Amanda Watkins

Identification and Characterization of MEDI4736, an Antagonistic Anti–PD-L1 Monoclonal Antibody

A human antibody to PD-L1, engineered to eliminate Fc effector functions, which potently inhibits PD-L1 function, is in phase III clinical trials. Its characterization here provides clinicians and researchers with a basis for understanding and interpreting clinical trial results. Programmed cell-death 1 ligand 1 (PD-L1) is a member of the B7/CD28 family of proteins that control T-cell activation. Many tumors can upregulate expression of PD-L1, inhibiting antitumor T-cell responses and avoiding immune surveillance and elimination. We have identified and characterized MEDI4736, a human IgG1 monoclonal antibody that binds with high affinity and specificity to PD-L1 and is uniquely engineered to prevent antibody-dependent cell-mediated cytotoxicity. In vitro assays demonstrate that MEDI4736 is a potent antagonist of PD-L1 function, blocking interaction with PD-1 and CD80 to overcome inhibition of primary human T-cell activation. In vivo MEDI4736 significantly inhibits the growth of human tumors in a novel xenograft model containing coimplanted human T cells. This activity is entirely dependent on the presence of transplanted T cells, supporting the immunological mechanism of action for MEDI4736. To further determine the utility of PD-L1 blockade, an anti-mouse PD-L1 antibody was investigated in immunocompetent mice. Here, anti-mouse PD-L1 significantly improved survival of mice implanted with CT26 colorectal cancer cells. The antitumor activity of anti–PD-L1 was enhanced by combination with oxaliplatin, which resulted in increased release of HMGB1 within CT26 tumors. Taken together, our results demonstrate that inhibition of PD-L1 function can have potent antitumor activity when used as monotherapy or in combination in preclinical models, and suggest it may be a promising therapeutic approach for the treatment of cancer. MEDI4736 is currently in several clinical trials both alone and in combination with other agents, including anti–CTLA-4, anti–PD-1, and inhibitors of IDO, MEK, BRAF, and EGFR. Cancer Immunol Res; 3(9); 1052–62. ©2015 AACR.

Rational selection of syngeneic preclinical tumor models for immunotherapeutic drug discovery

Murine syngeneic tumor models are used to study responses to antitumor immunotherapies. To rationalize model selection, the underlying genetic and immunologic biology of the models was analyzed, allowing parallels to be drawn between models and human disease phenotypes. Murine syngeneic tumor models are critical to novel immuno-based therapy development, but the molecular and immunologic features of these models are still not clearly defined. The translational relevance of differences between the models is not fully understood, impeding appropriate preclinical model selection for target validation, and ultimately hindering drug development. Across a panel of commonly used murine syngeneic tumor models, we showed variable responsiveness to immunotherapies. We used array comparative genomic hybridization, whole-exome sequencing, exon microarray analysis, and flow cytometry to extensively characterize these models, which revealed striking differences that may underlie these contrasting response profiles. We identified strong differential gene expression in immune-related pathways and changes in immune cell–specific genes that suggested differences in tumor immune infiltrates between models. Further investigation using flow cytometry showed differences in both the composition and magnitude of the tumor immune infiltrates, identifying models that harbor “inflamed” and “non-inflamed” tumor immune infiltrate phenotypes. We also found that immunosuppressive cell types predominated in syngeneic mouse tumor models that did not respond to immune-checkpoint blockade, whereas cytotoxic effector immune cells were enriched in responsive models. A cytotoxic cell–rich tumor immune infiltrate has been correlated with increased efficacy of immunotherapies in the clinic, and these differences could underlie the varying response profiles to immunotherapy between the syngeneic models. This characterization highlighted the importance of extensive profiling and will enable investigators to select appropriate models to interrogate the activity of immunotherapies as well as combinations with targeted therapies in vivo. Cancer Immunol Res; 5(1); 29–41. ©2016 AACR.

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.

The MEK inhibitor selumetinib complements CTLA-4 blockade by reprogramming the tumor immune microenvironment

Background T-cell checkpoint blockade and MEK inhibitor combinations are under clinical investigation. Despite progress elucidating the immuno-modulatory effects of MEK inhibitors as standalone therapies, the impact of MEK inhibition on the activity of T-cell checkpoint inhibitors remains incompletely understood. Here we sought to characterize the combined effects of MEK inhibition and anti-CTLA-4 mAb (anti-CTLA-4) therapy, examining effects on both T-cells and tumor microenvironment (TME). Methods In mice, the effects of MEK inhibition, via selumetinib, and anti-CTLA-4 on immune responses to keyhole limpet haemocyanin (KLH) immunization were monitored using ex vivo functional assays with splenocytes. In a KRAS-mutant CT26 mouse colorectal cancer model, the impact on the tumor microenvironment (TME) and the spleen were evaluated by flow cytometry. The TME was further examined by gene expression and immunohistochemical analyses. The combination and sequencing of selumetinib and anti-CTLA-4 were also evaluated in efficacy studies using the CT26 mouse syngeneic model. Results Anti-CTLA-4 enhanced the generation of KLH specific immunity following KLH immunization in vivo; selumetinib was found to reduce, but did not prevent, this enhancement of immune response by anti-CTLA-4 in vivo. In the CT26 mouse model, anti-CTLA-4 treatment led to higher expression levels of the immunosuppressive mediators, Cox-2 and Arg1 in the TME. Combination of anti-CTLA-4 with selumetinib negated this up-regulation of Cox-2 and Arg1, reduced the frequency of CD11+ Ly6G+ myeloid cells, and led to the accumulation of differentiating monocytes at the Ly6C+ MHC+ intermediate state in the tumor. We also report that MEK inhibition had limited impact on anti-CTLA-4-mediated increases in T-cell infiltration and T-cell activation in CT26 tumors. Finally, we show that pre-treatment, but not concurrent treatment, with selumetinib enhanced the anti-tumor activity of anti-CTLA-4 in the CT26 model. Conclusion These data provide evidence that MEK inhibition can lead to changes in myeloid cells and immunosuppressive factors in the tumor, thus potentially conditioning the TME to facilitate improved response to anti-CTLA-4 treatment. In summary, the use of MEK inhibitors to alter the TME as an approach to enhance the activities of immune checkpoint inhibitors warrants further investigation in clinical trials. Electronic supplementary material The online version of this article (doi:10.1186/s40425-017-0268-8) contains supplementary material, which is available to authorized users.BackgroundT-cell checkpoint blockade and MEK inhibitor combinations are under clinical investigation. Despite progress elucidating the immuno-modulatory effects of MEK inhibitors as standalone therapies, the impact of MEK inhibition on the activity of T-cell checkpoint inhibitors remains incompletely understood. Here we sought to characterize the combined effects of MEK inhibition and anti-CTLA-4 mAb (anti-CTLA-4) therapy, examining effects on both T-cells and tumor microenvironment (TME).MethodsIn mice, the effects of MEK inhibition, via selumetinib, and anti-CTLA-4 on immune responses to keyhole limpet haemocyanin (KLH) immunization were monitored using ex vivo functional assays with splenocytes. In a KRAS-mutant CT26 mouse colorectal cancer model, the impact on the tumor microenvironment (TME) and the spleen were evaluated by flow cytometry. The TME was further examined by gene expression and immunohistochemical analyses. The combination and sequencing of selumetinib and anti-CTLA-4 were also evaluated in efficacy studies using the CT26 mouse syngeneic model.ResultsAnti-CTLA-4 enhanced the generation of KLH specific immunity following KLH immunization in vivo; selumetinib was found to reduce, but did not prevent, this enhancement of immune response by anti-CTLA-4 in vivo. In the CT26 mouse model, anti-CTLA-4 treatment led to higher expression levels of the immunosuppressive mediators, Cox-2 and Arg1 in the TME. Combination of anti-CTLA-4 with selumetinib negated this up-regulation of Cox-2 and Arg1, reduced the frequency of CD11+ Ly6G+ myeloid cells, and led to the accumulation of differentiating monocytes at the Ly6C+ MHC+ intermediate state in the tumor. We also report that MEK inhibition had limited impact on anti-CTLA-4-mediated increases in T-cell infiltration and T-cell activation in CT26 tumors. Finally, we show that pre-treatment, but not concurrent treatment, with selumetinib enhanced the anti-tumor activity of anti-CTLA-4 in the CT26 model.ConclusionThese data provide evidence that MEK inhibition can lead to changes in myeloid cells and immunosuppressive factors in the tumor, thus potentially conditioning the TME to facilitate improved response to anti-CTLA-4 treatment. In summary, the use of MEK inhibitors to alter the TME as an approach to enhance the activities of immune checkpoint inhibitors warrants further investigation in clinical trials.

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 270: Preclinical assessment of combination therapy with selumetinib and CTLA-4 for cancer

Immune mediated therapies for Cancer (IMT-C), such as anti-CTLA-4 and anti-PD-1/PD-L1 monoclonal antibodies (mAb), are showing promise in the treatment of solid tumors. However, although these treatments demonstrate clinical activity in a range of tumors, the durable benefit of these agents appears to be limited to a subset of patients. One hypothesis is that activity in these patients is limited by a lack of immune priming or by additional immunosuppressive pathways. Combination with molecular targeted therapies offers the potential to explore additional treatment strategies for patients. However, in order to select the best combination partners, a greater understanding is needed as to how each therapy affects the immune system both directly, through effects on leukocytes, and indirectly, through effects on tumor immunogenicity and the microenvironment. Selumetinib is a potent inhibitor of the mitogen-activated protein kinase (MAPK) kinase 1/2 (MEK1/2), involved in the activation of signaling pathways which regulates cellular growth, proliferation and survival. Given that these pathways are often found aberrantly activated in human tumors, and also play key roles in the regulation of immunological processes, it is difficult to predict the combinatorial effect of MEK1/2 inhibition and IMT in cancer treatment. To assess the effect of selumetinib in a tumor model with an intact immune system, we have characterized the impact of dosing selumetinib with or without anti-CTLA-4 mAb on the immune system in the CT26 syngeneic tumor model. Flow cytometric analysis showed that this combination reduced T-cell proliferation and memory T-cell generation, but increased the frequency of splenic T cells. Selumetinib treatment decreased PD-L1 expression and increased MHC class II expression on the tumor cells, whilst decreasing myeloid-derived suppressor cell (MDSC)-like cells in the tumor. Despite the apparent ability of selumetinib to have both positive and negative effects on immune responses in vivo, the combination of selumetinib and anti-CTLA4 mAb was found to show anti-tumor activity in the CT26 colorectal tumor mouse model. These studies highlight the potential for enhancing anti-tumor immune response through combinations of molecular targeted therapies and IMT-Cs. Citation Format: Stefanie R. Mullins, Edmund Poon, Amanda Watkins, Paul D. Smith, Andrew Leishman, Ross Stewart, Robert Wilkinson. Preclinical assessment of combination therapy with selumetinib and CTLA-4 for cancer. [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 270. doi:10.1158/1538-7445.AM2015-270

Immune mediated therapy and MEK inhibition: preclinical assessment of immunobiology and combination activity in vitro and in vivo

Immune mediated therapies (IMT), such as anti-CTLA-4 and anti-PD-1/PD-L1, are showing significant promise in the treatment of solid tumors. However, although these treatments can show significant overall benefit, a subset of patients fails to respond. It is believed that activity in these patients is limited by a lack of immune priming or by immunosuppression. Combination with molecular targeted therapies has the potential to overcome these hurdles to response and maximize patient benefit. In order to select the best combination partners, a greater understanding is needed of how other therapies affect the immune system both directly, through effects on leukocytes, and indirectly, through effects on tumor immunogenicity and induction of tumor cell death. Selumetinib (AZD6244, ARRY-142886) is a potent inhibitor of the mitogen-activated protein/extracellular signal-regulated kinase kinases 1/2 (MEK1/2), involved in the activation of signaling pathways which regulates cellular growth, proliferation and survival. Given that these pathways are often found aberrantly activated in human tumours and also play key roles in the regulation of immunological processes, it is difficult to predict the combinatorial effect of MEK1/2 inhibition and IMT. Here we detail a systematic approach taken towards examining the effects of MEK1/2 inhibition on tumour immunogenicity in vitro and in vivo and the direct effects it may have on the function of the immune system. We also present preclinical data demonstrating the anti-tumour activity of combining selumetinib and IMT mAbs in a syngeneic mouse model and report on possible mechanisms of synergism in promoting an anti-tumour immune response through promotion of antigen presentation.