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Identification and characterisation of anti-IL-13 inhibitory single domain antibodies provides new insights into receptor selectivity and attractive opportunities for drug discovery.

ABSTRACT: Interleukin-13 (IL-13) is a cytokine involved in T-cell immune responses and is a well validated therapeutic target for the treatment of asthma, along with other allergic and inflammatory diseases. IL-13 signals through a ternary signalling complex formed with the receptors IL-13Rα1 and IL-4Rα. This complex is assembled by IL-13 initially binding IL-13Rα1, followed by association of the binary IL-13:IL-13Rα1 complex with IL-4Rα. The receptors are shared with IL-4, but IL-4 initially binds IL-4Rα. Here we report the identification and characterisation of a diverse panel of single-domain antibodies (VHHs) that bind to IL-13 (K_D 40 nM-5.5 μM) and inhibit downstream IL-13 signalling (IC₅₀ 0.2-53.8 μM). NMR mapping showed that the VHHs recognise a number of epitopes on IL-13, including previously unknown allosteric sites. Further NMR investigation of VHH204 bound to IL-13 revealed a novel allosteric mechanism of inhibition, with the antibody stabilising IL-13 in a conformation incompatible with receptor binding. This also led to the identification of a conformational equilibrium for free IL-13, providing insights into differing receptor signalling complex assembly seen for IL-13 compared to IL-4, with formation of the IL-13:IL-13Rα1 complex required to stabilise IL-13 in a conformation with high affinity for IL-4Rα. These findings highlight new opportunities for therapeutic targeting of IL-13 and we report a successful ¹⁹F fragment screen of the IL-13:VHH204 complex, including binding sites identified for several hits. To our knowledge, these ¹⁹F containing fragments represent the first small-molecules shown to bind to IL-13 and could provide starting points for a small-molecule drug discovery programme.

SUBMITTER: Walker K

PROVIDER: S-EPMC10359924 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Identification and characterisation of anti-IL-13 inhibitory single domain antibodies provides new insights into receptor selectivity and attractive opportunities for drug discovery.

Walker Kayleigh K Baravalle Roberta R Holyfield Rachel R Kalms Jacqueline J Wright Helena H Seewooruthun Chitra C Muskett Frederick W FW Scott-Tucker Anthony A Merritt Andy A Henry Alistair A Lawson Alastair D G ADG Hall Gareth G Prosser Christine C Carr Mark D MD

Frontiers in immunology 20230706

Interleukin-13 (IL-13) is a cytokine involved in T-cell immune responses and is a well validated therapeutic target for the treatment of asthma, along with other allergic and inflammatory diseases. IL-13 signals through a ternary signalling complex formed with the receptors IL-13Rα1 and IL-4Rα. This complex is assembled by IL-13 initially binding IL-13Rα1, followed by association of the binary IL-13:IL-13Rα1 complex with IL-4Rα. The receptors are shared with IL-4, but IL-4 initially binds IL-4Rα ...[more]

PMID: 37483614

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Project description:ABSTRACT Background: Proteases are one of the largest pharmaceutical targets for drug developments. Their dysregulations result in a wide variety of diseases. Because proteolytic networks usually consist of protease family members that share high structural and catalytic homology, distinguishing them using small molecule inhibitors is often challenging. To achieve specific inhibition, this study described a novel approach for the generation of protease inhibitory antibodies. As a proof of concept, we aimed to convert a matrix metalloproteinase (MMP)-14 specific inhibitor to MMP-9 specific inhibitory antibodies with high selectivity. Methods: An error-prone single-chain Fv (scFv) library of an MMP-14 inhibitor 3A2 was generated for yeast surface display. A dual-color competitive FACS was developed for selection on MMP-9 catalytic domain (cdMMP-9) and counter-selection on cdMMP-14 simultaneously, which were fused/conjugated with different fluorophores. Isolated MMP-9 inhibitory scFvs were biochemically characterized by inhibition assays on MMP-2/-9/-12/-14, proteolytic stability tests, inhibition mode determination, competitive ELISA with TIMP-2 (a native inhibitor of MMPs), and paratope mutagenesis assays. Results: We converted an MMP-14 specific inhibitor 3A2 into a panel of MMP-9 specific inhibitory antibodies with dramatic selectivity shifts of 690–4,500 folds. Isolated scFvs inhibited cdMMP-9 at nM potency with high selectivity over MMP-2/-12/-14 and exhibited decent proteolytic stability. Biochemical characterizations revealed that these scFvs were competitive inhibitors binding to cdMMP-9 near its reaction cleft via their CDR-H3s. Conclusions: This study developed a novel approach able to convert the selectivity of inhibitory antibodies among closely related protease family members. This methodology can be directly applied for mAbs inhibiting many proteases of biomedical importance. Statement of Significance To achieve high selectivity required for therapies, we developed a novel approach for the generation of protease inhibitory antibodies with nM potency and decent proteolytic stability. The methodology demonstrated here can be readily applied to many proteases of biomedical importance.

Project description:BackgroundA subset of Australians who have been bitten by ticks experience a complex of chronic and debilitating symptoms which cannot be attributed to the known pathogenic species of bacteria present in Australia. As a result, there has been a renewed effort to identify and characterise viruses in Australian terrestrial ticks. Recent transcriptome sequencing of Ixodes and Amblyomma ticks has revealed the presence of multiple virus sequences. However, without virus isolates our ability to understand the host range and pathogenesis of newly identified viruses is limited. We have established a successful method for high-throughput virus discovery and isolation in mosquitoes using antibodies to double-stranded RNA. In this study we sought to characterise five archival tick-borne viruses to adapt our virus discovery protocol for Australian ticks.MethodsWe performed virus characterisation using a combination of bioinformatic sequence analysis and in vitro techniques including replication kinetics, antigenic profiling, virus purification and mass spectrometry.ResultsOur sequence analysis of Nugget virus, Catch-me-Cave virus and Finch Creek virus revealed marked genetic stability in isolates collected from the same location approximately 30 years apart. We demonstrate that the Ixodes scapularis-derived ISE6 cell line supports replication of Australian members of the Flaviviridae, Nairoviridae, Phenuiviridae and Reoviridae families, including Saumarez Reef virus (SREV), a flavivirus isolated from the soft tick Ornithodoros capensis. While antibodies against double-stranded RNA could be used to detect replication of a tick-borne reovirus and mosquito-borne flavivirus, the tick-borne flaviviruses Gadgets Gully virus and SREV could not be detected using this method. Finally, four novel virus-like sequences were identified in transcriptome sequencing of the Australian native tick Ixodes holocyclus.ConclusionsGenetic and antigenic characterisations of archival viruses in this study confirm that three viruses described in 2002 represent contemporary isolates of virus species first identified 30 years prior. Our findings with antibodies to double-stranded RNA highlight an unusual characteristic shared by two Australian tick-borne flaviviruses. Finally, comparative growth kinetics analyses of Australian tick-borne members of the Flaviviridae, Nairoviridae, Phenuiviridae and Reoviridae families in ISE6 and BSR cells will provide a useful resource for isolation of Australian tick-borne viruses using existing cell lines.

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Identification and characterisation of anti-IL-13 inhibitory single domain antibodies provides new insights into receptor selectivity and attractive opportunities for drug discovery.

Publications

Identification and characterisation of anti-IL-13 inhibitory single domain antibodies provides new insights into receptor selectivity and attractive opportunities for drug discovery.

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