Development of monoclonal ScFv antibodies targeting the t-cell co-inhibitory molecules VISTA and PD-L1

Abstract

Tumour development is a multistep process characterized by aberrant monoclonal cell proliferation. Immune cells exert selective pressure on tumour populations, promoting the survival of low-immunogenic variants that can evade immune detection. One such mechanism involves the recruitment of immunosuppressive cells expressing inhibitory receptors and ligands that suppress T-cell activation. Currently, inhibitory receptors and ligands are being extensively studied as therapeutic targets. The food and drug administration (FDA) has approved several immune checkpoint inhibitors (ICIs) targeting Cytotoxic T-Lymphocyte-Associated Protein 4 (CTLA-4), Programmed Cell Death Protein 1 (PD-1), and its ligand Programmed Cell Death-Ligand 1 (PD-L1). These treatments have shown efficacy in limiting tumour progression and extending patientsurvival.However, due to dynamic intrinsic and extrinsic changes in the tumour microenvironment (TME), a high rate of acquired resistance to ICIs remains a major challenge. To address this, alternative co-inhibitory molecules are being explored for therapeutic targeting, either as standalone agents or in combination therapies. In this study, recombinant single-chain variable fragment (scFv) antibodies with high affinity for the co-inhibitory molecules V-domain Immunoglobulin Suppressor of T-cell Activation (VISTA) and PD-L1 were initially targeted. These co-inhibitory molecules play a critical role in immune regulation by inhibiting T-cell proliferation and cytokine production when interacting with their respective ligands. Both VISTA and PD-L1 have been shown to be overexpressed in various solid tumours including melanoma, non-small cell lung carcinoma (NSCLC) and ovarian cancer. The generation of recombinant scFv antibodies against these co-inhibitory molecules were pursued using the phage display biopanning technique. Due to technical challenges encountered during the biopanning phage display process, a statistically significant increase in binding towards either bait protein was not achieved after four rounds. As a result the project’s focus shifted toward characterizing 60 monoclonal antibodies derived from a previously successful VISTA-directed biopanning experiment conducted in a separate study. These clones were evaluated for specificity against VISTA and control bait proteins, and their binding affinities were analysed using the Enzyme-Linked Immunosorbent Assay (ELISA). ELISA analysis revealed that most of the clones exhibited strong binding affinity to VISTA with minimal cross-reactivity to control proteins. Notably, most clones demonstrated statistically significant preferential binding to their target bait, with p-values <0.000001. To assess sequence diversity, Sanger sequencing was performed on all clones. The results of Sanger sequencing showed that full-length sequencing was completed only on half of the, most of which had an identical sequence, suggesting potential amplification bias during phage display. Thus, in this project, 60 monoclonal scFvs with a high binding affinity towards VISTA and minimal cross-reactivity towards control bait proteins was achieved. Sanger sequencing of these clones showed minimal diversity indicating an amplification bias during biopanning phage display. Further confirmatory and validation techniques are required to validate the functional activity of the scFv binders. Future efforts should focus on optimizing the biopanning phage display protocol to enhance target-specific library generation and minimize bias.