IL-15 and costimulatory receptors of the tumor necrosis superfamily (TNFRSF) have shown great potential to support and drive an antitumor immune response. However, their efficacy as monotherapy is limited. Here, we present the development of a novel format for a trifunctional antibody-fusion protein that combines and focuses the activity of IL-15/TNFSF-ligand in a targeting-mediated manner to the tumor site. The previously reported format consisted of a tumor-directed antibody (scFv), IL-15 linked to an IL-15Ralpha-fragment (RD) and the extracellular domain of 4-1BBL, where non-covalent trimerization of 4-1BBL into its functional unit led to a homotrimeric molecule with three antibody and three IL-15-RD units. In order to reduce the size and complexity of the molecule, we have now designed a second format, where 4-1BBL is introduced as single-chain (sc), i.e. three consecutively linked 4-1BBL ectodomains. Thus, a monomeric trifunctional fusion protein presenting only one functional unit of each component was generated. Interestingly, the in vitro activity on T cell stimulation was conserved or even enhanced for the soluble and target-bound molecule, respectively. Also in a lung tumor mouse model comparable antitumor effects were observed. Furthermore, corroborating the concept, OX40L and GITRL were also successfully incorporated into the novel single-chain format and the advantage of target-bound trifunctional versus corresponding combined bifunctional fusion proteins demonstrated by measuring T cell proliferation and cytotoxic potential in vitro and antitumor effects of RD_IL-15_scFv_scGITRL in a lung tumor mouse model in vivo. Thus, the trifunctional antibody-fusion protein single-chain format constitutes a promising innovative platform for further therapeutic developments.