Multiple Syngeneic Tumor Models Research Articles

Abstract 2156: IL-18 pathway agonism expands intratumoral PD1+ Tcf1+ stem-like CD8+ cells and their polyfunctional effector progeny to promote anti-tumor immunity

Abstract Recombinant cytokines were the first modern immunotherapies to produce durable cures in metastatic cancer, but their application has been hampered by only modest efficacy and limited tolerability. A major limitation to cytokine therapies is their lack of selectivity for the small fraction of lymphocytes that specifically recognize tumors. To identify cytokine pathways with greater selectively for anti-tumor lymphocytes, we analyzed single-cell RNA-seq expression data and flow cytometric profiles of tumor-infiltrating lymphocytes (TILs). Among detectable cytokine pathways, Interleukin-18 (IL-18) and its receptors IL-18R1 and IL-18RAP were markedly upregulated in activated and dysfunctional TILs, suggesting the potential of IL-18R agonism to promote anti-tumor immunity. However, recombinant IL-18 therapy has consistently failed to demonstrate anti-tumor efficacy in clinical trials. We hypothesized this may be due to the upregulation of an ultra-high affinity inhibitory decoy receptor of IL-18, IL-18BP. Indeed, IL-18BP is highly expressed in the tumor microenvironment, which we found by immunohistochemical staining and qPCR analysis of IL-18BP levels in several human cancers and syngeneic murine tumor models. Using directed evolution with yeast-surface display, we engineered “decoy-resistant” IL-18 (DR-18), a variant of IL-18 that maintains signaling potential but is impervious to binding and inhibition by IL-18BP. In multiple syngeneic tumor models including YUMMER1.7, MC38, and CT26, we found that recombinant WT IL-18 was ineffective by itself or in combination with anti-PD-1 treatment, similar to the clinical experience of rIL-18 in patients. However, in the same models, DR-18 showed robust monoagent activity that was commensurate or superior to anti-PD-1 antibodies, and it produced added therapeutic synergism when combined with anti-PD-1. Single-cell RNA-seq profiling and flow cytometry analysis of DR-18 treated tumors indicated that DR-18 treatment drives the development of poly-functional effector CD8+ T cells and NK cells. Importantly, DR-18 expands the pool of intratumoral Tcf1+ memory precursor PD1+ CD8+ T cells and decreases the prevalence of exhausted CD8+ T cells that express TOX. Mechanistically, we demonstrate that tumor antigen specific CD8+ T cells were sufficient to mediate tumor regression using LCMV epitope GP33 specific P14 CD8+ T cells in a B16-GP33 model. Together, these results highlight DR-18 is able to remodel the tumor-immune landscape to bias immune cells towards potent anti-tumor effector cells and away from the T exhausted lineage. Therefore, we conclude that the IL-18 pathway is a powerful target for immunotherapeutic intervention and implicate the secreted checkpoint IL-18BP as an obstacle to effective IL-18 immunotherapy. Citation Format: Ting Zhou, Orr-El Weizman, William Damsky, Meaghan McGeary, Marcus W. Bosenberg, Aaron M. Ring. IL-18 pathway agonism expands intratumoral PD1+ Tcf1+ stem-like CD8+ cells and their polyfunctional effector progeny to promote anti-tumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2156.

Abstract C082: AGX-73, a novel small molecule multi-immunokinase inhibitor, has robust antitumor activity in murine syngeneic tumor models as a single agent and in combination with anti-PD1 checkpoint inhibitor

Abstract One of the central hypotheses of modern targeted cancer treatment paradigm is that aberrant activation and/or over-expression of oncogenic kinases drive intrinsic cancer cell proliferation and survival and is the causative event in cancer initiation and progression. The numerous examples of effective cancer therapies targeting specific kinases are testaments to its validity. In recent years, the demonstrations of profound and broad antitumor responses to immune checkpoint inhibitors, such as anti-PD1/PD-L1 or anti-CTLA-4, have confirmed and emphasized the crucial role immune evasion plays in creating a permissive condition for disease progression. Because protein kinases are known to regulate the majority of cellular functions, we hypothesize that Just as cancer cells frequently usurp the kinase signaling machinery to promote uncontrolled proliferation and survival, they may similarly hijack key immunokinase signaling machinery to suppress immune detection and elimination. We prioritized 6 key kinases that play profound roles in sustaining immunosuppressive cells in the tumor microenvironment (AXL, DDR1, DDR2, FMS, KIT and MER) and design inhibitors that potently, selectively and collectively counteract their action. AGX-73, optimized for drug-like properties, is a small molecule compound currently undergoing IND evaluation as potential clinical candidate for first-in-human trial. In in vitro assay, carried out at high ATP concentration (100 μM), AGX-73 inhibited the activity of recombinant human AXL, DDR1, DDR2, FMS, KIT and MER with IC50 values of 30, 12, 0.63, 96, 54, and 40 nM, respectively, but was highly selective against a panel of 374 kinases. Importantly, in in vitro primary macrophage assays, AGX-73 promoted M1 macrophage polarization and increased the secretion of IL-6 and TNFα. In contrast, AGX-73 suppressed the activation of M2 macrophages and decreased the secretion of IL-10. In vivo, in the MC38 colon carcinoma model, AGX-73 treatment reduced the number of tumor-associated macrophages (TAMs), from 46 to 22% of the total CD45+ cells in the tumors. Moreover, the M1/M2 ratio was favorably increased, from 1.3 to 1.8. AGX-73 demonstrated robust antitumor activity in multiple syngeneic tumor models. In the MC38 model, AGX-73 administered orally was active at multiple dose levels, from 30-150 mg/kg, QDx14, yielding %TGI that ranged from 55-92%. Importantly, AGX-73 treatment produced greater antitumor effects in immunocompetent mice than in immunodeficient mice (88 vs. 65% TGI at 90 mg/kg), suggesting the involvement of a functional immune system in its optimal antitumor activity. Of the 13 syngeneic tumor models tested in immunocompetent mice (150 mg/kg, QDx14), eleven were sensitive with TGI>50% (range 45-122%). In combination with anti-PD1 (10 mg/kg) in the MC38 model, AGX-73 produced synergistic activity at multiple dose levels evaluated when compared with either single agent alone. In nonclinical ADME studies, AGX-73 demonstrated favorable drug-like properties: including good bioavailability (F>50% in mouse, rat and monkey) and systemic exposure following oral administration in multiple species, acceptable metabolic stability, lack of significant drug-drug interaction liabilities (transporters, CYP inhibition, CYP induction). IND toxicologic studies are currently in progress. Citation Format: Francis Y.F. Lee, Wen-Lian Wu, Zhi-Qiang Yang, John Tan. AGX-73, a novel small molecule multi-immunokinase inhibitor, has robust antitumor activity in murine syngeneic tumor models as a single agent and in combination with anti-PD1 checkpoint inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C082. doi:10.1158/1535-7163.TARG-19-C082

Abstract LB-063: Validation of humanized PD-1 knock-in mice as an emerging model to evaluate human specific PD-1 therapeutics

Abstract Over the past decade there has been an increasing demand in the use of syngeneic models for evaluating the efficacy of checkpoint inhibition-based cancer immunotherapies. During tumor development, immune cells can became unresponsive to the presence of tumor cells due to chronic activation and expression of the programmed cell death protein-1 (PD-1) or the T-lymphocyte-associated antigen 4 (CTLA4) in T-cells or the presence of FoxP3+ Treg cells resulting in tumor immune-tolerance. Our previous studies have demonstrated that murine anti-PD-1 and CTLA-4 therapy can effectively re-activate the antitumor response against multiple syngeneic tumor models. While these models proved instrumental for evaluating murine immune-checkpoint inhibitors (ICI), there is a clear need for additional mouse models to evaluate the efficacy of ICI specific for human targets. To address this need, we describe the development of a humanized PD-1 knock-in (KI) mouse model. This mouse model has the advantage of expressing the human PD-1 protein in the context of a fully functional immune system. We also show the response to clinically relevant immune checkpoint inhibitors in two preclinical tumor models. We evaluated the anti-tumor activity of pembrolizumab in the MC38 colorectal carcinoma and the GL261 glioblastoma models. We observed significant tumor growth inhibition and growth delay in the MC38 tumor model when treated with pembrolizumab monotherapy, but not when treated with the murine counterpart (anti-PD-1 clone RPM1-14). To extend our validation studies to other tumor models, we implanted GL261 glioblastoma orthotopically in the brain of PD-1 KI mice and achieved a significant increased life span in the group treated with pembrolizumab compared to both the control group and the group treated with murine anti-PD-1 antibody. The immune profile from control and treated these animals were also characterized in these studies by flow cytometry. In summary, the results shown here underscore the value of the humanized PD-1 knock-in (KI) mouse model as a tool to evaluate human specific immune-checkpoint based therapeutics alone and in combination with other agents. Citation Format: Anya Avrutskaya, Fabiane Sonego, Jacob Hauser, Emily O’Koren, Gaëlle Martin, Julie Chaix, Robin Ball, Thi Bui, Ian Belle, Chassidy Hall, Kader Thiam, Paula L. Miliani De Marval. Validation of humanized PD-1 knock-in mice as an emerging model to evaluate human specific PD-1 therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-063.

Abstract 3206: Host immunity following near infrared photoimmunotherapy is enhanced with PD-1 checkpoint blockade to eradicate established highly antigenic tumors

Abstract Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that employs a targeted monoclonal antibody-photo-absorber conjugate (APC). Following antibody localization of the APC to a tumor cell surface antigen, NIR light is used to induce highly selective cytolysis. Extensive pre-clinical evidence demonstrates that NIR-PIT is effective at inducing tumor cell lysis using a number of different antibody-APC conjugates and NIR-PIT targeting EGFR with a cetuximab-APC conjugate has shown promising results in phase 2 clinical evaluation for the treatment of recurrent head and neck squamous cell carcinoma. NIR-PIT induces rapid, necrotic cell death that yields innate immune ligands that activate dendritic cells, consistent with immunogenic cell death. Yet, NIR-PIT treatment of syngeneic tumors in wild-type mice has mostly failed to induce durable regression of established tumors, suggesting the presence of one or more mechanisms of resistance to formation of meaningful anti-tumor immunity. In this study, we hypothesized that NIR-PIT could induce anti-tumor immunity that was being restricted by the PD-1/PD-L1 signaling axis, and that PD-1 could reverse innate immune resistance to induce durable, effective anti-tumor immune responses. Using a CD44-targeting APC, we demonstrated the ability of PD-1 immune checkpoint blockade to significantly enhance antigen-specific anti-tumor immunity induced by NIR-PIT in multiple syngeneic tumor models (MC38, LLC, and MOC1 cancer cell line). In two of three models, NIR-PIT monotherapy halted tumor growth, enhanced dendritic cell tumor infiltration, and induced de novo tumor antigen-specific T-cell responses absent at baseline. The addition of PD-1 blockade appeared to reverse adaptive immune resistance, resulting in both enhanced pre-existing tumor antigen-specific T-cell responses and enhanced de novo T-cell responses induced by NIR-PIT. Enhanced immune responses appeared to correlate with shared tumor antigen expression, suggesting that antigenicity is a major determinant of response to combination NIR-PIT and PD-1 blockade. Combination treatment induced complete rejection of MC38 tumors treated with NIR-PIT as well as untreated, distant tumors. Accordingly, tumor antigen-specific T-cell responses were measured in both treated and untreated tumors, validating the development of systemic anti-tumor immunity. Cured mice resisted tumor challenge, indicating the presence of systemic immune memory. Cumulatively these results demonstrate reversal of adaptive immune resistance following induction of innate and adaptive immunity by NIR-PIT, resulting in high rates of tumor rejection and/or significant tumor growth control in highly antigenic syngeneic models of cancer. Citation Format: Tadanobu Nagaya, Jay Friedman, Yasuhiro Maruoka, Fusa Ogata, Shuhei Okuyama, Paul E. Clavijo, Peter L. Choyke, Clint Allen, Hisataka Kobayashi. Host immunity following near infrared photoimmunotherapy is enhanced with PD-1 checkpoint blockade to eradicate established highly antigenic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3206.

Immuno-oncological Efficacy of RXDX-106, a Novel TAM (TYRO3, AXL, MER) Family Small-Molecule Kinase Inhibitor

Expression of the TAM (TYRO3, AXL, MER) family of receptor tyrosine kinases (RTK) has been associated with cancer progression, metastasis, and drug resistance. In immune cells, TAM RTKs can dampen inflammation in favor of homeostatic wound-healing responses, thus potentially contributing to the evasion of cancer cells from immune surveillance. Here we characterize the small-molecule RXDX-106 as a selective and potent pan-TAM RTK inhibitor with slow dissociation kinetics and significant antitumor activity in multiple syngeneic tumor models. Expression of AXL and MER on both immune and tumor cells increased during tumor progression. Tumor growth inhibition (TGI) following treatment with RXDX-106 was observed in wild-type mice and was abrogated in immunodeficient mice, suggesting that the antitumor activity of RXDX-106 is, in part, due to the presence of immune cells. RXDX-106-mediated TGI was associated with increased tumor-infiltrating leukocytes, M1-polarized intratumoral macrophages, and activation of natural killer cells. RXDX-106 proportionally increased intratumoral CD8+ T cells and T-cell function as indicated by both IFNγ production and LCK phosphorylation (pY393). RXDX-106 exhibited its effects via direct actions on TAM RTKs expressed on intratumoral macrophages and dendritic cells, leading to indirect activation of other immune cells in the tumor. RXDX-106 also potentiated the effects of an immune checkpoint inhibitor, α-PD-1 Ab, resulting in enhanced antitumor efficacy and survival. Collectively, these results demonstrate the capacity of RXDX-106 to inhibit tumor growth and progression and suggest it may serve as an effective therapy against multiple tumor types. SIGNIFICANCE: The pan-TAM small-molecule kinase inhibitor RXDX-106 activates both innate and adaptive immunity to inhibit tumor growth and progression, indicating its clinical potential to treat a wide variety of cancers.

Host Immunity Following Near-Infrared Photoimmunotherapy Is Enhanced with PD-1 Checkpoint Blockade to Eradicate Established Antigenic Tumors.

Near-infrared photoimmunotherapy (NIR-PIT) induces immunogenic cell death but has mostly failed to induce durable antitumor responses in syngenic tumor mouse models. We hypothesized that adaptive immune resistance could be limiting durable responses after treatmemt with NIR-PIT. We investigated the effects of combining NIR-PIT targeting cell-surface CD44 and PD-1 blockade in multiple syngeneic tumor models. In two of three models, NIR-PIT monotherapy halted tumor growth, enhanced dendritic cell tumor infiltration, and induced de novo tumor antigen-specific T-cell responses absent at baseline. The addition of PD-1 blockade reversed adaptive immune resistance, resulting in both enhanced preexisting tumor antigen-specific T-cell responses and enhanced de novo T-cell responses induced by NIR-PIT. Enhanced immune responses correlated with shared tumor antigen expression, suggesting that antigenicity is a major determinant of response to combination NIR-PIT and PD-1 blockade. Combination treatment induced complete rejection of MC38 tumors treated with NIR-PIT, as well as untreated, distant tumors. Accordingly, tumor antigen-specific T-cell responses were measured in both treated and untreated tumors, validating the development of systemic antitumor immunity. Mice that cleared tumors resisted subsequent tumor challenge, indicating the presence of systemic immune memory. Cumulatively, these results demonstrate reversal of adaptive immune resistance following induction of innate and adaptive immunity by NIR-PIT, resulting in high rates of tumor rejection and/or significant tumor growth control in antigenic syngeneic models of cancer.

Order of administration of combination cytokine therapies can decouple toxicity from efficacy in syngeneic mouse tumor models

ABSTRACTIn combination cancer immunotherapies, consideration should be given to designing treatment schedules that harmonize with the immune system’s natural timing. An efficacious temporally programmed combination therapy of extended half-life interleukin 2 (eIL2), tumor targeting antibody, and interferon (IFN) α was recently reported; however, tumor-ablative efficacy was associated with significant toxicity. In the current work, altering the order and timing of the three agents is shown to decouple toxicity from efficacy. Delaying the administration of eIL2 to be concurrent with or after IFNα eliminates toxicity without affecting efficacy in multiple syngeneic tumor models and mouse strains. The toxicity resulting from eIL2 administration before IFNα is dependent on multiple systemic inflammatory cytokines including IL6, IL10, IFNγ, and tumor necrosis factor α. Natural killer (NK) cells are the main cellular contributor to toxicity, but are not essential for tumor control in this system. When pre-conditioned with eIL2, splenic NK cells became hyper-activated and upregulate IFNα signaling proteins that cause an excessive, toxic response to subsequent IFNα exposure. This work illustrates an example where accounting for the temporal dynamics of the immune system in combination therapy treatment schedule can favorably decouple efficacy and toxicity.

Abstract 1751: MSC-1 is a first-in-class humanized monoclonal antibody that modulates the tumor microenvironment by inhibiting a novel cancer immunotherapy target, LIF

Abstract Leukemia Inhibitory Factor (LIF) is a member of the IL-6 family of cytokines and is involved in many physiological and pathological processes including the promotion of an immunosuppressive environment to support embryo implantation, down-regulation of autoimmune processes and the regulation of stem cell homeostasis and differentiation. In cancer, LIF is hypothesized to have a complex role tumor development and progression, creating an immunosuppressive tumor microenvironment as well as promoting the activity of cancer initiating cells (CICs). LIF is highly expressed in a subset of tumors across multiple tumor types, e.g. glioblastoma multiforme (GBM), non-small cell lung cancer (NSCLC), colon, ovarian, prostate, and pancreatic cancer, and correlates with poor prognosis. Given the pleiotropic role LIF is hypothesized to play in cancer, inhibition of LIF represents an exciting new therapeutic concept sitting at the intersection of two key therapeutic approaches in oncology: blockade of tumor evasion of the immune system and blockade of tumor growth via inhibition of CICs. We have identified and developed MSC-1, a first-in-class humanized IgG1 monoclonal antibody that is a potent and selective inhibitor of LIF. MSC-1 cross reacts with mouse and non-human primate LIF and inhibits LIF signaling by blocking the recruitment of gp130 to the LIF-LIFR-gp130 signaling complex. The efficacy of MSC-1 was evaluated in multiple mouse tumor models and the mechanism of action investigated. LIF inhibition with MSC-1 or shRNAs reduced tumor growth in multiple syngeneic tumor models (NSCLC, ovarian and colon), and clear target engagement was shown for MSC-1. Investigations into the mechanism of action identified that inhibition of LIF with MSC-1 reprogrammed the tumor microenvironment by decreasing immunosuppressive M2 macrophages and increasing the number of intratumoral NK cells and total/activated T cells. MSC-1 also decreased immunosuppressive M2 macrophages in an orthotopic GBM xenograft model and human GBM organotypic tumor slices in an ex vivo model. Similarly, immunosuppressive macrophage genes were decreased when monocytes were co-cultured with supernatants from a GBM cell line in which LIF expression had been knocked-down. Given the effects of MSC-1 on intratumoral immune cells, we hypothesized that MSC-1 could be effectively combined with checkpoint inhibitors and we are currently evaluating MSC-1/checkpoint inhibitor combination therapy. Taken together, these findings form the basis of a robust therapeutic hypothesis, whereby MSC-1 treatment will lead to clinical activity in multiple cancer indications. A Phase I dose-escalation and expansion study of MSC-1 is planned to initiate early 2018 in patients with advanced solid tumors that will incorporate target engagement and PD biomarkers, as well as safety and efficacy endpoints. Citation Format: Angus Sinclair, Robin Hallett, Patricia Giblin, Isabel Huber-Ruano, Judit Anido, Naimish Pandya, Kimberly Hoffman, Ada Sala, Monica Pascual, Vanessa Chigancas, Swetha Raman, Johan Fransson, Jean-Philippe Julien, Robert Wasserman, Jeanne Magram, Joan Seoane. MSC-1 is a first-in-class humanized monoclonal antibody that modulates the tumor microenvironment by inhibiting a novel cancer immunotherapy target, LIF [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1751.

Abstract 3771: Mechanistic and pharmacodynamic characterization of the immuno-oncological activity of RXDX-106, a novel TYRO3, AXL, and MER (TAM) inhibitor in clinical development

Abstract The TAM family of receptor tyrosine kinases (RTKs) includes TYRO3, AXL, and MER, and is a unique set of molecular targets for cancer immunotherapy. TAM RTKs play a key role in the tumor microenvironment (TME) as negative regulators of innate immune responses and contribute to the evasion of cancer cells from immunosurveillance. RXDX-106 is a novel small molecule TAM inhibitor with single-digit nanomolar kinase inhibition and slow dissociation binding kinetics resulting in potent and durable target inhibition. In preclinical studies, RXDX-106 has demonstrated immune-mediated single agent in vivo activity in multiple syngeneic tumor models and potentiated anti-tumor activity in combination with immune checkpoint inhibitors. To characterize the mechanism of action of and pharmacodynamic responses to RXDX-106 treatment, an integrated approach was employed to explore RXDX-106-mediated immune modulatory effects at the cellular, protein, and transcriptional levels. In syngeneic tumor models, RXDX-106 treatment resulted in the recruitment of both innate and adaptive immune cell subtypes to the TME. Specifically, RXDX-106 treatment resulted in increased immune cell infiltration, macrophage polarization towards an antitumor M1 phenotype, activation of T cell and NK cell function, and modulation of immune markers at both the protein and RNA level, indicative of a pro-inflammatory and anti-tumorigenic effect. Importantly, the immune modulatory effects of RXDX-106 were concordant across platforms. Immune modulation was further characterized in a dose-dependent manner, with increasing immune cell infiltration observed with escalating doses of RXDX-106. In summary, RXDX-106 has the potential to restore and enhance immune function by modulating the local immunosuppressive TME. The unique mechanism of activating both innate and adaptive immunity, and regulating cross-talk between immune cells and tumor cells, supports clinical evaluation of RXDX-106 as an immunomodulatory agent for the treatment of a variety of cancers. Citation Format: Amanda Albert, Erin D. Lew, Kristen Smith, Elizabeth A. Tindall, Yumi Yokoyama, Amy Diliberto, Heather Ely, Jack Lee, Robin Nevarez, Joanne Oh, Colin Walsh, Jason Christiansen, Gary Li, Robert Shoemaker. Mechanistic and pharmacodynamic characterization of the immuno-oncological activity of RXDX-106, a novel TYRO3, AXL, and MER (TAM) inhibitor in clinical development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3771.

Abstract 1758: Intratumoral IL-12 mRNA promotes cytotoxic T cell-dependent anti-tumor immunity and enhances responses to PD-L1 blockade in pre-clinical models

Abstract As a central mediator of TH1 immune responses, interleukin 12 (IL-12) in the tumor microenvironment (TME) plays a key role in driving anti-tumor immunity. Recombinant IL-12 has been reported to promote CD8+ cytotoxic T cell (CTL) dependent anti-tumor immunity in a wide variety of preclinical models. However, systemic recombinant IL-12 was poorly tolerated in early clinical trials. We are therefore investigating whether intra-tumoral (IT) delivery of a messenger RNA encoding IL-12 formulated in lipid nanoparticles can drive local IL-12 expression in solid tumors to induce TME transformation and anti-tumor immunity. Here we report that IT IL-12 mRNA induces dose-dependent local IL-12 protein expression in mice bearing both subcutaneous syngeneic and patient-derived xenograft tumors. In multiple syngeneic tumor models (e.g. MC38, A20), mouse IL-12 mRNA (mIL-12 mRNA) treatment promoted dose-dependent tumor regression and significantly improved overall survival. Flow cytometry and luminex analysis revealed that anti-tumor activity of mIL-12 mRNA was accompanied by transformation of the TME characteristic of a TH1 immune response. IFNγ induction and natural killer cell activation was accompanied by antigen presenting cell maturation, increased CTL numbers and activation, and increased PD-L1 expression. Furthermore, rejection of established MC38 tumors in response to mIL-12 mRNA was CTL-dependent, and animals demonstrated immunity to subsequent re-challenge with MC38 tumor cells. Since mIL-12 mRNA enhanced PD-L1 expression in the TME, we next investigated whether PD-L1 blockade would enhance responses to mIL-12 mRNA. We found that the combination of a single dose of mIL-12 mRNA and PD-L1 blockade led to significantly more complete responses compared to mIL-12 mRNA monotherapy in the PD-L1 resistant MC38 model. Furthermore, A single dose of mIL-12 mRNA induced rejection of un-injected MC38 tumors in a dual flank model, and this was further enhanced by combination with PD-L1 blockade. These preclinical data demonstrate the potential for IT IL-12 mRNA to drive TME transformation into a proinflammatory state consistent with a TH1 immune response and to ignite CTL-dependent anti-tumor immunity, even in a PD-L1 resistant setting. Citation Format: Susannah L. Hewitt, Nadia Luheshi, Dyane Bailey, John Zielinski, Ameya Apte, Faith Musenge, Russell Karp, Grace Adjei, Andrew Leinster, Sushma Gurumurthy, Ankita Mishra, Kristen Arnold, Darren Potz, Robert W. Wilkinson, Ronald Herbst, Joshua P. Frederick. Intratumoral IL-12 mRNA promotes cytotoxic T cell-dependent anti-tumor immunity and enhances responses to PD-L1 blockade in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1758.

Abstract 4614: Upregulation of memory T cell population and enhancement of Th1 response by lenvatinib potentiate antitumor activity of PD-1 signaling blockade

Abstract Introduction: Lenvatinib (LEN) selectively inhibits the kinase activity of VEGFR1-3, FGFR1-4, KIT, PDGFRα, and RET, which are involved in tumor angiogenesis and tumor cell proliferation in several cancer types. Currently, Phase 1b/2 clinical trials of the combination of LEN and pembrolizumab (a monoclonal antibody [mAb] that blocks the interaction between PD-1 and its ligands) are ongoing for selected types of cancer including renal cell carcinoma, melanoma and non-small cell lung carcinoma. We have reported that tumor associated macrophage and regulatory T cell population were downregulated by treatment of LEN. In this study, mechanism of LEN and PD-1 mAb combination was investigated by flow cytometory and RNA-seq analyses. Experimental Procedure: We examined antitumor activity of combination treatment of LEN (10mg/kg, qd) and anti-mouse PD-1 mAb (500μg/mouse, twice weekly) in Ct26 and B16F10 syngeneic mouse models. In order to investigate tumor-specific memory T cell response, re-inoculation of fresh CT26 cells into tumor-free mice was conducted. For immune cell population analyses, tumor or spleen samples were analyzed by flow cytometry. Changes of molecular immune response in tumors were examined by RNAseq followed by weighted gene co-expression network analysis (WGCNA). Summary of data: Combination of LEN with PD-1 mAb showed more potent anti-tumor growth activity in CT26 and B16F10 models compared with either treatment alone. Notably, complete tumor regression was detected in a portion of mice that received combination treatment only. Re-inoculation of fresh CT26 cells into these tumor-free mice did not grow tumor; in contrast, all naïve animals grew tumor aggressively in the same experimental condition. RNAseq analysis of treated CT26 tumors indicated that combination of LEN with PD-1 mAb synergistically up-regulates IFN signaling-related genes. Flow cytometory analysis revealed that LEN increased memory T cell population compared with vehicle treatment. Conclusions: The results indicate that the combination of LEN with PD-1 mAb was more effective than either single agent alone in multiple syngeneic tumor models and was accompanied with a potent antitumor immune response, especially the up-regulation of memory T cell and Th1 activation by LEN. Citation Format: Yu Kato. Upregulation of memory T cell population and enhancement of Th1 response by lenvatinib potentiate antitumor activity of PD-1 signaling blockade [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 4614. doi:10.1158/1538-7445.AM2017-4614

Abstract 2615: Preclinical evaluation and mechanistic characterization of M7824 (MSB0011359C), a novel bifunctional fusion protein targeting the PD-L1 and TGFβ pathways

Abstract Dual targeting of the programmed death ligand 1 (PD-L1) and transforming growth factor β (TGFβ) pathways, 2 key mediators of tumor immune evasion, may provide synergistic antitumor activity. The novel bifunctional fusion protein M7824 (MSB0011359C) is a fully human IgG1 monoclonal antibody against PD-L1, fused via a glycine-serine linker at the CH3-C terminus to the extracellular domain of human TGFβ receptor II, which functions as a TGFβ “trap.” Thus, M7824 is designed to exert independent and complementary anti-immunosuppressive functions by simultaneously blocking the PD-L1 and TGFβ pathways. The purpose of this study was to evaluate M7824 preclinically. M7824 was efficacious in multiple syngeneic tumor models (EMT-6, MC38, PANC02, 4T1, CT26, and GL261). In orthotopic EMT-6 and intramuscular MC38 models, M7824 showed superior additive antitumor effects compared to anti-PD-L1 antibody or TGFβ trap monotherapy. M7824 also inhibited spontaneous metastases in EMT-6 and 4T1 models. Furthermore, M7824 extended survival and conferred long-term protective antitumor immunity, as demonstrated by protection of cured mice against tumor cell rechallenge. Mechanistically, the dual anti-immunosuppressive activity of M7824 in the MC38 model resulted in unique activation of both the adaptive and innate immune systems, including increased tumor infiltration, proliferation, and cytotoxicity of CD8+ T cells; increased prevalence of splenic tumor antigen-specific CD8+ T cells; increased CD8+ TEM infiltration and CD8+ TEM/naïve ratio; increased tumor-infiltrated natural killer (NK) cells; increased ratio of M1/M2 macrophages; and decreased infiltration of tumor-associated neutrophils and myeloid-derived suppressor cells. Similar immunophenotypic changes were observed in the EMT-6 model. Activation of both the adaptive and innate immune systems contributed to the antitumor activity of M7824 in the MC38 model, as depletion of CD8+ T cells and NK cells led to a substantial decrease in M7824-mediated tumor regression; CD4+ T cells and antibody-dependent cellular cytotoxicity, however, appeared dispensable. Toxicologic evaluation in cynomolgus monkeys indicated M7824 was well tolerated, with only minor hematologic effects observed. Although M7824 is a bifunctional molecule, its antitumor activity can be further improved by combination with standard-of-care therapies: in the MC38 model, combining M7824 with radiotherapy or chemotherapy resulted in more effective inhibition of tumor growth and an increased frequency of tumor antigen-specific IFN-γ-producing CD8+ T cells vs either monotherapy. Collectively, our preclinical data suggest that M7824 elicits potent and synergistic antitumor activity via the simultaneous blockade of the PD-L1 and TGFβ pathways. Phase 1 clinical trials with M7824 are under way in patients with solid tumors. Citation Format: Yan Lan, Dong Zhang, Chunxiao Xu, Bo Marelli, Jin Qi, Huakui Qi, Guozhong Qin, Xiaomei Xu, Hong Wang, Aroop Sircar, Beatrice Brunkhorst, Eric Austin, Laszlo Radvanyi, Kin-Ming Lo. Preclinical evaluation and mechanistic characterization of M7824 (MSB0011359C), a novel bifunctional fusion protein targeting the PD-L1 and TGFβ pathways [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 2615. doi:10.1158/1538-7445.AM2017-2615

Abstract 5593: Inhibition of A2AR induces anti-tumor immunity alone and in combination with anti-PD-L1 in preclinical and clinical studies

Abstract Adenosine signaling via A2A receptor (A2AR) on immune cells suppresses anti-tumor immunity and limits the efficacy of immunotherapy, chemotherapy, CAR-T, and vaccines. CPI-444 is a potent and selective oral A2AR antagonist. Daily treatment of mice with CPI-444 led to dose-dependent inhibition of tumor growth in multiple syngeneic tumor models. Combining CPI-444 with anti-PD-L1 treatment synergistically eliminated tumors in up to 90% of treated mice, including restoration of immune responses in models that are poorly responsive to anti-PD-1 or anti-PD-L1 monotherapy. We have initiated a Phase 1/1b clinical trial to examine safety, tolerability, biomarkers, and preliminary efficacy of CPI-444 as a single agent and in combination with the anti-PD-L1 antibody, atezolizumab, in patients with non-small cell lung, melanoma, renal, triple negative breast, and other (bladder, prostate, head and neck, colorectal) tumors. Step 1 of the trial focused on determining the optimal dose and schedule for CPI-444; Step 2 is currently evaluating the efficacy of optimal CPI-444 dosing alone and with atezolizumab. In 48 patients treated in Step 1, CPI-444 was well tolerated with 1 Grade 3 or 4 treatment related adverse events. Preliminary evidence of clinical activity was observed in patients treated with single agent CPI-444, including patients who previously failed anti-PD-1 therapy. A pCREB-based pharmacodyamic assay showed that 100 mg, BID dose of CPI-444 resulted in a complete, sustained inhibition of A2AR on circulating immune cells. CPI-444 treatment alone or in combination with atezolizumab resulted in increased frequency of circulating CD8+PD-1+ cells and memory/effector subsets of CD4+ and CD8+ T cells. Substantial changes in TCR repertoire (Morisita Index <0.9) were observed in both anti-PD-1 native and refractory patients and correlated to patient responses. CD73 expression, CD8+ infiltration, TREG distribution, and gene expression signatures are currently being evaluated in serial tumor biopsy specimens and will be presented. In total, this shows that CPI-444 is well tolerated in cancer patients, exhibits functional inhibition of adenosine signaling, and treatment is associated with activation of anti-tumor immunity and clinical activity. Citation Format: Stephen Willingham, Andrew Hotson, Po Ho, Carmen Choy, Kim Walter, Erik Yusko, Sharon Benzeno, Ginna Laport, Richard Miller, Ian McCaffery. Inhibition of A2AR induces anti-tumor immunity alone and in combination with anti-PD-L1 in preclinical and clinical studies [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 5593. doi:10.1158/1538-7445.AM2017-5593

Abstract 573: Preclinical evaluation of JTX-2011, an anti-ICOS agonist antibody

Abstract ICOS (inducible co-stimulator molecule) is a co-stimulatory molecule and a member of the CD28 superfamily expressed primarily on T lymphocytes. Analysis of cancer patient samples as well as rodent preclinical data have implicated a role for the ICOS pathway in cancer immunotherapy. We have generated a panel of anti-ICOS monoclonal antibodies with in vitro agonistic properties. The anti-ICOS antibodies are efficacious as monotherapies and in combination with anti-PD1 in multiple syngeneic tumor models. Mechanistic studies demonstrate that tumor regression is associated with enhanced ratios of cytotoxic CD8:T regulatory (Treg) cells as well as preferential reduction in ICOS-high Tregs in the tumor microenvironment. JTX-2011, a species cross-reactive high affinity humanized agonist monoclonal antibody, has been selected for development. Evaluation of JTX-2011 in nonhuman primate models will be presented, including data informing safety and PK parameters. Our preclinical data provides rational for clinical development of JTX-2011 as a cancer immunotherapeutic to be tested as both a monotherapy as well as in combination with immunotherapies in solid tumor indications. Citation Format: Jennifer S. Michaelson, Christopher J. Harvey, Kutlu G. Elpek, Ellen Duong, Matthew Wallace, Chengyi J. Shu, Sriram Sathyanarayanan, Robert Mabry, Lindsey Shallberg, Tong Zi, Amit Deshpande, Stephen L. Sazinsky, Joshua Apgar, Deborah Law. Preclinical evaluation of JTX-2011, an anti-ICOS agonist antibody. [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 573.

Abstract 4904: The BET inhibitor INCB054329 enhances the activity of checkpoint modulation in syngeneic tumor models

Abstract Inhibitors of the BET family of bromodomain proteins have been shown to be growth inhibitory across a spectrum of tumor types due to their ability to regulate the expression of key survival and cell fate determining genes such as c-myc. In addition to their role in cancer, studies using genetic knockdown and small molecule inhibitors have demonstrated that targeting BET proteins controls the expression of pro-inflammatory cytokine genes in macrophages and is therapeutic in models of acute inflammation. These data suggest that in addition to their tumor intrinsic effects, BET inhibitors may also regulate the cytokine milieu within the tumor microenvironment and have immunomodulatory activity in cancer. To study this aspect, we evaluated INCB054329, a novel and selective BET inhibitor currently in Phase 1 trials, alone and in combination either with epacadostat, a highly selective IDO1 inhibitor, or with PD-1/PD-L1 axis blockade in syngeneic tumor models using immunocompetent animals. When used alone, INCB054329 suppressed a panel of cytokines and chemokines in a whole blood assay, confirming that INCB054329 can antagonize a pro-inflammatory response. The potency of INCB054329 in reducing the levels of these inflammatory mediators in the whole blood assay was similar to that for inhibition of c-myc, suggesting that the effects were on-target. INCB054329 was capable of inhibiting the growth of multiple syngeneic tumor models in immunocompetent mice, whereas only modest tumor growth inhibition was observed in immunodeficient mice and a lack of activity was observed in vitro, supporting the immunomodulatory activity of the compound. Because maximal in vivo tumor growth inhibition required an intact immune system, we investigated the impact of INCB054329 on various immune cell subsets, both in vitro and in vivo. Of note, increases in effector T cell populations were observed and efforts are ongoing to further characterize the tumor infiltrating immune cells following INCB054329 treatment. The mechanistic complimentarity of this novel BET inhibitor-mediated immunomodulation was also evaluated in combination with other therapeutically relevant mechanisms, including IDO1 inhibition and PD-1 axis blockade. Enhanced efficacy was observed with all INCB054329-containing regimens. These data demonstrate for the first time that BET inhibition can suppress tumor growth through both tumor-intrinsic and immune modulatory mechanisms, and support the potential of epigenetic-based, immunotherapy combinations as a novel approach to cancer therapy. Citation Format: Holly K. Koblish, Michael Hansbury, Leslie Hall, Liang-Chuan Wang, Yue Zhang, Maryanne Covington, Timothy Burn, Mark Rupar, Christine Gardiner, Thomas Condamine, Kerri Lasky, Matthew C. Stubbs, Eddy Yue, Richard Sparks, Richard Sparks, Thomas Maduskuie, Andrew P. Combs, Gregory Hollis, Reid Huber, Phillip CC Liu, Peggy Scherle. The BET inhibitor INCB054329 enhances the activity of checkpoint modulation in syngeneic tumor models. [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 4904.

CmFPA008, an anti-mouse CSF-1R antibody, combines with multiple immunotherapies to reduce tumor growth in nonclinical models

The colony stimulating factor 1 receptor (CSF1R) signaling pathway drives the recruitment, differentiation, and survival of tumor-associated macrophages (TAMs) in the neoplastic microenvironment, promoting tumor progression and modulation of the immune response. As such, CSF1R represents an intriguing therapeutic target for immuno-oncology. FivePrime has developed FPA008, an IgG4 antibody with high affinity (KD = 0.35 nM) for CSF1R and the ability to block binding of both CSF1 and IL-34 to this receptor. In order to interrogate the impact of CSF1R signaling inhibition on tumor growth and the immune environment, we generated a surrogate antibody, cmFPA008, which targets mouse CSF1R and demonstrates equivalent affinity and ligand-blocking ability as FPA008. Consistent with other reports on molecules targeting this pathway, cmFPA008 as monotherapy results in statistically significant but modest growth inhibition in multiple preclinical tumor models, including MC38 colon adenocarcinoma and B16 melanoma. Utilizing a combination of flow cytometric, immunohistochemical, and gene expression analyses, we show that CSF1R inhibition induces dramatic reduction of TAMs and an increase in the CD8+ T cell to regulatory T cell ratio in syngeneic tumor models. Given that the regulation an anti-tumor immune response is complex, effective cancer therapy may require combining multiple immunotherapy agents. We sought to determine whether inhibition of CSF1R when combined with other immuno-oncology therapeutics enhanced the anti-tumor impact. We observed increased PD-L1 (CD274) expression in the tumor after treatment with cmFPA008 monotherapy, thereby providing a rationale for a combination of FPA008 with a compound targeting the PD-1 pathway. Our results show that cmFPA008 significantly enhances anti-tumor efficacy when combined with an anti-PD1 therapeutic in multiple syngeneic tumor models. In addition, we show that co-administration of cmFPA008 with an agonist anti-CD40 significantly enhances tumor suppression compared to either therapy alone. Changes in tumor-infiltrating lymphocyte (TIL) populations upon treatment provide important insight into the mechanism of action of cmFPA008 monotherapy and in combination with other immunotherapies. These results provide support for ongoing clinical efforts to evaluate FPA008 as an anti-cancer immunotherapy, particularly in combination with other immuno-oncology therapeutics. FivePrime has planned a clinical trial in collaboration with Bristol-Myers Squibb (BMS) to investigate the efficacy of FPA008 in combination with the anti-PD1 therapeutic Opdivo® (nivolumab) in six tumor types.

Pre-clinical development of the human CD40 agonistic antibody ADC-1013

Activation of CD40 on dendritic cells increases cross-presentation of tumor antigens and as a result the number of activated tumor directed T effector cells. CD40 agonists have been shown to generate powerful systemic anti-tumor responses when administered in situ. The pre-clinical proof of concept data has been generated mainly using an hCD40 transgenic mouse (hCD40tg) strain. Anti-tumor effects has been demonstrated in multiple syngeneic tumor models including bladder tumor (MB49), melanoma (B16) and lymphoma (A20) models. The anti-tumor effects has been shown to induce long term, T cell dependent, tumor immunity. As expected, the anti-tumor effects depends on the immune status of the tumors as evaluated by immunohistochemistry and flow cytometry. Since CD40 agonistic antibodies mainly exert their effects upstream of the checkpoint inhibitors they have the potential to be ideal candidates for combination regimens including e.g. PD-1 antagonists and OX40 antibodies. Combining ADC-1013 with antibodies against these targets has been shown to improve the anti-tumor effects in B16.F10 melanoma. Toxicology studies were performed in cynomolgus monkeys using subcutaneous or intravenous administration. Doses of 1 to 10 mg/kg were evaluated in both studies. ADC-1013 was well tolerated at all dose levels and no major safety concerns were identified. Receptor saturation of approximately 95% was achieved and pharmacodynamic activity, notably B-cell depletion, was observed at all dose levels. In cynomolgus monkey, serum concentrations of ADC-1013 declined in a multi-phasic manner following a single intravenous dose. The mean terminal half-life was approximately 30 hours. Following subcutaneous administration the mean elimination half-life was found to be between 0.6 and 3.2 days. The clearance (CL_F) decreased and the half-life increased with increasing dose indicating target-mediated clearance. Alligator Bioscience has recently started dosing of the first patients in a clinical trial of the CD40 agonistic antibody ADC-1013. The study is a first-in-human, multicenter, open-label, multiple ascending dose Phase I study in patients with advanced solid tumors to determine the safety, pharmacokinetics and pharmacodynamics of intratumorally administered ADC-1013.

Abstract 5025: Dissecting the dynamics of anti-PD1 immunotherapy in preclinical tumor models

Abstract MK-3475 is an antagonistic humanized IgG4 anti-PD1 antibody currently in late stage clinical development for the treatment of cancer. MK-3475 exerts its anti-tumor effect through blocking the interaction of the immune inhibitory molecule PD-1 with its ligands. MK-3475 does not bind to mouse PD-1. Therefore, we generated a surrogate antibody, mDX400 for use in preclinical rodent models. In multiple syngeneic tumor models, treatment with mDX400 murinized anti-mouse antibody results in complete and durable tumor regression. We have used mDX400 as a mouse equivalent of MK-3475 to aid in our understanding of the cellular and molecular dynamics of anti-PD1 immunotherapy. In the current study, we examined peripheral and intratumoral lymphoid compartments pre- and post-mDX400 treatment. The data indicated that: 1. Anti-tumor efficacy was primarily driven by the level of PD-1 receptor blockade in tumor, but not blood. 2. An altered molecular signature, including significant upregulation of genes involved in monocyte, B- and T-cell activation and differentiation, was observed post-treatment in the tumors, but not peripheral blood or draining lymph nodes, of responding animals. 3. Tumor-specific immune memory was evident, as assessed by ultrahigh-throughput DNA sequencing of TCR CDR3 region, following successful treatment with mDX400. These data provide insights into the molecular dynamics following anti-PD1 immunotherapy and will guide hypothesis generation for biomarkers to inform our clinical strategy. In addition, the cellular and molecular pathways that predominate in preclinical tumor models insensitive to anti-PD1 immunotherapy are being evaluated. These types of studies may inform us on potential therapies that can be combined with MK-3475 to expand the therapeutic benefit of this biologic. Citation Format: Venkataraman Sriram, Michael E. Bigler, Holly Cherwinski, Erin Murphy, Terrill K. McClanahan, Joseph H. Phillips. Dissecting the dynamics of anti-PD1 immunotherapy in preclinical tumor models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5025. doi:10.1158/1538-7445.AM2014-5025