Duokines: a novel class of dual-acting co-stimulatory molecules acting in cis or trans

Abstract

Co-stimulatory signals induced by ligands of the tumor necrosis factor superfamily (TNFSF) play a central role in T cell activation and have emerged as a promising strategy in cancer immunotherapy. Here, we established a novel class of bifunctional co-stimulatory fusion proteins with the aim to boost T cell activation at the level of T cell – antigen-presenting cell (APC) interaction. These novel dual-acting cytokine fusion proteins were created by connecting two different homotrimeric TNFSF ligands to form homotrimeric bifunctional molecules (Duokines) or by connecting single-chain derivatives of two different homotrimeric TNFSF with a single, flexible linker (single-chain Duokines, scDuokines). By linking the TNFSF ligands 4-1BBL, OX40L and CD27L in all possible combinations, cis-acting Duokines were generated that act on the same or adjacent T cells, while combining CD40L with 4-1BBL, OX40L and CD27L resulted in trans-acting Duokines acting simultaneously on APCs and T cells. In vitro, co-stimulation of T cells was seen for cis- and trans-acting Duokines and scDuokines in an antigen-independent as well as antigen-specific setting. Trans-acting molecules furthermore activated B cells, which represent a subclass of APCs. In a pilot experiment using the syngeneic B16-FAP mouse tumor model scDuokines displayed antitumoral activity in vivo in combination with a primary T cell-activating bispecific antibody, evident from reduced number of lung metastasis compared to the antibody-only treated group. Our data show that the bifunctional, co-stimulatory duokines are capable to enhance T cell-mediated anti-tumor immune responses, suggesting that they can serve as a new class of immuno-stimulatory molecules for use in cancer immunotherapy strategies.

Fc-based Duokines: dual-acting costimulatory molecules comprising TNFSF ligands in the single-chain format fused to a heterodimerizing Fc (scDk-Fc)

“…Stable transfected HT1080-CD40, HT1080-CD27, HT1080-OX40 and HT1080-4-1BB were kindly provided by Dr. H. Wajant (University Hospital Würzburg, Germany) and were cultivated in RPMI-1640 (Thermo Fisher Scientific, 11875), 10% FBS (Pan Biotech, P30-3309). Mouse melanoma B16-FAP cells 13 were cultured in RPMI-1640, 5% FBS supplemented with 200 μg/mL zeocin (Thermo Fisher Scientific, R25001). Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy donors (Blood bank, Klinikum Stuttgart) by density gradient centrifugation (Lymphocyte Separation Medium 1077, Promocell, C-44010) and cultivated in RPMI-1640, 10% FBS.…”

“…This was achieved by combining two different members of the TNFSF into one molecule connecting either single subunits of two ligands (so-called Duokines, Dk) or single-chain derivatives of the ligand (single-chain Duokines, scDk) by flexible linkers. 13 We have demonstrated that these dual-acting costimulatory molecules can activate two different receptor types either on the same cell (i.e. acting in cis) or on different cell types (i.e.…”

“…In combination with a bispecific T-cell engager, these molecules mediated an enhanced T-cell proliferation in vitro and showed promising anti-tumor effects in vivo . 13 …”

Antibody-cytokine fusion proteins: A novel class of biopharmaceuticals for the therapy of cancer and of chronic inflammation

“…Dual-cytokine antibody fusions have been described for IL2 and IL12 [160][161][162], IL12 and GM-CSF [161], TNF and IL2 [57], as well as IL15 and 4-1BBL [40]. The group of Roland Kontermann has introduced the concept of Duokines, dual-acting cytokine fusion proteins generated by connecting two TNF superfamily members (combinations 4-1BBL, OX40L and CD27L) by a flexible peptide linker in different conformations [163].…”

Concepts for agonistic targeting of CD40 in immuno-oncology

“…The development of bispecific CD40 agonists is challenging due to the ambiguous expression profile of CD40, nevertheless attempts have been made to target CD40-expressing tumors using CD40/mesothelin-bispecific constructs (ABBV-428, NCT02955251, currently recruiting), CD40/HER2-bispecific constructs 78 and so-called duokines. 79 Additional CD40 bispecifics are on the way, including CEA and others as tumor-targeting antigen. 80…”