Search

Background Modulation of the PD-1/PD-L1 axis through antagonist antibodies that block either receptor or ligand has been shown to reinvigorate the function of tumor-specific T cells and unleash potent anti-tumor immunity, leading to durable objective responses in a subset of patients across multiple tumor types. Results Here we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ monoclonal antibody that binds to human PD-L1 with high affinity and inhibits interactions of PD-L1 with its two cognate receptors PD-1 and CD80. The functional activity of LY3300054 on primary human T cells is evaluated using a series of in vitro T cell functional assays and in vivo models using human-immune reconstituted mice. LY3300054 is shown to induce primary T cell activation in vitro, increase T cell activation in combination with anti-CTLA4 antibody, and to potently enhance anti-tumor alloreactivity in several xenograft mouse tumor models with reconstituted human immune cells. High-content molecular analysis of tumor and peripheral tissues from animals treated with LY3300054 reveals distinct adaptive immune activation signatures, and also previously not described modulation of innate immune pathways. Conclusions LY3300054 is currently being evaluated in phase I clinical trials for oncology indications.

Purpose: Combination strategies leveraging chemotherapeutic agents and immunotherapy have held the promise as a method to improve benefit for patients with cancer. However, most chemotherapies have detrimental effects on immune homeostasis and differ in their ability to induce immunogenic cell death (ICD). The approval of pemetrexed and carboplatin with anti-PD-1 (pembrolizumab) for treatment of non–small cell lung cancer represents the first approved chemotherapy and immunotherapy combination. Although the clinical data suggest a positive interaction between pemetrexed-based chemotherapy and immunotherapy, the underlying mechanism remains unknown. Experimental Design: Mouse tumor models (MC38, Colon26) and high-content biomarker studies (flow cytometry, Quantigene Plex, and nCounter gene expression analysis) were deployed to obtain insights into the mechanistic rationale behind the efficacy observed with pemetrexed/anti-PD-L1 combination. ICD in tumor cell lines was assessed by calreticulin and HMGB-1 immunoassays, and metabolic function of primary T cells was evaluated by Seahorse analysis. Results: Pemetrexed treatment alone increased T-cell activation in mouse tumors in vivo, robustly induced ICD in mouse tumor cells and exerted T-cell–intrinsic effects exemplified by augmented mitochondrial function and enhanced T-cell activation in vitro. Increased antitumor efficacy and pronounced inflamed/immune activation were observed when pemetrexed was combined with anti-PD-L1. Conclusions: Pemetrexed augments systemic intratumor immune responses through tumor intrinsic mechanisms including immunogenic cell death, T-cell–intrinsic mechanisms enhancing mitochondrial biogenesis leading to increased T-cell infiltration/activation along with modulation of innate immune pathways, which are significantly enhanced in combination with PD-1 pathway blockade. See related commentary by Buque et al., p. 6890

Background TGFβ signaling plays a pleotropic role in tumor biology, promoting tumor proliferation, invasion and metastasis, and escape from immune surveillance. Inhibiting TGFβ’s immune suppressive effects has become of particular interest as a way to increase the benefit of cancer immunotherapy. Here we utilized preclinical models to explore the impact of the clinical stage TGFβ pathway inhibitor, galunisertib, on anti-tumor immunity at clinically relevant doses. Results In vitro treatment with galunisertib reversed TGFβ and regulatory T cell mediated suppression of human T cell proliferation. In vivo treatment of mice with established 4T1-LP tumors resulted in strong dose-dependent anti-tumor activity with close to 100% inhibition of tumor growth and complete regressions upon cessation of treatment in 50% of animals. This effect was CD8+ T cell dependent, and led to increased T cell numbers in treated tumors. Mice with durable regressions rejected tumor rechallenge, demonstrating the establishment of immunological memory. Consequently, mice that rejected immunogenic 4T1-LP tumors were able to resist rechallenge with poorly immunogenic 4 T1 parental cells, suggesting the development of a secondary immune response via antigen spreading as a consequence of effective tumor targeting. Combination of galunisertib with PD-L1 blockade resulted in improved tumor growth inhibition and complete regressions in colon carcinoma models, demonstrating the potential synergy when cotargeting TGFβ and PD-1/PD-L1 pathways. Combination therapy was associated with enhanced anti-tumor immune related gene expression profile that was accelerated compared to anti-PD-L1 monotherapy. Conclusions Together these data highlight the ability of galunisertib to modulate T cell immunity and the therapeutic potential of combining galunisertib with current PD-1/L1 immunotherapy.

Combination strategies leveraging chemotherapy and immunotherapy have held the promise as a method to improve benefit to cancer patients. However, most chemotherapies have detrimental effects on immune homeostasis and do not induce immunogenic cell death. The positive phase III trial of pemetrexed/carboplatin with the PD-1 antibody pembrolizumab in NSCLC (Keynote-189) lead to the first chemotherapy/immunotherapy combination ever approved. While this suggests a positive interaction between pemetrexed-based chemotherapy and PD-1 therapy, the underlying mechanism is unknown. Therefore, it is important to understand the role of pemetrexed in modulating antitumor immune response to assure application of this therapy to the appropriate patients. To characterize the effects of pemetrexed on intra-tumor immune response, murine tumor models which were sensitive to pemetrexed and known to be sensitive to PD-L1, were treated with pemetrexed with or without carboplatin, or anti-mouse PD-L1. In MC38 tumors, pemetrexed monotherapy demonstrated a trend towards an increased frequency of intra-tumor leukocytes that was accompanied by immune-related gene expression changes indicative of enhanced T cell infiltration and/or activation. Gene expression induced by pemetrexed was largely unaffected by carboplatin. Treatment of both MC38 and Colon26 tumor cells in vitro with pemetrexed induced release of HMGB1, indicative of immunogenic cell death. Although proliferation of primary human T cells was slightly reduced by pemetrexed, at clinically relevant concentrations, treatment lead to an enhanced T cell activation phenotype, including upregulation of multiple interferon gamma-induced genes, and increased mitochondrial respiration. This correlated with improved antigen specific in vitro cytotoxic activity of OT-1 TCR transgenic CD8 T cells when treated with pemetrexed during priming with OVA peptide. Treatment with pemetrexed and PD-L1 demonstrated a combination benefit compared to either monotherapy in both tumor models. Pathway Analysis of gene expression data revealed that improved antigen presentation, enhanced T cell and cytokine signaling and an engagement of CD4+ T cell-mediated immunity during the combination. This correlated with upregulation of MHC-I & II on monocytes, macrophages and tumor cells, suggesting increased immune priming. Accordingly, treatment with S1P1R antagonist (FTY720, preventing T cell LN egress) after initiation of therapy resulted in a loss of combination efficacy. This data suggests that pemetrexed promotes intra-tumor T cell-mediated immune response through immunogenic tumor cell death and increased activation and metabolic fitness of T cells. The combination of these effects results in enhanced T cell function leading to an improved anti-tumor efficacy in combination with PD-L1 antibody Citation Format: David A. Schaer, Nelusha Amaladas, Zhao Hai Lu, Erik Rasmussen, Andreas Sonyi, Darin Chin, Andrew Capen, Yanxia Li, Catalina Meyer, Bonita Jones, Xiaodong Hong, Shuang Luo, Carmine Carpenito, Kenneth Roth, Alexander Nikolayev, Bo Tan, Manisha Brahmachary, Krishna Chodavarapu, Frank Charles Dorsey, Jason Manro, Thompson Doman, Greg Donoho, David Surguladze, Gerald Hall, Sandaruwan Geeganage, Michael Kalos, Ruslan Novosiadly. The folate pathway inhibitor pemetrexed pleiotropically enhances effects of cancer immunotherapy via immunogenic tumor cell death and T cell-intrinsic mechanisms [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 3945.

Background: Recently, pemetrexed and carboplatin in combination with PD–1 antibody (pembrolizumab) demonstrated markedly improved clinical outcome in NSCLC patients (KEYNOTE–021G trial) suggesting a positive interaction between pemetrexed–based chemotherapy and immunotherapy. However, the role of pemetrexed in modulating antitumor immune response is largely unknown. The objective of this study was to characterize the effects of pemetrexed on intra–tumor immune response in monotherapy and combination with carboplatin or PD–1 pathway blockade in preclinical models. Methods: Mice bearing syngeneic MC38 or Colon26 tumors were treated with pemetrexed with or without carboplatin or anti–mouse PD–L1 antibody (178G7). Immune cell subsets and immune–related changes in mouse tumor tissue and T cells were assessed by flow cytometry and gene expression analysis (Quantigene Plex and nanoString nCounter assays). Effects of pemetrexed on immunogenic cell death in tumor cells and mitochondrial respiration in T cells were evaluated by HMGB1 ELISA and Agilent Seahorse XF analysis, respectively. Results: In MC38 tumors, pemetrexed monotherapy demonstrated a trend towards an increased frequency of intratumoral leukocytes and total and cycling (Ki67+) T cells accompanied by immune–related gene expression changes indicative of enhanced antigen presentation, T cell infiltration and/or activation and down–modulation of the myeloid cell compartment. Immune gene expression signature induced by pemetrexed was largely unaffected by carboplatin. In both MC38 and Colon26 tumor cells, in vitro treatment with pemetrexed induced a robust release of HMGB1 indicative of immunogenic cell death. Proliferation of primary human T cells stimulated with CD3/CD28 was inhibited by pemetrexed in a dose–dependent manner. At clinically relevant concentrations pemetrexed also enhanced T cell activation phenotype exemplified by an increased frequency of CD137+, GITR+ and PD–L1+ T cells as well as upregulation of multiple interferon gamma–induced genes, and increased mitochondrial respiration. In both MC38 and Colon26 models, treatment with pemetrexed and 178G7 demonstrated a combination benefit compared to either monotherapy, and nCounter profiling of Colon26 tumors followed by Ingenuity Pathway Analysis revealed that improved antigen presentation, enhanced T cell and cytokine signaling and an engagement of CD4+ T cell–mediated immunity might underlie this combination effect. Conclusions: Pemetrexed promotes intra–tumor T cell–mediated immune response through immunogenic tumor cell death and increased activation and metabolic fitness of T cells leading to an enhanced anti–tumor efficacy in combination with PD–L1 antibody. Citation Format: Ruslan Novosiadly, David Schaer, Nelusha Amaladas, Erik Rasmussen, Zhao Hai Lu, Andreas Sonyi, Carmine Carpenito, Yanxia Li, Shuang Luo, Andrew Capen, Catalina Meyer, Xiaodong Huang, Jason Manro, Gregory Donoho, Thompson Doman, Gerald Hall, Sandaruwan Geeganage, Michael Kalos. Pemetrexed enhances anti-tumor efficacy of PD1 pathway blockade by promoting intra tumor immune response via immunogenic tumor cell death and T cell intrinsic mechanisms [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 4549.

Modulating the PD-L1/PD-1 axis in man through function blocking antibodies can release potent anti-tumor immunity, leading to durable objective responses across multiple tumor types. Here we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ antibody capable of binding human PD-L1 with high affinity and inhibiting binding to its two cognate receptors, PD-1 and CD80. LY3300054 is an antagonist monoclonal antibody recognizing human PD-L1 with high affinity (KD 0.08 nM), selected and derived from a scFv phage library. LY3300054 was engineered and expressed as an IgG1-Fc null monoclonal antibody to ablate immune effector function. Multiple in vitro assays, including mixed leukocyte reaction (MLR) and tetanus toxoid recall assay were utilized to demonstrate LY3300054 potent functional activity in enhancing the activation of primary human T cells in culture. The biological activity of LY3300054 on T cells was also shown to be enhanced by co-administration of anti-CTLA4 mAb (ipilimumab) in MLR. Further, we evaluated LY3300054 activity in vivo using xenograft mouse tumor models reconstituted with human immune cells. LY3300054 demonstrated anti-tumor activity in both NCI-H292 xenografts co-implanted with human PBMCs and HCC827 xenograft model upon infusion of human T cells. LY3300054 in vivo activity was also tested in HCC827- and Ov79-bearing immunodeficient NSG/NOG mice reconstituted with human hematopoietic stem cells. In this setting, LY3300054 displayed robust anti-tumor and immunomodulatory effects exemplified by T cell inflamed phenotype in the tumor and peripheral tissues. High-content molecular profiling identified distinct gene expression changes indicative of T cell activation in all models tested. A mutational strategy based on integrating the PD-1/PD-L1 structure data with the orthologous sequence data of PD-L1 has identified a residue on PD-L1, which is part of the PD-L1/PD-1 interface, that is critical for the species specificity of LY3300054. This study demonstrates that LY3300054 is novel anti-PD-L1 monoclonal antibody, capable of potently enhancing human T cell function both in vitro and in vivo, and provides previously not described insights into the effects of PD-L1 blockade on the intra- and extra-tumoral immune response. LY3300054 is currently under clinical evaluation in monotherapy and combination with other therapeutic modalities in multiple tumor types (NCT02791334; NCT03099109; NCT02791334; NCT02791334) Citation Format: Carmine Carpenito, Yiwen Li, George X. Wang, Maria S. Malabunga, Jaafar N. Haidar, Amelie Forest, Mary Y. Murphy, Gerald E. Hall, Cindy Wang, Leyi Shen, Andreas Sonyi, Darin Chin, Anthony L. Pennello, Ivan V. Inigo, David Surguladze, Yung-mae Yao, Douglas Burtrum, Ruslan D. Novosiadly, Kris Persaud, Dale L. Ludwig, Michael D. Kalos. Preclinical characterization of the anti-PD-L1 monoclonal antibody LY3300054 [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 2730.

The combination of tumor targeted therapeutics with PD-L1 checkpoint blockade is being explored as a method to increase the clinical benefits of immunotherapy, and expand response to additional cancer types. Merestinib (Mer) is a kinase inhibitor targeting several oncokinases1 (including MET, MST1R, AXL, MERTK, and MKNK1/2) that can potentially modulate immune function, angiogenesis, as well as target the tumor 1-5. To determine the combinatorial potential with immunotherapy, the effects of Mer were evaluated in vitro on human T cells, PBMCs and murine tumor lines CT26 colon carcinoma (harbors KRASmt G12D expresses low Met/no p-Met/high Axl/p-Axl) and B16F10 melanoma (expressing high Met/pMet/peIF4E). Additionally, the anti-tumor effect of Mer was tested in vivo on established CT26 and B16F10 tumors compared to MET specific TKIs (savolitinib, PF4217903) alone or in combination with PD-L1 antibody (Ab) blockade. In vitro, Mer showed no significant effects on either T cells or PBMCs, but was able to inhibit downstream signaling in both CT26 and B16F10 showing activity on murine tumor cell lines. In vivo, daily Mer monotherapy (6, 12 or 24 mg/kg) showed significant anti-tumor effect at all doses in both CT26 and B16F10, that was not seen with either savolitinib or PF4217903. Concurrent combination of Mer (12 mg/kg) and anti-PD-L1 Ab (0.5 mg qw) in CT26 was found to have anti-tumor activity that was synergistic as compared to each single agent alone. While the effect of Mer monotherapy was lost when treatment ended, tumors continued to regress in the combination group even upon cessation of therapy. The combination was well tolerated and resulted in 90% complete responders compared to 30% with anti-PD-L1 Ab alone, 35 days after completing dosing. To test the ability to generate immunologic memory, complete responders were re-challenged with CT26 cells on the contralateral side. All mice in the combination group resisted re-challenge, showing that Mer/PD-L1 Ab combination was triggering immunologic memory. Although there was no significant change in intra-tumor immune cell populations between groups, combination therapy showed an enhanced and unique intra-tumor immune activation/inflammation gene expression signature compared to PD-L1 Ab monotherapy. The enhanced immune activation of the combination therapy, leading to synergistic anti-tumor efficacy, demonstrates that merestinib has the potential to augment immunotherapy while targeting the tumor directly. This preclinical data provides the rationale for the clinical investigation of merestinib in combination with checkpoint therapies targeting the PD-L1/PD1 axis (NCT02791334). 1 - Yan et al. Invest New Drugs 2013;31:833-44 2 - Balan et al. J Biol Chem 2015;290:8110-20 3 - Eyob et al. Cancer Discov 2013;3:751-60 4 - Lemke G. CSH Persp Biol 2013;5:a009076 5 - Piccirillo et al. Nat Immunol 2014;15:503-11 Citation Format: Sau-Chi Betty Yan, Victoria L. Peek, Jennifer R. Stephens, Um L. Um, Amaladas Nelusha, Colleen A. Burns, Kelly M. Credille, Thompson N. Doman, Scott W. Eastman, Beverly L. Falcon, Gerald E. Hall, Philip W. Iversen, Bruce W. Konicek, Jason R. Manro, Any T. Pappas, Julie A. Stewart, Michael B. Topper, Swee-Seong Wong, Michael Kalos, Ruslan D. Novosiadly, Richard A. Walgren, David Schaer. Combination of an oncokinase inhibitor merestinib with anti-PD-L1 results in enhanced immune mediated antitumor activity in CT26 murine tumor model [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 5590. doi:10.1158/1538-7445.AM2017-5590

TGFb signaling is known to play a central role in tumor biology, via inducing and/or enhancing tumor cell growth and differentiation, modulating the extracellular matrix in the stroma, inducing epithelial to mesenchymal transition, modulating angiogenesis, and inhibiting immune surveillance and anti-tumor immunity. Galunisertib is a pharmacological inhibitor of the TGFb pathway which acts by inhibiting signaling though TGFbRI. Galunisertib is currently being evaluated clinically in several Phase I and II studies; as a monotherapy, galunisertib has shown antitumor activity against a variety of tumors, including durable and long-term responses in patients with glioma. To explore the impact of Galunisertib monotherapy on anti-tumor T cell immunity, we utilized murine tumor models. Treatment of mice with well-established 4T1-LP (poorly immunogenic 4T1 breast tumor engineered to express luciferase) implanted in the mammary fat pad resulted in strong dose-dependent anti-tumor activity with nearly 100% inhibition of tumor growth across doses during the dosing period, with complete tumor responses upon cessation of treatment in ~50% of animals at the highest dose tested; depletion studies demonstrated that regression of 4T1-LP was dependent on the presence of CD8+ T cells. Rechallenge of treated, tumor free mice resulted in complete rejection of 4T1-LP tumor cells but no rejection of EMT6-LM2 tumor cells, demonstrating the establishment of a durable response and immunological memory. Treatment of mice bearing established parental 4T1 tumors in the mammary fat pad resulted in no significant inhibition of tumor growth, indicating that the presence of a foreign antigen (i.e. LP), potentially enhanced the ability to regress the 4T1-LP derivative. Animals that rejected the immunogenic 4T1-LP tumors were able to also reject 4T1 parental cells upon rechallenge, suggesting the development of a secondary immune response via antigen spreading as a consequence of effective tumor targeting. In the CT26 murine colon carcinoma model, treatment of established tumors with galunisertib or anti-PD-L1 as monotherapies resulted in tumor growth inhibition compared to control of 75% and 86%, respectively (T/C values of 25% and 14%); complete responders were observed in about 20% of treated animals in both monotherapy groups. Combination of galunisertib with anti-PD-L1 resulted in an enhanced tumor growth inhibition of 98% (T/C value of ~2%), and a complete response rate of ~50%, suggesting at least additive activity with potential for synergy when targeting the TGFb and PD-1 pathways. Taken together, these data demonstrate the potential for galunisertib treatment to enhance the development of anti- tumor T cell immunity, which can be enhanced by combinations with immune check point inhibitors. Citation Format: David Schaer, Yanxia Li, Stephen Castaneda, Ivan Inigo, David Surguladze, Xiaohong Xu, Desiree Nugent, Mary Murphy, Gerald Hall, Karim Benhadji, Susan Guba, Yiwen Li, Michael Kalos, Kyla Driscoll. Targeting the TGFb pathway with galunisertib, a TGFbRI SMI, promotes antitumor immunity leading to durable, complete responses, as monotherapy and in combination with checkpoint inhibition. [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 A091.