Target agnostic phage display for nanobodies that alter immune cell function

Introduction: The discovery of novel cancer immunotherapies is currently limited by a reliance on known antigens and screens that prioritize binding affinity over function. To overcome this, we developed INSPIRE-seq, a platform integrating NGS and phage display to unbiasedly select for nanobodies against cell subtypes within the tumor microenvironment (TME). Here, we advance this technology to address a critical unmet need: the direct isolation of nanobodies that functionally modulate immune cell behavior. Our objective is to move beyond simple binding and deploy a strictly target-agnostic strategy to identify therapeutic candidates based solely on their ability to enhance T cell efficacy, without prior knowledge of their molecular targets. Experimental Procedures: To validate this functional selection strategy, we established an ex vivo model using bead-purified naïve CD8 T cells. Cells were activated with alphaCD3 and alphaCD28 antibodies in the presence of recombinant PD-L1 to mimic TME inhibitory signaling. Simultaneously, the cells were co-incubated with a library of nanobody-displaying bacteriophage. The top 10% of live T cells expressing the highest levels of the activation marker CD69 were sorted via flow cytometry to capture nanobodies capable of enhancing T cell activation, independent of their binding target. Enrichment dynamics were tracked via Oxford Nanopore sequencing. Individual phage clones were subsequently amplified and re-tested for both binding and functional modulation within the same assay. New Data: We initiated the screen using a library with confirmed in vivo efficacy, verifying that this activity translated to detectable baseline modulation in our ex vivo model to ensure the presence of enrichable candidates. From this validated starting point, we performed four rounds of biopanning and sorting for increased CD69 expression. We isolated individual clones that, based on amplicon sequencing, collectively represented 25% of the starting library population. These dominant clones were individually tested for activity. Notably, clone 9-1-12 exhibited significant binding to CD8 T cells compared to controls. Critically, this binding was functionally linked to activation: the specific population of T cells bound by the nanobody displayed a significantly increased frequency of CD69 positivity despite the presence of PD-L1 inhibition. This demonstrates the feasibility of recovering a functional modulator solely through phenotypic selection. Conclusions: Our findings provide evidence that INSPIRE-seq can select and enrich for nanobodies that alter cell function in a target-agnostic manner. This supports our central hypothesis that phenotypic screening can identify immunomodulators without prior antigen knowledge, offering a novel pipeline to uncover therapeutic candidates that overcome TME immunosuppression.