Omalizumab is a widely used therapeutic anti-IgE antibody. Here we report the crystal structure of the omalizumab–Fab in complex with an IgE-Fc fragment. This structure reveals the mechanism of omalizumab-mediated inhibition of IgE interactions with both high- and low-affinity IgE receptors, and explains why omalizumab selectively binds free IgE. The structure of the complex also provides mechanistic insight into a class of disruptive IgE inhibitors that accelerate the dissociation of the high-affinity IgE receptor from IgE. We use this structural data to generate a mutant IgE-Fc fragment that is resistant to omalizumab binding. Treatment with this omalizumab-resistant IgE-Fc fragment, in combination with omalizumab, promotes the exchange of cell-bound full-length IgE with omalizumab-resistant IgE-Fc fragments on human basophils. This combination treatment also blocks basophil activation more efficiently than either agent alone, providing a novel approach to probe regulatory mechanisms underlying IgE hypersensitivity with implications for therapeutic interventions.
Structural basis of omalizumab therapy and omalizumab-mediated IgE exchange
1. Structural basis of omalizumab therapy and omalizumab-
mediated IgE exchange
Luke F. Pennington, Svetlana Tarchevskaya, Daniel Brigger, Karthik Sathiyamoorthy,
Michelle T. Graham, Kari Christine Nadeau, Alexander Eggel & Theodore S. Jardetzky
Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305, USA.
Progam in Immunology, Stanford University School of Medicine, Stanford, California 94305, USA.
Sean N. Parker Center for Allergy Research at Stanford University, Stanford University School of Medicine, Stanford, California 94305, USA.
Department of Rheumatology, Immunology and Allergology, University Hospital Bern, Bern 3012, Switzerland.
Department of Clinical Research, University of Bern, Bern 3012, Switzerland.
Department of Medicine, Stanford University School of Medicine, Stanford, California 94305, USA.
Dated: 20180516
Presenter: Gul Muneer
NATURE COMMUNICATIONS
19 May, 2016
DOI: 10.1038/ncomms11610
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2. How does the body produce allergic response?
Induction
of Th2 cells
APC Tₕ0 Tₕ2
IL-4
IL-13
B cell
Mast Cell Basophil
Histamine
Leukotrienes
Cytokines
Prostaglandins
Allergic Reactions
IL-3, IL-5
IL-3, IL-5
Eosinophil
PAF, LTC
FcɛRI
CD40/L
Allergen
IgE
Antibody
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4. Conformational changes in the IgE-Fc upon binding its receptors
Jon D. Wright et al., Scientific
Reports volume 5, Article
number: 11581 (2015)4
5. What is Omalizumab?
Humanized, anti-IgE monoclonal Antibody
5% murine sequence & 95% human sequence
Binds to Cε3 domain of Fc portion of IgE molecule,
where IgE binds to FcεRI
Omalizumab, sold under the trade name Xolair.
Used to treat severe allergic asthma (not treatable by corticosteroids)
FDA approved antibody drug in 2003.
JACI. 2001; 108(2):S77-S83. doi:10.1067/mai.2001.116434
Genentech/Novartis
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6. 6
Omalizumab is US FDA approved antibody drug.
Yet, we don’t have structural information about Omalizumab:IgE complex!
~the structure can provide insight into a wide range of IgE inhibitors that can accelerate the
dissociation of the receptor from IgE.
Aims and motivations of the study
It’s ability to inhibit FceRI & CD23 binding?
QJM. 2004;97(5):247-257. doi:10.1093/qjmed/hch052
7. Purified Protein
Insect cells
(IgE-G335C-Fc₃₋₄)
Omalizumab Digestion &
Purification
Purification
Ni-NTA & GF
Hanging drop
Crystallization
Method
X-rays
X-ray detector
Phase Problem
¿φ?
Electron Density
P(xyz)
Phaser-MR
Mod.building
Mod.building
Validation
Final Model
Resolution: 2.5 Å
Refinement
Schematic View of the Experimental Approach
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9. ~Omalizumab approaches perpendicularly to Cε3 domain
~Contacts formed b/w Omalizumab-Fab and Cε3 β-sheet residues
Jon D. Wright et al., Scientific Reports
volume 5, Article number: 11581 (2015)
Perpendicular binding orientation of the omalizumab Fab
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10. Omalizumab binds between FcεRIα and CD23 complexes
~Alignment shows no major perturbations.
~Omalizumab can bind preformed IgE-Fc₃₋₄: FcεRIα complexes
but not full length.
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11. Omlalizumab contacts Cε3 residues of IgE
~Majority of contacts extend along the length of Cε3 on IgE.
~IgE mutants (reported previously), correspond well with the
binding interface observed in the crystal structure of IgE.
Mutant residues are colour-coded by their relative binding to Omalizumab (green) (magenta 0–14%, blue 15–44% and orange 44–75% of wild-type IgE)
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12. Residues implicated in Omalizumab:IgE interface
~a network of hydrogen bonds, salt bridges and extensive hydrophobic interactions facilitate
omalizumab:IgE interactions.
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13. Omalizumab inhibition of FcεRIα & CD23 binding
~Significant clashes b/w Omalizumab LC and FcεRIα but no
clashes exit at site 1.
~Heavy steric overlap b/w Omalizumab-HC and CD23
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14. Steric clashes b/w protein backbone of Omalizumab and CHO
groups on FcεRIα
~Extensive overlap b/w Omalizumab epitope and CD23 a drive omalizumab
inhibition of CD23:IgE interactions.
~DARPin (IgE inhibitor) has 3 times less
atomic overlap with FcεRIα
~Steric clashes occur with N-linked glycans
found on FceRIa.
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19. IgE-R419N-Fc and Omalizumab act synergistically to block
basophil activation
~Omalizumab and IgE-R419N-Fc completely inhibited basophil activation
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20. Conclusion
Crystal structure of the Omalizumab-fab in complex with an IgE-Fc fragment was solved at 2.5A.
A mutant IgE-R419N-Fc was created that is resistant to omalizumab binding.
Combination of IgE-R419N-Fc with omalizumab, promotes the exchange of IgE with omalizumab-
resistant variant on human basophils.
This combination treatment also blocks basophil activation more efficiently than either agent alone.
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21. Future perspective
IgE-R419N-Fc may elicit immune response in human body.
IgE-R419N-Fc could also bind to other cells in which FcɛRI and CD23 are expressed.
IgE-R419N-Fc was tested in ex-vivo and IgE-R419N-Fc has no role upon binding may cause
problem when combined with Omalizumab.
Future studies are needed to clarify the role of IgE-R419N-Fc in vivo and how this mutant with no
role could survive in body? and how long these mutants are able to bind with receptors?
Such approach could be tolerated if human body can tolerate functionless mutant IgE-Fc.
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Editor's Notes
Degranulation will cause the release of inflammatory molecules