{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T06:55:25Z","timestamp":1791356125395,"version":"4.3.1"},"reference-count":57,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T00:00:00Z","timestamp":1791331200000},"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:p>Antibody fusion proteins incorporating immunomodulatory molecules, such as cytokines of the common \u03b3-chain receptor family and costimulatory members of the TNF superfamily, hold great potential for cancer immunotherapy, with many candidates already advancing into clinical studies. However, the efficacy as single agent with one payload is limited and combinatorial approaches are required to fully exploit their immunomodulatory potential. Here, we describe a novel format of a trifunctional antibody fusion protein, composed of a tumor-directed Fab antibody fragment, specific for the fibroblast activation protein (FAP), fused to single-chain 4-1BBL in combination with either IL-15 connected to an IL-15R\u03b1 fragment or IL-21. Binding to target cells was confirmed, and activity was assessed on activated PBMCs either bound to target cells or in an untargeted setting. Combinatorial effects enhancing IFN-\u03b3 release were found to be targeting-dependent for both trifunctional antibody fusion proteins and superior to the activity observed with combined bifunctional antibody-fusion proteins carrying single payloads. In contrast, significant combined activity enhancing granzyme B expression was observed only for the trifunctional fusion protein containing sc4-1BBL\/IL-21 and appeared to be targeting-independent. In 3D coculture settings of fibroblast spheroids and PBMCs, both trifunctional fusion proteins enhanced the cytotoxic effect of an EGFR-directed T cell engager (TCE) and cetuximab (CTX), whereby sc4-1BBL\/IL-15 was more effective than sc4-1BBL\/IL-21, and comparable effects were obtained by the respective bifunctional antibody-cytokines. The activity of antibody fusion proteins containing IL-15 was associated with strong activation of CD8+T cells, CD3+CD56+ T cells and NK cells, as well as enhanced proliferation, particularly in combination with TCE, expanding CD8+ T cells and NKT-like cells. Activation by fusion proteins containing IL-21 became mainly apparent in combination with TCE on CD8+ T cells and with CTX on NK cells, while proliferation was restricted to NK cells. Thus, the trifunctional antibody fusion proteins presenting sc4-1BBL\/IL-15 and sc4-1BBL\/IL-21 exhibit distinct patterns of combined activity and immune cell stimulation, both demonstrating combinatorial potential with TCE and CTX.<\/jats:p>","DOI":"10.3389\/fimmu.2026.1903525","type":"journal-article","created":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T06:42:06Z","timestamp":1791355326000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Trifunctional antibody fusion proteins for targeted co-delivery of 4-1BBL with IL-15 or IL-21 to enhance antitumor immune responses"],"prefix":"10.3389","volume":"17","author":[{"given":"Annika M.","family":"M\u00f6ller","sequence":"first","affiliation":[{"name":"Institute of Cell Biology and Immunology, University of Stuttgart","place":["Stuttgart, 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