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- EMDB-16113: Cryo-EM structure of the CODV-IL13-RefAb triple complex -

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Basic information

Entry
Database: EMDB / ID: EMD-16113
TitleCryo-EM structure of the CODV-IL13-RefAb triple complex
Map dataPre-sharpened, unmasked map of CODV-IL13-RefAb after non-uniform refinement
Sample
  • Complex: Ternary complex of CODV-Fab with IL13 and RefAb
    • Protein or peptide: CODV-Fab, heavy chain
    • Protein or peptide: CODV-Fab, light chain
    • Protein or peptide: RefAb, light chain
    • Protein or peptide: Interleukin-13, human
    • Protein or peptide: RefAb, heavy chain
KeywordsCytosolic / bispecific / Fab / therapeutical / IMMUNE SYSTEM
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.97 Å
AuthorsFernandez-Martinez D / Kandiah E
Funding support France, 1 items
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, 2022-
Header (metadata) releaseJun 7, 2023-
Map releaseJun 7, 2023-
UpdateJun 7, 2023-
Current statusJun 7, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16113.png

Downloads & links

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Map

FileDownload / File: emd_16113.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPre-sharpened, unmasked map of CODV-IL13-RefAb after non-uniform refinement
Voxel sizeX=Y=Z: 0.827 Å
Density
Contour LevelBy AUTHOR: 0.135
Minimum - Maximum-0.08114001 - 0.447086
Average (Standard dev.)0.0003670783 (±0.0123683335)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 330.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16113_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Final sharpened and masked map of CODV-IL13-RefAb

Fileemd_16113_additional_1.map
AnnotationFinal sharpened and masked map of CODV-IL13-RefAb
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B of the pre-sharpened, unmasked map...

Fileemd_16113_half_map_1.map
AnnotationHalf map B of the pre-sharpened, unmasked map of CODV-IL13-RefAb during non-uniform refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A of the pre-sharpened, unmasked map...

Fileemd_16113_half_map_2.map
AnnotationHalf map A of the pre-sharpened, unmasked map of CODV-IL13-RefAb during non-uniform refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of CODV-Fab with IL13 and RefAb

EntireName: Ternary complex of CODV-Fab with IL13 and RefAb
Components
  • Complex: Ternary complex of CODV-Fab with IL13 and RefAb
    • Protein or peptide: CODV-Fab, heavy chain
    • Protein or peptide: CODV-Fab, light chain
    • Protein or peptide: RefAb, light chain
    • Protein or peptide: Interleukin-13, human
    • Protein or peptide: RefAb, heavy chain

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Supramolecule #1: Ternary complex of CODV-Fab with IL13 and RefAb

SupramoleculeName: Ternary complex of CODV-Fab with IL13 and RefAb / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: CODV-Fab, heavy chain

MacromoleculeName: CODV-Fab, heavy chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 36.907098 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: VQLQQSGPEL VKPGASVKIS CKASGYSFTS YWIHWIKQRP GQGLEWIGMI DPSDGETRLN QRFQGRATLT VDESTSTAYM QLRSPTSED SAVYYCTRLK EYGNYDSFYF DVWGAGTLVT VSSGEVQLKE SGPGLVAPGG SLSITCTVSG FSLTDSSINW V RQPPGKGL ...String:
VQLQQSGPEL VKPGASVKIS CKASGYSFTS YWIHWIKQRP GQGLEWIGMI DPSDGETRLN QRFQGRATLT VDESTSTAYM QLRSPTSED SAVYYCTRLK EYGNYDSFYF DVWGAGTLVT VSSGEVQLKE SGPGLVAPGG SLSITCTVSG FSLTDSSINW V RQPPGKGL EWLGMIWGDG RIDYADALKS RLSISKDSSK SQVFLEMTSL RTDDTATYYC ARDGYFPYAM DFWGQGTSVT VS SGGASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLG TQTYIC NVNHKPSNTK VDKKVEP

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Macromolecule #2: CODV-Fab, light chain

MacromoleculeName: CODV-Fab, light chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 35.726516 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DIVLTQSPAS LAVSLGQRAT ISCRASESVD SYGQSYMHWY QQKAGQPPKL LIYLASNLES GVPARFSGSG SRTDFTLTID PVQAEDAAT YYCQQNAEDS RTFGGGTKLE IKGGGGGGGD IQMTQSPASL SVSVGDTITL TCHASQNIDV WLSWFQQKPG N IPKLLIYK ...String:
DIVLTQSPAS LAVSLGQRAT ISCRASESVD SYGQSYMHWY QQKAGQPPKL LIYLASNLES GVPARFSGSG SRTDFTLTID PVQAEDAAT YYCQQNAEDS RTFGGGTKLE IKGGGGGGGD IQMTQSPASL SVSVGDTITL TCHASQNIDV WLSWFQQKPG N IPKLLIYK ASNLHTGVPS RFSGSGSGTG FTLTISSLQP EDIATYYCQQ AHSYPFTFGG GTKLEIKGGG GGRTVAAPSV FI FPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVT HQGLSS PVTKSFNR

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Macromolecule #3: RefAb, light chain

MacromoleculeName: RefAb, light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.51002 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: YVLTQPPSVS VAPGKTARIT CGGNIIGSKL VHWYQQKPGQ APVLVIYDDG DRPSGIPERF SGSNSGNTAT LTISRVEAGD EADYYCQVW DTGSDPVVFG GGTKLTVLGQ PKAAPSVTLF PPSSEELQAN KATLVCLISD FYPGAVTVAW KADSSPVKAG V ETTTPSKQ ...String:
YVLTQPPSVS VAPGKTARIT CGGNIIGSKL VHWYQQKPGQ APVLVIYDDG DRPSGIPERF SGSNSGNTAT LTISRVEAGD EADYYCQVW DTGSDPVVFG GGTKLTVLGQ PKAAPSVTLF PPSSEELQAN KATLVCLISD FYPGAVTVAW KADSSPVKAG V ETTTPSKQ SNNKYAASSY LSLTPEQWKS HRSYSCQVTH EGSTVEKTVA PTEC

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Macromolecule #4: Interleukin-13, human

MacromoleculeName: Interleukin-13, human / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 12.077022 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
PPSTALRELI EELVNITQNQ KAPLCNGSMV WSINLTAGMY CAALESLINV SGCSAIEKTQ RMLSGFCPHK VSAGQFSSLH VRDTKIEVA QFVKDLLLHL KKLFREGQFN

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Macromolecule #5: RefAb, heavy chain

MacromoleculeName: RefAb, heavy chain / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.751457 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: VQLVQSGAEV KKPGASVKVS CKASGYTFTN YGLSWVRQAP GQGLEWMGWI SANNGDTNYG QEFQGRVTMT TDTSTSTAYM ELRSLRSDD TAVYYCARDS SSSWARWFFD LWGRGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT V SWNSGALT ...String:
VQLVQSGAEV KKPGASVKVS CKASGYTFTN YGLSWVRQAP GQGLEWMGWI SANNGDTNYG QEFQGRVTMT TDTSTSTAYM ELRSLRSDD TAVYYCARDS SSSWARWFFD LWGRGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT V SWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPK

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 46.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.97 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 218792
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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