6G1E

BEAT Fc with improved heterodimerization (Q3A-D84.4Q)


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.88 Å
  • R-Value Free: 0.272 
  • R-Value Work: 0.231 
  • R-Value Observed: 0.233 

wwPDB Validation 3D Report Full Report


This is version 1.1 of the entry. See complete history


Literature

A single mutation increases heavy chain heterodimer assembly of bispecific antibodies by inducing structural disorder in one homodimer species.

Stutz, C.Blein, S.

(2020) J Biol Chem 

  • DOI: 10.1074/jbc.RA119.012335
  • Structures With Same Primary Citation

  • PubMed Abstract: 
  • We previously reported efficient heavy chain (Hc) assembly of heterodimeric bispecific antibodies (bsAbs) by exchanging the inter-domain protein interface of the human IgG1 CH3 dimer with the protein interface of the constant α (Ca) and β (Cb) domain ...

    We previously reported efficient heavy chain (Hc) assembly of heterodimeric bispecific antibodies (bsAbs) by exchanging the inter-domain protein interface of the human IgG1 CH3 dimer with the protein interface of the constant α (Ca) and β (Cb) domains of the human T-cell receptor (TCR), a technology known as BEAT (Bispecific Engagement by Antibodies based on the T-cell receptor). Efficient heterodimerization in mammalian cell transient transfections was observed, but levels were influenced by the nature of the binding arms, particularly in the Fab-scFv-Fc format. In this study, we report a single amino acid change that significantly and consistently improved the heterodimerization rate of this format (≥95%) by inducing partial disorder in one homodimer species without affecting the heterodimer. Correct folding and assembly of the heterodimer were confirmed by the high-resolution (1.88-1.98 Å) crystal structure presented here. Thermal stability and 1-anilinonaphthalene-8-sulfonic acid (ANS)-binding experiments, comparing original BEAT, mutated BEAT and "knobs-into-holes" (KiH) interfaces, suggested a cooperative assembly process of Hcs in heterodimers. The observed gain in stability of the interfaces could be classified in the following rank order: mutated BEAT > original BEAT > KiH. We therefore propose that the superior cooperativity found in BEAT interfaces is the key driver of their greater performance. Furthermore, we show how the mutated BEAT interface can be exploited for the routine preparation of drug candidates, with minimal risk of homodimer contamination using a single Protein A chromatography step.


    Organizational Affiliation

    Antibody Engineering, Ichnos Sciences SA, Switzerland.



Macromolecules

Find similar proteins by: Sequence  |  Structure

Entity ID: 1
MoleculeChainsSequence LengthOrganismDetails
Immunoglobulin heavy constant gamma 1,Immunoglobulin heavy constant gamma 3
A
233Homo sapiensMutation(s): 11 
Gene Names: IGHG1
Find proteins for P01857 (Homo sapiens)
Go to UniProtKB:  P01857
NIH Common Fund Data Resources
PHAROS  P01857
Find proteins for P01860 (Homo sapiens)
Go to UniProtKB:  P01860

Find similar proteins by: Sequence  |  Structure

Entity ID: 2
MoleculeChainsSequence LengthOrganismDetails
Immunoglobulin gamma-1 heavy chain
B
227Homo sapiensMutation(s): 16 
Find proteins for P0DOX5 (Homo sapiens)
Go to UniProtKB:  P0DOX5
Small Molecules
Ligands 5 Unique
IDChainsName / Formula / InChI Key2D Diagram3D Interactions
NAG
Query on NAG

Download CCD File 
A, B
N-ACETYL-D-GLUCOSAMINE
C8 H15 N O6
OVRNDRQMDRJTHS-FMDGEEDCSA-N
 Ligand Interaction
BMA
Query on BMA

Download CCD File 
A, B
BETA-D-MANNOSE
C6 H12 O6
WQZGKKKJIJFFOK-RWOPYEJCSA-N
 Ligand Interaction
MAN
Query on MAN

Download CCD File 
A, B
ALPHA-D-MANNOSE
C6 H12 O6
WQZGKKKJIJFFOK-PQMKYFCFSA-N
 Ligand Interaction
GAL
Query on GAL

Download CCD File 
A, B
BETA-D-GALACTOSE
C6 H12 O6
WQZGKKKJIJFFOK-FPRJBGLDSA-N
 Ligand Interaction
FUL
Query on FUL

Download CCD File 
A, B
BETA-L-FUCOSE
C6 H12 O5
SHZGCJCMOBCMKK-KGJVWPDLSA-N
 Ligand Interaction
Modified Residues  1 Unique
IDChainsTypeFormula2D DiagramParent
MLY
Query on MLY
AL-PEPTIDE LINKINGC8 H18 N2 O2LYS
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.88 Å
  • R-Value Free: 0.272 
  • R-Value Work: 0.231 
  • R-Value Observed: 0.233 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 49.65α = 90
b = 73.02β = 90
c = 140.93γ = 90
Software Package:
Software NamePurpose
REFMACrefinement
MOSFLMdata reduction
SCALAdata scaling
PHASERphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

  • Deposited Date: 2018-03-21 
  • Released Date: 2019-04-10 
  • Deposition Author(s): Stutz, C., Blein, S.

Revision History 

  • Version 1.0: 2019-04-10
    Type: Initial release
  • Version 1.1: 2020-06-03
    Changes: Data collection, Database references