Many key immune checkpoint interactions are not yet fully characterised – hampering efforts to discover and develop new, targeted immuno-therapies – due to the low success rates of standard techniques in identifying specific ligand-receptor pairing. Retrogenix’s cell microarray technology (as known as plasma membrane protein array screening) has been successful in identifying key receptors for both orphan ligands and for phenotypic molecules discovered using functional studies.
Here we demonstrate the power of the Retrogenix approach in uncovering immune checkpoint interactions using the ligands CTLA4 and CD27.
Both CTLA4-hFc and CD27-hFc were screened for binding with approx 4,500 human plasma membrane proteins that were individually over-expressed in HEK293 cells as outlined below.
Primary screens: 13 slides were spotted in duplicate with expression vectors encoding full-length, unfused plasma membrane proteins. Human HEK293 cells were overlaid and reverse-transfected. Slides were then fixed. Each Fc-tagged test ligand was added to the slides and gain of binding detected using an Alexafluor647 anti-hIgGFc secondary antibody.
Confirmation screen: Positive ‘hits’ were confirmed by sequencing, re-expressed on fresh slides and tested for specificity along with appropriate experimental controls.