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- PDB-8so3: CryoEM structure of a therapeutic antibody (favezelimab) bound to... -

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Basic information

Entry
Database: PDB / ID: 8so3
TitleCryoEM structure of a therapeutic antibody (favezelimab) bound to human LAG3
Components
  • Lymphocyte activation gene 3 protein
  • favezelimab Fab heavy chain
  • favezelimab Fab light chain
KeywordsIMMUNOSUPPRESSANT / Lymphocyte activation gene 3 protein / favezelimab-LAG3 complex / cryoEM / Single particle reconstruction.
Function / homology
Function and homology information


plasmacytoid dendritic cell activation / negative regulation of regulatory T cell differentiation / positive regulation of natural killer cell mediated cytotoxicity / MHC class II protein binding / natural killer cell mediated cytotoxicity / negative regulation of interleukin-2 production / regulation of immune response / T cell activation / MHC class II antigen presentation / antigen binding ...plasmacytoid dendritic cell activation / negative regulation of regulatory T cell differentiation / positive regulation of natural killer cell mediated cytotoxicity / MHC class II protein binding / natural killer cell mediated cytotoxicity / negative regulation of interleukin-2 production / regulation of immune response / T cell activation / MHC class II antigen presentation / antigen binding / transmembrane signaling receptor activity / adaptive immune response / cell surface receptor signaling pathway / external side of plasma membrane / extracellular region / plasma membrane
Similarity search - Function
Interleukin-1 receptor family / Immunoglobulin subtype / Immunoglobulin / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Lymphocyte activation gene 3 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.61 Å
AuthorsMishra, A.K. / Shahid, S. / Karade, S.S. / Mariuzza, R.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)AI144422 United States
CitationJournal: Structure / Year: 2023
Title: CryoEM structure of a therapeutic antibody (favezelimab) bound to human LAG3 determined using a bivalent Fab as fiducial marker.
Authors: Arjun K Mishra / Salman Shahid / Sharanbasappa S Karade / Pragati Agnihotri / Alexander Kolesnikov / S Saif Hasan / Roy A Mariuzza /
Abstract: Lymphocyte activation gene 3 protein (LAG3) is an inhibitory receptor that is upregulated on exhausted T cells in tumors. LAG3 is a major target for cancer immunotherapy with many anti-LAG3 ...Lymphocyte activation gene 3 protein (LAG3) is an inhibitory receptor that is upregulated on exhausted T cells in tumors. LAG3 is a major target for cancer immunotherapy with many anti-LAG3 antibodies in clinical trials. However, there is no structural information on the epitopes recognized by these antibodies. We determined the single-particle cryoEM structure of a therapeutic antibody (favezelimab) bound to LAG3 to 3.5 Å resolution, revealing that favezelimab targets the LAG3-binding site for MHC class II, its canonical ligand. The small size of the complex between the conventional (monovalent) Fab of favezelimab and LAG3 (∼100 kDa) presented a challenge for cryoEM. Accordingly, we engineered a bivalent version of Fab favezelimab that doubled the size of the Fab-LAG3 complex and conferred a highly identifiable shape to the complex that facilitated particle selection and orientation for image processing. This study establishes bivalent Fabs as new fiducial markers for cryoEM analysis of small proteins.
History
DepositionApr 28, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 6, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2023Group: Database references / Refinement description / Category: citation / em_3d_fitting_list
Item: _citation.journal_volume / _citation.page_first / _em_3d_fitting_list.initial_refinement_model_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: Lymphocyte activation gene 3 protein
H: favezelimab Fab heavy chain
L: favezelimab Fab light chain
X: Lymphocyte activation gene 3 protein
Y: favezelimab Fab heavy chain
Z: favezelimab Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)222,22410
Polymers221,3396
Non-polymers8854
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Lymphocyte activation gene 3 protein / LAG-3


Mass: 57508.270 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LAG3, FDC / Cell line (production host): Expi-HEK293 / Production host: Homo sapiens (human) / References: UniProt: P18627
#2: Antibody favezelimab Fab heavy chain


Mass: 27245.727 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): Expi-HEK293 / Production host: Homo sapiens (human)
#3: Antibody favezelimab Fab light chain


Mass: 25915.627 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): Expi-HEK293 / Production host: Homo sapiens (human)
#4: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN

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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: therapeutic antibody (favezelimab) bound to human LAG3
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: Expi-HEK293
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 3.2 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9508

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 171144 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 7TZG

7tzg


Pdb chain-ID: C / Accession code: 7TZG / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0048979
ELECTRON MICROSCOPYf_angle_d0.65412296
ELECTRON MICROSCOPYf_dihedral_angle_d5.391295
ELECTRON MICROSCOPYf_chiral_restr0.0451407
ELECTRON MICROSCOPYf_plane_restr0.0061596

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