Immunosuppression In Glioblastoma Research Articles

Enhancing Glioblastoma Immunotherapy with Integrated Chimeric Antigen Receptor T Cells through the Re-Education of Tumor-Associated Microglia and Macrophages.

Glioblastoma (GBM) is an aggressive brain cancer that is highly resistant to treatment including chimeric antigen receptor (CAR)-T cells. Tumor-associated microglia and macrophages (TAMs) are major contributors to the immunosuppressive GBM microenvironment, which promotes tumor progression and treatment resistance. Hence, the modulation of TAMs is a promising strategy for improving the immunotherapeutic efficacy of CAR-T cells against GBM. Molecularly targeting drug pexidartinib (PLX) has been reported to re-educate TAMs toward the antitumorigenic M1-like phenotype. Here, we developed a cell-drug integrated technology to reversibly conjugate PLX-containing liposomes (PLX-Lip) to CAR-T cells and establish tumor-responsive integrated CAR-T cells (PLX-Lip/AZO-T cells) as a combination therapy for GBM. We used a mouse model of GBM to show that PLX-Lip was stably maintained on the surface of PLX-Lip/AZO-T cells in circulation and these cells could transmigrate across the blood-brain barrier and deposit PLX-Lip at the tumor site. The uptake of PLX-Lip by TAMs effectively re-educated them into the M1-like phenotype, which in turn boosted the antitumor function of CAR-T cells. GBM tumor growth was completely eradicated in 60% of the mice after receiving PLX-Lip/AZO-T cells and extended their overall survival time beyond 50 days; in comparison, the median survival time of mice in other treatment groups did not exceed 35 days. Overall, we demonstrated the successful fusion of CAR-T cells and small-molecule drugs with the cell-drug integrated technology. These integrated CAR-T cells provided a superior combination strategy for GBM treatment and presented a reference for the construction of integrated cell-based drugs.

Abstract 2650: IGFBP2 assists EGFRvIII-positive glioblastoma to evade immune elimination

Abstract In recent years, immunotherapy has achieved significant success in the treatment of a variety of cancer types, including melanoma, non-small cell lung cancer, and colorectal cancer. However, in stark contrast, patients with glioblastoma (GBM) show remarkably poor responses to immunotherapy, failing to derive any therapeutic benefit. Thus, an understanding of the immunosuppressive microenvironment of GBM, which is considered an immunotherapy-cold tumor, is of paramount importance. One common and distinctive genetic mutation found in gliomas is that of EGFRvIII, which is linked with enhanced cell proliferation, invasion, migration, and angiogenesis in GBM. Intriguingly, we found that this mutation induced a significantly more severe immunosuppressive microenvironment compared with wild-type EGFR. To unravel the mechanisms underlying EGFRvIII mutation-induced immunosuppression in GBM, we posited that the mutant variant secretes an array of immunosuppression-related proteins into the glioma microenvironment. To investigate this, we conducted proteomic analysis of conditioned media from EGFRvIII mutant cell lines and discovered significant changes in the expression of insulin-like growth factor-binding protein-2 (IGFBP2), a well-documented oncogenic protein. Although IGFBP2 is known to be upregulated in various tumor types and is associated with malignant behaviors such as proliferation and invasion in gliomas, its role in glioma immunology remains largely unexplored. In line with our earlier bioinformatic analyses, our study revealed significant enrichment of IGFBP2 in pathways related to immunosuppression in GBM. Thus, in the present investigation, we analyzed the mechanisms by which IGFBP2 induces an immunosuppressive microenvironment. Using co-culture models, we found that IGFBP2-overexpressing glioma cells as well as glioma cells treated with exogenous IGFBP2 recruited immune cells through binding to integrin αvβ5. Furthermore, we demonstrated that IGFBP2 promoted the secretion of the classical macrophage polarization factor IL-10 by phosphorylation of STAT3. These findings indicate the significant influence of IGFBP2 in steering glioma-associated macrophages toward a pro-tumor M2-like phenotype. We also observed that IGFBP2 upregulated the expression of PD-L1 on tumor cell surfaces. These results were verified in animal experiments. Intriguingly, our preliminary data indicated that exogenous IGFBP2 translocated to the cell nucleus within hours, and immunoprecipitation assays confirmed its interaction with STAT3 in vitro. These findings collectively underscore the pivotal role of IGFBP2 in the creation of the immunosuppressive microenvironment induced by EGFRvIII mutation. Consequently, targeting IGFBP2 holds great promise for advancing immunotherapeutic approaches in EGFRvIII-mutant glioblastomas, offering an exciting avenue for potential treatments of GBM. Citation Format: Jikang Fan, Xisen Wang, Tao Li, Xuejun Yang, Wei Zhang. IGFBP2 assists EGFRvIII-positive glioblastoma to evade immune elimination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2650.

Systemic and local immunosuppression in glioblastoma and its prognostic significance.

The effectiveness of tumor therapy, especially immunotherapy and oncolytic virotherapy, critically depends on the activity of the host immune cells. However, various local and systemic mechanisms of immunosuppression operate in cancer patients. Tumor-associated immunosuppression involves deregulation of many components of immunity, including a decrease in the number of T lymphocytes (lymphopenia), an increase in the levels or ratios of circulating and tumor-infiltrating immunosuppressive subsets [e.g., macrophages, microglia, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs)], as well as defective functions of subsets of antigen-presenting, helper and effector immune cell due to altered expression of various soluble and membrane proteins (receptors, costimulatory molecules, and cytokines). In this review, we specifically focus on data from patients with glioblastoma/glioma before standard chemoradiotherapy. We discuss glioblastoma-related immunosuppression at baseline and the prognostic significance of different subsets of circulating and tumor-infiltrating immune cells (lymphocytes, CD4+ and CD8+ T cells, Tregs, natural killer (NK) cells, neutrophils, macrophages, MDSCs, and dendritic cells), including neutrophil-to-lymphocyte ratio (NLR), focus on the immune landscape and prognostic significance of isocitrate dehydrogenase (IDH)-mutant gliomas, proneural, classical and mesenchymal molecular subtypes, and highlight the features of immune surveillance in the brain. All attempts to identify a reliable prognostic immune marker in glioblastoma tissue have led to contradictory results, which can be explained, among other things, by the unprecedented level of spatial heterogeneity of the immune infiltrate and the significant phenotypic diversity and (dys)functional states of immune subpopulations. High NLR is one of the most repeatedly confirmed independent prognostic factors for shorter overall survival in patients with glioblastoma and carcinoma, and its combination with other markers of the immune response or systemic inflammation significantly improves the accuracy of prediction; however, more prospective studies are needed to confirm the prognostic/predictive power of NLR. We call for the inclusion of dynamic assessment of NLR and other blood inflammatory markers (e.g., absolute/total lymphocyte count, platelet-to-lymphocyte ratio, lymphocyte-to-monocyte ratio, systemic immune-inflammation index, and systemic immune response index) in all neuro-oncology studies for rigorous evaluation and comparison of their individual and combinatorial prognostic/predictive significance and relative superiority.

The need for paradigm shift: prognostic significance and implications of standard therapy-related systemic immunosuppression in glioblastoma for immunotherapy and oncolytic virotherapy.

Despite significant advances in our knowledge regarding the genetics and molecular biology of gliomas over the past two decades and hundreds of clinical trials, no effective therapeutic approach has been identified for adult patients with newly diagnosed glioblastoma, and overall survival remains dismal. Great hopes are now placed on combination immunotherapy. In clinical trials, immunotherapeutics are generally tested after standard therapy (radiation, temozolomide, and steroid dexamethasone) or concurrently with temozolomide and/or steroids. Only a minor subset of patients with progressive/recurrent glioblastoma have benefited from immunotherapies. In this review, we comprehensively discuss standard therapy-related systemic immunosuppression and lymphopenia, their prognostic significance, and the implications for immunotherapy/oncolytic virotherapy. The effectiveness of immunotherapy and oncolytic virotherapy (viro-immunotherapy) critically depends on the activity of the host immune cells. The absolute counts, ratios, and functional states of different circulating and tumor-infiltrating immune cell subsets determine the net immune fitness of patients with cancer and may have various effects on tumor progression, therapeutic response, and survival outcomes. Although different immunosuppressive mechanisms operate in patients with glioblastoma/gliomas at presentation, the immunological competence of patients may be significantly compromised by standard therapy, exacerbating tumor-related systemic immunosuppression. Standard therapy affects diverse immune cell subsets, including dendritic, CD4+, CD8+, natural killer (NK), NKT, macrophage, neutrophil, and myeloid-derived suppressor cell (MDSC). Systemic immunosuppression and lymphopenia limit the immune system's ability to target glioblastoma. Changes in the standard therapy are required to increase the success of immunotherapies. Steroid use, high neutrophil-to-lymphocyte ratio (NLR), and low post-treatment total lymphocyte count (TLC) are significant prognostic factors for shorter survival in patients with glioblastoma in retrospective studies; however, these clinically relevant variables are rarely reported and correlated with response and survival in immunotherapy studies (e.g., immune checkpoint inhibitors, vaccines, and oncolytic viruses). Our analysis should help in the development of a more rational clinical trial design and decision-making regarding the treatment to potentially improve the efficacy of immunotherapy or oncolytic virotherapy.

Small Molecule Immunomodulators as Next-Generation Therapeutics for Glioblastoma.

Glioblastoma (GBM), the most aggressive astrocytic glioma, remains a therapeutic challenge despite multimodal approaches. Immunotherapy holds promise, but its efficacy is hindered by the highly immunosuppressive GBM microenvironment. This review underscores the urgent need to comprehend the intricate interactions between glioma and immune cells, shaping the immunosuppressive tumor microenvironment (TME) in GBM. Immunotherapeutic advancements have shown limited success, prompting exploration of immunomodulatory approaches targeting tumor-associated macrophages (TAMs) and microglia, constituting a substantial portion of the GBM TME. Converting protumor M2-like TAMs to antitumor M1-like phenotypes emerges as a potential therapeutic strategy for GBM. The blood-brain barrier (BBB) poses an additional challenge to successful immunotherapy, restricting drug delivery to GBM TME. Research efforts to enhance BBB permeability have mainly focused on small molecules, which can traverse the BBB more effectively than biologics. Despite over 200 clinical trials for GBM, studies on small molecule immunomodulators within the GBM TME are scarce. Developing small molecules with optimal brain penetration and selectivity against immunomodulatory pathways presents a promising avenue for combination therapies in GBM. This comprehensive review discusses various immunomodulatory pathways in GBM progression with a focus on immune checkpoints and TAM-related targets. The exploration of such molecules, with the capacity to selectively target key immunomodulatory pathways and penetrate the BBB, holds the key to unlocking new combination therapy approaches for GBM.

Potential roles for efferocytosis in glioblastoma immune evasion.

Glioblastoma is an aggressive and incurable brain cancer. This cancer establishes both local and systemic immunosuppression that creates a major obstacle to effective immunotherapies. Many studies point to tumor-resident myeloid cells (primarily microglia and macrophages) as key mediators of this immunosuppression. Myeloid cells exhibit a high level of plasticity with respect to their phenotype and are capable of both stimulating and repressing immune responses. How glioblastomas recruit myeloid cells and exploit them to avoid the immune system is an active area of research. Macrophages can acquire an immunosuppressive phenotype as a consequence of exposure to cytokines such as TGFB1 or IL4; in addition, macrophages can acquire an immunosuppressive phenotype as a consequence of the engulfment of apoptotic cells, a process referred to as efferocytosis. There is substantial evidence that glioblastoma cells are able to secrete cytokines and other factors that induce an immunosuppressive phenotype in macrophages and microglia. However, less is known about the contribution of efferocytosis to immunosuppression in glioblastoma. Here I review the literature in this area and discuss the potential of efferocytosis inhibition to improve glioblastoma response to immunotherapy.

Chimeric Exosomes Functionalized with STING Activation for Personalized Glioblastoma Immunotherapy.

A critical challenge of existing cancer vaccines is to orchestrate the demands of antigen-enriched furnishment and optimal antigen-presentation functionality within antigen-presenting cells (APCs). Here, a complementary immunotherapeutic strategy is developed using dendritic cell (DC)-tumor hybrid cell-derived chimeric exosomes loaded with stimulator of interferon genes (STING) agonists (DT-Exo-STING) for maximized tumor-specific T-cell immunity. These chimeric carriers are furnished with broad-spectrum antigen complexes to elicit a robust T-cell-mediated inflammatory program through direct self-presentation and indirect DC-to-T immunostimulatory pathway. This chimeric exosome-assisted delivery strategy possesses the merits versus off-the-shelf cyclic dinucleotide (CDN) delivery techniques in both the brilliant tissue-homing capacity, even across the intractable blood-brain barrier (BBB), and the desired cytosolic entry for enhanced STING-activating signaling. The improved antigen-presentation performance with this nanovaccine-driven STING activation further enhances tumor-specific T-cell immunoresponse. Thus, DT-Exo-STING reverses immunosuppressive glioblastoma microenvironments to pro-inflammatory, tumoricidal states, leading to an almost obliteration of intracranial primary lesions. Significantly, an upscaling option that harnesses autologous tumor tissues for personalized DT-Exo-STING vaccines increases sensitivity to immune checkpoint blockade (ICB) therapy and exerts systemic immune memory against post-operative glioma recrudesce. These findings represent an emerging method for glioblastoma immunotherapy, warranting further exploratory development in the clinical realm.

The missing link? LGMN connects hypoxia and immunosuppression in glioblastoma

In this issue, Pang and colleagues1 identify the protease legumain as a potential immunotherapy target in glioblastoma that drives tumor-associated macrophages in response to hypoxia.

Comparison of peripheral leukocyte parameters in patients receiving conventionally and hypofractionated radiotherapy schemes for the treatment of newly diagnosed glioblastoma.

Treatment for glioblastomas, aggressive and nearly uniformly fatal brain tumors, provide limited long-term success. Immunosuppression by myeloid cells in both the tumor microenvironment and systemic circulation are believed to contribute to this treatment resistance. Standard multi-modality therapy includes conventionally fractionated radiotherapy over 6 weeks; however, hypofractionated radiotherapy over 3 weeks or less may be appropriate for older patients or populations with poor performance status. Lymphocyte concentration changes have been reported in patients with glioblastoma; however, monocytes are likely a key cell type contributing to immunosuppression in glioblastoma. Peripheral monocyte concentration changes in patients receiving commonly employed radiation fractionation schemes are unknown. To determine the effect of conventionally fractionated and hypofractionated radiotherapy on complete blood cell leukocyte parameters, retrospective longitudinal concentrations were compared prior to, during, and following standard chemoradiation treatment. This study is the first to report increased monocyte concentrations and decreased lymphocyte concentrations in patients treated with conventionally fractionated radiotherapy compared to hypofractionated radiotherapy. Understanding the impact of fractionation on peripheral blood leukocytes is important to inform selection of dose fractionation schemes for patients receiving radiotherapy.

Hypoxia-driven protease legumain promotes immunosuppression in glioblastoma

Glioblastoma (GBM) is a hypoxic and "immune-cold" tumor containing rich stromal signaling molecules and cell populations, such as proteases and immunosuppressive tumor-associated macrophages (TAMs). Here, we seek to profile and characterize the potential proteases that may contribute to GBM immunosuppression. Legumain (LGMN) emerges as the key protease that is highly enriched in TAMs and transcriptionally upregulated by hypoxia-inducible factor 1-alpha (HIF1α). Functionally, the increased LGMN promotes TAM immunosuppressive polarization via activating the GSK-3β-STAT3 signaling pathway. Inhibition of macrophage HIF1α and LGMN reduces TAM immunosuppressive polarization, impairs tumor progression, enhances CD8+ Tcell-mediated anti-tumor immunity, and synergizes with anti-PD1 therapy in GBM mouse models. Thus, LGMN is a key molecular switch connecting two GBM hallmarks of hypoxia and immunosuppression, providing an actionable therapeutic intervention for this deadly disease.

SYST-32 A NOVEL APPROACH TO TARGET TREG-MEDIATED IMMUNOSUPPRESSION IN GBM

Abstract Macrophage inflammatory protein-3, or MIP-3α, is a chemokine that is highly expressed in gliomas, with expression progressively increasing with tumor grade. It is one of the top expressed genes in pseudopalisading and perinecrotic zones of glioblastoma (GBM) and as a chemokine, can greatly influence tumor-immune interactions. The receptor for MIP-3α, CCR6, is found on a variety of GBM-infiltrating immune subsets such as γδ T cells, conventional CD4 T cells and most notably, Tregs, which is disproportionately increased in GBM patients. Patients with lower CCR6-MIP3α gene signatures in tumors have improved overall survival and disease-free progression. We have found that circulating MIP-3α is also increased in the presence of intracranial glioma in mice, and can be normalized with immunotherapy. We note that ablation of CCR6 remarkably improves response of glioma-bearing mice to anti-PD-1 therapy. Mechanistically, we observed a reduction in regulatory T cell immunosuppressive phenotype with CCR6 ablation, which improves CD8 T cell effector function against tumor cells. Specifically, we found that CCR6 expression marks highly glycolytic Tregs, which is a known mechanism of immunosuppression of CD8 T cells, and that ablation of CCR6 from Tregs reduces their glycolytic capacity and immunosuppressivity. Targeting the CCR6:MIP-3α axis can therefore be a promising approach to alleviate CD8 T cell immunosuppression in GBM and to improve anti-tumor response.

Abstract LB097: Armored bicistronic CAR T cells with dominant-negative TGF-β receptor II to alleviate antigenic heterogeneity and suppressive immune microenvironment in glioblastoma

Abstract Introduction: We have completed two CAR T cell clinical trials for glioblastoma (GBM) and have identified several key challenges to therapeutic efficacy, including the inherently heterogenous genomic landscape and the immunosuppressive tumor microenvironment (TME) found in GBM. Our previous study showed that EGFR variant III (EGFRvIII)-targeting monovalent CAR T cells reduced target-positive tumor cell populations, but tumor recurrence resulted from target-negative tumor cells, highlighting the limitation of single-target approaches in heterogenous tumors. With regards to the highly immunosuppressive TME in GBM, we found that transforming growth factor-β (TGFβ) was present in the GBM TME as a major driver of suppression of the anti-GBM response in clinical samples. TGFb is consistently highly expressed in both GBM tumor cell lines and patient tumor tissues. Methods: We used two parallel scFv constructs, independently targeting both IL13Rα2 and EGFRvIII, in combination with a truncated dominant negative (dn) TGFβ receptor II. This trivalent construct was designed to explore possible additive effects in both in vitro and in vivo GBM model systems to limit tumor escape and overcome the immunosuppressive GBM TME. The CART-EGFR-IL13Rα2-dnTGFb construct broadened the targeted tumor cell repertoire, blocked TGFβ signaling, and served as a sink for free TGFβ in the GBM TME to overcome the suppressive function of TGFβ. Results: The tri-modular CAR T construct had an enhanced proliferative response when compared with the CART-EGFR-IL13Rα2 construct, in vitro. In co-culture assays, this construct led to reduced PD-1 expression and increased central memory phenotype, when compared to the bicistronic CAR T construct, which suggested a lower fraction of exhausted T cells. Tri-modular CAR T cells blocked the suppressive pSmad2/3 signaling pathway, leading to the increased tumor killing activity in co-culture experiments with both adherent and suspension GBM cell lines. In an immunodeficient mouse model, tri-modular CAR T cells eradicated tumor cells efficiently and mice had a longer median survival when compared those treated with the bicistronic CART-EGFR-IL13Rα2 cells, lacking the dnTGFb receptor II. Conclusion: Overcoming the adaptive changes in the local TME and addressing antigen heterogeneity will be required to improve the clinical efficacy of CAR T-directed strategies. Our combination work showed that bicistronic CART constructs cooperate with truncated TGFβ receptor II efficiently. In summary, the dominant-negative TGFβ RII CART-EGFR-IL13Rα2 structure is a promising strategy to address the clinical challenges of antigenic heterogeneity and the immunosuppressive TME in GBM we have observed in our two GBM CART cell trials at UPenn. Citation Format: Nannan Li, Jesse Rodriguez, Zev Binder, Donald O’Rourke. Armored bicistronic CAR T cells with dominant-negative TGF-β receptor II to alleviate antigenic heterogeneity and suppressive immune microenvironment in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB097.

886 Stereotactic Radiosurgery Reprograms Macrophage, Microglia, and T Cell Populations in the Glioblastoma Immune Microenvironment

INTRODUCTION: Daily ionizing radiation, the first-line treatment for glioblastoma, prevents durable immune infiltration of the tumor microenvironment. Hypofractionated radiotherapy is used to treat glioblastomas at recurrence, but the impact of hypofractionated radiotherapy on the glioblastoma immune microenvironment is incompletely understood. METHODS: Syngeneic GL261 (3x105 cells/mouse) or SB28 glioblastoma cells (3 x 104 cells/mouse) were implanted into the frontal lobe of immunocompetent C57BL/6J mice. Bioluminescence was used to assess glioblastoma growth. After tumor engraftment, glioblastomas were treated with conformal SRS (18Gy/1Fx) or sham. Glioblastomas were collected for histologic, single-cell, or molecular analyses 5 days after treatment or at the time of euthanasia after monitoring for survival. Glioblastoma immune microenvironment responses were assessed using (1) H&E, (2) single cell mass cytometry (CyTOF) or IHC to define or validate immune cell changes, respectively, or (3) multiplexed cytokine assays to elucidate molecular mechanisms reprograming the glioblastoma immune microenvironment in response to SRS. RESULTS: SRS attenuated glioblastoma growth and prolonged survival compared to sham treatment in both immunocompetent intracranial mouse models (GL261: 14 days versus 27 days, p < 0.001, SB28: 19 days versus 22 days, p = 0.001). CyTOF showed SRS decreased immunosuppressive macrophage infiltration and increased microglia or CD8+ T cell infiltration of the glioblastoma immune microenvironment in GL261 tumors , however, there were very few changes in the lymphoid compartment of SB28 tumors . Histologic analyses validated T cell and microglia infiltration after SRS . Glioblastoma cytokine analysis revealed inhibition of pro-tumor/anti-inflammatory cytokines (IL6, LIF) after SRS in SB28 tumors . CONCLUSIONS: Single-fraction SRS reprograms glioblastoma macrophage, microglia, and CD8+ T cell populations in preclinical models, suggesting SRS or inhibition of pro-tumor/anti-inflammatory mechanisms underlying the immunosuppressive glioblastoma microenvironment represent immunogenic therapies that may offer a benefit to patients with glioblastoma.

Immunotoxin-αCD40 therapy activates innate and adaptive immunity and generates a durable antitumor response in glioblastoma models.

D2C7-immunotoxin (IT), a dual-specific IT targeting wild-type epidermal growth factor receptor (EGFR) and mutant EGFR variant III (EGFRvIII) proteins, demonstrates encouraging survival outcomes in a subset of patients with glioblastoma. We hypothesized that immunosuppression in glioblastoma limits D2C7-IT efficacy. To improve the response rate and reverse immunosuppression, we combined D2C7-IT tumor cell killing with αCD40 costimulation of antigen-presenting cells. In murine glioma models, a single intratumoral injection of D2C7-IT+αCD40 treatment activated a proinflammatory phenotype in microglia and macrophages, promoted long-term tumor-specific CD8+ T cell immunity, and generated cures. D2C7-IT+αCD40 treatment increased intratumoral Slamf6+CD8+ T cells with a progenitor phenotype and decreased terminally exhausted CD8+ T cells. D2C7-IT+αCD40 treatment stimulated intratumoral CD8+ T cell proliferation and generated cures in glioma-bearing mice despite FTY720-induced peripheral T cell sequestration. Tumor transcriptome profiling established CD40 up-regulation, pattern recognition receptor, cell senescence, and immune response pathway activation as the drivers of D2C7-IT+αCD40 antitumor responses. To determine potential translation, immunohistochemistry staining confirmed CD40 expression in human GBM tissue sections. These promising preclinical data allowed us to initiate a phase 1 study with D2C7-IT+αhCD40 in patients with malignant glioma (NCT04547777) to further evaluate this treatment in humans.

Regulation of Macrophage Polarization and Tumor Immunosuppression in Glioblastoma

Regulation of Macrophage Polarization and Tumor Immunosuppression in Glioblastoma

TMIC-78. CIRCADIAN REGULATOR CLOCK DRIVES IMMUNOSUPPRESSION IN GLIOBLASTOMA

Abstract The symbiotic interactions between cancer stem cells and the tumor microenvironment (TME) are critical for tumor progression. However, the molecular mechanism underlying this symbiosis in glioblastoma (GBM) remains enigmatic. Here, we show that circadian locomotor output cycles kaput (CLOCK) and its heterodimeric partner brain and muscle ARNT-like 1 (BMAL1) in glioma stem cells (GSCs) drive immunosuppression in GBM. Integrated analyses of the data from transcriptome profiling, single-cell RNA sequencing, and TCGA datasets, coupled with functional studies, identified legumain (LGMN) as a direct transcriptional target of the CLOCK–BMAL1 complex in GSCs. Moreover, CLOCK-directed olfactomedin-like 3 (OLFML3) upregulates LGMN in GSCs via the hypoxia-inducible factor 1-alpha (HIF1A) signaling. Consequently, LGMN promotes microglial infiltration into the GBM TME via upregulating CD162, and polarizes infiltrating microglia towards an immune-suppressive phenotype. In GBM mouse models, inhibition of the CLOCK–OLFML3–HIF1A–LGMN–CD162 axis reduces intratumoral immune-suppressive microglia, increases CD8+ T cell infiltration, activation and cytotoxicity, and synergizes with anti-PD1 therapy. In human GBM, the CLOCK-regulated LGMN signaling correlates positively with microglial level and poor prognosis. Together, these findings uncover the CLOCK–OLFML3–HIF1A–LGMN axis as a molecular switch that controls microglial biology and immunosuppression, thus revealing potential new therapeutic targets for GBM patients.

CTIM-23. DOSE ESCALATION TRIAL OF FC-ENGINEERED ANTI-CD40 MONOCLONAL ANTIBODY (2141-V11) ADMINISTERED INTRATUMORALLY WITH D2C7-IT VIA CONVECTION-ENHANCED DELIVERY (CED) FOR RECURRENT MALIGNANT GLIOMAS (RMGS)

Abstract BACKGROUND D2C7-IT, a novel immunotoxin-based cytotoxic therapy, targets epidermal growth factor receptor (EGFR) and mutant EGFR variant III. In preclinical studies, D2C7-IT kills tumor cells and prolongs survival, but is unable to generate cures in all animals. We hypothesized that immunosuppression in glioblastoma limits D2C7-IT efficacy. Eliminating glioblastoma immunosuppression via CD40 co-stimulation is anticipated to enhance D2C7-IT-induced antitumor responses. In murine glioma models, CED of D2C7-IT+αCD40 generated cures and long-term tumor-specific adaptive immunity. Hence, we are conducting a phase 1 trial of D2C7-IT+2141-V11 (αhuman-CD40) administered via CED in rMG patients. METHODS Eligibility includes adult patients with solitary supratentorial rMG (WHO grade 3/4); ≥ 4 weeks after chemotherapy, bevacizumab, or study drug; adequate organ function; and KPS ≥ 70%. Cohorts of 3 patients are treated with increasing doses of 2141-V11 to determine the maximum tolerated dose when administered sequentially following D2C7-IT (166,075 ng) via CED at 0.5 mL/hr. Five dose levels (DLs) are evaluated (2141-V11 at: #1: 0.70 mg; #2: 2.0 mg; #2**: 3.0 mg #2*: 4.0 mg; #3: 7.0 mg). RESULTS As of May 29, 2022, 13 patients were treated (3 patients on DL1, DL2, and DL2**; 2 patients on DL3 and DL2*). No dose-limiting toxicities were observed; however, lower DLs were added due to higher frequency of adverse events (AEs) expected with D2C7-IT+2141-V11 with DL3 and DL2* (fever, neurologic symptoms). All patients remain alive 0.7-10.6 months after therapy. Grade ≥ 2 AEs due to D2C7-IT+2141-V11 include: headache (grade 3, n = 1; grade 2, n = 4); pyramidal tract disorder (grade 3, n = 1; grade 2, n = 2); paresthesia (grade 3, n = 1); dysphasia (grade 3, n = 1); seizures (grade 2; n = 2); fever (grade 2; n = 2); and one each of grade 2 depressed level of consciousness, fatigue, and concentration impairment. Enrollment is ongoing. CONCLUSIONS Intratumoral administration of D2C7-IT+2141-V11 via CED is safe, encouraging efficacy results are observed.

CNSC-13. TUMOR INFILTRATION OF GAMMA-DELTA T CELLS IN GLIOBLASTOMA

Abstract Gamma-delta (γδ) T cells are innate immunity effector lymphocytes with known prominent anti-tumor reactivity against aggressive glioblastoma (GBM). However, therapeutic approaches have had limited success due to the protective blood-brain-barrier and the immunosuppressive GBM tumor microenvironment. In this study, we determined Vδ1 a Vδ2 γδ T cell populations in peripheral blood and paired tumor tissue samples in patients (n=40) following the resection and throughtout the therapy follow-up. Tumor samples were processed using enzymatic kits and gentleMACSTM Dissociator (Miltenyi Biotec Inc.) and tumor-infiltrating γδ T lymphocytes (TILs) were analyzed by flow cytometry.We found infiltration of both intratumoral CD3+ γδ T cell subsets in 68% tumor samples. We detected Vδ1 γδ T cells in the range 0-0.8% (median 0.26%). Majority of GBM patients presented the Vδ2 subset among TILs in the range 0-13.8% (median 1.5%). Functional studies showed prominent cytotoxicity of magnetically sorted Vδ1 a Vδ2 γδ T cells against GBM cell lines and more importantly against primary tumors. Detailed phenotypic profiling and single-cell sequencing of Vδ2 γδ T cells is currently underway. Next, we identified the EphA2 receptor as one of the targets for tumor-reactive Vδ1 γδ T cells. Specifically, we found that blocking of EphA2 expression resulted in significant inhibition of GBM killing mediated by Vδ1 γδ T cells. Furthermore, Luminex xMAP technology identified significantly elevated levels of stress ligand MICA and check-point inhibitor ligands PD-L1 (B7-H1, CD274) and B7-H3 (CD276) and galectin-9 in patients‘ plasma samples at diagnosis compared to age-matched controls.The patient’s clinical course and therapeutic protocols will be discussed.This study was supported by Ministry of Health, Czech Republic (grant NV19-05-00410 to AK) by Ministry of Health, Czech Republic-conceptual development of research organization (FNBr, 65269705). All rights reserved.

RBIO-04. STEREOTACTIC RADIOSURGERY REPROGRAMS MACROPHAGE, MICROGLIA, AND CD8+ T CELL POPULATIONS IN THE GLIOBLASTOMA IMMUNE MICROENVIRONMENT

Abstract INTRODUCTION Fractionated radiotherapy is a first-line treatment for glioblastoma, but daily ionizing radiation prevents durable immune infiltration of the tumor microenvironment. Hypofractionated radiotherapy is used to treat glioblastomas at recurrence, but the impact of hypofractionated radiotherapy on the glioblastoma immune microenvironment is incompletely understood. Here we define immune microenvironment changes across multiple immunocompetent intracranial mouse models of glioblastoma after treatment with ionizing radiation mimicking stereotactic radiosurgery (SRS) in humans. METHODS Syngeneic GL261 (3x105 cells/mouse) or SB28 glioblastoma cells (3x104 cells/mouse) were implanted into the frontal lobe of immunocompetent C57BL/6J mice (18 mice/arm x 4 arms). Intracranial bioluminescence was used to assess glioblastoma growth. After tumor engraftment, glioblastomas were treated with conformal SRS (18Gy/1Fx) or sham. Glioblastomas were collected for histologic, single-cell, or molecular analyses 5 days after treatment (6 mice/arm) or at the time of euthanasia after monitoring for survival (12 mice/arm). Glioblastoma immune microenvironment responses were assessed using (1) H&E, (2) single cell mass cytometry (CyTOF) or IHC to define or validate immune cell changes, respectively, or (3) multiplexed cytokine assays to elucidate molecular mechanisms reprograming the glioblastoma immune microenvironment in response to SRS. RESULTS SRS attenuated glioblastoma growth and prolonged survival compared to sham treatment in both immunocompetent intracranial mouse models (GL261: 14 days versus 27 days, p< 0.001, SB28: 19 days versus 22 days, p=0.001). CyTOF showed SRS decreased immunosuppressive macrophage infiltration and increased microglia or CD8+ T cell infiltration of the glioblastoma immune microenvironment. Histologic analyses validated T cell and microglia infiltration after SRS. Glioblastoma cytokine analysis revealed inhibition of pro-tumor/anti-inflammatory cytokines (IL6, LIF) after SRS. CONCLUSIONS Single-fraction SRS durably reprograms glioblastoma macrophage, microglia, and CD8+ T cell populations in preclinical models, suggesting SRS or inhibition of pro-tumor/anti-inflammatory mechanisms underlying the immunosuppressive glioblastoma microenvironment represent immunogenic therapies that may offer a benefit to patients with glioblastoma.

BIOM-31. TEMPOROSPATIAL PROTEIN PROFILING OF HUMAN GLIOBLASTOMAS REVEALS MOLECULAR MECHANISMS AND BIOMARKERS UNDERLYING RESPONSES TO IMMUNE CHECKPOINT INHIBITION

Abstract Glioblastoma responses to immune checkpoint inhibition (ICI) are rare, and the molecular mechanisms underlying ICI responses are incompletely understood. Thus, serial glioblastoma samples are valuable resources for identifying biomarkers or therapeutic targets to increase the efficacy of ICI in patients with glioblastoma. We obtained paired glioblastoma samples from 7 patients who underwent sequential surgery, ICI, and eventual salvage surgery for recurrence. Patients were distinguished as ICI responders (n=3) or non-responders (n=4) based on (1) MRI evidence of tumor stability/reduction over 6+ months after ICI, or (2) pathologic evidence of predominant treatment effect at the time of salvage surgery after ICI. FFPE sections from each tumor (n=14) were stained using H&E or IHC/IF for macrophages/microglia (CD68) or T cells (CD3) and analyzed using light or fluorescence microscopy. Six regions-of-interest (ROIs) comprising viable tumor were selected neuropathologist from each sample (n=84 ROIs). ROIs were analyzed using quantitative spatial profiling of 72 proteins on the Nanostring Digital Profiler platform. Glioblastomas responding to ICI were enriched in T-cell proteins (CD3, CD4, CD8) and T-cell activation markers (CD25) at the time of salvage compared to initial surgery. Markers of MAPK signaling were suppressed in pre-ICI samples compared to post-ICI samples in responders. p-ERK was suppressed in post-ICI samples compared to pre-ICI samples in non-responders. Myeloid proteins (CD68, CD163, CD11c) were enriched in post-ICI samples compared to pre-ICI samples in non-responders. Principle components analysis revealed p-ERK and immune proteins (CD3, CD4, CD8, CD20, CD11c, CTLA4, CD68, CD45, CD56, and CD127) accounted for 62% of the variance among pre-ICI and post-ICI samples in responders. In conclusion, temporospatial protein profiling of human glioblastomas reveals molecular mechanisms and biomarkers underlying responses to immune checkpoint inhibition. These data establish a foundation for functional studies to reprogram the immunosuppressive glioblastoma microenvironment and sensitize tumors to immune checkpoint inhibition.