Lesley Young

A Novel Murine GITR Ligand Fusion Protein Induces Antitumor Activity as a Monotherapy That Is Further Enhanced in Combination with an OX40 Agonist

Purpose: To generate and characterize a murine GITR ligand fusion protein (mGITRL-FP) designed to maximize valency and the potential to agonize the GITR receptor for cancer immunotherapy. Experimental Design: The EC50 value of the mGITRL-FP was compared with an anti-GITR antibody in an in vitro agonistic cell–based reporter assay. We assessed the impact of dose, schedule, and Fc isotype on antitumor activity and T-cell modulation in the CT26 tumor model. The activity of the mGITRL-FP was compared with an agonistic murine OX40L-FP targeting OX40, in CT26 and B16F10-Luc2 tumor models. Combination of the mGITRL-FP with antibodies targeting PD-L1, PD-1, or CTLA-4 was analyzed in mice bearing CT26 tumors. Results: The mGITRL-FP had an almost 50-fold higher EC50 value compared with an anti-murine GITR antibody. Treatment of CT26 tumor-bearing mice with mGITRL-FP–mediated significant antitumor activity that was dependent on isotype, dose, and duration of exposure. The antitumor activity could be correlated with the increased proliferation of peripheral CD8+ and CD4+ T cells and a significant decrease in the frequency of intratumoral Tregs. The combination of mGITRL-FP with mOX40L-FP or checkpoint inhibitor antagonists enhanced antitumor immunity above that of monotherapy treatment. Conclusions: These results suggest that therapeutically targeting GITR represents a unique approach to cancer immunotherapy and suggests that a multimeric fusion protein may provide increased agonistic potential versus an antibody. In addition, these data provide, for the first time, early proof of concept for the potential combination of GITR targeting agents with OX40 agonists and PD-L1 antagonists. Clin Cancer Res; 23(13); 3416–27. ©2017 AACR.

MEDI1873, a potent, stabilized hexameric agonist of human GITR with regulatory T-cell targeting potential

ABSTRACT Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) is part of a system of signals involved in controlling T-cell activation. Targeting and agonizing GITR in mice promotes antitumor immunity by enhancing the function of effector T cells and inhibiting regulatory T cells. Here, we describe MEDI1873, a novel hexameric human GITR agonist comprising an IgG1 Fc domain, a coronin 1A trimerization domain and the human GITRL extracellular domain (ECD). MEDI1873 was optimized through systematic testing of different trimerization domains, aglycosylation of the GITRL ECD and comparison of different Fc isotypes. MEDI1873 exhibits oligomeric heterogeneity and superiority to an anti-GITR antibody with respect to evoking robust GITR agonism, T-cell activation and clustering of Fc gamma receptors. Further, it recapitulates, in vitro, several aspects of GITR targeting described in mice, including modulation of regulatory T-cell suppression and the ability to increase the CD8+:CD4+ T-cell ratio via antibody-dependent T-cell cytotoxicity. To support translation into a therapeutic setting, we demonstrate that MEDI1873 is a potent T-cell agonist in vivo in non-human primates, inducing marked enhancement of humoral and T-cell proliferative responses against protein antigen, and demonstrate the presence of GITR- and FoxP3-expressing infiltrating lymphocytes in a range of human tumors. Overall our data provide compelling evidence that MEDI1873 is a novel, potent GITR agonist with the ability to modulate T-cell responses, and suggest that previously described GITR biology in mice may translate to the human setting, reinforcing the potential of targeting the GITR pathway as a therapeutic approach to cancer.

Abstract 1475: Local immune activation resulting in tumor growth inhibition with MEDI9197 - an intratumorally administered TLR7/8 agonist

MEDI9197 (formerly 3M-052) is a sustained-release imidazoquinoline toll like receptor (TLR) TLR7/8 agonist designed with a lipid tail that, when injected, is retained within the tumor. This has been shown in pharmacokinetic studies in mice and rats injected with MEDI9197 via the subcutaneous (SC) or intratumoral (IT, mouse only) routes. Stimulation of TLR7 and TLR8 in primary human dendritic cells induces the release of interferon-alpha (IFN-a) from plasmacytoid dendritic cells (pDCs) and interleukin 12 (IL-12) from myeloid dendritic cells (mDCs). Intratumoral administration of MEDI9197 induces a local immune response characterized by upregulation of genes involved in activation of innate and adaptive immunity both from the injected tumor and tumor draining lymph node. Furthermore, flow cytometric analysis of tumor infiltrating lymphocytes (TILs) show increased expression of activation markers, such as CD69, on natural killer (NK) cells and CD8 cytotoxic T cells. This local stimulation of immune cells with MEDI9197 results in tumor growth inhibition as shown in the B16F10 luc syngeneic mouse tumor model using in vivo imaging system (IVIS) equipment. Additionally, combination of MEDI9197 with immune checkpoint inhibitors enhances the efficacy observed in syngeneic mouse tumor models. The data presented shows intratumoral administration of MEDI9197 induces local immune activation leading to tumor growth inhibition in preclinical models of cancer. MEDI9197 is currently being evaluated as a monotherapy for safety and efficacy in human clinical trials. Citation Format: Stefanie Mullins, Iwen Grigsby, Lester I. Harrison, Song Ren, Serguei Soukharev, Lesley Young, James M. Elvecrog, Robert W. Wilkinson, Mark A. Tomai, Ronald Herbst, John P. Vasilakos, Andrew J. Leishman. Local immune activation resulting in tumor growth inhibition with MEDI9197 - an intratumorally administered TLR7/8 agonist. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1475.

Abstract 561: MEDI1873: A novel hexameric GITRL fusion protein with potent agonsitic and immunomodulatory activities in preclinical systems

Glucocorticoid-induced TNFR-related protein (GITR) is a member of the tumor necrosis factor receptor (TNFR) superfamily. GITR is expressed constitutively on regulatory T cells (Tregs) and is up-regulated on other T cells following activation. Agonistic antibodies to GITR have demonstrated significant activity in preclinical models of cancer. Here we describe the generation and characterisation of a GITR ligand (GITRL) fusion protein (FP) (MEDI1873), currently in phase 1 clinical trials. Protein engineering was used to generate a series of GITRL FPs, which were screened using a high throughput reporter gene assay for GITR signalling. The most potent fusion protein resulted in a 20 times greater maximal signal and a 5 times higher EC50 when compared to a GITR targeting antibody. This increased potency was considered to be a result of the enhanced valency achieved by the hexameric format. Two versions of GITRL FP, MEDI1873 and MEDI5607, bearing an IgG1 and IgG4 Fc respectively, both demonstrated equivalent potency in a reporter assay and were able to enhance T-cell activation, with respect to proliferation and cytokine release, and to overcome the suppressive effect of Tregs, in primary human cell based assays. Assessment of two surrogate mouse GITRL FPs in the CT26 model of colorectal cancer indicated that the version with increased binding to Fc gamma receptors resulted in increased activity, coincident with an increased depletion of intratumoral Tregs, likely through Fc mediated effector functions. A comparison of GITR expression on Tregs and effector T cells in mouse and human, via flow cytometry, indicated a similar pattern of expression across species, with significantly higher expression observed on Tregs. Immunohistochemical analysis indicated the presence of high levels of both GITR and FoxP3 in sections from human tumors; suggesting that the intratumoral Treg depletion observed in mice could also occur in humans. Both MEDI1873 and MEDI5607 demonstrated enhanced binding to Fc gamma receptors when compared to antibody controls of the same isotype, again considered to be a result of their increased valency. However, only MEDI1873 was able to mediate ADCC against activated T cells in vitro; resulting in an increase in the CD8:CD4 ratio within the culture. As a result of these studies, MEDI1873 was selected as an optimal GITR targeting agent that possessed the ability to both agonise GITR and to modulate Tregs through suppression and/or depletion. MEDI1873 is currently being assessed in a phase 1 clinical study (NCT02583165) in patients with solid tumors.

Abstract 4604: MEDI1873, a GITR ligand fusion protein (GITRL FP), induces effector T-cell proliferation, modulates T-regulatory cell function and has the potential to combine with checkpoint inhibitors

Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) is part of a system of signals involved in controlling T-cell activation. MEDI1873 is a novel hexameric human GITR agonist comprising an IgG1 Fc domain, a coronin 1A trimerisation domain and the human GITRL extracellular domain (ECD) that is currently being assessed in a Phase 1 clinical study (NCT02583165) in patients with solid tumors. MEDI1873 exhibits in vitro superiority to an anti-GITR antibody with respect to evoking robust GITR agonism, T-cell activation and clustering of Fc gamma receptors. Using in vitro assay systems, MEDI1873 recapitulates aspects of GITR targeting previously described in mice, including modulation of regulatory T-cell (Treg) suppression and the ability to increase the CD8:CD4 T-cell ratio via antibody-dependent T-cell cytotoxicity. Pharmacodynamic assessment of an agonistic mouse GITRL FP (mGITRL FP) in the CT26 model of colorectal cancer demonstrated activation and proliferation of peripheral CD4+ and CD8+ T cells coincident with an increased depletion of intratumoral Tregs, likely through Fc mediated effector functions. Furthermore, CT26 tumor growth studies indicated the mGITRL FP could result in significant antitumor activity. These data provide evidence that MEDI1873 is a novel, potent GITR agonist with the potential to modulate T-cell responses and enhance anti-tumor immunity. Combinations of immunotherapies are generating exciting results in the clinic, therefore, we sought to assess the potential for GITRL FPs to combine with antibodies targeting either anti-PD-L1 (durvalumab) or anti-CTLA-4 (tremelimumab) using both in vitro and in vivo systems. In vitro studies where MEDI1873 was combined with either durvalumab or tremelimumab showed that both combinations have the potential to enhance interleukin-2 release in a superantigen-stimulation of human peripheral blood mononuclear cells (PBMCs) compared to checkpoint blockade alone. Further evidence to support the potential for combinatorial antitumor activity was generated in the CT26 model where either 0.2mg/kg mGITRL combined with 10mg/kg anti-mouse PD-L1 or 0.1mg/kg mGITRL combined with 5mg/kg anti-mouse CTLA-4 antibodies resulted in enhanced antitumor activity versus monotherapies alone. Overall, our data suggest that therapeutically targeting GITR with a multimeric fusion protein, GITRL FP, may provide increased agonistic potential versus an antibody, and have the ability to both activate effector T-cells and modulate Tregs through suppression and/or depletion. Finally, combination studies provide preclinical evidence to support the rationale for combination of MEDI1873 with anti-PD-L1 or anti-CTLA-4 antibodies further reinforcing the potential of targeting the GITR pathway as a therapeutic approach to treating patients with cancer. Citation Format: Michelle Morrow, Rebecca Leyland, James Hair, Ross Stewart, Natalie Tigue, Lisa Bamber, Samantha Ireland, Nicholas Holoweckyi, Michael Oberst, Amanda Watkins, Emily Offer, David Perez-Martinez, Ching Ching Leow, Lesley Young, Tristan Vaughan, Philip Mallinder, Robert Wilkinson. MEDI1873, a GITR ligand fusion protein (GITRL FP), induces effector T-cell proliferation, modulates T-regulatory cell function and has the potential to combine with checkpoint inhibitors [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 4604. doi:10.1158/1538-7445.AM2017-4604

Abstract 4902: A mouse GITRL fusion protein drives T-cell activation and antitumor activity in preclinical mouse models of cancer

GITR is a member of the TNFR superfamily of proteins and is expressed on resting regulatory T-cells and on other T-cells following activation. Signals through GITR have been shown to drive increased T-cell activity and reduced regulatory T-cell function. In order to explore the potential of therapeutically targeting GITR in a cancer setting, we generated a mouse GITRL fusion protein (mGITRL FP) consisting of the extracellular domain of mGITRL linked to a structural domain and an IgG Fc domain. The antitumor activity and pharmacodynamic effects of this molecule were then explored in the colorectal syngeneic model of cancer (CT26). Treatment of mice with mGITRL FP mediated anti-tumor activity that was dependent on isotype and exposure. The anti-tumor activity could be attributed at least in part to the increased activation and proliferation status of CD8+ and CD4+ T-cells, as evidenced by increases in Ki67 expression and ICOS upregulation. Intratumourally we observed a significant decrease in the frequency of CD4+ T-cells (including T regs), but a corresponding increase in cytotoxic CD8+ T-cells. OX40 is another member of the TNFR superfamily, that has similar expression and functions to GITR. In order to better understand the potential differences between targeting of these two pathways, the activity and pharmacodynamic effects of the mGITRL FP were additionally compared and contrasted to those of a mOX40L FP and the observed differences will be discussed.

A mouse GITRl fusion protein drives T cell activation and antitumor activity in preclinical mouse models of cancer

GITR is a member of the TNFR superfamily of proteins and is expressed on resting regulatory T cells and on other T cells, and NK cells, following activation. Signals through GITR have been shown to drive increased T cell activity and reduced regulatory T cell function. In order to explore the potential of therapeutically targeting GITR in a cancer setting, we generated a mouse GITRL fusion protein (mGITRL FP) consisting of the extracellular domain of mGITRL linked to a structural domain and an IgG Fc domain. The antitumor activity and pharmacodynamic effects of this molecule were then explored in the colorectal syngeneic model of cancer (CT26). Treatment of mice with mGITRL FP mediated anti-tumor activity that was dependent on isotype and exposure. The anti-tumour activity could be attributed at least in part to the increased activation and proliferation status of CD8+ and CD4+ T cells, as evidenced by increases in Ki67 expression, ICOS upregulation and increased cytokine secretion of these cells. Intratumourally we observed a significant decrease in the frequency of CD4+ T cells (including T regs), but a corresponding increase in cytotoxic CD8+ T cells. OX40 is another member of the TNFR superfamily, that has similar expression and functions to GITR. In order to better understand the potential differences between targeting of these two pathways, the activity and pharmacodynamic effects of the mGITRL FP were additionally compared and contrasted to those of a mOX40L FP and the observed differences will be discussed.

Abstract 427: Host and tumoral CXCR2 signaling contributes to tumor growth

Chemokines are essential mediators of leukocyte migration and inflammation. Additionally they play an important role in tumour growth. The G-protein coupled receptor, CXCR2, and its ligands (CXCL 1,2,3,5,6,7 and IL8) have been shown to promote tumour initiation and growth, chemo-resistance, angiogenesis and immune cell infiltration (neutrophil, myeloid derived suppressor cells (MDSCs) and macrophages) into the tumour microenvironment. However the contribution of host and tumour CXCR2 has not been elucidated. We have developed both murine and human specific anti-CXCR2 antibodies alongside a humanised CXCR2 transgenic mouse to address the contribution of host and tumour CXCR2 signalling to tumour growth and maintenance. In pre-clinical tumours, granulocytic cells (neutrophils and MDSCs) within the tumour increase with size, in parallel with alteration in cell numbers observed within the spleen, peripheral blood and bone marrow. Inhibition of host CXCR2 has a dramatic impact on peripheral neutrophil levels, as well as their ability to become activated. In pre-clinical tumour models, inhibition of CXCR2 results in tumour growth inhibition. Contribution of host or tumour cells to the effect of CXCR2 blockade was model dependent. In the EL4 murine lymphoma model, only inhibition of host/peripheral CXCR2 was able to impact tumour growth. Whereas, in other murine syngeneic models (CT26 and B16), both host and tumour CXCR2 play a role in tumour growth. This was further demonstrated in an NSCLC patient derived xenograft, where the contribution of tumour CXCR2 was stronger than the host. Collectively our data show that CXCR2 inhibition (alone or in combination) has potential to influence growth of a number of tumour types. Future work is focused on understanding the mechanisms underlying the effects of CXCR2 inhibition, which remain key to developing it as an effective anti-cancer therapeutic. Citation Format: Danielle Carroll, James Harper, Karen McDaid, Ruth Franks, Catherine Eberline, Jane Kendrew, Richard Sainson, Judith Anderton, Chris Rossant, Karen Coffman, Ching Ching Leow, Ivan Inigo, Mitchell Reville, Jacintha Shenton, Lesley Young, Simon Barry. Host and tumoral CXCR2 signaling contributes to tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 427. doi:10.1158/1538-7445.AM2015-427