding / negative regulation of endothelial cell apoptotic process / immune response / extracellular region
Similarity search - Function
Interleukin-13 / Interleukin-13 / Interleukin-4/interleukin-13 / Interleukin-4/interleukin-13, conserved site / Interleukins -4 and -13 signature. / Interleukins 4 and 13 / Four-helical cytokine-like, core
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.97 Å
AuthorsFernandez-Martinez, D. / Kandiah, E.
Funding support France, 1items
OrganizationGrant numberCountry
Other government2017/1309 France
CitationJournal: Sci Rep / Year: 2023
Title: Structural insights into the bi-specific cross-over dual variable antibody architecture by cryo-EM.
Authors: David Fernandez-Martinez / Mark D Tully / Gordon Leonard / Magali Mathieu / Eaazhisai Kandiah /
Abstract: Multi-specific antibodies (msAbs) are being developed as next generation antibody-based therapeutics. Knowledge of the three-dimensional structures, in the full antibody context, of their fragment ...Multi-specific antibodies (msAbs) are being developed as next generation antibody-based therapeutics. Knowledge of the three-dimensional structures, in the full antibody context, of their fragment antigen-binding (Fab) moieties with or without bound antigens is key to elucidating their therapeutic efficiency and stability. However, the flexibility of msAbs, a feature essential for their multi specificity, has hindered efforts in this direction. Cross-Over Dual Variable immunoglobulin (CODV) is a promising bispecific antibody format, designed to simultaneously target the interleukins IL4 and IL13. In this work we present the biophysical and structural characterisation of a CODV:IL13 complex in the full antibody context, using cryo-electron microscopy at an overall resolution of 4.2 Å. Unlike the 1:2 stoichiometry previously observed for CODV:IL4, CODV:IL13 shows a 1:1 stoichiometry. As well as providing details of the IL13-CODV binding interface, including the residues involved in the epitope-paratope region, the structure of CODV:IL13 also validates the use of labelling antibody as a new strategy for the single particle cryo-EM study of msAbs in complex with one, or more, antigens. This strategy reduced the inherent flexibility of the IL13 binding domain of CODV without inducing either structural changes at the epitope level or steric hindrance between the IL4 and IL13 binding regions of CODV. The work presented here thus also contributes to the development of methodology for the structural study of msAbs, a promising platform for cancer immunotherapy.
History
DepositionNov 10, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 7, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CODV-Fab, heavy chain
B: CODV-Fab, light chain
C: RefAb, light chain
D: Interleukin-13, human
E: RefAb, heavy chain


Theoretical massNumber of molelcules
Total (without water)130,9725
Polymers130,9725
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS, IL-13 and RefAb are bound to a full-antibody CODV with a 1:1:1 stoichiometry., electron microscopy, Most particles in negative-stain EM suggest a 1:1:1 stoichiometry, gel filtration, ...Evidence: SAXS, IL-13 and RefAb are bound to a full-antibody CODV with a 1:1:1 stoichiometry., electron microscopy, Most particles in negative-stain EM suggest a 1:1:1 stoichiometry, gel filtration, Clear shift observed compared to apo- and IL13-bound CODV elution peaks.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12090 Å2
ΔGint-77 kcal/mol
Surface area54430 Å2

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Components

#1: Antibody CODV-Fab, heavy chain


Mass: 36907.098 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell (production host): HEK-293-FS / Production host: Homo sapiens (human)
#2: Antibody CODV-Fab, light chain


Mass: 35726.516 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell (production host): HEK-293-FS / Production host: Homo sapiens (human)
#3: Antibody RefAb, light chain


Mass: 22510.020 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster)
#4: Protein Interleukin-13, human /


Mass: 12077.022 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli) / References: UniProt: Q4VB50
#5: Antibody RefAb, heavy chain


Mass: 23751.457 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Ternary complex of CODV-Fab with IL13 and RefAb / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Image recordingElectron dose: 46 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.97 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 218792 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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