Screening
set up to search for cancer immunotherapy targets utilised phage display using DARPins to select anti- body-like molecules that showed preferential bind- ing to human regulatory T (Treg) cells. The cDNA expression target deconvolution that followed rapidly identified TNFR2 as the primary receptor for all molecules screened. Subsequent tests on spe- cific molecules that were categorised as TNFR2- agonists resulted in tumour inhibition in mouse models, highlighting the importance of TNFR2 as an immunotherapy target2.
The recent advances in target deconvolution is providing researchers with the necessary tools to support the development of promising phenotypic molecules while also uncovering novel receptor tar- gets that can provide a real competitive advantage.
Strengths of cDNA microarray technology
The physiological relevance of the system and the vast cDNA library of unmodified (untagged) pro- teins is the core strength of the cell microarray technology. Numerous target deconvolution pro- jects with industry partners have allowed the rate of success in identifying the membrane target of a compatible phenotypic antibody to be quantified at around 70%. This is significantly higher than the success rates of standard protein arrays and proteomics-based approaches, with results deliv- ered in as little as two weeks.
An additional advantage of cDNA cell microar- ray screening is that, as well as identifying a pri- mary target, there is the opportunity for concomi- tant identification of potential off-targets for molecules tested. A low incidence of off-target binding among phenotypic antibodies may be anticipated; however, detecting any potential cross-reactivity at this early stage can provide valuable additional information that can help guide lead selection and optimisation. The sensitivity of the technology does depend on the level of expression of the target receptor rela- tive to endogenous expression in HEK cells. Although specific target receptors are identified, the technology does not quantitate the affinity of interactions that are observed. However, without any amplification strategies, a known antibody:tar- get interaction with predicted Kd of 10uM has been detected using cell microarrays, indicating the sensitivity of the technology.
Cell microarray screening is extremely versatile and suited to a wide variety of molecules including human and non-human antibodies, ScFvs, DARPins, proteins, peptides and small molecules, among others. Beyond its applications in pheno-
Drug Discovery World Fall 2017
typic screening, the technology is also being used to great success in identifying the human cell sur- face receptors of disease relevant orphan ligands4.
Current limitations
There is a very high likelihood of detecting an interaction with the test ligand among the >75% of membrane proteins that are already represented within cDNA-expression cell microarrays. It is inevitable that, even despite rigorous transfection controls, some receptors may be poorly expressed to the extent where interactions may not be readily detectable. In these cases, strategies to amplify the binding signal or create an avidity gain for the test molecule have been shown to increase the sensitiv- ity of the technology and help to minimise the threat of false negative results.
The library of membrane proteins continues to expand; however, not every membrane protein – nor every protein isoform – is currently represent- ed. In addition, although the over-expression in HEK293 cells provides a native human system, in some cases it may not result in the correct protein conformation, the particular post-translational modifications or the disease-relevant epitopes that would be necessary to facilitate binding in vivo. In some cases – for example, ion channels – ligands may only interact with multi-subunit receptors. Therefore, over-expression of a single subunit may be insufficient to provide a fully functional protein within the system. Despite these theoretical limitations, there is evidence that the human cell expression system can, in some instances, make allowances for single subunits through the interplay with endogenously- expressed receptors and the provision of the addi- tional co-factors where necessary. This has been observed in a target deconvolution study involv- ing integrin receptors where the target was identi- fied despite the underlying cDNA vector only encoding one monomer of a heterodimeric recep- tor. A possible future direction for cDNA expres- sion cell microarrays could be a hypothesis-driv- en, focused effort on creating multi-subunit arrays through co-transfection of multiple con- structs in order to ensure full representation of receptor complexes and multi-subunit entities. Cell microarrays are very well-suited to screen- ing antibodies and protein ligands using a wide variety of detection systems from direct fluorescent labelling through to using specific secondary anti- bodies and protein tags (such as His-, Flag-, Fc- and biotin, among others). However, small molecules do need to be radiolabelled which can prove impractical in early screening. Development
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