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The TGF-β inhibitory activity of antibody 37E1B5 depends on its H-CDR2 glycan.

ABSTRACT: 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.

SUBMITTER: Tsui P

PROVIDER: S-EPMC5240645 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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The TGF-β inhibitory activity of antibody 37E1B5 depends on its H-CDR2 glycan.

Tsui Ping P Higazi Daniel R DR Wu Yanli Y Dunmore Rebecca R Solier Emilie E Kasali Toyin T Bond Nicholas J NJ Huntington Catherine C Carruthers Alan A Hood John J Borrok M Jack MJ Barnes Arnita A Rickert Keith K Phipps Sandrina S Shirinian Lena L Zhu Jie J Bowen Michael A MA Dall'Acqua William W Murray Lynne A LA

mAbs 20161111 1

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 ...[more]

PMID: 27834568

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Project description:BackgroundTherapeutic antibodies targeting programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis induce potent and durable anti-tumor responses in multiple types of cancers. However, only a subset of patients benefits from anti-PD-1/PD-L1 therapies. As a negative regulator of anti-tumor immunity, TGF-β impairs the efficacy of anti-PD-1/PD-L1 and induces drug resistance. Developing a novel treatment strategy to simultaneously block PD-1/PD-L1 and TGF-β would be valuable to enhance the effect of anti-PD-1/PD-L1 and relieve drug resistance.MethodsBased on the Check-BODY™ technology platform, we developed an anti-TGF-β/PD-L1 bispecific antibody YM101. The bioactivity of the anti-TGF-β moiety was determined by Smad-luciferase reporter assay, transwell assay, western blotting, CCK-8, and flow cytometry. The bioactivity of the anti-PD-L1 moiety was measured by T cell activation assays. EMT-6, CT26, and 3LL tumor models were used to investigate the anti-tumor activity of YM101 in vivo. RNA-seq, immunohistochemical staining, and flow cytometry were utilized to analyze the effect of YM101 on the tumor microenvironment.ResultsYM101 could bind to TGF-β and PD-L1 specifically. In vitro experiments showed that YM101 effectively counteracted the biological effects of TGF-β and PD-1/PD-L1 pathway, including activating Smad signaling, inducing epithelial-mesenchymal transition, and immunosuppression. Besides, in vivo experiments indicated the anti-tumor activity of YM101 was superior to anti-TGF-β and anti-PD-L1 monotherapies. Mechanistically, YM101 promoted the formation of 'hot tumor': increasing the numbers of tumor infiltrating lymphocytes and dendritic cells, elevating the ratio of M1/M2, and enhancing cytokine production in T cells. This normalized tumor immune microenvironment and enhanced anti-tumor immune response might contribute to the robust anti-tumor effect of YM101.ConclusionOur results demonstrated that YM101 could simultaneously block TGF-β and PD-L1 pathways and had a superior anti-tumor effect compared to the monotherapies.

Dataset Information

The TGF-β inhibitory activity of antibody 37E1B5 depends on its H-CDR2 glycan.

Publications

The TGF-β inhibitory activity of antibody 37E1B5 depends on its H-CDR2 glycan.

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