Treatment with a VEGFR-2 antibody results in intra-tumor immune modulation and enhances anti-tumor efficacy of PD-L1 blockade in syngeneic murine tumor models.

Prolonged activation of vascular endothelial growth factor receptor-2 (VEGFR-2) due to mis-regulation of the VEGF pathway induces aberrant blood vessel expansion, which supports growth and survival of solid tumors. Therapeutic interventions that inhibit the VEGFR-2 pathway have therefore become a mainstay of cancer treatment. Non-clinical studies have recently revealed that blockade of angiogenesis can modulate the tumor microenvironment and enhance the efficacy of concurrent immune therapies. Ramucirumab is an FDA-approved anti-angiogenic antibody that inhibits VEGFR-2 and is currently being evaluated in clinical studies in combination with anti-programmed cell death (PD-1) axis checkpoint inhibitors (pembrolizumab, durvalumab, or sintilimab) across several cancer types. The purpose of this study is to establish a mechanistic basis for the enhanced activity observed in the combined blockade of VEGFR-2 and PD-1-axis pathways. Pre-clinical studies were conducted in murine tumor models known to be responsive to anti-PD-1 axis therapy, using monoclonal antibodies that block mouse VEGFR-2 and programmed death-ligand 1 (PD-L1). Combination therapy resulted in enhanced anti-tumor activity compared to anti-PD-L1 monotherapy. VEGFR-2 blockade at early timepoints post-anti-PD-L1 therapy resulted in a dose-dependent and transient enhanced infiltration of T cells, and establishment of immunological memory. VEGFR-2 blockade at later timepoints resulted in enhancement of anti-PD-L1-driven immune cell infiltration. VEGFR-2 and PD-L1 monotherapies induced both unique and overlapping patterns of immune gene expression, and combination therapy resulted in an enhanced immune activation signature. Collectively, these results provide new and actionable insights into the mechanisms by which concurrent VEGFR-2 and PD-L1 antibody therapy leads to enhanced anti-tumor efficacy.

The Folate Pathway Inhibitor Pemetrexed Pleiotropically Enhances Effects of Cancer Immunotherapy.

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.

FcγRIIIa-dependent IFN-γ release in whole blood assay is predictive of therapeutic IgG1 antibodies safety.

Immunomodulatory monoclonal IgG1 antibodies developed for cancer and autoimmune disease have an inherent risk of systemic release of pro-inflammatory cytokines. In vitro cytokine release assays are currently used to predict cytokine release syndrome (CRS) risk, but the validation of these preclinical tools suffers from the limited number of characterized CRS-inducing IgG1 antibodies and the poor understanding of the mechanisms regulating cytokine release. Here, we incubated human whole blood from naïve healthy volunteers with four monoclonal IgG1 antibodies with different proven or predicted capacity to elicit CRS in clinic and measured cytokine release using a multiplex assay. We found that, in contrast to anti-CD52 antibodies (Campath-1H homolog) that elicited high level of multiple inflammatory cytokines from human blood cells in vitro, other IgG1 antibodies with CRS-inducing potential consistently induced release of a single tested cytokine, interferon (IFN)-γ, with a smaller magnitude than Campath. IFN-γ expression was observed as early as 2-4 h after incubation, mediated by natural killer cells, and dependent upon tumor necrosis factor and FcγRIII. Importantly, the magnitude of the IFN-γ response elicited by IgG1 antibodies with CRS-inducing potential was determined by donor FcγRIIIa-V158F polymorphism. Overall, our results highlight the importance of FcγRIIIa-dependent IFN-γ release in preclinical cytokine release assay for the prediction of CRS risk associated with therapeutic IgG1 antibodies.

Targeting the TGFß pathway with galunisertib, a TGFßRI small molecule inhibitor, promotes anti-tumor immunity leading to durable, complete responses, as monotherapy and in combination with checkpoint blockade.

BACKGROUND: TGFß signaling plays a pleotropic role in tumor biology, promoting tumor proliferation, invasion and metastasis, and escape from immune surveillance. Inhibiting TGFß'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 TGFß pathway inhibitor, galunisertib, on anti-tumor immunity at clinically relevant doses. RESULTS: In vitro treatment with galunisertib reversed TGFß 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 TGFß 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.

The CDK4/6 Inhibitor Abemaciclib Induces a T Cell Inflamed Tumor Microenvironment and Enhances the Efficacy of PD-L1 Checkpoint Blockade.

Abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4/6), has recently been approved for the treatment of hormone receptor-positive breast cancer. In this study, we use murine syngeneic tumor models and in vitro assays to investigate the impact of abemaciclib on T cells, the tumor immune microenvironment and the ability to combine with anti-PD-L1 blockade. Abemaciclib monotherapy resulted in tumor growth delay that was associated with an increased T cell inflammatory signature in tumors. Combination with anti-PD-L1 therapy led to complete tumor regressions and immunological memory, accompanied by enhanced antigen presentation, a T cell inflamed phenotype, and enhanced cell cycle control. In vitro, treatment with abemaciclib resulted in increased activation of human T cells and upregulated expression of antigen presentation genes in MCF-7 breast cancer cells. These data collectively support the clinical investigation of the combination of abemaciclib with agents such as anti-PD-L1 that modulate T cell anti-tumor immunity.

Blockade of surface-bound TGF-ß on regulatory T cells abrogates suppression of effector T cell function in the tumor microenvironment.

Regulatory T cells (Tregs) suppress antitumor immunity by inhibiting the killing of tumor cells by antigen-specific CD8+ T cells. To better understand the mechanisms involved, we used ex vivo three-dimensional collagen-fibrin gel cultures of dissociated B16 melanoma tumors. This system recapitulated the in vivo suppression of antimelanoma immunity, rendering the dissociated tumor cells resistant to killing by cocultured activated, antigen-specific T cells. Immunosuppression was not observed when tumors excised from Treg-depleted mice were cultured in this system. Experiments with neutralizing antibodies showed that blocking transforming growth factor-ß (TGF-ß) also prevented immunosuppression. Immunosuppression depended on cell-cell contact or cellular proximity because soluble factors from the collagen-fibrin gel cultures did not inhibit tumor cell killing by T cells. Moreover, intravital, two-photon microscopy showed that tumor-specific Pmel-1 effector T cells physically interacted with tumor-resident Tregs in mice. Tregs isolated from B16 tumors alone were sufficient to suppress CD8+ T cell-mediated killing, which depended on surface-bound TGF-ß on the Tregs Immunosuppression of CD8+ T cells correlated with a decrease in the abundance of the cytolytic protein granzyme B and an increase in the cell surface amount of the immune checkpoint receptor programmed cell death protein 1 (PD-1). These findings suggest that contact between Tregs and antitumor T cells in the tumor microenvironment inhibits antimelanoma immunity in a TGF-ß-dependent manner and highlight potential ways to inhibit intratumoral Tregs therapeutically.

Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy.

With the success of ipilimumab and promise of programmed death-1 pathway-targeted agents, the field of tumor immunotherapy is expanding rapidly. Newer targets for clinical development include select members of the tumor necrosis factor receptor (TNFR) family. Agonist antibodies to these co-stimulatory molecules target both T and B cells, modulating T-cell activation and enhancing immune responses. In vitro and in vivo preclinical data have provided the basis for continued development of 4-1BB, OX40, glucocorticoid-induced TNFR-related gene, herpes virus entry mediator, and CD27 as potential therapies for patients with cancer. In this review, we summarize the immune response to tumors, consider preclinical and early clinical data on select TNFR family members, discuss potential translational challenges and suggest possible combination therapies with the aim of inducing durable antitumor responses.

Biomarkers for immunostimulatory monoclonal antibodies in combination strategies for melanoma and other tumor types.

Modulation of the immune system by targeting coinhibitory and costimulatory receptors has become a promising new approach of immunotherapy for cancer. The recent approval of the CTLA-4-blocking antibody ipilimumab for the treatment of melanoma was a watershed event, opening up a new era in the field of immunotherapy. Ipilimumab was the first treatment to ever show enhanced overall survival (OS) for patients with stage IV melanoma. However, measuring response rates using standard Response Evaluation Criteria in Solid Tumors (RECIST) or modified World Health Organization criteria or progression-free survival does not accurately capture the potential for clinical benefit for ipilimumab-treated patients. As immunotherapy approaches are translated into more tumor types, it is important to study biomarkers, which may be more predictive of OS to identify the patients most likely to have clinical benefit. Ipilimumab is the first-in-class of a series of immunomodulating antibodies that are in clinical development. Anti-PD1 (nivolumab and MK-3475), anti-PD-L1 (BMS-936 559, RG7446, and MEDI4736), anti-CD137 (urelumab), anti-OX40, anti-GITR, and anti-CD40 monoclonal antibodies are just some of the agents that are being actively investigated in clinical trials, each having the potential for combination with the ipilimumab to enhance its effectiveness. Development of rational combinations of immunomodulatory antibodies with small-molecule pathway inhibitor therapies such as vemurafenib makes the discovery of predictive biomarkers even more important. Identifying reliable biomarkers is a necessary step in personalizing the treatment of each patient's cancer through a baseline assessment of tumor gene expression and/or immune profile to optimize therapy for the best chance of therapeutic success.

GITR pathway activation abrogates tumor immune suppression through loss of regulatory T cell lineage stability.

Ligation of GITR (glucocorticoid-induced tumor necrosis factor (TNF) receptor-related gene, or TNFRSF18) by agonist antibody has recently entered into early phase clinical trials for the treatment of advanced malignancies. Although the ability of GITR modulation to induce tumor regression is well-documented in preclinical studies, the underlying mechanisms of action, particularly its effects on CD4(+)foxp3(+) regulatory T cells (Treg), have not been fully elucidated. We have previously demonstrated that GITR ligation in vivo by agonist antibody DTA-1 causes a >50% reduction of intra-tumor Treg with down modulation of Foxp3 expression. Here we show that the loss of Foxp3 is tumor-dependent. Adoptively-transferred Foxp3(+)Treg from tumor-bearing animals lose Foxp3 expression in the host when treated with DTA-1, whereas Treg from naïve mice maintain Foxp3 expression. GITR ligation also alters the expression of various transcription factors and cytokines important for Treg function. Complete Foxp3 loss in intra-tumor Treg correlates with a dramatic decrease in Helios expression and is associated with the upregulation of transcription factors T-Bet and Eomes. Changes in Helios correspond with a reduction in IL-10 and an increase in IFNγ expression in DTA-1-treated Treg. Together, these data show that GITR agonist antibody alters Treg lineage stability inducing an inflammatory effector T cell phenotype. The resultant loss of lineage stability causes Treg to lose their intra-tumor immune suppressive function, making the tumor susceptible to killing by tumor-specific effector CD8(+) T cells.

Modulation of GITR for cancer immunotherapy.

Modulation of co-inhibitory and co-stimulatory receptors of the immune system has become a promising new approach for immunotherapy of cancer. With the recent FDA approval of CTLA-4 blockade serving as an important proof of principal, many new targets are now being translated into the clinic. Preclinical research has demonstrated that targeting glucocorticoid-induced tumor necrosis factor (TNF) receptor related gene (GITR), a member of TNF receptor superfamily, by agonist antibodies or natural ligand, can serve as an effective anti-tumor therapy. In this review, we will cover this research and the rationale that has led to initiation of two phase 1 clinical trials targeting GITR as a new immunotherapeutic approach for cancer.

Hiding the road signs that lead to tumor immunity.

Tumors exploit many strategies to evade T cell-mediated destruction. For example, tumors can prevent T cell infiltration by modifying gene expression in the endothelial cells and pericytes that form their vasculature. New work showing that the T cell-attracting chemokine CCL2 can be posttranslationally modified in the tumor microenvironment adds another mechanism to the already formidable arsenal of immunoevasion tactics used by solid tumors.

Detection of intra-tumor self antigen recognition during melanoma tumor progression in mice using advanced multimode confocal/two photon microscope.

Determining how tumor immunity is regulated requires understanding the extent to which the anti-tumor immune response "functions" in vivo without therapeutic intervention. To better understand this question, we developed advanced multimodal reflectance confocal/two photon fluorescence intra-vital imaging techniques to use in combination with traditional ex vivo analysis of tumor specific T cells. By transferring small numbers of melanoma-specific CD8+ T cells (Pmel-1), in an attempt to mimic physiologic conditions, we found that B16 tumor growth alone was sufficient to induce naive Pmel-1 T cell proliferation and acquisition of effector phenotype. Tumor -primed Pmel-1 T cells, are capable of killing target cells in the periphery and secrete IFNγ, but are unable to mediate tumor regression. Within the tumor, Pmel-1 T cells have highly confined mobility, displaying long term interactions with tumor cells. In contrast, adoptively transferred non tumor-specific OT-I T cells show neither confined mobility, nor long term interaction with B16 tumor cells, suggesting that intra-tumor recognition of cognate self antigen by Pmel-1 T cells occurs during tumor growth. Together, these data indicate that lack of anti-tumor efficacy is not solely due to ignorance of self antigen in the tumor microenvironment but rather to active immunosuppressive influences preventing a protective immune response.

Anti-GITR antibodies--potential clinical applications for tumor immunotherapy.

Since the development of the first vaccines, modern medicine has been consistently aiming to improve the efficacy of immune responses. Traditionally, adjuvants have been used as non-specific immune modulators to enhance recognition and activation against a desired antigen. By providing 'danger' signals to the immune system, adjuvants activate innate immunity, which enhances the development of protective and therapeutic adaptive immune responses. The newest class of immune modulators bypasses the innate response and targets cells of the adaptive response directly. Targeted immunomodulatory therapy is focused primarily on the activation of costimulatory receptors (eg, 4-1BB, OX40 and GITR [glucocorticoid-induced TNF receptor-related gene]) or the blockade of co-inhibitory receptors (eg, CTLA-4, PD-1 and PD-L1) on T-cells during activation and/or effector responses. With promising clinical results obtained to date, immunomodulatory therapy is becoming an integral part of immunotherapeutic approaches. The modulation of GITR is listed as one of the top 25 most promising research areas by the NCI, and has demonstrated potential in both antitumor and vaccine settings. This review discusses the role of GITR as a potential target for immunomodulatory therapy, as well as the research involved in understanding the mechanisms of anti-GITR therapy and current progress in translation into the clinic.

Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation.

In vivo GITR ligation has previously been shown to augment T-cell-mediated anti-tumor immunity, yet the underlying mechanisms of this activity, particularly its in vivo effects on CD4+ foxp3+ regulatory T cells (Tregs), have not been fully elucidated. In order to translate this immunotherapeutic approach to the clinic it is important gain better understanding of its mechanism(s) of action. Utilizing the agonist anti-GITR monoclonal antibody DTA-1, we found that in vivo GITR ligation modulates regulatory T cells (Tregs) directly during induction of melanoma tumor immunity. As a monotherapy, DTA-1 induced regression of small established B16 melanoma tumors. Although DTA-1 did not alter systemic Treg frequencies nor abrogate the intrinsic suppressive activity of Tregs within the tumor-draining lymph node, intra-tumor Treg accumulation was significantly impaired. This resulted in a greater Teff:Treg ratio and enhanced tumor-specific CD8+ T-cell activity. The decreased intra-tumor Treg accumulation was due both to impaired infiltration, coupled with DTA-1-induced loss of foxp3 expression in intra-tumor Tregs. Histological analysis of B16 tumors grown in Foxp3-GFP mice showed that the majority of GFP+ cells had lost Foxp3 expression. These "unstable" Tregs were absent in IgG-treated tumors and in DTA-1 treated TDLN, demonstrating a tumor-specific effect. Impairment of Treg infiltration was lost if Tregs were GITR(-/-), and the protective effects of DTA-1 were reduced in reconstituted RAG1(-/-) mice if either the Treg or Teff subset were GITR-negative and absent if both were negative. Our results demonstrate that DTA-1 modulates both Teffs and Tregs during effective tumor treatment. The data suggest that DTA-1 prevents intra-tumor Treg accumulation by altering their stability, and as a result of the loss of foxp3 expression, may modify their intra-tumor suppressive capacity. These findings provide further support for the continued development of agonist anti-GITR mAbs as an immunotherapeutic strategy for cancer.