Parney, Ian F.

TPS2085 Background: Glioblastoma (GBM) is the most common and aggressive form of brain tumor in adults. Despite maximal surgical resection, irradiation, and chemotherapy, median overall survival (OS) remains at only 30 weeks for recurrent GBM (rGBM) patients, emphasizing the need for novel treatments. GBM tumors are immunologically ‘cold,’ in that they have an immune-suppressive microenvironment with a low number of tumor-infiltrating lymphocytes (TIL). Interleukin 7 (IL-7) plays a key role in T cell development and survival. The physiological level of IL-7 increases to stimulate T cell expansion in lymphopenic condition. Previous studies have shown that lymphopenic GBM patients have shorter survival. However, their IL-7 levels are unexpectedly low. Efineptakin alfa (NT-I7), a long-acting IL-7, has been shown to increase effector CD8 T cells and reduce suppressor T-regulatory cells in murine GBM models, with subsequently improved OS. Further, we recently completed a Phase I study which showed that NT-I7 is safe, well- tolerated, and increased absolute lymphocyte counts (ALC) in newly diagnosed GBM patients. Phase II dosing was determined through this Phase I study reaching maximum tolerated dose. Thus, we hypothesized that NT-I7 may reverse GBM-associated immune suppression and potentiate the effect of an immune checkpoint inhibitor, leading to an increase in TILs and improved survival. Our preliminary data suggests that combining NT-I7 with pembrolizumab, an anti-PD-1 antibody, improves survival in murine glioma models. The purpose of this study is to evaluate the safety and efficacy of combining NT-I7 and pembrolizumab in rGBM patients. Methods: This is an open-label single arm one-stage Phase II study of NT-I7 and pembrolizumab as neoadjuvant and adjuvant therapy with surgery for adult patients with rGBM. We plan to enroll 30 evaluable patients, including a 6-patient safety run-in. Key eligibility criteria include candidates willing to undergo clinically indicated resection or biopsy, age ≥ 18 years, Karnofsky performance status ≥ 70, and dexamethasone dose > 2 mg/day ≤ 2 days prior to registration. The primary endpoint is a 9-month OS rate. Secondary endpoints include progression-free survival, objective response rate, changes in ALC, and adverse events profile. The correlative endpoints include studies to assess the anti-glioma immune response, changes in T cell subsets, T cell effector function, cytokine analysis, and immunohistochemical analysis of GBM tissue. Longitudinal cerebrospinal fluid proteomics is optional. This study opened on 1/20/2023. Two of the planned 30 patients had been enrolled by submission on 2/14/2023. Clinical trial information: NCT05465954 .

TPS2081 Background: Murine double minute 2 (MDM2) directly regulates the stability of the tumor suppressor p53, and dysregulation of this pathway can critically disrupt cellular response to genomic stress and lead to cell death. BI 907828 is a highly potent inhibitor of the MDM2-p53 interface that triggers accumulation of p53 and subsequent apoptosis in multiple GBM patient-derived xenografts (PDXs) with wild-type p53. Moreover, the combination of BI 907828 and radiotherapy provides significant survival extension in multiple orthotopic GBM PDXs. Methods: An integrated Phase 0/1 trial design is being used to evaluate the potential for BI 907828 to achieve a therapeutic concentration in brain tumor tissue and to test the safety of the combination with radiation. The co-primary endpoints for the Phase 0 study are i) measured total concentration of BI 907828 and ii) calculated unbound concentration of BI 907828 in contrast and non-contrast enhancing regions of tumor. Key inclusion criteria for the Phase 0 study are patients at least 18 years of age, ECOG performance status of 0 or 1, histologic or radiologic new diagnosis of GBM, and planned surgical resection. The first approximately 6 patients enrolled in the Phase 0 will be treated at the starting dose of 30 mg, and the future patients enrolled will be dosed at higher levels (45 mg or 60 mg) provided there are no safety concerns. If dose-limiting toxicities (DLTs) are encountered, then a Bayesian Logistic Regression Model (BLRM) with overdose control will be used to guide the dose level decisions for the additional patients. Multiple, image-registered tumor samples and intra-operative plasma samples will be analyzed for BI 907828 by LC-MS/MS. Intra-tumoral variation in BI 907828 will be correlated with pharmacodynamic effects of MDM2 inhibition (elevation of p53, p21, MDM2, GDF15, PUMA). Those patients completing the Phase 0 study and who meet additional eligibility criteria can enroll on the Phase 1a portion of the study. The pathologic inclusion criteria are TP53 wild-type, IDH1/2 wild-type, MGMT unmethylated GBM. Co-primary endpoints for the Phase 1a trial are i) occurrence of DLTs during and within 21 days after completion of radiation therapy, and ii) occurrence of adverse events throughout the treatment period. Tolerability of inter-patient dose-escalation of BI 907828 (30, 45, 60 mg) combined with radiation therapy (60 Gy in 30 fractions) will be evaluated with dose-escalation guided by a two-parameter BLRM with overdose control. Patients will remain on BI 907828 monotherapy following completion of radiotherapy until progression. To date, two patients have enrolled on the Phase 0 study, and one patient went on to enroll on the Phase 1a portion of the study. Clinical trial information: NCT05376800 .

2062 Background: While cancer therapeutics have made tremendous progress within the past several decades, this benefit has not been seen in many primary cancers of the brain. Particularly confounding have been high-grade gliomas (HGG), which retain a dismal prognosis. Currently, novel therapies are being explored to rise to this unmet, critical need. One such therapy are CAR T cells, immune cells which have been engineered to target malignancy-specific antigens. Unfortunately, the efficacy of CAR T cell therapies against solid tumors is significantly limited, in large part due to impaired expansion/persistence in the immune suppressive tumor microenvironment (TME). Here we show that in vivo reactivation of CAR T cells through their native T Cell Receptor (TCR) by an oncolytic virus (OV) has therapeutic benefit in HGG. Methods: An EGFRvIII third-generation MSGV1 retroviral CAR construct containing the CD28, 4-1BB, and CD3z moieties, in tandem with the scFv derived from the human monoclonal antibody 139 and the marker Thy1.1 (38) was used to generate our CAR T cells. C57Bl/6 mice were used for in vivo experiments and both B16- and CT2A-EGFRvIII murine glioma cell lines were injected in the brain to model HGG. OVs and CAR T cells were given systemically by tail vein. OVs used include reovirus, vesicular stomatitis virus, and adenovirus. Results: By using OV in combination with EGFRvIII CAR T cells, we were able to generate a CD8 CAR population with TCR specificity for both the EGFRvIII and OV epitopes. These dual-specific (DS) CAR T expressed a memory phenotype and persisted for much longer than conventional CAR T cells. Further, we showed that these DS CAR T cells are more cytotoxic and can respond more rapidly than their conventional counterparts. We created a novel delivery mechanism for this combination OV + CAR T therapy using virus-loaded CAR T cells to bypass initial antiviral clearance from the immune system. Treatment with these OV-loaded CAR T cells lead to significant benefit in mice with HGG tumors which could be further enhanced by a systemic boost with OV, which rapidly re-activated DS CAR T cells against tumor and resulted in long-term cures of greater than 80% of treated animals. Conclusions: These promising results show that DS CAR T cells can overcome the critical therapeutic challenge of CAR T as a treatment for solid tumors. Given these promising results, we will go on to develop a clinical trial in which CAR T cells will be pre-loaded with OV and administered intravenously to patients with HGG, followed by systemic boosting with virus to re-activate DS CAR T cells against their tumor.

Abstract Glioblastoma (GBM), a lethal astrocytic brain tumor, has limited available treatment options. A major hindrance to the progress of new therapies is the selectivity of the blood brain barrier (BBB) which limits drug access to the tumor. Further, there are currently no minimally-invasive tests that can quickly and accurately determine whether novel therapeutics have crossed the BBB during clinical trials. Therefore, development of a non-invasive test that can identify drug delivery and target engagement in GBM represents an opportunity to enhance knowledge during early phase trials of a new compound’s pharmacological suitability and potential efficacy. This study proposes that cargo contained in GBM-derived extracellular vesicles (EVs), which are small membrane-bound particles released from cells, may serve as one such pharmacodynamic reporter. We analyzed RNA-seq data for EVs isolated from two GBM patient-derived xenograft models grown as short-term in vitro cultures, GBM10 and GBM120, following treatment with either temozolomide (TMZ) or pevonedistat (MLN). We assessed whether EV RNA payloads vary with different drug concentrations (EC50 vs EC80) and at different time points (4hr vs 24hr) post treatment. In the samples treated with MLN, a few long non-coding RNAs are commonly upregulated across the different time points and two models. Under different TMZ concentrations, differentially expressed genes are overrepresented in biological processes such as cytoplasmic translation and cellular macromolecule biosynthesis. We also found a negative correlation between the TMZ concentration and Y RNA expression level, which are functionally involved with DNA replication. Additionally, using CIRI2, we predicted the amount of circular RNAs in the EV cargo; we found that the ratio of circular RNAs to their linear counterparts (CLR) varies under different treatments. In GBM10, the percentage of circRNA with CLR > 1 decreases with increasing drug effect. Thus, our preliminary results indicate that EV cargo post-treatment may be a novel pharmacodynamic reporter in GBM. To apply such an assessment to patients, we have simultaneously pursued the development of an immunoprecipitation (IP) approach to enrich GBM-derived EVs that can then undergo cargo profiling. Potential biomarkers were screened across isolated GBM EVs from in vitro supernatant by western blot and ExoView analysis. B7H3 has emerged as a lead candidate for GBM-EV enrichment. To validate the ability of EV cargo to reflect drug distribution across the BBB in patients, current efforts are focused on applying IP enrichment of GBM-EVs in longitudinally-acquired cerebrospinal fluid samples from patients during their disease course. Overall, we anticipate that the results of this study will lead to development of a clinical test that reflects BBB penetration and tumor response, which will likely aid in novel drug development efforts. Citation Format: Valerie DeLuca, Nanyun Tang, Yue Hao, Anjali Raju, Charles Shaffer, Cecile Riviere-Cazaux, Terry C. Burns, Jann Sarkaria, Ian Parney, Patrick Pirrotte, Kendall Van Keuren-Jensen, Michael E. Berens. Extracellular vesicles as a pharmacodynamic reporter in glioblastoma [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 2788.

Glioblastoma (GBM) is the most common malignant primary brain tumor and confers a dismal prognosis. With only two FDA-approved therapeutics showing modest survival gains since 2005, there is a great need for the development of other disease-targeted therapies. Due, in part, to the profound immunosuppressive microenvironment seen in GBMs, there has been a broad interest in immunotherapy. In both GBMs and other cancers, therapeutic vaccines have generally yielded limited efficacy, despite their theoretical basis. However, recent results from the DCVax-L trial provide some promise for vaccine therapy in GBMs. There is also the potential that future combination therapies with vaccines and adjuvant immunomodulating agents may greatly enhance antitumor immune responses. Clinicians must remain open to novel therapeutic strategies, such as vaccinations, and carefully await the results of ongoing and future trials. In this review of GBM management, the promise and challenges of immunotherapy with a focus on therapeutic vaccinations are discussed. Additionally, adjuvant therapies, logistical considerations, and future directions are discussed.

Abstract Glioblastoma (GBM) is the most common malignant brain tumor in adults, responsible for approximately 225,000 deaths per year. Despite preclinical successes, most interventions have failed to extend patient survival by more than a few months. Treatment with anti—programmed cell death protein 1 (anti–PD-1) immune checkpoint blockade (ICB) monotherapy has been beneficial for malignant tumors such as melanoma and lung cancers but has yet to be effectively employed in GBM. This study aimed to determine whether supplementing anti–PD-1 ICB with engineered extended half-life IL2, a potent lymphoproliferative cytokine, could improve outcomes. This combination therapy, subsequently referred to as enhanced checkpoint blockade (ECB), delivered intraperitoneally, reliably cures approximately 50% of C57BL/6 mice bearing orthotopic GL261 gliomas and extends median survival of the treated cohort. In the CT2A model, characterized as being resistant to CBI, ECB caused a decrease in CT2A tumor volume in half of measured animals similar to what was observed in GL261-bearing mice, promoting a trending survival increase. ECB generates robust immunologic responses, features of which include secondary lymphoid organ enlargement and increased activation status of both CD4 and CD8 T cells. This immunity is durable, with long-term ECB survivors able to resist GL261 rechallenge. Through employment of depletion strategies, ECB's efficacy was shown to be independent of host MHC class I–restricted antigen presentation but reliant on CD4 T cells. These results demonstrate ECB is efficacious against the GL261 glioma model through an MHC class I–independent mechanism and supporting further investigation into IL2-supplemented ICB therapies for tumors of the central nervous system.

Extracellular vesicles (EVs) are membrane-bound particles released by all cells. Previous research has found that these microscopic vesicles contribute to intercellular signaling and communication. EVs carry a variety of cargo, including nucleic acids, proteins, metabolites, and lipids. The composition of EVs varies based on cell of origin. Therefore, EVs can serve as an important biomarker in the diagnosis and treatment of various cancers. EVs derived from glioblastoma (GBM) cells carry biomarkers, which could serve as the basis for a potential diagnostic strategy known as liquid biopsy. Multiple EV isolation techniques exist, including ultrafiltration, size exclusion chromatography, flow field-flow fractionation, sequential filtration, differential ultracentrifugation, and density-gradient ultracentrifugation. Recent and ongoing work aims to identify cellular markers to distinguish GBM-derived EVs from those released by noncancerous cells. Strategies include proteomic analysis of GBM EVs, identification of GBM-specific metabolites, and use of Food and Drug Administration-approved 5-aminolevulinic acid—an oral agent that causes fluorescence of GBM cells—to recognize GBM EVs in a patient's blood. In addition, accurately and precisely monitoring changes in EV cargo concentrations could help differentiate between pseudoprogression and GBM recurrence, thus preventing unnecessary surgical interventions.

The NCCN Guidelines for Central Nervous System (CNS) Cancers focus on management of the following adult CNS cancers: glioma (WHO grade 1, WHO grade 2–3 oligodendroglioma [1p19q codeleted, IDH- mutant], WHO grade 2–4 IDH- mutant astrocytoma, WHO grade 4 glioblastoma), intracranial and spinal ependymomas, medulloblastoma, limited and extensive brain metastases, leptomeningeal metastases, non–AIDS-related primary CNS lymphomas, metastatic spine tumors, meningiomas, and primary spinal cord tumors. The information contained in the algorithms and principles of management sections in the NCCN Guidelines for CNS Cancers are designed to help clinicians navigate through the complex management of patients with CNS tumors. Several important principles guide surgical management and treatment with radiotherapy and systemic therapy for adults with brain tumors. The NCCN CNS Cancers Panel meets at least annually to review comments from reviewers within their institutions, examine relevant new data from publications and abstracts, and reevaluate and update their recommendations. These NCCN Guidelines Insights summarize the panel’s most recent recommendations regarding molecular profiling of gliomas.

Abstract Glioblastoma multiforme (GBM) is a deadly CNS malignancy with an average survival of ~12–18 months post diagnosis. The unique immune environment of the brain and the immunosuppressive features of GBM complicate the use of immunotherapies against these tumors. We aimed to promote the efficacy of αPD-1 against the GL261 murine model of GBM by supplementing this therapy with an engineered IL-2 cytokine. IL-2 therapy has been used clinically for metastatic melanoma and renal cancer, but its short half-life in circulation necessitated high doses to keep the drug concentration sufficiently high. Fusing IL-2 to the mouse serum albumin molecule (MSA-IL-2), extends the cytokine’s half-life. Neither αPD-1 nor MSA-IL-2 are effective monotherapies for GL261 tumors, but the combination provides durable tumor clearance and prevents subsequent rechallenge. Most strikingly, this combination therapy cleared established GL261 tumors even in mice incapable of MHC class I restricted antigen presentation. Instead, therapeutic efficacy was abrogated by the depletion of CD4 T cells. Our combination therapy also reversed multiple key features of GBM-associated peripheral immune suppression, including depressed CD4 T cell counts and dampened MHC class II expression. Our combination therapy’s ability to overcome these immune derangements associated with glioma growth and to function independent of MHC class I restricted antigen presentation enhance the appeal of this therapy for future translational adaptation. This work was supported by the National Institutes of Health (R01 NS103212) and the Mayo Clinic-Koch Institute Cancer Solutions Team Grant

Abstract Glioblastoma is the most common and aggressive primary brain tumor. Median survival is 15 months despite surgery, radiation, and chemotherapy. Immunotherapy is promising but glioblastoma-mediated immunosuppression remains a barrier. Recently, extracellular vesicles (EVs) have been implicated in glioblastoma-mediated immunosuppression through expression of the immune checkpoint molecules PD-L1 and IDO1 which have been implicated in T cell function suppression and decreased proliferation in some cancers. Our data demonstrate that PD-L1 and IDO1 expression are increased in most nucleated cells following IFN-γ exposure. IFN-γ exposure did not alter EV production or expression of common EV markers but increased PD-L1 and IDO1 expression in EVs from differentiated but not stem-like GBM cells. Differentiated glioblastoma cell derived EVs induced myeloid-derived suppressor cell and non-classical monocyte differentiation from normal monocytes. This was increased following IFN-γ exposure and was dependent upon both PD-L1 and IDO1 expression but not stem-like GBM cell. Monocytes exposed to differentiated but not stem-like GBM cell EVs inhibited T cell proliferation in a similar manner (increased with IFN-γ exposure, decreased with PD-L1 or IDO1 knockdown). Thus, IFN-γ exposure results in super-induction of immunosuppressive EVs from differentiated but not stem-like GBM cells that increase MDSC and NCM differentiation in normal monocytes and increase their ability to inhibit T cell proliferation. These effects are dependent upon PD-L1 and IDO1 up-regulation induced by IFN-γ. This may be an important mechanism GBMs utilize to suppress anti-tumor T cell responses that are typically accompanied by increased IFN-γ expression.