Title: A platform for extracellular interactome discovery identifies novel functional binding partners for the immune receptors B7-H3/CD276 and

Receptors expressed on the plasma membrane and their interacting partners critically regulate cellular communication during homeostasis and disease, and as such represent main therapeutic targets. Despite its importance for drug development, receptor-ligand proteomics has remained a daunting field, in part due to the challenges associated to the study of membrane-expressed proteins. Here, to enable sensitive detection of receptor-ligand interactions in high throughput, we implemented a new platform, the Conditioned Media AlphaScreen, for interrogation of a library consisting of most single transmembrane human proteins. Utilizing this method to study key immune receptors, we identify and further validate the interleukin receptor IL20RA as the first binding partner for the checkpoint inhibitor B7-H3. Furthermore, KIR2DL5, a natural killer cell protein that had remained orphan, is uncovered as a functional binding partner for the poliovirus receptor (PVR). This interaction is characterized using orthogonal assays, which demonstrate that PVR specifically engages KIR2DL5 on natural killer cells leading to inhibition of cytotoxicity. Altogether, these results reveal unappreciated links between protein families that may importantly influence receptor-driven functions during disease. Applicable to any target of interest, this technology represents a versatile and powerful approach for elucidation of receptor-ligand interactomes, which is essential to understand basic aspects of the biology of the plasma membrane proteins and ultimately inform the development of novel therapeutic strategies. expected PVR interactors we identified a previously unrecognized binding partner, KIR2DL5, an orphan immune receptor expressed in natural killer (NK) cells (16, 17). This interaction was further characterized using orthogonal technologies that demonstrate a specific interaction between PVR and KIR2DL5, but not related family members. Moreover, our functional studies show that KIR2DL5 controls NK cytotoxicity in a PVR-dependent fashion, altogether uncovering a new association between the PVR/nectin and KIR families that may influence NK responses. In summary, here we describe a versatile technology that can be used to study any extracellular target of interest, enabling elucidation of receptor interactomes with remarkable sensitivity and minimal background. Utilizing this technology, we identify IL20RA as the first receptor discovered for the immune checkpoint B7-H3, and uncover the orphan receptor KIR2DL5 as a previously unappreciated modulator of NK cell functions via direct engagement of PVR. These findings suggest potential targets with therapeutic potential, and provide unique insights into immune receptor biology. was for detection of PVR binding to the cell surface. Together, these results illustrated the sensitivity of the miniaturized AlphaScreen method for detection of transient ePPIs, including those that depend on receptor glycosylation, and demonstrated that these interactions can be detected when proteins are expressed in the conditioned media from human cell cultures. inset). validate identified interaction between KIR2LD5 and PVR by BLI using purified recombinant These results confirmed PVR binding to KIR2DL5A and not to other members of the family closely related KIR2DL4 Next, to fully assess binding specificity for the KIR receptor family, we analyzed PVR binding to the cell surface of cells transiently the KIR receptors. These results confirmed that PVR selectively binds to KIR2DL5A on the cell surface 5 C ), consistent with our receptor discovery results (Fig. 5 A ), and corroborated binding to PVRL4, a receptor highly related to the known PVR binder PVRL3, to our knowledge describing a new interaction within the PVR/PVRL4 family. this receptor have not been explored in depth, primarily due to the lack of physiological ligands. Here, we show that KIR2DL5 plays an inhibitory function in NK cytolysis through direct interaction with PVR expressed on tumor cells. It is plausible that, similarly to TIGIT, KIR2DL5 also delivers a signal in the PVR-expressing cells triggering a functional response that should be addressed in future studies. The PVR-KIR2DL5 interaction characterized in this work establishes a new association between the nectin and KIR receptor families, otherwise unrelated immune molecules. In fact, to our knowledge, this study is the first to reveal receptor-ligand interactions unrelated to MHC for a KIR receptor, suggesting functions for this family beyond MHC recognition. In healthy individuals, the expression of KIR2DL5 seems to be restricted to a low percentage of NK cells and to a lower extent CD8 + T cells (16). Nevertheless, whether KIR2DL5 expression is upregulated in NK or T cells infiltrating the tumors remains to be addressed. Emerging preclinical evidence has shown promise in modulating the PVR/nectin family, and understanding the dynamic interaction between these receptors will be crucial to further develop efficacious therapies. Notably, a recent phase 1 trial has shown a safety profile and potential for reinvigoration of NK cell responses upon administration of an anti-KIR2DL-1,-2 and -3 antibody for the treatment of solid tumors (45). Much remains to be understood about the molecular mechanisms by which the nectin and KIR families regulate immune function, and whether the crosstalk with the KIR family can be exploited to enhance immune responses. Notwithstanding these challenges, here we present a new workflow for extracellular interactome discovery and provide important insights into the biology of these receptor families that may ultimately inform therapeutic development. using a throughput for cell transfection. ii, in following incubation with acceptor beads. iii, biotinylated query iv, v, Query