{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,17]],"date-time":"2026-04-17T05:58:53Z","timestamp":1776405533544,"version":"3.51.2"},"reference-count":43,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,4,17]],"date-time":"2026-04-17T00:00:00Z","timestamp":1776384000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec>\n                    <jats:title>Introduction<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      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\n                      <jats:sub>50<\/jats:sub>\n                      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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Discussion<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  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