Tri-functional antibody- fusion proteins for cancer immunotherapy
Final Report Abstract
Immunotherapeutic strategies that involve the administration of cytokines in order to support and drive an antitumor immune response have shown high potential, but face the challenge of systemic toxicity and limitations as monotherapy. To address these problems, we propose a targeted approach with trifunctional antibody‐fusion proteins, combining a tumor‐directed antibody with a stimulatory cytokine and a costimulatory ligand of the TNF‐superfamily in a single molecule. According to the concept, the antibody targets the cytokine to the tumor site, locally promoting an antitumor response. In addition, co‐presentation of the costimulatory ligand further supports and modulates this effect in a combinatorial approach. The proof of concept was shown in the preceding project by means of a trifunctional antibody fusion protein with RD_IL‐15 and 4‐1BBL. Antibody‐mediated targeting of RD_IL‐15 and 4‐1BBL to the tumor cell mimics their physiological cell surface presentation, whereby IL‐15 activity was further enhanced by the connection to a fragment of the IL‐15R chain (RD). Given that IL‐15 is also active in soluble form, but the costimulatory activity of 4‐1BBL is strongly targeting‐dependent, combined effects on immune cells were only observed for the tumor‐targeted form of the fusion protein. From a protein engineering part of view, the inherent property of TNF‐superfamily members to trimerize into one functional unit, leads to the assembly of three independent polypeptide chains. In a conventional arranged fusion protein the consequence is a bulky homotrimeric molecule carrying three antibody moieties and three RD_IL‐15 units, respectively. In this project we focused on the optimization of the fusion protein format by introducing the TNFSF‐ligand in a single‐chain arrangement, i.e. fusing three ligand domains in a row, enabling trimerization within one polypeptide chain. Thus, a smaller and simpler molecule type was generated comprising only one functional unit of each component. We analyzed and compared both formats for bi‐ and trifunctional antibody‐fusion proteins confirming similar or even improved costimulatory activity. Furthermore, using the new format, the concept of the trifunctional antibody‐fusion protein was demonstrated to be extendable to the combination of RD_IL‐ 15 with other costimulatory members of the TNF‐superfamily (OX40L, GITRL, LIGHT). In this context we observed differences in the stimulatory effect on different T cell subsets and NK cells in terms of proliferation and cytotoxic potential in vitro. Here, in terms of proliferation, trifunctional fusion proteins were at lower concentrations generally more effective than the combination of the respective bifunctional antibody‐fusion proteins. Advantages were in particular evident for the trifunctional antibody‐fusion protein with GITRL, enhancing efficiently the proliferation of T cells and NK cells and the cytotoxic potential of CD8+ T cells in vitro. Thus, we generated the corresponding mouse compatible fusion proteins for in vivo analysis. Indeed, antitumor effects were demonstrated for the trifunctional fusion protein with GITRL in a lung metastasis mouse model, demonstrating the importance of targeting and advantages in comparison to the combined application of bi‐functional antibody‐fusion proteins. Hence, we have successfully introduced a new format for the trifunctionalantibody‐fusion protein approach and confirmed and extended the concept to further TNF‐superfamily members, where GITRL emerged as a promising candidate to be considered for further immunotherapeutic development.
Publications
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(2012) An antibody fusion protein for cancer immunotherapy mimicking IL‐15 trans‐presentation at the tumor site. Mol Cancer Ther. 11(6):1279‐88
Kermer V, Baum V, Hornig N, Kontermann RE, Müller D
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(2012) Targeted cancer immunotherapy: mimicking physiological trans‐presentation of IL‐15. OncoImmunology 1(7):1213‐1214
Müller D.
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(2014) Antibody‐cytokine fusion proteins for cancer immunotherapy: an update on recent developments. BioDrugs. 28(2):123‐31
Müller D.
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(2014) Combining antibody‐ directed presentation of IL‐15 and 4‐1BBL in a trifunctional fusion protein for cancer immunotherapy. Mol Cancer Ther. 13(1):112‐21
Kermer V, Hornig N, Harder M, Bondarieva A, Kontermann RE, Müller D
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(2015) Antibody fusions with immunomodulatory proteins for cancer therapy. (2015) Pharmacol Ther. 154:57‐66
Müller D.
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(2016) Advancing targeted co‐stimulation with antibody‐fusion proteins by introducing TNF superfamily members in a single‐chain format. Oncoimmunology. 5(11):e1238540
Fellermeier S, Beha N, Meyer JE, Ring S, Bader S, Kontermann RE, Müller D
