IL15-Based Trifunctional Antibody-Fusion Proteins with Costimulatory TNF-Superfamily Ligands in the Single-Chain Format for Cancer Immunotherapy.

IL15 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 IL15/TNFSF-ligand in a targeting-mediated manner to the tumor site. The previously reported format consisted of a tumor-directed antibody (scFv), IL15 linked to an IL15Rα-fragment (RD), and the extracellular domain of 4-1BBL, where noncovalent trimerization of 4-1BBL into its functional unit led to a homotrimeric molecule with 3 antibody and 3 IL15-RD units. 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), that is 3 consecutively linked 4-1BBL ectodomains. Thus, a monomeric trifunctional fusion protein presenting only 1 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_IL15_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.