Download Specificity screening of antibodies and related

Specificity screening of antibodies and
related molecules using human cell
microarray technology
Kingsley E, Freeth J & Soden J
Correspondence: [email protected]
Retrogenix 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.
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.
HEK cells grown
over spots
Test mAbs 1& 2: The primary target receptor for the two test mAbs
screened 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.
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.
ZsGreen1
ERBB2 (HER2)
Trastuzumab
biosimilar
ERBB2 (HER2)
EGFR
EGFR
Cetuximab
biosimilar
Alemtuzumab
biosimilar
CD52
Figure 3. Initial hits from cell microarray screening for trastuzumab, cetuximab & alemtuzumab
biosimilars. Left hand panels (GFP) show the duplicated microarray pattern; right hand panels show
specific interactions detected using Alexafluor647 labelled secondary antibody.
Trastuzumab
biosimilar
FCGR1A
Cetuximab
biosimilar
FCGR1A
Alemtuzumab
biosimilar
EGFR
FCGR1A
ERBB2
ERBB2
Cells over-express
individual plasma
membrane proteins
IGHG3
FCGR2A
>4,500 unique plasma
membrane proteins as
cDNA spots
CD52
IGHG3
FCGR2A
IGHG3
FCGR2A
EGFR
Test molecule added
Target receptor
discovered through ‘gainof-binding’
Figure 1. Overview of the
Retrogenix technology
AlexaFluor647
Full data package
generated
RESULTS
ZsGreen1
AlexaFluor647
AlexaFluor647
AlexaFluor647
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 biosimilar screened (shown for trastuzumab, cetuximab & alemtuzumab biosimilars) with no additional
specific hits identified.
Biosimilar of:
Rituximab
Trastuzumab
Cetuximab
Alemtuzumab
Daclizumab
Specific primary
target identified?
Y
Y
Y
Y
Y
Specific cell surface secondary
‘targets’ identified
N
N
N
N
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 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.
www.retrogenix.com
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. Nature
498:502–505.
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