The TGF-β inhibitory activity of antibody 37E1B5 depends on its H-CDR2 glycan.
Excessive transforming growth factor (TGF)-β is associated with pro-fibrotic responses in lung disease, yet it also plays essential roles in tissue homeostasis and autoimmunity. Therefore, selective inhibition of excessive and aberrant integrin-mediated TGF-β activation via targeting the α-v family of integrins is being pursued as a therapeutic strategy for chronic lung diseases, to mitigate any potential safety concerns with global TGF-β inhibition. In this work, we reveal a novel mechanism of inhibiting TGF-β activation utilized by an αvβ8 targeting antibody, 37E1B5. This antibody blocks TGF-β activation while not inhibiting cell adhesion. We show that an N-linked complex-type Fab glycan in H-CDR2 of 37E1B5 is directly involved in the inhibition of latent TGF-β activation. Removal of the Fab N-glycosylation site by single amino acid substitution, or removal of N-linked glycans by enzymatic digestion, drastically reduced the antibody's ability to inhibit latency-associated peptide (LAP) and αvβ8 association, and TGF-β activation in an αvβ8-mediated TGF-β signaling reporter assay. Our results indicate a non-competitive, allosteric inhibition of 37E1B5 on αvβ8-mediated TGF-β activation. This unique, H-CDR2 glycan-mediated mechanism may account for the potent but tolerable TGF-b activation inhibition and lack of an effect on cellular adhesion by the antibody.
a Antibody Discovery and Protein Engineering, Medimmune LLC , Gaithersburg , MD , USA.
Ping
P
Tsui
b Biopharmaceutical Development, MedImmune Ltd , Cambridge , UK.
Daniel R
DR
Higazi
a Antibody Discovery and Protein Engineering, Medimmune LLC , Gaithersburg , MD , USA.
Yanli
Y
Wu
c Respiratory, Inflammation and Autoimmunity, MedImmune Ltd , Cambridge , UK.
Rebecca
R
Dunmore
b Biopharmaceutical Development, MedImmune Ltd , Cambridge , UK.
Emilie
E
Solier
b Biopharmaceutical Development, MedImmune Ltd , Cambridge , UK.
Toyin
T
Kasali
b Biopharmaceutical Development, MedImmune Ltd , Cambridge , UK.
Nicholas J
NJ
Bond
d Antibody Discovery and Protein Engineering, MedImmune Ltd , Cambridge , UK.
Catherine
C
Huntington
c Respiratory, Inflammation and Autoimmunity, MedImmune Ltd , Cambridge , UK.
Alan
A
Carruthers
e Translational Sciences, Medimmune Ltd ., Cambridge , UK.
John
J
Hood
a Antibody Discovery and Protein Engineering, Medimmune LLC , Gaithersburg , MD , USA.
M Jack
MJ
Borrok
a Antibody Discovery and Protein Engineering, Medimmune LLC , Gaithersburg , MD , USA.
Arnita
A
Barnes
a Antibody Discovery and Protein Engineering, Medimmune LLC , Gaithersburg , MD , USA.
Keith
K
Rickert
a Antibody Discovery and Protein Engineering, Medimmune LLC , Gaithersburg , MD , USA.
Sandrina
S
Phipps
a Antibody Discovery and Protein Engineering, Medimmune LLC , Gaithersburg , MD , USA.
Lena
L
Shirinian
a Antibody Discovery and Protein Engineering, Medimmune LLC , Gaithersburg , MD , USA.
Jie
J
Zhu
a Antibody Discovery and Protein Engineering, Medimmune LLC , Gaithersburg , MD , USA.
Michael A
MA
Bowen
a Antibody Discovery and Protein Engineering, Medimmune LLC , Gaithersburg , MD , USA.
William
W
Dall'Acqua
c Respiratory, Inflammation and Autoimmunity, MedImmune Ltd , Cambridge , UK.
Lynne A
LA
Murray
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology* / Q000493:pharmacokinetics; D022801:Complementarity Determining Regions / Q000737:chemistry* / Q000276:immunology; D006031:Glycosylation; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology; D016023:Integrins / Q000037:antagonists & inhibitors*; D051379:Mice; D011134:Polysaccharides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors*
D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D007140:Immunoglobulin Fab Fragments; D016023:Integrins; D011134:Polysaccharides; D016212:Transforming Growth Factor beta; C403414:integrin alphavbeta8
ITGB8; allosteric inhibition; fab glycosylation; integrin; mAb; αvβ8