bluuebunny/crossref_metadata_2025_split huggingface dataset:
EXTH-44. ENHANCING GLIOBLASTOMA CLEARANCE: EMPOWERING ANTI-EGFRVIII CAR T CELLS WITH A PARACRINE SIRPΓ-DERIVED CD47 BLOCKER
Authors: Martins Tomas , Tatari Nazanin , Kaymak Deniz , Bartoszek Ewelina M , Hogan Sabrina , Buck Alicia , Ritz Marie-Françoise , Wieboldt Ronja , McDaid Marta , Gerber Alexandra , Shekarian Tala , Hedad Hayget Mohamed Seid , Schmassmann Philip , Weiss Tobias , Laeubli Heinz , Hutter Gregor 
Published: November 2023

Abstract
A major challenge for chimeric antigen receptor (CAR) T cell therapy against glioblastoma (GBM) is the immunosuppressive microenvironment (iTME), which is densely populated by protumoral glioma-associated microglia and macrophages (GAMs). Blockade of CD47, a “don’t-eat-me” signal overexpressed by GBM cells, disrupts the CD47-SIRPα axis, and regulates the phagocytic function of GAMs. However, systemic anti-CD47 monotherapy has been ineffective against human solid tumors to date. Here, we combined local CAR T therapy with paracrine GAM modulation to synergistically eliminate GBM. We engineered an armored CAR T cell against EGFRvIII that constitutively secretes a SIRPγ-related protein (SGRP) with high affinity to CD47. Anti-EGFRvIII-SGRP CAR T cells eliminated EGFRvIII+ GBM in vitro, and eradicated EGFRvIII-heterogeneous (EGFRvIII+ and EGFRvIII- mosaic) orthotopically xenografted tumors by locoregional application in vivo. This resulted in significant tumor-free long-term survival, followed by partial tumor control upon re-challenge. Combination of anti-CD47 antibodies with anti-EGFRvIII-CAR T cells failed to achieve synergy, pointing towards the importance of sustained paracrine GAM conversion. Paracrine SGRP enhanced CAR T infiltration, tumor clearance and GAM reconversion to an antitumor phenotype as shown by multidimensional brain immunofluorescence microscopy and in-depth spectral flow cytometry multiplexing. Moreover, the plasma of anti-EGFRvIII-SGRP CAR T-treated mice displayed a distinct signature of innate immune activation including TNFα and CCL3. Validation of the anti-EGFRvIII-SGRP CAR T in human explants is ongoing. Furthermore, armoring anti-CD19 CAR T cells with SGRP resulted in superior efficacy in a peripheral lymphoma mouse model compared to anti-CD19 CAR T treatment alone, most problably due to innate immune activation. Thus, local anti-EGFRvIII-SGRP CAR T cell therapy combines the potent antitumor effect of engineered T cells with the modulation of the surrounding innate iTME, inducing a synergistic clearance of GBM in a manner that overcomes known mechanisms of CAR T therapy evasion, such as tumor immune suppression and antigen escape.
EXTH-49. A NOVEL MULTIVALENT DRUG CONJUGATE FOR HIGH-GRADE GLIOMA TREATMENT
Authors: Debinski Waldemar , Fink Kaitlin N , Herpai Denise , Rossmeisl John 
Published: November 2023

Abstract
Interleukin 13 receptor alpha 2 (IL-13RA2), EphA2, EphA3 and EphB2 receptors are over-expressed collectively in almost all patients with glioblastoma (GBM), and also in spontaneous canine gliomas, the most faithful model of human disease. The four receptors are widely present in various compartments of GBM tumors, including tumor core, infiltrating cells, tumor neovasculature and tumor-associated macrophages. Thus, the four receptors are expressed in several GBM compartments involved in tumor progression and/or resistance to therapies, covering almost 100% of the tumor microenvironment. In first-of-a-kind approach, we performed Phase I clinical trial in dogs with gliomas using a cocktail of cytotoxins targeting IL-13RA2 and EphA2 receptor. The cocktail was administered loco-regionally through convection-enhanced delivery, using anti-reflux catheters and real-time MRI monitoring of drugs distribution. We observed prominent anti-tumor responses, including several near complete regressions, prolongation of survival and excellent quality of life, although we did not achieve dose-limiting toxicity (Neuro-Oncol 2021;23(3):422-434). Encouraged by these exceptional results, we pursued the novel idea of targeting the four GBM-associated receptors with one pharmaceutical compound. To this end, we designed, produced, and tested a multivalent ligand binding to all four receptors of our interest, termed QUAD, including murine and canine receptors. However, we found that a conjugate of QUAD with DM1, a microtubule-disrupting agent, demonstrated an extremely high (in low picomolar range) and specific killing potency on GBM cells. The IC50s of QUAD-DM1 are ~50 times higher than that for all other QUAD-based drug conjugates tested thus far, having a favorable toxicity profile in rodents when injected either intracranially or intravenously. QUAD-DM1 has already produced up to 95% tumor reduction in dogs with spontaneous brain tumors. QUAD-DM1 is a highly promising agent for the treatment of human and canine high-grade gliomas.
EXTH-53. VEGFR2-SPECIFIC CAR-T CELLS SHOW IMPROVED TUMOR INFILTRATION IN SYNGENEIC MOUSE GLIOMA MODELS
Authors: Mastall Maximilian , Dunkel Gina , Silginer Manuela , Okada Naoki , Weiss Tobias , Weller Michael , Weigelin Bettina , Roth Patrick 
Published: November 2023

Abstract

BACKGROUND
Chimeric antigen receptor (CAR-)T cell therapy, a powerful immunotherapy against hematological malignancies, has so far limited efficiency in solid tumors. Also in glioblastoma, a major obstacle remains limited infiltration of CAR-T cells into the tumor. Vessel-targeting CAR-T cells have been shown to infiltrate solid tumors more efficiently. Vascular endothelial growth factor receptor 2 (VEGFR2) is expressed in the glioma vasculature and VEGFR2-specific CAR-T cells prolong the survival of xenograft glioma-bearing mice. In this study, we investigated the efficacy of murine VEGFR2-specific CAR-T cells against syngeneic experimental gliomas and assessed the CAR-T cell infiltration of this approach.

METHODS
VEGFR2-CAR-T cells were generated by retroviral transduction to express a second generation CAR construct. Their activity was assessed in co-culture assays in vitro against murine endothelial and glioma cells, respectively. Syngeneic orthotopic mouse glioma models were used to explore the infiltration of intravenously administered CAR-T cells by in vivo fluorescence molecular tomography (FMT), ex vivo flow cytometry as well as ex vivo 3D light-sheet fluorescence microscopy (LSFM).

RESULTS
VEGFR2-CAR-T cells showed anti-endothelial and anti-glioma activity in vitro. Intravenous treatment with fluorescently labeled CAR-T or mock-transduced T cells of tumor-bearing mice resulted in a higher FMT signal at the tumor site. This was confirmed by ex vivo flow cytometry, wherein CAR-T cells represented a larger fraction of all T cells within the tumor (26.5% CAR versus 5.4% Mock, p = 0.0013) resulting in a higher donor T cell to tumor cell ratio. Similarly, significantly more VEGFR2-CAR-T cells were detected at the tumor site compared to tumor-targeting (but not vessel-targeting) control CAR-T cells. Currently, VEGFR2-CAR-T cell infiltration patterns as well as vasculature density are further investigated and visualized by LSFM.

CONCLUSION
VEGFR2-CAR-T cells show improved glioma infiltration in syngeneic mouse models. Therefore, VEGFR2 might be a relevant target that could improve CAR-T cell infiltration in glioblastoma.
EXTH-63. MTA-COOPERATIVE PRMT5 INHIBITORS ARE EFFICACIOUS IN MTAP-DELETED MALIGNANT PERIPHERAL NERVE SHEATH TUMOR MODELS
Authors: Borcherding Dana , Zhang Xiaochun , Zhang Minjie , Bhatia Himanshi , Lyu Yang , He Kevin , Yang Liuzhan , Yang Kuangying , DiBenedetto Heather , Tsai Alice , Huang Alan , Maxwell John , Cottrell Kevin , Briggs Kimberly , Hirbe Angela 
Published: November 2023

Abstract

OBJECTIVES
Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive sarcomas with limited treatment options and poor survival rates, necessitating the development of novel therapeutics. Approximately 25-50% of MPNST harbor loss of the enzyme methylthioadenosine phosphorylase (MTAP) due to a passenger deletion driven by loss of the proximal tumor suppressor gene, CDKN2A. PRMT5 was identified as a selective dependence in MTAP-deleted cells due to the accumulation of the substrate methylthioadenosine (MTA), which is itself an endogenous PRMT5 inhibitor. TNG908 and TNG462 are clinical stage MTA-cooperative PRMT5 inhibitors that demonstrate selectivity for MTAP-deleted cells over MTAP-intact cells of 15X and 45X, respectively. Previous reports show both molecules drive durable tumor regressions in xenograft models of various MTAP-deleted cancer histologies. Here, our objectives are to examine the activity of TNG908 and TNG462 in preclinical MPNST models.

METHODS
The proliferation effects of MTA-cooperative PRMT5 inhibitors, TNG908 or TNG462, and a SAM-cooperative PRMT5 inhibitor, GSK3326595, on MTAP-deleted and MTAP-intact MPNST cell lines were determined using CellTiter-Glo (CTG) assays. TNG908 and TNG462 were further profiled in two MTAP-deleted MPNST patient-derived xenograft (PDX) models, WU-356 and WU-386.

RESULTS
Incubation with the MTA-cooperative PRMT5 inhibitors, TNG908 and TNG462, selectively decreased the proliferation of MTAP-deleted MPNST cell lines relative to MTAP-intact MPNST cell lines. TNG908 and TNG462 drove dose-dependent antitumor activity including tumor regressions in the MTAP-deleted MPNST PDX models, WU-356 and WU-386, at well-tolerated doses.

CONCLUSIONS
The clinical stage MTA-cooperative PRMT5 inhibitors TNG908 (NCT05275478) and TNG462 (NCT05732831) are efficacious in MPNST models in vitro and in vivo and are therefore promising therapeutic agents for patients with MTAP-deleted MPNST.
EXTH-64. ESTABLISHING A CLINICALLY RELEVANT MOUSE MODEL OF MESENCHYMAL GLIOBLASTOMA FOR STUDYING TME TARGETING TREATMENTS
Authors: Connor Kate , White Kieron , Clerkin James , Shiels Liam , Sweeney Kieron , Van Brussel Thomas , Arijs Ingrid , Lambrechts Diether , Shankar Gautam , de Smet Frederik , Zopf Dieter , Maher Stephen , Marignol Laure , Dicker Patrick , Prehn Jochen , O'Brien David , Byrne Annette 
Published: November 2023

Abstract
Historically, pre-clinical GBM models have largely failed to predict response in the clinical setting. Here, we establish a more clinically faithful model using the syngeneic NFpp10a-Luc2 mesenchymal-GBM cell line to study response to resection and temozolomide (TMZ), adjuvant/neo-adjuvant anti-PD1 and Regorafenib (REGO) therapy. Cells were orthotopically implanted in C57BL/6-mice. Response to surgical and therapeutic interventions was assessed using bioluminescence imaging (BLI). Murine Microenvironment cell-population (mMCP)-counter, GSEA and multiple iteractive labelling by antibody neodeposition (MILAN, high-dimensional single-cell multiplex analysis) were employed to characterise treatment-related TME effects. We observed survival advantage in aged mice undergoing resection (resection:33.5 days vs non-resection:18 days; p = 0.0166). TMZ/anti-PD1 had no impact on tumour growth (TMZ: p = 0.9001, anti-PD1: p = 0.7933) or survival (TMZ:p = 0.3035, anti-PD1-:p = 0.6328). Neo-adjuvant anti-PD1 also conferred no survival advantage in young mice (33 vs 35 days; p = 0.9429). REGO/REGO+TMZ treatment conferred no survival benefit in young mice (REGO:p = 0.0735 and REGO+TMZ:p = 0.3945). mMCP-counter (which estimates the abundance of TME-cell populations from gene-expression data) showed resection upregulated B-cells and mast-cells, whereas TMZ caused a decreased abundance of vessels. Anti-PD1 treatment caused an enrichment of B-cells, mast-cells, and CXCR3 expression (p = 0.0045). REGO/TMZ+REGO treatment upregulated cytotoxic-lymphocyte populations. MILAN analysis showed resection increased cytotoxic-T-cells (20.3% vs pre-resection:7%). In contrast, TMZ/REGO monotherapy increased tumour-cells and decreased cytotoxic-T-cells. Anti-PD1 decreased macrophage abundance (39.8% vs control:14.5%). Likewise, TMZ+REGO decreased macrophages (11.3% vs control:35.3%) but upregulated B-cells and vessels. Overall, we have characterised response of the NFpp10a mouse model to resection, TMZ, anti-PD1 and REGO. We have shown that the model is insensitive to chemotherapy and TME-targeting therapies, mirroring patient-response patterns. Nevertheless, we observe transcriptomic and proteomic changes following TME-targeting treatments. Further analyses of these TME-associated resistance properties may help guide novel combinatorial treatment regimens. Overall, the NFpp10a model of mesenchymal GBM may be employed in future pre-clinical studies to accurately guide future clinical trials.
EXTH-67. LP-184, AN MGMT-AGNOSTIC SMALL MOLECULE, HAS POTENT SYNERGY WITH SPIRONOLACTONE TO EFFECTIVELY INHIBIT ORTHOTOPIC GBM XENOGRAFT TUMORS
Authors: Lal Bachchu , Kulkarni Aditya , McDermott Joseph , Lopez-Bertoni Hernando , Bhatia Kishor , Sharma Panna , Laterra John 
Published: November 2023

Abstract
Temozolomide (TMZ) is currently the most effective standard-of-care chemotherapy based on its ability to extend survival of patients with newly diagnosed MGMT-methylated glioblastoma (GBM). Blood-brain barrier (BBB) permeable agents effective against TMZ-resistant GBMs (i.e. recurrent GBM, MGMT-unmethylated GBM) are needed. Lantern Pharma is advancing LP-184, an acylfulvene prodrug activated by the enzyme PTGR1. In MGMT-expressing TMZ-resistant GBM cultures, LP-184 was found to be ~5000X more potent than TMZ with LP-184 IC50s ranging from 20-200 nM. DNA damage induced by acylfulvenes is primarily repaired via transcription-coupled nucleotide excision repair (TC-NER) mediated by ERCC complexes. ERCC3 is proteolytically degraded in response to the FDA approved BBB permeable diuretic, spironolactone (SP). We found that SP induces rapid ERCC3 degradation by up to 95% in GBM cells and validated that SP-induced ERCC3 loss led to substantial increases in LP-184 responses (DNA damage, tumor cell death) in vitro and in subcutaneous (s.c.) tumor xenografts. Co-treatment of GBM cells with LP-184 and SP resulted in a 3-6 fold decrease in LP-184 IC50s in PDX-derived (M1123 and Mayo39) and cell line (U87) cultures in vitro. Systemic SP sensitized pre-established s.c. and orthotopic (i.c.) GBM xenografts to LP-184 therapy. Pre-established s.c. U87 tumors recurred after initial regression in 5/5 animals following treatment with LP-184 alone whereas tumors showed durable complete regression without recurrence in 4/5 animals following treatment with LP-184 + SP. Pre-established M1123 i.c. xenografts treated with LP-184 + SP were ~3-fold smaller than those treated with LP-184 alone. LP-184 is a promising agent with potential clinical translation in GBM patients. Our results indicate that LP-184 is MGMT-agnostic and effective in TMZ-resistant preclinical GBM models. The first-in-human LP-184 dose escalation Phase 1A trial enrolling solid tumor patients including GBM is planned to launch Q3 2023.
EXTH-69. REGULATION OF N1/N2 NEUTROPHIL PHENOTYPE BY A MANZAMINE-DERIVED COMPOUND FOR BRAIN TUMOR TREATMENT
Authors: Lin Ya-Jui , Huang Chiungyin , Wei Kuo-Chen , Chen Pin-Yuan 
Published: November 2023

Abstract
In cancer microenvironment, the presence of neutrophils is associated with tumor proliferation, invasion and immunomodulation. The tumor associated neutrophils (TAN) can be polarized toward N1 (anti-tumor) or N2 (pro-tumor) phenotypes. Therefore, modulating neutrophil phenotype may serve as brain tumor treatment strategy. We use immunosuppressive and normal mice bearing brain tumor cell xenograft to study the role of TANs in tumor progression. A manzamine-derived compound 1-(9’-propyl-3’-carbazole)-1, 2, 3, 4-tetrahydro-β-carboline (PCTC) was used to treat brain tumor mice. In brain tumor tissue, flowcytometry study revealed the amount of pro-tumor N2 TAN is significantly greater than anti-tumor N1 subtype. While PCTC was intracranial administrated into brain tumor, the ratio of N1/N2 is reversed. Immunochemical staining showed increasing neutrophils in tumor at day 1 post treatment, followed by subsequent T cell and macrophage gathering at day 5. Cytokine array results revealed the expression level of CXCL9/CXCL10 were upregulated in PCTC treatment glioma cells and brain tumor tissues. Cell study also demonstrated T cell migration ability was significantly increased when co-cultured with neutrophil and PCTC treated glioma cells. In mice tumor model, NU/NU mouse lack of T cell represented no response to PCTC treatment, while significant tumor shrunken was observed in B6 mice. Compensation study by intravenous inoculation of T cell and PCTC to NU/NU glioma mice showed flavor response to animal survival. In this study, we demonstrated the N1/N2 ratio of TAN can be regulated by PCTC, and be beneficial to glioma treatment. Furthermore, PCTC acts on tumor cells, together with TAN to attract T cell by increase CXCL9/CXCL10. Anti-tumor N1 and subsequent T cell gathering both contribute to tumor cell killing. As a conclusion, neutrophil phenotype can be modulated, and cooperated with T cells to serve as a new glioma treatment strategy.
EXTH-73. IN VIVO EFFECTS OF EPCAM-DIRECTED CAR T CELLS IN COMBINATION WITH PD-1 CHECKPOINT BLOCKADE IN LUNG CANCER BRAIN METASTASIS
Authors: Blobner Jens , Dengler Laura , Eberle Constantin , Herold Julika J , Xu Tao , Teske Nico , Karschnia Philipp , Ishikawa-Ankerhold Hellen , Subklewe Marion , Thon Niklas , Tonn Joerg-Christian , Buchholz Veit , von Baumgarten Louisa 
Published: November 2023

Abstract

BACKGROUND
Metastatic lung cancer is incurable once it spreads to the brain. Chimeric antigen receptor (CAR) T cells show promise in hematologic malignancies but face challenges in solid tumors, particularly in brain tumors, due to a highly immunosuppressive tumor microenvironment (TME), tumor antigen (TA) heterogeneity and impeded infiltration and persistence at the tumor site. To test the efficacy of CAR T cells and to find strategies for potentially enhancing its efficacy, CAR T cells were applied with and without checkpoint-blockade to treat lung cancer brain metastasis.

METHODS
We used an immunocompetent syngeneic orthotopic cerebral metastasis model combined with a chronic cranial window and repetitive intracerebral two-photon laser scanning microscopy (TPLSM). This allowed us to observe fluorescent tumor cells and CAR T cells in vivo at a single cell level over time. We injected red fluorescent EpCAM-transduced Lewis Lung carcinoma cells (EpCAM-LL/2tdT) into the brain tissue, followed by injection of EpCAM-directed or mock CAR T cells. Animals received anti-PD-1 and the respective isotype control via intraperitoneal injections.

RESULTS
EpCAM-directed CAR T cells were generated and showed substantial anti-tumor effects in vitro. Local injection into EpCAM-LL/2 tumor-bearing mice led to intratumoral accumulation of CAR T cells, resulting in reduced tumor growth compared to undirected CAR T cells translating into long-term survival in a fraction of animals. However, intratumoral CAR T cell numbers decrease over time pointing towards insufficient persistence. Interestingly, anti-PD-1 treatment did not enhance intratumoral CAR T accumulation, persistence and anti-tumor efficacy.

CONCLUSION
Our findings demonstrate the great potential of cell-based treatment approaches for the treatment of lung cancer brain metastases and underlines the necessity of further investigations to enhance infiltration and persistence in solid tumors.
EXTH-80. A SYSTEMIC COMPARISON OF CAR T CELLS, CAR NK CELLS AND CAR MACROPHAGES FOR GLIOMA TREATMENT
Authors: Look Thomas , Fazio Serena , Binder Niklas , Sentman Charles L , Roth Patrick , Arrieta Cesar Nombela , Sankowski Roman , Pascolo Steve , Weller Michael , Weiss Tobias 
Published: November 2023

Abstract
Adoptive transfer of engineered immune cells expressing chimeric antigen receptors (CAR) is an emerging cancer immunotherapy that is also being investigated for glioblastoma. Many separate studies focus on either CAR T or CAR NK cells, and CAR macrophages are in early stages of development. However, a systemic cross-comparison of engineered immune effector cells against solid tumors, including glioblastoma, is lacking. Here, we generated second-generation NKG2D CAR mouse and human T cells, NK cells and macrophages and characterized them in vitro in co-culture assays with glioma cells and in vivo in orthotopic glioma mouse models. In vitro, mouse and human CAR T cells and CAR NK cells exhibited the highest tumor-killing activity. In contrast, in vivo, only CAR T cells exhibited anti-tumor activity in two immunocompetent orthotopic glioma-bearing mouse models, whereas CAR NK cells and CAR macrophages failed to prolong survival. More in-depth characterizations using flow cytometry, in vivo imaging and 3D microscopy, revealed that CAR T cells had the best tumor migration properties upon i.v. administration with distinct spatial distribution within the tumor compared to CAR NK cells and CAR macrophages. To profile the effects on bystander immune cells within the tumor microenvironment, we employed flow cytometry and single-cell RNA sequencing. This revealed effector cell-specific effects on the tumor microenvironment and provided insights into why the effective in vitro activity did not translate to in vivo. To unleash the anti-tumor potential of CAR immune effector cells, we engineered them to co-express the pro-inflammatory cytokines IL-12/IFNα2. This improved the in vivo anti-tumor activity of all effector cells and led to the highest fraction of long-term surviving mice with CAR NK cells. This study offers the first cross-comparison of different CAR immune effector cells against gliomas, providing valuable insights for the design of future adoptive cell therapies targeting malignant brain tumors.
EXTH-84. INTRATUMORAL IL-12 DELIVERY VIA MESENCHYMAL STEM CELLS COMBINED WITH PD-1 BLOCKADE LEADS TO LONG-TERM ANTITUMOR IMMUNITY IN A MOUSE GLIOBLASTOMA MODEL
Authors: Ahn Stephen 
Published: November 2023

Abstract
Although PD-1 blockade is effective for treating several types of cancer, the efficacy of this agent in glioblastoma is largely limited. To overcome non-responders and the immunosuppressive tumor microenvironment, combinational immunotherapeutic strategies with anti-PD-1 need to be considered. Here, we developed IL-12-secreting mesenchymal stem cells (MSC_IL-12) with glioblastoma tropism and evaluated the therapeutic effects of anti-PD-1, MSC_IL-12, and their combination against glioblastoma using an immunocompetent mouse orthotopic model. Anti-PD-1 and MSC_IL-12 showed complete tumor remission in 25.0% (4/16) and 23.1% (3/13) of glioblastoma-implanted mice, respectively, and their combination yielded synergistic antitumor efficacy indicated by 50.0% (6/12) of complete tumor remission. Analyses of tumor-infiltrating lymphocytes using immunofluorescent imaging revealed that anti-PD-1 increased CD8+ T cells, while MSC_IL-12 led to infiltration of CD4+ T cells and NK cells. Both therapies reduced frequencies of Tregs. All these aspects observed in each monotherapy group were superimposed in the combination group. Notably, no tumor growth was observed upon rechallenge in cured mice, indicating long-term immunity against glioblastoma provoked by the therapies. Single-cell RNA-seq of mouse brains confirmed these results and revealed that the combined treatment led to immune-favorable tumor microenvironment–CD4+, CD8+ T cells, effector memory T cells, and activated microglia were increased, whereas exhausted T cells, Tregs, and M2 polarized microglia were reduced. Taken together, anti-PD-1 and MSC_IL-12 monotherapies show long-term therapeutic responses, and their combination further enhances antitumor efficacy against glioblastoma via inducing immune-favorable tumor microenvironment.