Lucien, Fabrice

Abstract Description Combination of radiotherapy (RT) and immunotherapy has gained popularity to turn up the heat on cold tumors. However, intrinsic resistance is prevalent, hence better therapeutic strategies are needed to improve the outcome of patients. We recently demonstrated in metastatic prostate cancer that circulating levels of tumor-derived extracellular vesicles (tdEVs) increased shortly after RT and was associated with poor survival. TdEVs are mediators of antitumor immunity but their role in response to RT remains unknown. Our study elucidates the impact of RT on immunomodulatory roles of tdEVs. Mouse tumors models expressing a bioluminescent EV reporter were treated with clinically used doses of RT. TdEV organotropism was measured by ex-vivo imaging at different timepoints. TdEVs were detected in blood, spleen, lymph nodes and immune cells. OT-1 transgenic mice and in vitro co-culture assays showed a reduction in CD8 T-cell cytotoxic activity in presence of tdEVs from irradiated cells. To identify key players of tdEV-mediated immunosuppression in response to RT, proteomic analysis revealed the immune checkpoint molecule B7-H3 was overexpressed in tdEVs after irradiation. Genetic deletion of B7-H3 inhibited the suppressive activity of tdEVs from irradiated cells resulting in enhanced T-cell cytotoxicity. This study describes tdEVs as a mediator of systemic immunosuppression in response to radiotherapy and B7-H3 as a potential therapeutic target to boost antitumor immunity. Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description Therapies targeting the PD-1/PD-L1 axis are among the leading treatments of cancer, but only a minority of patients benefit. Many therapies focus on preventing PD-L1 from binding to its receptor, PD-1, but do not address PD-L1’s ability to be recycled to the cell surface and any intrinsic functions it may possess. Our lab has developed an antibody (H1A) that binds to PD-L1 and prevents its association with a stabilizing protein, CMTM6, therefore targeting it for lysosomal degradation. Consequently, H1A limits any PD-L1 intrinsic function while also decreasing interactions with PD-1. Here, we use human-derived immune cells to test H1A’s effect on immune responses through flow cytometry, multiplex screening, and cytotoxicity assays. Additionally, we have developed a humanized PD-1/PD-L1 mouse model that allows us to test H1A’s effect on tumor growth inhibition in an immunocompetent host. Our results show that H1A specifically targets human myeloid cells, resulting in increased co-stimulation, antigen presentation, and cytokine secretion. H1A demonstrated enhanced T cell cytotoxicity in a whole PBMC culture. Finally, H1A resulted in greater tumor growth inhibition and survival in our in vivo tumor models. This work highlights the importance of targeting beyond PD-1/PD-L1 ligation and that myeloid cells are instrumental to enhancing the immune response against cancer. H1A is emerging as a new immunotherapy tool to target PD-L1 and CMTM6 in myeloid cells for treatment of cancers. Funding Sources NIH R01 92389022; NIH T32 AI170478; Mayo Clinic Graduate School of Biomedical Sciences Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description A durable T cell immunity against cancer is dependent on a continually refilling of effector CD8+ T cells. Thymic output has been correlated with favorable prognosis in cancer patients of advanced ages, suggesting thymus is an important source for refilling T cells that are capable of controlling cancer progression in the periphery. However, it remains unclear how PD-1 signals regulate the cytotoxic potential of CD8+ T cells in thymus before they egress to the periphery to establish durable anti-tumor immunity. Here we report PD-1 signaling prelimits thymic single positive (SP) CD8+ T cells to acquire cytotoxic potential by downregulating Gzmb and Nkg7, which are required for CD8+ T cell-mediated cytotoxicity. Unexpectedly, we found the absence of Pdcd1 also led to upregulation of Tox and Tim3 in thymic SP CD8+ T cells, thereby pre-programming these cells for exhaustion within the tumor. Thus, the absence of PD-1 signals in CD8+ T cells only partially delayed tumor growth in peripheral tissues. To improve therapeutic effects of PD-1 inhibition, we observed that tumor growth in CD8-specific Pdcd1 conditional knockout mice was further suppressed by treatment with a PD-L1 antibody that blocks PD-L1 and CD80 interaction. Our results imply that PD-1 inhibition promotes the expansion of effector cytotoxic CD8+ T cells but also retains T cell exhaustion potential, which can be mitigated by PD-L1 blockade that allows more CD80 to provide costimulatory signals to T cells via CD28. Funding Sources NCI R01CA256927 NCI R01CA261932 NCI P30CA15083 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description Background Castration-resistant prostate cancers (CRPC) do not respond to immunotherapy.1,2 Prostate cancer-derived immune factors (IF) like sPD-L1 drive immunosuppression and resistance.3 Circulating tumor-reactive T cells (TTR; CD8+CD11ahigh) and other immune subsets predict progression-free survival in oligometastatic CRPC after radiotherapy.4 A better understanding of the levels and changes in IF in CRPC—a tumor immune “circulome”—may lead to better immunotherapeutic approaches. Methods 79 men with oligometastatic castration-resistant prostate cancer were treated with radiation and blood was collected before and after treatment (NCT02816983). Peripheral blood mononuclear cells (PBMC) were subjected to immunophenotyping, and plasma was subjected to the Olink Explore panel assay measuring levels of 5,416 IFs using a proximity extension assay technology. Results High baseline soluble levels of PD-L1, PD-L2, TIM-3, and other selected immunotherapy-related IFs predicted inferior outcomes, including overall survival. Conversely, high soluble CTLA-4 levels strongly predicted superior outcomes (p < 0.0001). These results additionally correlated with changes in circulating tumor-reactive (TTR) T cells in these patients. Discussion IF influence systemic immunity in CRPC. These results suggest a multifactorial mechanism by which prostate cancers drive systemic immunosuppression and resistance to immunotherapy. Ongoing efforts to address IFs may help overcome CRPC immunotherapy resistance.5 Funding Sources Department of Defense HT9425-24-1-0622, An Atlas of the Tumor Immune Circulome (PI: Orme) Prostate Cancer Foundation Young Investigator Award (PI: Orme) National Cancer Institute K12 CA90628-23 (PI: Orme) Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Background Immune checkpoint therapies targeting programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) have been met with limited clinical responses in the treatment of patients of varying cancer types. Current US Food and Drug Administration (FDA)-approved therapies function to prevent PD-1 binding to PD-L1, thereby preventing T-cell suppression. However, these therapies fail to prevent PD-L1 intrinsic signaling and recycling, which can render these treatments ineffective. Moreover, most research primarily focuses on how these antibodies affect tumor cells and their interactions with T cells. Myeloid cells, which also express PD-L1 and influence T-cell responses, are integral to the efficacy of these therapies. However, strategies that can target these cells to boost antitumor responses have not been effective. Methods Our group has developed a new anti-PD-L1 antibody (H1A), which disrupts PD-L1 recycling and redirects it towards degradation. Due to the loss of available PD-L1 protein, H1A effectively prevents PD-L1 intrinsic signaling. Using in vivo humanized PD-1/PD-L1 mouse tumor models, we evaluated the therapeutic efficacy of H1A against current FDA-approved PD-L1 blocking antibodies. Using human-derived immune cell in vitro assays, we pinpoint the biological consequences of H1A-induced PD-L1 degradation in myeloid cells. Results H1A demonstrated improved tumor control and established immunological memory responses in vivo in the treatment of tumors with moderate immunogenicity that are less responsive to current immunotherapies. This was supported by enhanced activation of myeloid cells (major histocompatibility complex (MHC)-II and CD80) and frequencies of effector T-cell populations found intratumorally. Human myeloid cells treated with H1A were also observed to have increased activation (MHC-II and CD80) and cytokine secretion. Similar to our in vivo findings, human peripheral blood lymphocyte cultures demonstrated increased frequencies of effector T-cell populations and greater tumor cell killing. Conclusions This work highlights the importance of targeting PD-L1 recycling and intrinsic signaling, specifically in myeloid cells, to boost antitumor responses. Our studies suggest that H1A can provide a solution to the lack of responses seen with current therapeutics, while also revealing previously unknown intrinsic functions of PD-L1 in myeloid cells.

Abstract The effectiveness of T cell–based immunotherapy depends on durable T-cell responses that can efficiently eliminate tumor cells. NKG7 was discovered three decades ago as a protein associated with lytic granules. However, only studies published over the past 5 years have contributed substantially to our understanding of NKG7 in T-cell biology. NKG7 has been recognized as an important T-cell functional marker in responses to immune checkpoint inhibitor therapy and in the prognosis of certain cancers. Besides its role in the generation, trafficking, and release of lytic granules, which is critical for efficient T-cell cytotoxicity against tumor cells, NKG7 has been identified as a key negative regulator of mTORC1 activity. By restraining mTORC1 activity, NKG7 promotes T-cell longevity and memory generation after infection. Importantly, NKG7 upregulation has demonstrated therapeutic potential in preclinical T-cell therapy for cancer. Collectively, NKG7 is emerging as a promising biomarker and therapeutic addition to T cell–based immunotherapies.

Abstract Extracellular vesicles (EV) are phospholipid‐encapsulated nanoparticles secreted by cells into their surrounding environment. EVs can transfer a variety of biomolecules that mediate intercellular communication and play a key role in physiological and pathological processes. Therefore, EVs are emerging as new biomarkers for diseases, therapeutic targets, and drug delivery vehicles. The isolation and detection of EVs requires time‐consuming and labor‐intensive processes, first to extract EVs from biological and physiological fluids and then to detect EV‐associated molecules with high sensitivity. The methodologies and instruments commonly used for EV analysis are not widely accessible outside of dedicated research laboratories, creating practical barriers for the study of EV‐associated molecules in biology research and clinical applications. To bridge this gap, we developed a proof‐of‐concept digital microfluidic device that can automatedly extract EVs from 20 µL of culture and human plasma samples within 25 min and detect EV‐bound proteins (e.g PD‐L1) on‐chip using an electrochemical sensor. This work serves as a framework for the development of streamlined EV analysis in both research and diagnostics.

ABSTRACT Despite remarkable interest in the biomarker potential of urinary extracellular vesicles (uEVs) and the identification of numerous promising candidates, their clinical translation still presents multiple challenges. The opportunities for successful translation are obvious, yet the main roadblocks on the way have hardly been systematically considered and more coordinated approaches are needed to overcome them. In the present review article, we have identified the most relevant roadblocks of clinical translation of urinary EV‐based biomarkers and discuss possible solutions to overcome them. These roadblocks are categorized as fundamental and technical but also related to development of novel biomarker assays and clinical acceptance. In addition, hurdles within the regulatory approval process are discussed. It is clear that various roadblocks to clinical translation of urinary EV biomarkers exist; however, they are addressable by promoting rigor and reproducibility as well as collaboration between basic and clinical scientists, clinicians, industry and regulatory bodies. Moreover, knowledge of obstacles for assay development and regulatory requirements should already be considered when developing a new biomarker to maximize the chance of successful translation. This review presents not only a status quo, but also a roadmap for the further development of the field.

Background/Objective: Noninvasive PET imaging-based assessment of PD-L1 expression is of high clinical value for better patient selection and treatment response rates to PD-L1 immunotherapies. Due to their shorter biological half-life and faster clearance from the blood pool, radiolabeled antibody fragments are an attractive alternative for imaging than their full-length IgG counterpart. This work investigated the radiosynthesis and in vitro cell uptake of anti-PD-L1-B11-nanobody radiolabeled with 44Sc (t1/2 = 4.04 h) as an alternative to anti-PD-L1-B11-IgG, better suited for longer half-life radioisotopes such as 89Zr (t1/2 = 78.41 h). Methods: The proteins were conjugated with p-SCN-Bn-DTPA and radiolabeled at room temperature with 44Sc, achieving a radiochemical yield of a RCY of 94.8 ± 3.1% (n = 3) for [44Sc]Sc-B11-IgG and 73.6 ± 12.1% (n = 3) for [44Sc]Sc-B11-nanobody, before purification. Results: Significantly higher uptake in the PD-L1+ cells than PD-L1KO cells was observed for both probes. However, high non-specific uptake, particularly of the radiolabeled B11-nanobody, was also observed which may negatively impact its potential as a molecular imaging probe. Conclusions: Due to the high non-specific uptake in vitro, the 44Sc radiolabeled nanobody was not progressed to further in vivo evaluation. These results should, however, not discourage future evaluations of other nanobody based probes radiolabeled with 44Sc, due to their well-matched biological and physical half-life.