Quantifying Immune Checkpoint engagement in Patient Biopsies

Development of effective and durable antitumor immunity requires the activation, expansion and maintenance of function of tumor antigen-specific effector T cells. The mechanism of action of recently FDA approved monoclonal antibody (mAb) therapies which target immune checkpoints such as PD-1 and CTLA-4 and uncouple inhibitory pathways from the activation of antigen-specific T cells underlines the critical requirement for antigen-specific priming to elicit potent and long-lasting anti-tumor immunity. However, immune checkpoint blockade results in significant clinical benefit largely in malignancies with high mutational burden, where the tumor itself can initiate T cell priming, but is less effective when used as a single agent among cancers with lower mutational burden, associated typically with a low level of lymphocyte infiltration into the tumor microenvironment (TME). We are evaluating clinical immunotherapy regimens combining immune checkpoint blockade with recombinant live-attenuated double-deleted strains of the intracellular bacterium Listeria monocytogenes (LADD), based on a hypothesis that effective immunotherapy will result from agents that in combination re-polarize the TME to facilitate immune effector cell function, prime functional tumor-specific T cells in the appropriate context, and block inhibitory signaling pathways. Here we show that tumor antigen-expressing LADD therapy enhanced CD8+ T cell effector function, resulting in significant tumor eradication in several preclinical mouse models. LADD treatment in addition induced favorable changes in the TME, as shown by enhanced CD8+ T effector function, recruitment of antigen presenting cells and reduction of regulatory T cells. Treatment regimens combining LADD-based immunotherapy with PD-1 immune checkpoint blockade significantly enhanced antitumor efficacy in CT26, 4T1 and MC38 tumor models. Together these data support the rationale for integrating LADD-based immunotherapy into clinical regimens with immune checkpoint blockade on the basis that TME modification and priming of tumor Ag-specific T cells significantly enhances the activity of mAb therapies blocking T cell inhibitory pathways. Citation Format: Weiwen Deng, Weiqun Liu, Thomas Hudson, Chris S. Rae, Ed Lemmens, Anthony Desbien, Bill Hanson, Pete Lauer, Thomas W. Dubensky, Jr., Meredith Leong. Synergistic antitumor efficacy in mice with immunotherapy regimens combining recombinant live-attenuated Listeria with immune checkpoint inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B50.

Immune checkpoint blockade (ICB) therapy for tumors has arisen in growing interest. However, the low response rate of tumors to ICB is mainly attributed to the inhibitory infiltration of immune cells in the tumor microenvironment (TME). Despite the promising benefits of ICB, the therapeutic effects of antibodies are dependent on a high dose and long-term usage in the clinic, thereby leading to immune-related adverse effects. Accordingly, ICB combined with nano-delivery systems could be used to overcome T cell exhaustion, which reduces the side effects and the usage of antibodies with higher response rates in patients. In this review, the authors aim to overcome T cell exhaustion in TME via immune checkpoint modulation with nano-delivery systems for enhanced immunotherapy. Several strategies are summarized to combine ICB and nano-delivery systems to further enhance immunotherapy: a) expressing immune checkpoint on the surface of nano-delivery systems; b) loading immune checkpoint inhibitors into nano-delivery systems; c) loading gene-editing technology into nano-delivery systems; and d) nano-delivery systems mediated immune checkpoint modulation. Taken together, ICB combined with nano-delivery systems might be a promising strategy to overcome T cell exhaustion in TME for enhanced immunotherapy.

BackgroundThe immune suppressive tumor microenvironment (TME) that inhibits T cell infiltration, survival, and anti-tumor activity has posed a major challenge for developing effective immunotherapies for solid tumors. Chimeric antigen receptor...

New insights into cancer immune checkpoints landscape from single-cell RNA sequencing.

CC-122 Repairs T Cell Activation in Chronic Lymphocytic Leukemia That Results in a Concomitant Increase in PD-1:PD-L1 and CTLA-4 Immune Checkpoint Expression at the Immunological Synapse

Introduction: Neurofibromatosis Type 1 (NF1) is caused by mutations in the NF1 gene that encodes neurofibromin, a RAS GTPase-Activating Protein. Inactivating NF1 mutations result in hyperactivation of RAS-mediated signaling. NF1 patients develop benign tumors on the skin and along nerves in the spinal cord and brain, and ~10% of these progress to Malignant Peripheral Nerve Sheath Tumors (MPNST). Attempts to inhibit RAS-signaling to treat MPNST have not worked in clinical trials. Surgical removal is the only cure for MPNST, which is challenging due to large tumor size and/or proximity to nerves. MPNSTs often recur, metastasize, and respond poorly to chemo- and radiotherapy. As a result, MPNSTs are the leading cause of death in NF1 patients. Immune Checkpoint Blockade (ICB) is an approach to treat inoperable, undruggable cancers. Immune checkpoint proteins (ICPs) terminate normal immune responses to prevent collateral tissue damage. Cancer cells hijack this mechanism and express ICPs to avoid immune detection. ICB disrupts this process, enhancing antitumor immunity. Monoclonal antibodies (mABs) that disrupt the interaction between Programmed Cell Death Receptor 1 (PD-1) and its ligand Programmed Cell Death Ligand 1 (PD-L1) are a widely used ICB therapy. Having a T cell-enriched hot tumor microenvironment (TME) is required for successful ICB. However, MPNSTs lack a T cell-inflamed TME and respond poorly to ICB therapy. Activation of the protein “stimulator of interferon genes” (STING) enhances anti-tumor immunity via induction of pro-inflammatory cytokines, thus increasing T cell infiltration into the TME. We hypothesized that reprogramming the MPNST TME by STING pathway activation would turn cold MPNSTs into hot tumors amenable to ICB. Methods: We treated mouse MPNSTs with STING agonist or STING agonist + anti-PD-1/anti-PD-L1 mAb. Tumor size was measured throughout treatment. The mice were sacrificed and tumor tissue was analyzed for STING pathway activation, T cell infiltration, cell proliferation, and apoptosis. MPNST allograft tumors in athymic mice were controls. Results: STING pathway activation and increased T cell presence was observed in STING agonist-treated MPNSTs compared to vehicle-treated. MPNSTs treated with STING agonist alone or STING agonist + PD-1/PD-L1 mAbs showed less tumor growth relative to vehicle. Importantly, STING agonist + PD-1/PD-L1 mAb-treated tumors showed significant cell death increase while cell proliferation remained unchanged. Interestingly, tumors in athymic mice continued to grow despite treatment suggesting the antitumor effects seen in immunocompetent mice were mediated by T cells. Conclusions: Our data show that MPNSTs can become T cell-rich tumors by activating STING signaling. Reprogramming the TME made MPNSTs susceptible to immune destruction by ICB therapy. Our studies support this novel treatment strategy for MPNST, an aggressive, deadly tumor for which no molecular therapy currently exists. Citation Format: Nipunika Somatilaka, Laasya Madana, Ali Sadek, Renee M. McKay, Lu Q. Le. STING inflames NF1 malignancies for immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5170.

Temporal dynamics in cancer immunotherapy: the interplay between circadian rhythms, tumor microenvironment, and immune checkpoint blockade.

Chemoimmunotherapy by combining oxaliplatin with immune checkpoint blockades reduced tumor burden in colorectal cancer animal model

Cancer immunotherapy is one of the new prospective treatments for head and neck squamous cell carcinoma (HNSCC). This strategy can effectively induce anti-tumor effects. However, despite surveillance by the host immune system, tumor cells can develop and acquire malignant phenotypes, for example, invasion into stromal tissue, and metastasis to lymph nodes and distant organs. Tumors with oncogenic mutations and cellular heterogeneity actively suppress the host immune system with assistance from the tumor microenvironment, including regulatory T cells, myeloid-derived suppressor cells, and type II macrophages, leading to immunoediting and immunosuppression of the anti-tumor response in vivo . Accumulating evidence, obtained through the use of advanced immunological technology, has elucidated the interaction between tumor cells and the host immune system. Greater understanding of this interaction has given rise to new therapeutic interventions, including cancer immunotherapy. In this review, we compile recent findings from experimental and clinical studies of cancer immunotherapy and discuss whether cancer immunotherapy has been determined to be beneficial in HNSCC patients. Cancer immunotherapy, such as cancer vaccine, dendritic cell immunotherapy, and blockade of immune checkpoints, also plays a crucial role in treatments that have contributed to improving overall survival in HNSCC patients. Moreover, due to the direct improvement of tumor- or tumor microenvironment–mediated immunosuppression in HNSCC, cancer immunotherapy in combination with targeted therapy appears to be an effective and efficient therapeutic strategy. In Context The advanced genetic and immunological technologies have provided fresh insights into molecular mechanisms of tumor progression in head and neck squamous cell carcinoma (HNSCC). Sufficient effector T cell activation induces effective antitumor immune response in cancer patients. However, recent researches have shown that immune checkpoints and tumor microenvironment in which regulatory T cell and myeloid-derived suppressor cells exist repress antitumor immune response. The knowledge of cancer biology and immunology helps to develop a therapeutic theory that cancer immunotherapy induces an antitumor immune response by modulating the host immune system. In addition, identification of these immunosuppressive functions has promoted the development of the next generation of cancer immunotherapy. In this review, we summarized data from experimental models and clinical trials associated with cancer immunotherapy in several types of cancer. These data indicated that cancer immunotherapy is capable of enhancing the anti-tumor immune response in patient. Moreover, we also discussed and explored the possibility of innovative cancer immunotherapy in HNSCC. This therapeutic approach may help us to improve survival rate in patients with HNSCC. Keywords: immunosurveillance; immunosuppression; tumor microenvironment; cancer vaccine; dendritic cell; immune checkpoints; chimeric antigen receptor therapy; targeted therapy (Published: 16 June 2015) Citation: Advances in Cellular and Molecular Otolaryngology 2015, 3: 27690 - http://dx.doi.org/10.3402/acmo.v3.27690

Boosting natural immunity against malignant cells has had a major breakthrough in clinical cancer therapy. This is mainly due to the successful development of immune checkpoint blocking antibodies, which release a break on cytolytic anti-tumor-directed T-lymphocytes. However, immune checkpoint blockade is only effective for a proportion of cancer patients, and a major challenge in the field is to understand and overcome treatment resistance. Immune checkpoint blockade relies on successful trafficking of tumor-targeted T-lymphocytes from the secondary lymphoid organs, through the blood stream and into the tumor tissue. Resistance to therapy is often associated with a low density of T-lymphocytes residing within the tumor tissue prior to treatment. The recruitment of leukocytes to the tumor tissue relies on up-regulation of adhesion molecules and chemokines by the tumor vasculature, which is denoted as endothelial activation. Tumor vessels are often poorly activated due to constitutive pro-angiogenic signaling in the tumor microenvironment, and therefore constitute barriers to efficient leukocyte recruitment. An emerging possibility to enhance the efficiency of cancer immunotherapy is to combine pro-inflammatory drugs with anti-angiogenic therapy, which can enable tumor-targeted T-lymphocytes to access the tumor tissue by relieving endothelial anergy and increasing adhesion molecule expression. This would pave the way for efficient immune checkpoint blockade. Here, we review the current understanding of the biological basis of endothelial anergy within the tumor microenvironment, and discuss the challenges and opportunities of combining vascular targeting with immunotherapeutic drugs as suggested by data from key pre-clinical and clinical studies.

The role of the microbiome in modulating immune responses has garnered significant attention in cancer research, particularly in the context of immune checkpoint mechanisms. The microbiome influences the tumor micro- environment (TME) and the efficacy of immune checkpoint inhibitors (ICIs) by shaping immune cell function and responses. Immune checkpoints, such as PD-1, PD-L1, LAG-3, and CTLA-4, are critical in regulating immune tolerance and preventing excessive immune activation. However, in cancer, these checkpoints often suppress anti-tumor immunity, contributing to tumor immune evasion and therapeutic resistance. Emerging evidence suggests that the microbiome plays a pivotal role in regulating these checkpoints, enhancing or inhibiting the effectiveness of ICIs. Studies have shown that a balanced microbiome can promote a more favorable immune response, while dysbiosis may lead to increased inflammation and tumor progression. Additionally, the combination of microbiome modulation through probiotics, prebiotics, or dietary interventions, with immune checkpoint blockade has shown potential in overcoming resistance mechanisms and improving treatment outcomes. This comprehensive review explores the complex relationship between the microbiome and immune checkpoint mechanisms, highlighting how microbiome composition impacts immune checkpoint expression and function in cancer. It also discusses the therapeutic potential of microbiome-based interventions in conjunction with ICI therapies, emphasizing the need for further research to fully understand these interactions. Integrating microbiome science with cancer immunotherapy holds the promise of more effective, personalized treatment strategies that could improve patient outcomes and survival rates across various cancer types

The clinical promise of cancer immunotherapy relies on the immune system recognizing and eliminating tumor cells identified as non-self. However, the tumor microenvironment (TME) can greatly impede a tumor targeted immune response via increasing immunosuppressive cells or hampering T and NK cell maturation, recruitment, and function through numerous pathways, including upregulation of immune checkpoints such as PD-L1. Hence, there is an unmet clinical need to develop effective therapeutic strategies that can reprogram the TME to restore tumor immune recognition and reverse immune evasion. We recently demonstrated that Entinostat, a class I histone deacetylase (HDAC) inhibitor, reverses tumor immune escape to T cell-mediated lysis. We hypothesize that the immune-mediated tumor elimination promoted by the IL-15/IL-15Rα superagonist ALT803 in combination with PD-L1 checkpoint blockade or a therapeutic adenoviral vaccine targeting CEA (Ad-CEA) will be augmented by the epigenetic reprograming of the TME induced by Entinostat. In preclinical studies, ALT803 has been shown to exhibit potent antitumor activity in multiple murine models of cancer through the expansion of NK and CD8+ T cells with high effector function. Here, we demonstrate that Entinostat increased extracellular expression of immune-relevant proteins on murine colon and breast carcinoma cells in vitro. Additionally, the frequency of T cells with an activated phenotype were increased in non-tumor bearing mice. In the MC38-CEA murine model of colon carcinoma, the addition of Entinostat augmented the antitumor activity promoted by ALT803 plus Ad-CEA resulting in increased survival. Furthermore, in the 4T1 murine model of triple-negative breast cancer, the combination of Entinostat with ALT803 and a monoclonal antibody targeting PD-L1 significantly reduced primary tumor weight relative to ALT803 plus anti-PD-L1 therapy and resulted in a significant reduction of the number of 4T1 tumor-forming cells in the lung. The immune mechanism associated with antitumor efficacy of these multivalent therapies was examined in both the periphery and TME. Overall, these studies provide a rationale for combining Entinostat with multivalent immunotherapy combinations, including cytokines, antibodies targeting PD-L1, and therapeutic cancer vaccines. Citation Format: Kristin C. Hicks, Karin M. Knudson, Frank R. Jones, Peter Ordentlich, Shahrooz Rabizadeh, Hing C. Wong, James W. Hodge, Jeffrey Schlom, Sofia R. Gameiro. Epigenetic reprogramming of the tumor microenvironment by entinostat increases tumor sensitivity to multivalent immunotherapy combinations with an IL-15 superagonist plus vaccine or immune checkpoint blockade [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 1740.

Simple SummaryNewcastle disease virus (NDV) is an RNA virus belonging to the Paramyxoviridae family. In nature, NDV primarily infects birds, but poses no threat to human health. Multiple studies have demonstrated that NDV caries oncolytic potential due to its predilection for infection and replication in human cancer cells while sparing normal cells. In addition to its direct lytic effects, the virus triggers both innate and adaptive immune responses. In animal models, NDV injection into a tumor has been demonstrated to result in local inflammation and the recruitment of tumor-specific T cells, an effect that can be further potentiated through the use of viruses encoding immunomodulatory ligands and through combinations with immune checkpoint blockade. Initial clinical trials with naturally occurring NDV administered intravenously demonstrated durable responses across a number of cancer types. Clinical studies utilizing recombinant NDV in combination with immune checkpoint inhibitors are ongoing.Preclinical and clinical studies dating back to the 1950s have demonstrated that Newcastle disease virus (NDV) has oncolytic properties and can potently stimulate antitumor immune responses. NDV selectively infects, replicates within, and lyses cancer cells by exploiting defective antiviral defenses in cancer cells. Inflammation within the tumor microenvironment in response to NDV leads to the recruitment of innate and adaptive immune effector cells, presentation of tumor antigens, and induction of immune checkpoints. In animal models, intratumoral injection of NDV results in T cell infiltration of both local and distant non-injected tumors, demonstrating the potential of NDV to activate systemic adaptive antitumor immunity. The combination of intratumoral NDV with systemic immune checkpoint blockade leads to regression of both injected and distant tumors, an effect further potentiated by introduction of immunomodulatory transgenes into the viral genome. Clinical trials with naturally occurring NDV administered intravenously demonstrated durable responses across numerous cancer types. Based on these studies, further exploration of NDV is warranted, and clinical studies using recombinant NDV in combination with immune checkpoint blockade have been initiated.

BackgroundImmune-positron emission tomography (PET) imaging with tracers that target CD8 and granzyme B has shown promise in predicting the therapeutic response following immune checkpoint blockade (ICB) in immunologically “hot” tumors. However, immune dynamics in the low T-cell infiltrating “cold” tumor immune microenvironment during ICB remain poorly understood. This study uses molecular imaging to evaluate changes in CD4 + T cells and CD8 + T cells during ICB in breast cancer models and examines biomarkers of response.Methods[89Zr]Zr-DFO-CD4 and [89Zr]Zr-DFO-CD8 radiotracers were used to quantify changes in intratumoral and splenic CD4 T cells and CD8 T cells in response to ICB treatment in 4T1 and MMTV-HER2 mouse models, which represent immunologically “cold” tumors. A correlation between PET quantification metrics and long-term anti-tumor response was observed. Further biological validation was obtained by autoradiography and immunofluorescence.ResultsFollowing ICB treatment, an increase in the CD8-specific PET signal was observed within 6 days, and an increase in the CD4-specific PET signal was observed within 2 days in tumors that eventually responded to immunotherapy, while no significant differences in CD4 or CD8 were found at the baseline of treatment that differentiated responders from nonresponders. Furthermore, mice whose tumors responded to ICB had a lower CD8 PET signal in the spleen and a higher CD4 PET signal in the spleen compared to non-responders. Intratumoral spatial heterogeneity of the CD8 and CD4-specific PET signals was lower in responders compared to non-responders. Finally, PET imaging, autoradiography, and immunofluorescence signals were correlated when comparing in vivo imaging to ex vivo validations.ConclusionsCD4- and CD8-specific immuno-PET imaging can be used to characterize the in vivo distribution of CD4 + and CD8 + T cells in response to immune checkpoint blockade. Imaging metrics that describe the overall levels and distribution of CD8 + T cells and CD4 + T cells can provide insight into immunological alterations, predict biomarkers of response to immunotherapy, and guide clinical decision-making in those tumors where the kinetics of the response differ.

Cancer immunotherapies by immune checkpoint blockade (CKI) has shown great therapeutic efficacy by helping the immune system to recognize and attack cancer cells. However, a significant percentage of patients do not respond or develop resistance. T-cell exclusion from tumor core, mediated by myeloid cells and fibroblast colonizing the tumor micro-environment, has been described as an important factor of resistance to checkpoint inhibitors. Myeloid cells represent one of the most abundant immune cell types in many solid tumors and are often associated with a poor outcome. Interaction of SIRPalpha (SIRPá), expressed by myeloid cells, with the ubiquitous receptor CD47 is an important immune checkpoint of the innate response, involved in the regulation of macrophages and dendritic cells functions (e.g. phagocytosis, antigen presentation). Here we evaluated the impact of selective SIRPá blockade on the function of myeloid cells from tumor micro-environment. In vivo, in an orthotopic and syngeneic hepatocellular carcinoma (HCC) mouse model, anti-SIRPá antagonist monoclonal antibody in combination with an adaptive immune checkpoint inhibitor (anti-PD-L1) dramatically enhanced overall survival and lead to complete remission in up to 60% of immunocompetent mice (p<0.001; n=11/18). A robust memory immune response developed since a second tumor challenge after drug elimination was always rejected (p<0.01). Transcriptional analysis of tumor at early stage (10 days after treatment) using Nanostring technology revealed a significant enrichment of a multiple chemokine signature in animals that received anti-SIRPá and anti-PD-L1 combination. Histological analysis of HCC tumors revealed that anti-PDL1 mAb alone increased T-cell infiltrates, but T-cell remained excluded from tumor core. In contrast, tumor nodules were strongly reduced, and T-cell significantly enriched in tumor nest in combination with the SIRPá antagonist (p<0.01). In vitro, we next observed that CD47-induced signals inhibited secretion of chemokines by immature human macrophages. This inhibition could be reversed by a selective anti-SIRPá antagonist mAb. Ex-vivo, selective anti- SIRPá antagonist mAbs significantly increased the chemokine transcriptomic expression signature of human myeloid cells purified from ovarian cancer ascites or of HCC tumor explant fragment maintained in culture for 48 hours. Finally, we found in vivo in a humanized mice model of transmigration that SIRPá blockade significantly increased human T cell migration in microenvironment locally enriched by human macrophages. In conclusion, we showed that selective SIRPá antagonist mAbs synergized with T-cell CKI by modifying the tumor microenvironment, in particular the chemokine secretion by mouse or human myeloid cells which contributes to limit T cell exclusion and favors efficient and robust anti-tumor immune responses. Citation Format: Vanessa Gauttier, Sabrina Pengam, Justine Durand, Kévin Biteau, Mélanie Néel, Sophie Conchon, Dominique Costantini, Bernard Vanhove, Nicolas Poirier. SIRPa blockade reinvigorates myeloid cells in the tumor microenvironment and reverses T-cell exclusion [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 3238.