Natalie Tigue

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.

Use of the Site-Specific Retargeting Jump-In Platform Cell Line to Support Biologic Drug Discovery

Biologics represent a fast-growing class of therapeutics in the pharmaceutical sector. Discovery of therapeutic antibodies and characterization of peptides can necessitate high expression of the target gene requiring the generation of clonal stably transfected cell lines. Traditional challenges of stable cell line transfection include gene silencing and cell-to-cell variability. Our inability to control these can present challenges in lead isolation. Recent progress in site-specific targeting of transgene to specific genomic loci has transformed the ability to generate stably transfected mammalian cell lines. In this article, we describe how the use of the Jump-In platform (Life Technologies, Carlsbad, CA) has been applied to drug discovery projects. It can easily and rapidly generate homogeneous high-expressing cell pools with a high degree of reproducibility. Their use in cell-based screening to identify specific binders, identify binding to relevant species variants, or detect functionally relevant therapeutic antibodies is central in driving drug discovery.

Identification of RNA-binding proteins in exosomes capable of interacting with different types of RNA: RBP-facilitated transport of RNAs into exosomes

The RNA that is packaged into exosomes is termed as exosomal-shuttle RNA (esRNA); however, the players, which take this subset of RNA (esRNA) into exosomes, remain largely unknown. We hypothesized that RNA binding proteins (RBPs) could serve as key players in this mechanism, by making complexes with RNAs and transporting them into exosomes during the biosynthesis of exosomes. Here, we demonstrate the presence of 30 RBPs in exosomes that were shown to form RNA–RBP complexes with both cellular RNA and exosomal-RNA species. To assess the involvement of these RBPs in RNA-transfer into exosomes, the gene transcripts encoding six of the proteins identified in exosomes (HSP90AB1, XPO5, hnRNPH1, hnRNPM, hnRNPA2B1, and MVP) were silenced by siRNA and subsequent effect on esRNA was assessed. A significant reduction of total esRNA was observed by post-transcriptional silencing of MVP, compared to other RBPs. Furthermore, to confirm the binding of MVP with esRNA, a biotinylated-MVP was transiently expressed in HEK293F cells. Higher levels of esRNA were recovered from MVP that was eluted from exosomes of transfected cells, as compared to those of non-transfected cells. Our data indicate that these RBPs could end up in exosomes together with RNA molecules in the form of RNA–ribonucleoprotein complexes, which could be important for the transport of RNAs into exosomes and the maintenance of RNAs inside exosomes. This type of maintenance may favor the shuttling of RNAs from exosomes to recipient cells in the form of stable complexes.

Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids

The expression and subsequent purification of mammalian recombinant proteins is of critical importance to many areas of biological science. To maintain the appropriate tertiary structure and post-translational modifications of such proteins, transient mammalian expression systems are often adopted. The successful utilisation of these systems is, however, not always forthcoming and some recombinant proteins prove refractory to expression in mammalian hosts. In this study we focussed on the role of different N-terminal signal peptides and residues immediately downstream, in influencing the level of secreted recombinant protein obtained from suspension HEK293 cells. Using secreted alkaline phosphatase (SEAP) as a model protein, we identified that the +1/+2 downstream residues flanking a heterologous signal peptide significantly affect secreted levels. By incorporating these findings we conducted a comparison of different signal peptide sequences and identified the most productive as secrecon, a computationally-designed sequence. Importantly, in the context of the secrecon signal peptide and SEAP, we also demonstrated a clear preference for specific amino acid residues at the +1 position (e.g. alanine), and a detrimental effect of others (cysteine, proline, tyrosine and glutamine). When proteins that naturally contain these “undesirable” residues at the +1 position were expressed with their native signal peptide, the heterologous secrecon signal peptide, or secrecon with an additional alanine at the +1 or +1 and +2 position, the level of expression differed significantly and in an unpredictable manner. For each protein, however, at least one of the panel of signal peptide/adjacent amino acid combinations enabled successful recombinant expression. In this study, we highlight the important interplay between a signal peptide and its adjacent amino acids in enabling protein expression, and we describe a strategy that could enable recombinant proteins that have so far proved refractory to expression in HEK293 cells, to be produced in sufficient quantities to answer important biological questions.

MHC class I chain-related protein A and B (MICA and MICB) are predominantly expressed intracellularly in tumour and normal tissue

Background:Major histocompatibility complex (MHC) class I chain-related protein A (MICA) and MHC class I chain-related protein B (MICB) are polymorphic proteins that are induced upon stress, damage or transformation of cells which act as a ‘kill me’ signal through the natural-killer group 2, member D receptor expressed on cytotoxic lymphocytes. MICA/B are not thought to be constitutively expressed by healthy normal cells but expression has been reported for most tumour types. However, it is not clear how much of this protein is expressed on the cell surface.Methods:Using a novel, well-characterised antibody and both standard and confocal microscopy, we systematically profiled MICA/B expression in multiple human tumour and normal tissue.Results:High expression of MICA/B was detected in the majority of tumour tissues from multiple indications. Importantly, MICA/B proteins were predominantly localised intracellularly with only occasional evidence of cell membrane localisation. MICA/B expression was also demonstrated in most normal tissue epithelia and predominantly localised intracellularly. Crucially, we did not observe qualitative differences in cell surface expression between tumour and MICA/B expressing normal epithelia.Conclusions:This demonstrates for the first time that MICA/B is more broadly expressed in normal tissue and that expression is mainly intracellular with only a small fraction appearing on the cell surface of some epithelia and tumour cells.

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.

Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab

Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab.

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.