Selection of Antibodies to Transiently Expressed Membrane Proteins Using Phage Display

Jones, Martina L.; Mahler, Stephen M.; Kumble, Sumukh

doi:10.1007/978-1-4939-8648-4_10

Cited by 4 publications

(2 citation statements)

References 16 publications

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“…Indeed, it is well known that successful isolation of novel antibodies by phage display is critically limited by the presentation of a correctly folded antigen. The discovery of new and more selective antibodies capable of interfering with connexin hemichannels will undoubtedly benefit from improved methods targeting membrane proteins in their native conformation and optimized for whole cell biopanning (Jones et al, 2016, 2018).…”

Section: Discussionmentioning

confidence: 99%

A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function

et al. 2019

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Connexin hemichannels, which are plasma membrane hexameric channels (connexons) composed of connexin protein protomers, have been implicated in a host of physiological processes and pathological conditions. A number of single point pathological mutations impart a “leaky” character to the affected hemichannels, i.e., make them more active or hyperactive, suggesting that normal physiological condition could be recovered using selective hemichannel inhibitors. Recently, a human-derived monoclonal antibody named abEC1.1 has been shown to inhibit both wild type and hyperactive hemichannels composed of human (h) connexin 26 (hCx26) subunits. The aims of this work were (1) to characterize further the ability of abEC1.1 to selectively modulate connexin hemichannel function and (2) to assess its in vitro stability in view of future translational applications. In silico analysis of abEC1.1 interaction with the hCx26 hemichannel identified critically important extracellular domain amino acids that are conserved in connexin 30 (hCx30) and connexin 32 (hCx32). Patch clamp experiments performed in HeLa DH cells confirmed the inhibition efficiency of abEC1.1 was comparable for hCx26, hCx30 and hCx32 hemichannels. Of note, even a single amino acid difference in the putative binding region reduced drastically the inhibitory effects of the antibody on all the other tested hemichannels, namely hCx30.2/31.3, hCx30.3, hCx31, hCx31.1, hCx37, hCx43 and hCx45. Plasma membrane channels composed of pannexin 1 were not affected by abEC1.1. Finally, size exclusion chromatography assays showed the antibody does not aggregate appreciably in vitro . Altogether, these results indicate abEC1.1 is a promising tool for further translational studies.

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Section: Discussionmentioning

confidence: 99%

A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function

et al. 2019

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“…Transiently expressing the target receptor might also assist in increasing the density of the cell surface receptor [198,308].…”

Section: Monoclonal Phage Elisa For Human Epha2 Scfv-phage Bindersmentioning

confidence: 99%

Isolation and characterisation of antibodies against human and canine EphA2_ targets for comparative studies in brain cancer

Hagh¹

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Glioblastoma multiforme (GBM) is one of the high-grade gliomas, with a patient median survival of around 15 months from the time of diagnosis. Current treatments for this brain cancer initially comprise of surgery and radiotherapy, followed by administration of Temozolomide. Despite many advances in preclinical studies of GBM in mice, the outcomes have not been translated to the clinical level for humans. This is because the mouse model of GBM does not recapitulate the human disease and is not a suitable model for comparative studies of spontaneous tumours in humans. The canine model represents a powerful, large animal model of gliomas, since canine brain tumours occur spontaneously, with an incidence rate and patient age profile quite similar to human populations. EphA2 is one of the multi-domain receptors of the most significant tyrosine kinase family of receptors, and is highly expressed in both human and canine glioblastoma. Overexpression of EphA2 has been shown to correlate with tumour stage, progression and patient survival. Since EphA2 is not expressed in normal brain tissue, but is over-expressed on GBM cells, it is potentially a highly useful receptor for antibody-based therapy of brain cancer in both humans and dogs. Thus, monoclonal antibodies (mAbs) which cross-react with both human and canine EphA2 would be valuable molecular entities with diagnostic and therapeutic potential. To generate mAbs specific for EphA2 with cross-reactivity to both canine and human EphA2 receptors, phage display technology was employed using various strategies for the presentation of the EphA2 antigen to the phage antibody library. This included biopanning against firstly recombinant whole extracellular domain (ECD) of both human and canine EphA2, secondly recombinant ligandbinding domain (LBD) of human and canine EphA2, and lastly against cells expressing the native EphA2. Biopanning against recombinant EphA2-ECD generated one promising antibody (mAb AGH001) which showed cross-reactivity to both recombinant human and canine EphA2-ECD. However, mAb AGH001 was shown to also bind to EphA2 Knock Down (KD) cells, suggesting mAb AGH001 may bind to a domain within EphA2-ECD, which is conserved among EphA family receptors, other than the unique LBD of EphA2. To isolate specific anti-EphA2 antibodies for the LBD on EphA2-ECD, the human and canine LBD was utilised as an antigen for a biopanning campaign. Utilising this strategy of biopanning against the LBD of EpahA2, mAb HuB1 was isolated and showed binding to both human and canine EphA2-LBD and EphA2-ECD as well; however, mAb HuB1 also showed binding to the negative EphA2 cell line. This could be due to partially overlapping epitopes on other Eph receptors. mAb AGH001 and a positive control antibody, murine mAb 4B3 did not bind to EphA2-ECD subdomains. It can be concluded that the recombinant ECD sub-domains did not recapitulate the native conformation of EphA2. Furthermore, we demonstrated that mAb AGH001 has a conformational III epitope, therefore relies on a structural conform...

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Methods for transient expression and purification of monoclonal antibodies in mammalian cells

Kamle

Lee

et al. 2022

Advances in Protein Molecular and Structural Biology Methods

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Selection of Antibodies to Transiently Expressed Membrane Proteins Using Phage Display

Cited by 4 publications

References 16 publications

A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function

A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function

Isolation and characterisation of antibodies against human and canine EphA2_ targets for comparative studies in brain cancer

Methods for transient expression and purification of monoclonal antibodies in mammalian cells

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