Abstract
Introduction Inducible T cell co-stimulator (ICOS) is a co-stimulatory receptor that is important for promoting immune activation and function. Despite reported clinical activity and a wide ...range of non-clinical studies supporting a role for ICOS in lymphocyte activation, proliferation and pro-inflammatory cytokine secretion, little is known regarding the potential of monoclonal agonist antibody-mediated ICOS signaling to drive cytotoxic T cell infiltration into tumors. Feladilimab (GSK3359609) is a non-depleting IgG4 ICOS agonist antibody currently being evaluated in pivotal clinical trials. Here, we used PET/CT imaging to evaluate CD8+ T cell infiltration following treatment with a rodent surrogate of feladilimab (7E.17G9 mouse m IgG1) alone or in combination with anti-PD-1 mAb (RMP-14 rat IgG2a) in a syngeneic model of breast cancer (EMT6).
Method Female BALB/c mice with established tumors (~150 mm3) received 10µg, IP of either IgG control mAbs, ICOS mAb, or ICOS + PD-1 mAbs on day 0, 3, 5, 7, 9, 10, or 14. Imaging was performed at 24 & 48 hrs post IV dose of 89Zr labeled CD8 minibody (IAB42M1-14, ImaginAb, CA) on day 0, 3, 5, 9, or 14. In addition to the CD8 minibody uptake in tumor & tumor-draining lymph node (TDLN), 3D radiomic features were extracted from PET/CT images. Top ranked features were used for hierarchical clustering to identify treatment effects.
Results Tumor size regressed in all treated groups relative to IgG control, with a number of mice clearing tumors (ICOS: 4 mice, ICOS + PD-1: 9 mice). The in vivo uptake of CD8 minibody in TDLN was significantly higher in the ICOS + PD-1 group on day 4, 6, & 7 relative to IgG control P<0.05. The CD8 minibody uptake in tumor was significantly higher in the ICOS group on day 6, 11, & 16, and in the ICOS + PD-1 group on day 11 compared to IgG control P<0.05. Top ranked CT radiomic features were predictive for treatment effects at earlier days (day 3 - 5), while PET features were predictive at later days (6 - 10). Texture features in TDLN were consistently selected at earlier days and shape features in tumor were consistently selected in later days.
Conclusions Herein, we demonstrated for the first time that treatment of tumor-bearing mice with ICOS agonist mAb alone or in combination with PD-1 blockade can increase CD8+ T cell infiltration into tumors & TDLN, and is correlated with reduced tumor burden. Notably, radiomics features predicted an effect of treatment on CD8+ T cell infiltration earlier than the detection of absolute changes in the CD8 minibody uptake in tumor & TDLN. Overall, these data support the ongoing pivotal investigation of feladilimab. Moreover, this translational imaging method may be a useful tool to non-invasively monitor CD8+ T cell in response to immunotherapies and understand the temporal relationship between CD8+ T cell flux in tumor and in TDLN.
Citation Format: Hasan Alsaid, Shih-Hsun Cheng, Meixia Bi, Mary V. Rambo, Tinamarie Skedzielewski, Bao Hoang, Sunish Mohanan, Andrew Gehman, Chih-Yang Hsu, Minh Doan, Fang Xie, M. Reid Groseclose, Christopher Hopson, Sara Brett, Ian A. Wilson, Andrew Nicholls, Marc Ballas, Jeremy D. Waight, Beat M. Jucker, Axel Hoos. Immuno-PET monitoring of CD8+ T cell infiltration post anti-ICOS agonist antibody treatment alone and in combination with PD-1 blocking antibody using a 89Zr anti-CD8+ mouse minibody in EMT 6 syngeneic tumor mouse abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2816.
Gastrin is a known growth/differentiation factor for the gastric mucosa. Its effects are likely mediated by the induction of heparin-binding epidermal-like growth factor (HB-EGF), a member of the EGF ...family of growth factors that is expressed by gastric parietal cells. In this study, we investigated the regulation of the HB-EGF promoter by gastrin in a human gastric cancer cell line. Serial human HB-EGF promoter-luciferase reporter deletion constructs and heterologous promoter constructs were transfected into AGS-E cells and stimulated with gastrin (10-7 M) with or without various signal transduction inhibitors. EMSA were also performed. Gastrin stimulation resulted in a fivefold increase in HB-EGF-luciferase activity. The cis-acting element mediating gastrin responsiveness was mapped to the -69 to -58 region of the HB-EGF promoter. Gastrin stimulation was PKC dependent and at least partially mediated by activation of the EGF receptor. PUBLICATION ABSTRACT
Abstract
The purpose of these studies was to determine the effect of Ipilimumab treatment on T cell expansion, activation, cytokine production, tumor infiltration, and expression level of ...co-stimulatory and co-inhibitory receptors in a tumor bearing humanized NSG mouse model. Antibody therapies for immune modulation are proving to be effective in the oncology setting, as demonstrated by anti-CTLA-4 Ipilimumab and anti-PD-1 Pembrolizumab clinical activity. In order to assess efficacy of new therapies in the context of immune activation and tumor response, there is a need for suitable preclinical in vivo models. One approach to study immune cell function is to inject human peripheral blood mononuclear cells (PBMCs) into adult immunodeficient NSG (NOD/SCID/IL-2Rγnull) mice. This model, known as Hu-PBMC NSG, induces a Graft-versus-Host Disease (GvHD) state and has been used to study effector and memory T cell activity. Here we utilized the Hu-PBMC NSG model implanted with human cancer cell lines to investigate the effect of Ipilimumab on T cells and tumor growth in vivo. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee at GSK. The human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents. We confirmed human PBMC engraftment in NSG mice, validated tumor growth of human A2058 melanoma and 786-O renal adenocarcinoma cell lines, and dosed Ipilimumab in tumor bearing NSG mice with different human PBMC donors. Naïve NSG mice were intravenously injected with 20x106 human PBMCs and the kinetics of human cell engraftment was monitored weekly. For studies including Ipilimumab dosing, mice were inoculated with a subcutaneous injection of 2.5x106 A2058 or 1x106 786-O tumor cells. As tumor size reached 100 mm3, mice were randomized and injected with 20x106 human PBMCs. Two days later, mice were intraperitoneally treated with Ipilimumab or human IgG1 isotype control twice weekly for 6 doses total. Tumor growth and body weight was evaluated over time. Peripheral blood was collected weekly for analysis of T cell activation, receptor expression levels, and human cytokine production until mice developed GvHD or tumor volume reached 2,000 mm3. Select tumors were also harvested for tumor infiltrating lymphocyte (TIL) analysis by flow cytometry. Our studies showed NSG mice demonstrated similar human CD45+ cell engraftment in blood with various PBMC donors. The frequency of circulating human CD45+ lymphocytes in mouse peripheral blood increased to 50% or greater until week 4. 95% of the CD45+ cells were CD3+ T cells, containing both CD4+ and CD8+ subsets. Signs of GvHD were observed at week 4, and serum cytokine analysis showed high levels of GvHD markers e.g. IL5, IL10, and TNF-alpha. Ipilimumab treatment delayed tumor growth, increased the expansion of human CD45+ cells, and induced higher levels of TNF-alpha, IL-12p70, IL-13, and IL-5 cytokines compared to isotype control. Ipilimumab also increased the surface expression level of CD69, PD-1, OX40, ICOS, CD137, TIM3, and LAG3 on circulating T cells, and increased the number of A2058 tumor infiltrating lymphocytes. TIL analysis showed that compared to isotype control, Ipilimumab increased expression of CD69, PD-1, OX40, and ICOS on tumor infiltrating lymphocytes. In conclusion, Ipilimumab treatment increased T cell expansion, activation, expression of co-stimulatory and co-inhibitory receptors, and cytokine production in this tumor-bearing Hu-PBMC NSG model. It also increased the number of tumor infiltrating lymphocytes with a corresponding tumor growth delay. Based on Ipilimumab activity, this model can be utilized to assess pre-clinical efficacy of novel immunotherapies.
Citation Format: Meixia Bi, Chris Hopson, Tianqian Zhang, James Smothers, Axel Hoos. In vivo characterization of Ipilimumab T cell modulation and antitumor activity in a tumor bearing humanized NSG mouse model. abstract. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A054.
Abstract
Inducible T-cell costimulator (ICOS) is a costimulatory receptor that is upregulated on activated CD4 and CD8 T cells and plays an important role in T cell survival, differentiation, ...regulation of memory and regulatory T cell pools and humoral responses. Preclinically, augmenting signaling through the ICOS pathway has been reported to induce anti-tumor activity and enhance responses to CTLA4 blockade.
Here we present non-clinical data evaluating ICOS agonist antibody activity in human and mouse model systems using a different antibody for each species. GSK3359609 is a novel, selective anti-human ICOS agonist. GSK3359609 induces ICOS signaling through phosphorylation of intermediates in the Pi3K pathway leading to lymphocyte activation, proliferation and pro-inflammatory cytokine secretion in human PBMC in-vitro. A robust increase in CD4 effector T cell proliferation and Granzyme B secreting CD8 T cells was observed with GSK3359609 treatment in in-vitro assays utilizing PBMC from healthy donors, cancer patients or tumor infiltrating lymphocytes (TIL). Modest induction of regulatory T cell proliferation and IL-10 secretion were also observed. Significant increase in IFNγ (p<0.05) and TNFα secretion was observed in both primary PBMC and TIL based assays. Gene expression analysis of GSK3359609 treated human T cells confirmed changes in genes associated with T and B cell activation. In mice, an ICOS surrogate antibody was utilized in immune competent mouse tumor models. Tumor regressions were observed in 10-40% of mice and were associated with a robust increase in effector memory T cells in periphery as well as increases in T cell activation and proliferation in lymphoid tissues and tumor. Robust increases in PD1, PD-L1 and PD-L2 gene expression were observed in the tumors from ICOS antibody treated mice along with an increase in cytotoxic T cell signature and induction of an IFNγ gene signature. Changes in regulatory T cell proliferation were also observed in the blood and tumor of mice treated with the mouse ICOS agonist however changes were consistently less in magnitude than corresponding functional changes in cytotoxic CD8+ and effector CD4 cells.
We further explored treatment settings where a combination therapy may condition the tumor immune microenvironment to a more favorable context for ICOS agonist therapy. Treatment with an anti-PD1 antibody resulted in strong upregulation of ICOS expression on tumor infiltrating CD8, CD4 effector and regulatory T cells while decreasing ICOS+ Tregs relative to CD8 and CD4 effectors in the tumor microenvironment. Synergistic anti-tumor activity was observed for the combination of PD-1 with ICOS agonist antibodies in preclinical studies. These studies provide a strong rationale for the ongoing FTIH Phase I study of GSK3359609 administered alone and in combination with pembrolizumab to patients with selected advanced solid tumors.
Citation Format: Sapna Yadavilli, Tianqian Zhang, Ashleigh Hahn, Laura M. Seestaller-Wehr, Hong Shi, Yao-Bin Liu, M.Phillip DeYoung, David J. Kilian, Meixia Bi, Michael P. Adam, Shu-Yun Zhang, Sabyasachi Bhattacharya, Yuliya Katlinskaya, Christina Blackwell, Christopher B. Hopson, Niranjan Yanamandra, Roopa Srinivasan, Patrick A. Mayes, Axel Hoos. ICOS agonism induces potent immune activation and anti-tumor response in non-clinical models 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 1637. doi:10.1158/1538-7445.AM2017-1637
The presence and functional competence of intratumoral CD8
T cells is often a barometer for successful immunotherapeutic responses in cancer. Despite this understanding and the extensive number of ...clinical-stage immunotherapies focused on potentiation (co-stimulation) or rescue (checkpoint blockade) of CD8
T cell antitumor activity, dynamic biomarker strategies are often lacking. To help fill this gap, immuno-PET nuclear imaging has emerged as a powerful tool for in vivo molecular imaging of antibody targeting. Here, we took advantage of immuno-PET imaging using
Zr-IAB42M1-14, anti-mouse CD8 minibody, to characterize CD8
T-cell tumor infiltration dynamics following ICOS (inducible T-cell co-stimulator) agonist antibody treatment alone and in combination with PD-1 blocking antibody in a model of mammary carcinoma.
Female BALB/c mice with established EMT6 tumors received 10 µg, IP of either IgG control antibodies, ICOS agonist monotherapy, or ICOS/PD-1 combination therapy on days 0, 3, 5, 7, 9, 10, or 14. Imaging was performed at 24 and 48 h post IV dose of
Zr IAB42M1-14. In addition to
Zr-IAB42M1-14 uptake in tumor and tumor-draining lymph node (TDLN), 3D radiomic features were extracted from PET/CT images to identify treatment effects. Imaging mass cytometry (IMC) and immunohistochemistry (IHC) was performed at end of study.
Zr-IAB42M1-14 uptake in the tumor was observed by day 11 and was preceded by an increase in the TDLN as early as day 4. The spatial distribution of
Zr-IAB42M1-14 was more uniform in the drug treated vs. control tumors, which had spatially distinct tracer uptake in the periphery relative to the core of the tumor. IMC analysis showed an increased percentage of cytotoxic T cells in the ICOS monotherapy and ICOS/PD-1 combination group compared to IgG controls. Additionally, temporal radiomics analysis demonstrated early predictiveness of imaging features.
To our knowledge, this is the first detailed description of the use of a novel immune-PET imaging technique to assess the kinetics of CD8
T-cell infiltration into tumor and lymphoid tissues following ICOS agonist and PD-1 blocking antibody therapy. By demonstrating the capacity for increased spatial and temporal resolution of CD8
T-cell infiltration across tumors and lymphoid tissues, these observations underscore the widespread potential clinical utility of non-invasive PET imaging for T-cell-based immunotherapy in cancer.
In recent years, there has been considerable interest in mAb-based induction of costimulatory receptor signaling as an approach to combat cancer. However, promising nonclinical data have yet to ...translate to a meaningful clinical benefit. Inducible T-cell costimulator (ICOS) is a costimulatory receptor important for immune responses. Using a novel clinical-stage anti-ICOS immunoglobulin G4 mAb (feladilimab), which induces but does not deplete ICOS+ T cells and their rodent analogs, we provide an end-to-end evaluation of the antitumor potential of antibody-mediated ICOS costimulation alone and in combination with programmed cell death protein 1 (PD-1) blockade. We demonstrate, consistently, that ICOS is expressed in a range of cancers, and its induction can stimulate growth of antitumor reactive T cells. Furthermore, feladilimab, alone and with a PD-1 inhibitor, induced antitumor activity in mouse and humanized tumor models. In addition to nonclinical evaluation, we present three patient case studies from a first-time-in-human, phase I, open-label, dose-escalation and dose-expansion clinical trial (INDUCE-1; ClinicalTrials.gov: NCT02723955), evaluating feladilimab alone and in combination with pembrolizumab in patients with advanced solid tumors. Preliminary data showing clinical benefit in patients with cancer treated with feladilimab alone or in combination with pembrolizumab was reported previously; with example cases described here. Additional work is needed to further validate the translation to the clinic, which includes identifying select patient populations that will benefit from this therapeutic approach, and randomized data with survival endpoints to illustrate its potential, similar to that shown with CTLA-4 and PD-1 blocking antibodies.
Significance:
Stimulation of the T-cell activation marker ICOS with the anti-ICOS agonist mAb feladilimab, alone and in combination with PD-1 inhibition, induces antitumor activity across nonclinical models as well as select patients with advanced solid tumors.
Abstract
The explosion of research in the cancer immunotherapy field has led to a quest for better animal models to validate and develop various immunotherapies. Syngeneic mouse tumor models are one ...of the best options currently available to select cancers where clinical efficacy may be expected, to test mechanism of action hypotheses, in providing clues for possible biomarkers of immune pharmacodynamic (PD) activity and most successfully for predicting best partners for synergistic combinations. In order to select an appropriate model to evaluate a specific immunotherapy, it is essential to ensure sufficient understanding of the tumor model especially the immunogenicity and the characteristics of the immune infiltrates in the tumor microenvironment. It is also important to understand the dynamics of tumor growth and its effect on the immune landscape in the various peripheral tissues compartments during cancer progression.
We embarked on this study to systematically evaluate the dynamics and kinetics of phenotypic changes in immune cells in tumor, circulation and lymphoid organs with tumor progression in several syngeneic mouse tumor models. Pharmacodynamic evaluations of immune responses in all lymphoid compartments including the TME were made at various time points of tumor progression. Multicolor flow cytometry was performed on TILs, splenocytes and lysed whole blood to evaluate various T cells and the cell surface markers of activation, regulation and exhaustion. Additionally changes in cytokine levels in serum were also analyzed.
Our results show 1) significant changes in the tumor microenvironment (TME) with reference to the various types and functional status of immune cells in the various models and while tumor progression 2) Comparison between the different compartments within each model suggests significant changes in the co-stimulatory and co-inhibitory markers expressed on the TILs and their relative numbers and function in the periphery and other lymphoid compartments. 3) In addition, the dynamic changes observed with tumor progression in this study urge for a continuous assessment of these changes to obtain a complete kinetic picture rather than a snapshot when performing in-vivo biomarker studies in mice.
The information obtained from this study about the TIL and the expression of co-stimulatory/co-inhibitory molecules on these tumor models will enable us to employ a methodical approach when choosing a syngeneic model for efficacy and PD biomarker studies of immuno-modulatory agents under investigation.
All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed.
Citation Format: Sapna Yadavilli, Sabyasachi Bhattacharya, Ashleigh Hahn, Laura Seestaller-Wehr, David Kilian, Meixia Bi, Shu-Yun Zhang, Nicholas Vitali, Michael Adam, Yufeng Li, Niranjan Yanamandra, Roopa Srinivasan, Axel Hoos. Evaluating immune contexture in syngeneic mouse models of cancer. abstract. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A149.
Interleukin 12 (IL-12) is a potent and pleiotropic cytokine with applications in treatment of several infectious and malignant diseases. Clinical development of IL-12 as a recombinant protein was ...halted due to systemic toxicity resulting in patient deaths. The RTSTM (Rheoswitch Therapeutic System) represents a novel regulation system that allows control of gene expression using an orally bioavailable small molecule activator. In vitro transduction of the human fibrosarcoma cell line, HT1080, with an RTS-regulated adenoviral vector encoding human IL-12 (Ad-RTS-hIL12) at an MOI of 400 pfu, resulted in a >6000-fold induction of human IL-12 expression above background levels in the presence of the activator molecule, while no induction of IL-12 was detected when the activator molecule was omitted. Collectively, the results of these studies indicate that direct intratumoral injection of Ad-RTS-mIL12 results in anti-tumor activity and upregulation of IL-12-regulated immune mediators. These effects may provide a rationale for the use of intratumoral injection of Ad-RTS-IL-12 in patients with metastatic melanoma.
Interleukin-12 (IL-12) has been shown to be a key cytokine that enhances the ability of dendritic cells (DCs) to elicit anti-tumor T cells including TH1 cells and cytotoxic T lymphocytes (CTLs) as ...well as activation of natural killer (NK) cells. Komita et al Cancer Gene Therapy (2009) reported that gene therapy with intra-tumoral injection of syngeneic DCs transduced by an adenovirus vector, Ad-RTS-mIL-12, with the mIL-12 gene under the control of the Rheoswitch Therapeutic System(RTS)TM, led to therapeutic efficacy against B16 melanoma. To evaluate cell types transduced by direct adenovirus injection, adenovirus expressing green fluorescent protein (GFP) under a strong constitutive promoter was injected into established B16F0 melanoma tumors, and uptake of the vector after intratumoral injection was mainly in DCs and tumor cells. The analogous immunological mechanism of action with Ad-RTS-IL-12 in the mouse tumor model provides a good rationale for now translating the logistically simpler approach of direct injection of the Ad vector, compared to generation of the patient-specific transduced DC product, into accessible tumor lesions in combination with AL for clinical therapy.
Abstract
The Unfolded Protein Response (UPR) is a cellular homeostatic program initiated by an excess of unfolded/misfolded client proteins in the Endoplasmic Reticulum (ER) lumen, with a primarily ...cytoprotective effect. We have previously shown that tumor cell survival under hypoxic and nutrient deprivation stress is dependent on the ER resident protein and UPR effector PERK since the growth of PERK-null tumors is significantly inhibited in vivo. PERK deficient tumors are unable to tolerate ER stress induced by microenvironmental challenges such as hypoxia, and therefore, the tumors are unable to grow as ER-stress-induced apoptosis is induced. Similar findings have been confirmed for tumors lacking XBP1 (Romero-Ramirez et al). In addition to microenvironmental stresses such as hypoxia, oncogenes are known to activate cellular stress responses, including metabolic stress, apoptosis, DNA damage responses, and cellular senescence.
MYC is the target of chromosomal translocation or gene amplification during the development of many human cancers. Most notably, almost every case of Burkitt's lymphoma involves the translocation of the MYC proto-oncogene to the enhancer region of the heavy or light chain immunoglobulin loci resulting in the constitutive overexpression of c-Myc in B lymphocytes. c-Myc expression has been associated with a two-fold increase in both total cellular proteins and the rates of protein synthesis in the Eµ-myc mouse model. This finding raises the possibility that c-Myc-transformed cells are exposed to a higher than normal level of ER stress. Therefore, we hypothesized that the increase in protein burden in cells overexpressing c-Myc corresponds to increased ER stress and activation of the UPR.
Using multiple genetic models of regulated c-Myc and N-Myc activation, we demonstrate that Myc activates the PERK/eIF2α/Atf4 arm of the UPR. Activation of UPR leads to increased cell survival via the induction of cytoprotective autophagy as evidenced by upregulated LC3 processing, p62 degradation and increased double-membraned autophagosomes when analyzed by electron microscopy. Ablation of PERK significantly reduced Myc-induced autophagy, cell transformation and tumor formation in nude mice. Samples from Eμ-Myc mice and human lymphomas demonstrate higher levels of UPR activation (eIF2α phosphorylation and XBP1 splicing) compared to corresponding normal tissues and B-cells. Mechanistically, we genetically demonstrate an important link between Myc-dependent increases in protein synthesis and UPR activation. Specifically, by employing a mouse minute, which results in wild-type levels of protein synthesis and attenuation of Myc-induced lymphomagenesis, we show that Myc-induced UPR activation is reversed.
Our findings establish a novel role of UPR activation as an enhancer of c-Myc-induced transformation and suggest that inhibition of the UPR may be particularly effective against malignancies characterized by c-Myc overexpression. The identification of cellular stress response pathways activated by c-Myc represents an opportunity to exploit such pathways in the development of novel therapeutics targeting oncogene-dependent malignancies.
Supported by NCI grants CA94214 and CA 139362 to C.K.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY06-02. doi:10.1158/1538-7445.AM2011-SY06-02
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