Abstract 4963: Novel hexavalent GITR agonists stimulate T cells and enhance memory formation
Authors: Thiemann Meinolf , Gieffers Christian , Richards David M. , Merz Christian , Heinonen Karl , Mueller Mauricio Redondo , Marschall Viola , Sykora Jaromir , Fricke Harald , Hill Oliver 
Published: July 2017

Abstract
Introduction: The co-stimulatory receptor GITR plays an important role in initiating the immune response in the lymph nodes and in maintaining the immune response in the tumor tissue. Binding of GITR to its natural ligand directly leads to increased anti-tumor T cell activation and their survival. It also reduces the suppressive abilities of Treg cells, further increasing the anti-tumor immune response. The HERA-technology developed by Apogenix targets the TNF-receptor superfamily and generates fully human hexavalent fusion proteins with high clustering capacity for the cognate receptor. Hexavalent HERA-ligands are pure agonists whose signaling capacity is independent of secondary Fcγ-receptor crosslinking. Here we report in vitro and in vivo properties of novel hexavalent HERA-GITRL constructs.
Experimental procedures: For the assessment of in vivo stability, serum samples from a PK study with three HERA-GITRL constructs in CD1-mice were analyzed with respect to their drug levels employing a specific ELISA assay. For functional characterization of HERA-GITRL in vitro, immune cells were isolated from healthy-donor blood samples and profiled by multicolor flow cytometry (MC-FC). Subsequently, immune cells were cultured in growth media containing different HERA-GITRL constructs and anti-CD3. Changes in activation and memory markers on T cells (e.g. CD25, CD69, CD45RA, CD45RO), their proliferation rate (CFSE assay) and the intracellular staining of cytokines (e.g. TNF-α and IFN-γ) was assessed by MC-FC.
Results: Minor modifications led to three HERA-GITRL drug candidates with unique pharmacokinetic properties / in vivo stability as explored in mice. Terminal half-life was between 61.7 and 200.6 hours. Stimulation of pan T cells as well as naïve CD4+ T-lymphocytes by anti-CD3 was further augmented by HERA-GITRL as demonstrated by CD69 and CD25 expression. This effect was accompanied by an increased proliferation and an increased memory formation. Furthermore, we observed an increased level of intracellular TNF-α and IFN-γ in naïve CD4+ T-lymphocytes incubated with anti-CD3 that could be further raised by the addition of HERA-GITRL.
Conclusion: HERA-GITRL demonstrate excellent in vivo stability. Their ability to enhance proliferation and activation of naïve CD4+ T cells and to induce memory formation render them as attractive candidates for immunotherapeutic treatments of cancer.
Citation Format: Meinolf Thiemann, Christian Gieffers, David M. Richards, Christian Merz, Karl Heinonen, Mauricio Redondo Mueller, Viola Marschall, Jaromir Sykora, Harald Fricke, Oliver Hill. Novel hexavalent GITR agonists stimulate T cells and enhance memory formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4963. doi:10.1158/1538-7445.AM2017-4963