HERA-GITRL activates T cells and promotes anti-tumor efficacy independent of FcγR-binding functionality

J Immunother Cancer. 2019 Jul 19;7(1):191. doi: 10.1186/s40425-019-0671-4.


Background: Glucocorticoid-induced TNFR-related protein (TNFRSF18, GITR, CD357), expressed by T cells, and its ligand (TNFSF18, GITRL), expressed by myeloid populations, provide co-stimulatory signals that boost T cell activity. Due to the important role that GITR plays in regulating immune functions, agonistic stimulation of GITR is a promising therapeutic concept. Multiple strategies to induce GITR signaling have been investigated. The limited clinical efficacy of antibody-based GITR agonists results from structural and functional characteristics of antibodies that are unsuitable for stimulating the well-defined trimeric members of the TNFRSF.

Methods: To overcome limitations of antibody-based TNFRSF agonists, we have developed HERA-GITRL, a fully human hexavalent TNF receptor agonist (HERA) targeting GITR and mimicking the natural signaling concept. HERA-GITRL is composed of a trivalent but single-chain GITRL-receptor-binding-domain (scGITRL-RBD) unit fused to an IgG1 derived silenced Fc-domain serving as dimerization scaffold. A specific mouse surrogate, mmHERA-GITRL, was also generated to examine in vivo activity in respective mouse tumor models.

Results: For functional characterization of HERA-GITRL in vitro, human immune cells were isolated from healthy-donor blood and stimulated with anti-CD3 antibody in the presence of HERA-GITRL. Consistently, HERA-GITRL increased the activity of T cells, including proliferation and differentiation, even in the presence of regulatory T cells. In line with these findings, mmHERA-GITRL enhanced antigen-specific clonal expansion of both CD4+ (OT-II) and CD8+ (OT-I) T cells in vivo while having no effect on non-specific T cells. In addition, mmHERA-GITRL showed single-agent anti-tumor activity in two subcutaneous syngeneic colon cancer models (CT26wt and MC38-CEA). Importantly, this activity is independent of its FcγR-binding functionality, as both mmHERA-GITRL with a functional Fc- and a silenced Fc-domain showed similar tumor growth inhibition. Finally, in a direct in vitro comparison to a bivalent clinical benchmark anti-GITR antibody and a trivalent GITRL, only the hexavalent HERA-GITRL showed full biological activity independent of additional crosslinking.

Conclusion: In this manuscript, we describe the development of HERA-GITRL, a true GITR agonist with a clearly defined mechanism of action. By clustering six receptor chains in a spatially well-defined manner, HERA-GITRL induces potent agonistic activity without being dependent on additional FcγR-mediated crosslinking.

Keywords: Agonist; CD357; GITR; HERA; Single-chain GITRL; TNFSF; scGITRL-RBD.

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Humans
  • Immunoglobulin Fc Fragments / immunology
  • Lymphocyte Activation
  • Macaca fascicularis
  • Mice
  • Receptors, Tumor Necrosis Factor / agonists*
  • Recombinant Fusion Proteins / immunology
  • Signal Transduction
  • Single-Chain Antibodies / administration & dosage*
  • Single-Chain Antibodies / immunology
  • T-Lymphocytes, Regulatory / immunology*
  • Tumor Necrosis Factors / chemistry*
  • Tumor Necrosis Factors / metabolism


  • Immunoglobulin Fc Fragments
  • Receptors, Tumor Necrosis Factor
  • Recombinant Fusion Proteins
  • Single-Chain Antibodies
  • TNFSF18 protein, human
  • Tumor Necrosis Factors