Specificity screening of antibodies and related molecules using human cell microarray technologyKingsley E, Freeth J & Soden J Correspondence: jim.freeth@retrogenix.comRetrogenix Limited, Crown House, Bingswood Estate, Whaley Bridge, High Peak, SK23 7LY, UK

BACKGROUND

Biologics such as antibodies are widely favoured in drug discovery due to their inherent potential for specificity to a single target antigen. Despite this, off-target mediated toxicity could present an issue for a subset of antibodies and other molecules in development. Arguably, the consequences of any adverse events are being underestimated by the widely-held assumption that off-target risks for antibodies are minimal or non-existent. Furthermore, traditional protein array methodologies for exploring off-target interactions are not only limited by their low rates of success in uncovering real target binding but also by the threat of extensive false positive reporting which can tie up valuable resources in investigating numerous non-relevant hits. Human cell microarray screening is a powerful tool that has been successfully used to identify key receptors for both orphan ligands1and for phenotypic molecules discovered using functional studies2.

Test mAbs 1& 2: The primary target receptor for the two test mAbsscreened was not disclosed prior to this study. Cell microarray screening detected binding to three different isoforms of the primary receptor for both mAbs (target X, figure 2). In addition, specific binding to a further, unrelated receptor, target Y, was detected for test mAb 2 only. Target Y is in a different sub-class of plasma membrane protein to target X and represents a previously unknown off-target for test mAb 2 providing key information for study sponsors looking to differentiate between promising lead candidates.

IMPACT

These results demonstrate human cell microarray screening as a powerful tool that can be used in early discovery to assist with lead selection, as well as later in development to better understand any potential off-target liabilities of lead biotherapeutic molecules.

References:1. Turner L. et al. (2013). Severe malaria is associated with parasite binding to endothelial protein C receptor. Nature498:502–505.2. Sandercock AM et al. (2015) Identification of anti-tumour biologics using primary tumour models, 3-D phenotypic screening and image-based multi-parametric profiling. Molecular Cancer 14:147

Here describe how the cell microarray approach is being applied to safety screening for biologics, identifying specific and relevant off-targets with a very low incidence of false positives.

MATERIALS AND METHODS

Two antibodies provided by a commercial sponsor (‘test mAb 1&2’) along with biosimilars to five marketed antibodies (rituximab, trastuzumab, cetuximab, alemtuzumab and daclizumab) that were not anticipated to have any secondary cell surface targets were selected for this case study. Each antibody was screened for binding against 4,500+ human plasma membrane proteins that were individually over-expressed in HEK293 cells as outlined in figure 1. An AlexaFluor647 anti-hIgGFc detection antibody was used to identify gain-of-binding which was then confirmed by repeat screening of all initial hits re-expressed on custom arrays.

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>4,500 unique plasma

membrane proteins as

cDNA spots

HEK cells grown

over spotsCells over-express

individual plasma

membrane proteins

Target receptor

discovered through ‘gain-

of-binding’

Full data package

generated

Test molecule added

Figure 1. Overview of the Retrogenix technology

Biosimilars: Binding to its known primary target was detected for all biosimilars (figure 3; table 1). As expected, no additional specific receptor binding was detected (figure 4). This provides validation of the high specificity of the cell microarray approach and very low rate of false positives reported.

RESULTS

Figure 2. Confirmation screen results. Left hand panel shows all initial hits and controls spotted in duplicate (GFP detection). Test mAbs: Specific, reproducible hits for each mAb are indicated in green. Three different isoforms of target X were detected for both mAbs; target Y was specific to test mAb 2.

Figure 3. Initial hits from cell microarray screening for trastuzumab, cetuximab & alemtuzumabbiosimilars. Left hand panels (GFP) show the duplicated microarray pattern; right hand panels show specific interactions detected using Alexafluor647 labelled secondary antibody.

ZsGreen1 AlexaFluor647

Trastuzumabbiosimilar

ERBB2 (HER2)

ERBB2 (HER2)

Cetuximabbiosimilar

EGFREGFR

Alemtuzumabbiosimilar

CD52

Biosimilar of:Specific primary

target identified?Specific cell surface secondary

‘targets’ identified

Rituximab Y N

Trastuzumab Y N

Cetuximab Y N

Alemtuzumab Y N

Daclizumab Y N

Table 1. Summary of results for cell microarray screening. Only the known, specific primary target was identified for each of the five biosimilars screened with no false positives reported.

Figure 4. Confirmation screen results. Left hand panel shows all initial hits and controls spotted in duplicate (GFP detection). The known primary receptors were confirmed as specific hits for each bio-similar screened (shown for trastuzumab, cetuximab & alemtuzumab biosimilars) with no additional specific hits identified.

Trastuzumabbiosimilar

FCGR1A

IGHG3FCGR2A

ERBB2

Cetuximabbiosimilar

FCGR1A

IGHG3FCGR2A

EGFR

EGFRERBB2

Alemtuzumabbiosimilar

FCGR1A

IGHG3FCGR2A

CD52

AlexaFluor647 AlexaFluor647ZsGreen1 AlexaFluor647