Abstract
INTRODUCTION: CD28 is a critical co-stimulatory receptor expressed on T cells. Alongside the T-cell receptor (TCR), it provides a dual-signal requirement that is necessary for the complete activation of T cells. Developing co-stimulatory agonists targeting CD28 therefore represents a promising strategy to enhance the efficacy of T cell based anti-tumor immunotherapy. RESULTS: In this study, we isolated 34 VHHs (VHHs) from a phage-display immune library generated using PBMCs of a Bactrian camel immunized with recombinant human CD28 protein. These VHHs bound to cell surface CD28 with EC(50) values ranging from 8.5 nM to 130 nM and exhibited varied co-stimulatory activity. When incorporated as building blocks into a trispecific T cell engager (Tri-TCE) targeting DLL3, CD3, and CD28, the anti- CD28 VHHs conferred superior tumor specific cytotoxicity compared to a bispecific T cell engagers (Bi-TCEs) targeting only DLL3 and CD3. Among all candidates, the Tri-TCE containing VHH38 demonstrated the most potent antitumor efficacy. Structural mapping and alanine-scanning mutagenesis revealed that VHH38 engages the CD28 epitope, involving residues Arg34, Tyr51, and Tyr61, a region similar to that recognized by the published superagonistic antibody TGN1412. Unlike TGN1412, however, VHH38 exhibits a lateral paratope orientation that likely spatially hinder the cross-linking of CD28 molecules on the same T cell, probiding a structural basis for its co-stimulatory activity rather than superagonistic activity. DISCUSSION: In summary, through an integrated process of affinity screening, functional agonism ranking, and therapeutic efficacy evaluation, we have identified a panel of co-stimulatory anti-CD28 VHHs with strong potential as functional modules in Tri-TCEs for cancer immunotherapy.