Screening the secretome for off-target binding using human cell microarray technology

Background

Human cell microarray technology is a powerful and highly validated method for off-target screening of biotherapeutics. As such, it is now the standard tool for lead selection and for safety assessment, providing supportive data for IND submissions. Many molecule types can be screened, including mono- and bi-specific antibodies, ScFvs, ADCs, proteins, peptides, and whole CAR T cells. The high success rates and low false positive rates result from the expression of a large, high quality library of full-length human plasma membrane proteins in the context of human cells.

Off-target binding to secreted proteins also has the potential to cause clinical toxicity or alter the pharmacokinetic profile of biotherapeutics in development. Here we describe the adaptation of the cell microarray technology – beyond the plasma membrane proteome – to screen for interactions with proteins that are usually secreted from the cell.

Tethering secreted proteins

As secreted proteins are not naturally localised on the cell surface, novel constructs were developed allowing expression of ‘secreted’ proteins that are tethered to the plasma membrane by fusing to an inert transmembrane and intracellular domain. Three lengths of tether were tested – short, medium and long (Figure 1).  A V5-tag was also incorporated to verify expression of the secreted protein. Co-transfected GFP allowed for transfected cell clusters to be mapped on the microarrays. Binding to tethered secreted proteins was tested using the standard human cell microarray approach.

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Figure 1 – Schematic of the tethered secreted protein constructs tested using human cell microarray technology

 

Library selection

>2,000 candidate secreted proteins were identified and prioritised based upon manual curation of subcellular location, the presence of a signal peptide sequence and/or prediction algorithms. Toxicology and bioinformatics teams from four major pharmaceutical companies also advised in order to prioritise key secreted off-targets for inclusion. Currently, Retrogenix has built a library of 1,400 of these ‘high confidence’ proteins, with a further 600 currently under development.

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Figure 2 – Functional classification of proteins currently represented within the Retrogenix cell microarray secreted library

Validation of expression & interactions

Multiple antibody:secreted target pairs were used to determine optimal construct length, and to establish proof of principle. Data is shown for a pateclizumab biosimilar against its secreted target LTA (TNFb) (Figure 3). Transfection efficiency and spot positions were confirmed using ZsGreen fluorescence (left-hand panel). Pateclizumab showed binding to the medium & long tethered forms of LTA, but not to untethered LTA, nor the short tethered form of LTA. Pateclizumab showed no cross-reactivity against the related family member TNFa (a transmembrane protein), despite confirming its expression by Infliximab. The proof-of-principle results on a subset of secreted targets led to the selection of long-tether constructs for development of the full secreted library.

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Figure 3 – Proof of principle using the interaction between pateclizumab and its secreted target LTA.

Impact

Retrogenix’s cell microarray libraries now have >5,500 proteins for screening which is the largest set of plasma membrane and secreted proteins expressed in human cells. This novel secreted library complements Retrogenix’s existing membrane protein screening capability, providing an even more powerful solution for off-target assessment of biotherapeutics.

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