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INTRODUCTION: EGFRvIII, a constitutively active EGFR deletion driver mutation, is associated with poor long-term survival in glioblastoma (GB). The investigational vaccine rindopepimut consists of a peptide sequence unique to EGFRvIII conjugated to keyhole limpet hemocyanin (KLH), delivered intradermally with granulocyte macrophage colony-stimulating factor. Three phase II studies in newly diagnosed, resected, EGFRvIII+ GB demonstrated encouraging progression-free survival (PFS), overall survival (OS), and safety profile. Compassionate-use experience suggests that rindopepimut may also provide benefit in relapsed GB, particularly with agents such as bevacizumab (BV). METHODS: In the Phase II "ReACT" study, BV-naïve patients in 1st or 2nd relapse with EGFRvIII+ GB were randomly assigned 1:1 to BV plus double-blinded injection of rindopepimut or control (KLH). END POINTS: 6-month PFS (PFS6; primary), objective response rate (ORR), PFS, OS, and safety. RESULTS: Accrual is complete (n = 72); study follow-up continues (n = 30). Primary rindopepimut toxicity is grade 1 to 2 injection site reaction. For rindopepimut + BV vs KLH + BV (per investigator; RANO criteria): PFS6 = 27% (9/33) vs 11% (4/35) (P = .048, 1-side χ test); ORR = 24% (7/29) vs 17% (5/30). Central PFS/ORR assessment is underway. Median (95% CI) OS = 12.0 (9.7, -) vs 8.8 (6.8, 11.4) months (HR = 0.47 [0.25, 0.91]; P = .0208), with 8 vs 4 patients progression-free. OS analyses favor rindopepimut including when adjusted for various prognostic factors. Rindopepimut induced robust anti-EGFRvIII titers (1:12800-1:6553600) in 80% of patients. Rapid titer generation was associated with prolonged OS (HR = 0.47 [0.18, 1.27]; P = .128) within the rindopepimut arm, and was most frequent in patients with KPS ≥ 90 (odds ratio = 9.75; P = .007). Evaluation of humoral response quality and HLA typing vs outcome are underway. In an additional cohort of BV-exposed patients (n = 53), four patients experienced objective tumor response. CONCLUSION: These near-final data show that rindopepimut induces potent EGFRvIII-specific immune response and tumor regression, and appears to significantly prolong survival when administered with BV in patients with relapsed GB.

Background: The current study was a phase 1 clinical trial conducted with patients who had recurrent or progressive malignant glioma (MG). The trial was designed to determine the maximum tolerated dose (MTD) and toxicity of irinotecan (CPT-11) when administered with temozolomide (TMZ) and O(6)-benzylguanine (O(6)-BG).Methods: All 3 drugs, CPT-11, TMZ, and O(6)-BG, were administered on Day 1 of a 21-day treatment. First, patients were treated with a 1-hour bolus infusion of O(6)-BG at a dose of 120 mg/m(2) followed immediately by a 48-hour continuous infusion of O(6)-BG at a dose of 30 mg/m(2)/d. Second, within 60 minutes of the end of the 1-hour bolus infusion of O(6)-BG, TMZ was administered orally at a dose of 355 mg/m(2). Third, 1 hour after administration of TMZ, CPT-11 was infused over 90 minutes. Patients were accrued to 1 of 2 strata based on CYP3A1- and CYP3A4-inducing antiepileptic drug (EIAED) use; dose escalation was conducted independently within these strata.Results: Fifty-five patients were enrolled. In both strata, the dose-limiting toxicities were hematologic and included grade 4 neutropenia, febrile neutropenia, leukopenia, and/or thrombocytopenia. For Stratum 1 (EIAEDs), when TMZ was administered at a dose of 355 mg/m(2), the MTD of CPT-11 was determined to be 120 mg/m(2). In contrast, for Stratum 2 (no EIAEDs), when TMZ was administered at a dose of 200 mg/m(2), the MTD of CPT-11 was determined to be 80 mg/m(2).Conclusions: The authors believe that the results of the current study provide the foundation for a phase 2 trial of O(6)-BG in combination with CPT-11 and TMZ in patients with MG. [ABSTRACT FROM AUTHOR]

Despite remarkable advancements in imaging modalities and treatment options available to patients diagnosed with malignant brain tumors, the prognosis for those with high-grade lesions remains poor. The imprecise mechanisms of currently available treatments to manage these tumors do not spare damage to the normal surrounding brain and often result in major cognitive and motor deficits. Immunotherapy holds the promise of offering a potent, yet targeted, treatment to patients with brain tumors, with the potential to eradicate the malignant tumor cells without damaging normal tissues. The T cells of the immune system are uniquely capable of recognizing the altered protein expression patterns within tumor cells and mediating their destruction through a variety of effector mechanisms. Adoptive T-cell therapy is an attempt to harness and amplify the tumor-eradicating capacity of a patients' own T cells and then return these effectors to the patient in such a state that they effectively eliminate residual tumor. Although this approach is not new to the field of tumor immunology, new advancements in our understanding of T-cell activation and function and breakthroughs in tumor antigen discovery hold great promise for the translation of this modality into a clinical success. [ABSTRACT FROM AUTHOR]

Objective: Preoperative assessment is important for neurosurgical risk stratification, but the level of evidence for individual screening tests is low. In preoperative urinalysis (UA), testing may significantly increase costs and lead to inappropriate antibiotic treatment. We prospectively evaluated whether eliminating preoperative UA was noninferior to routine preoperative UA as measured by 30-day readmission for surgical site infection in adult elective neurosurgical procedures., Methods: A single-institution prospective, pragmatic study of patients receiving elective neurosurgical procedures from 2018 to 2020 was conducted. Patients were allocated based on same-day versus preoperative admission status. Rates of preoperative UA and subsequent wound infection were measured along with detailed demographic, surgical, and laboratory data., Results: The study included 879 patients. The most common types of surgery were cranial (54.7%), spine (17.4%), and stereotactic/functional (19.5%). No preoperative UA was performed in 315 patients, while 564 underwent UA. Of tested patients, 103 (18.3%) met criteria for suspected urinary tract infection, and 69 (12.2%) received subsequent antibiotic treatment. There were 14 patients readmitted within 30 days (7 without UA [2.2%] vs. 7 with UA [1.2%]) for subsequent wound infection with a risk difference of 0.98% (95% confidence interval -0.89% to 2.85%). The upper limit of the confidence interval exceeded the preselected noninferiority margin of 1%., Conclusions: In this prospective study of preoperative UA for elective neurosurgical procedures using a pragmatic, real-world design, risk of readmission due to surgical site infection was very low across the study cohort, suggesting a limited role of preoperative UA for elective neurosurgical procedures., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The accepted paradigm for both cellular and anti-tumor immunity relies upon tumor cell killing by CD8 + T cells recognizing cognate antigens presented in the context of target cell major histocompatibility complex (MHC) class I (MHC-I) molecules. Likewise, a classically described mechanism of tumor immune escape is tumor MHC-I downregulation. Here, we report that CD8 + T cells maintain the capacity to kill tumor cells that are entirely devoid of MHC-I expression. This capacity proves to be dependent instead on interactions between T cell natural killer group 2D (NKG2D) and tumor NKG2D ligands (NKG2DLs), the latter of which are highly expressed on MHC-loss variants. Necessarily, tumor cell killing in these instances is antigen independent, although prior T cell antigen-specific activation is required and can be furnished by myeloid cells or even neighboring MHC-replete tumor cells. In this manner, adaptive priming can beget innate killing. These mechanisms are active in vivo in mice as well as in vitro in human tumor systems and are obviated by NKG2D knockout or blockade. These studies challenge the long-advanced notion that downregulation of MHC-I is a viable means of tumor immune escape and instead identify the NKG2D-NKG2DL axis as a therapeutic target for enhancing T cell-dependent anti-tumor immunity against MHC-loss variants., (© 2023. The Author(s).)

Background: MDNA55 is an interleukin 4 receptor (IL4R)-targeting toxin in development for recurrent GBM, a universally fatal disease. IL4R is overexpressed in GBM as well as cells of the tumor microenvironment. High expression of IL4R is associated with poor clinical outcomes., Methods: MDNA55-05 is an open-label, single-arm phase IIb study of MDNA55 in recurrent GBM (rGBM) patients with an aggressive form of GBM (de novo GBM, IDH wild-type, and nonresectable at recurrence) on their 1st or 2nd recurrence. MDNA55 was administered intratumorally as a single dose treatment (dose range of 18 to 240 ug) using convection-enhanced delivery (CED) with up to 4 stereo-tactically placed catheters. It was co-infused with a contrast agent (Gd-DTPA, Magnevist®) to assess distribution in and around the tumor margins. The flow rate of each catheter did not exceed 10μL/min to ensure that the infusion duration did not exceed 48 h. The primary endpoint was mOS, with secondary endpoints determining the effects of IL4R status on mOS and PFS., Results: MDNA55 showed an acceptable safety profile at doses up to 240 μg. In all evaluable patients (n = 44) mOS was 11.64 months (80% one-sided CI 8.62, 15.02) and OS-12 was 46%. A subgroup (n = 32) consisting of IL4R High and IL4R Low patients treated with high-dose MDNA55 (>180 ug) showed the best benefit with mOS of 15 months, OS-12 of 55%. Based on mRANO criteria, tumor control was observed in 81% (26/32), including those patients who exhibited pseudo-progression (15/26)., Conclusions: MDNA55 demonstrated tumor control and promising survival and may benefit rGBM patients when treated at high-dose irrespective of IL4R expression level.Trial Registration: Clinicaltrials.gov NCT02858895., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Chimeric antigen receptor (CAR) T cell therapy in glioblastoma faces many challenges including insufficient CAR T cell abundance and antigen-negative tumor cells evading targeting. Unfortunately, preclinical studies evaluating CAR T cells in glioblastoma focus on tumor models that express a single antigen, use immunocompromised animals, and/or pre-treat with lymphodepleting agents. While lymphodepletion enhances CAR T cell efficacy, it diminishes the endogenous immune system that has the potential for tumor eradication. Here, we engineered CAR T cells to express IL7 and/or Flt3L in 50% EGFRvIII-positive and -negative orthotopic tumors pre-conditioned with non-lymphodepleting irradiation. IL7 and IL7 Flt3L CAR T cells increased intratumoral CAR T cell abundance seven days after treatment. IL7 co-expression with Flt3L modestly increased conventional dendritic cells as well as the CD103+XCR1+ population known to have migratory and antigen cross-presenting capabilities. Treatment with IL7 or IL7 Flt3L CAR T cells improved overall survival to 67% and 50%, respectively, compared to 9% survival with conventional or Flt3L CAR T cells. We concluded that CAR T cells modified to express IL7 enhanced CAR T cell abundance and improved overall survival in EGFRvIII heterogeneous tumors pre-conditioned with non-lymphodepleting irradiation. Potentially IL7 or IL7 Flt3L CAR T cells can provide new opportunities to combine CAR T cells with other immunotherapies for the treatment of glioblastoma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Swan, Mehta, Ilich, Shen, Wilkinson, Anderson, Segura, Sanchez-Perez, Sampson and Bellamkonda.)

Purpose: Hypofractionated stereotactic radiosurgery (HF-SRS) with or without surgical resection is potentially a preferred treatment for larger or symptomatic brain metastases (BMs). Herein, we report clinical outcomes and predictive factors following HF-SRS., Methods and Materials: Patients undergoing HF-SRS for intact (iHF-SRS) or resected (rHF-SRS) BMs from 2008 to 2018 were retrospectively identified. Linear accelerator-based image-guided HF-SRS consisted of 5 fractions at 5, 5.5, or 6 Gy per fraction. Time to local progression (LP), time to distant brain progression (DBP), and overall survival (OS) were calculated. Cox models assessed effect of clinical factors on OS. Fine and Gray's cumulative incidence model for competing events examined effect of factors on LP and DBP. The occurrence of leptomeningeal disease (LMD) was determined. Logistic regression examined predictors of LMD., Results: Among 445 patients, median age was 63.5 years; 87% had Karnofsky performance status ≥70. Fifty-three % of patients underwent surgical resection, and 75% received 5 Gy per fraction. Patients with resected BMs had higher Karnofsky performance status (90-100, 41 vs 30%), less extracranial disease (absent, 25 vs 13%), and fewer BMs (multiple, 32 vs 67%). Median diameter of the dominant BM was 3.0 cm (interquartile range, 1.8-3.6 cm) for intact BMs and 4.6 cm (interquartile range, 3.9-5.5 cm) for resected BMs. Median OS was 5.1 months (95% confidence interval [CI], 4.3-6.0) following iHF-SRS and 12.8 months (95% CI, 10.8-16.2) following rHF-SRS ( P < .01). Cumulative LP incidence was 14.5% at 18 months (95% CI, 11.4-18.0%), significantly associated with greater total GTV (hazard ratio, 1.12; 95% CI, 1.05-1.20) following iFR-SRS, and with recurrent versus newly diagnosed BMs across all patients (hazard ratio, 2.28; 95% CI, 1.01-5.15). Cumulative DBP incidence was significantly greater following rHF-SRS than iHF-SRS ( P  = .01), with respective 24-month rates of 50.0 (95% CI, 43.3-56.3) and 35.7% (95% CI, 29.2-42.2). LMD (57 events total; 33% nodular, 67% diffuse) was observed in 17.1% of rHF-SRS and 8.1% of iHF-SRS cases (odds ratio, 2.46; 95% CI, 1.34-4.53). Any radionecrosis and grade 2+ radionecrosis events were observed in 14 and 8% of cases, respectively., Conclusions: HF-SRS demonstrated favorable rates of LC and radionecrosis in postoperative and intact settings. Corresponding LMD and RN rates were comparable to those of other studies., (© 2022 The Authors.)

Background: Early hypotension after severe traumatic brain injury (sTBI) is associated with increased mortality and poor long-term outcomes. Current guidelines suggest the use of intravenous vasopressors, commonly norepinephrine and phenylephrine, to support blood pressure after TBI. However, guidelines do not specify vasopressor type, resulting in variation in clinical practice. We describe early vasopressor utilization patterns in critically ill patients with TBI and examine the association between utilization of norepinephrine, compared to phenylephrine, with hospital mortality after sTBI., Methods: We conducted a retrospective cohort study of US hospitals participating in the Premier Healthcare Database between 2009 and 2018. We examined adult patients (>17 years of age) with a primary diagnosis of sTBI who were treated in an intensive care unit (ICU) after injury. The primary exposure was vasopressor choice (phenylephrine versus norepinephrine) within the first 2 days of hospital admission. The primary outcome was in-hospital mortality. Secondary outcomes examined included hospital length of stay (LOS) and ICU LOS. We conducted a post hoc subgroup analysis in all patients with intracranial pressure (ICP) monitor placement. Regression analysis was used to assess differences in outcomes between patients exposed to phenylephrine versus norepinephrine, with propensity matching to address selection bias due to the nonrandom allocation of treatment groups., Results: From 2009 to 2018, 24,718 (37.1%) of 66,610 sTBI patients received vasopressors within the first 2 days of hospitalization. Among these patients, 60.6% (n = 14,991) received only phenylephrine, 10.8% (n = 2668) received only norepinephrine, 3.5% (n = 877) received other vasopressors, and 25.0% (n = 6182) received multiple vasopressors. In that time period, the use of all vasopressors after sTBI increased. A moderate degree of variation in vasopressor choice was explained at the individual hospital level (23.1%). In propensity-matched analysis, the use of norepinephrine compared to phenylephrine was associated with an increased risk of in-hospital mortality (OR, 1.65; CI, 1.46-1.86; P < .0001)., Conclusions: Early vasopressor utilization among critically ill patients with sTBI is common, increasing over the last decade, and varies across hospitals caring for TBI patients. Compared to phenylephrine, norepinephrine was associated with increased risk of in-hospital mortality in propensity-matched analysis. Given the wide variation in vasopressor utilization and possible differences in efficacy, our analysis suggests the need for randomized controlled trials to better inform vasopressor choice for patients with sTBI., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 International Anesthesia Research Society.)

Glioblastoma (GBM) is notorious for its immunosuppressive tumor microenvironment (TME) and is refractory to immune checkpoint blockade (ICB). Here, we identify calmodulin-dependent kinase kinase 2 (CaMKK2) as a driver of ICB resistance. CaMKK2 is highly expressed in pro-tumor cells and is associated with worsened survival in patients with GBM. Host CaMKK2, specifically, reduces survival and promotes ICB resistance. Multimodal profiling of the TME reveals that CaMKK2 is associated with several ICB resistance-associated immune phenotypes. CaMKK2 promotes exhaustion in CD8 + T cells and reduces the expansion of effector CD4 + T cells, additionally limiting their tumor penetrance. CaMKK2 also maintains myeloid cells in a disease-associated microglia-like phenotype. Lastly, neuronal CaMKK2 is required for maintaining the ICB resistance-associated myeloid phenotype, is deleterious to survival, and promotes ICB resistance. Our findings reveal CaMKK2 as a contributor to ICB resistance and identify neurons as a driver of immunotherapeutic resistance in GBM., (© 2022. The Author(s).)

Subunit vaccines inducing antibodies against tumor-specific antigens have yet to be clinically successful. Here, we use a supramolecular α-helical peptide nanofiber approach to design epitope-specific vaccines raising simultaneous B cell, CD8 + T cell, and CD4 + T cell responses against combinations of selected epitopes and show that the concurrent induction of these responses generates strong antitumor effects in mice, with significant improvements over antibody or CD8 + T cell-based vaccines alone, in both prophylactic and therapeutic subcutaneous melanoma models. Nanofiber vaccine-induced antibodies mediated in vitro tumoricidal antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). The addition of immune checkpoint and phagocytosis checkpoint blockade antibodies further improved the therapeutic effect of the nanofiber vaccines against murine melanoma. These findings highlight the potential clinical benefit of vaccine-induced antibody responses for tumor treatments, provided that they are accompanied by simultaneous CD8 + and CD4 + responses, and they illustrate a multiepitope cancer vaccine design approach using supramolecular nanomaterials.

Background: The phase 1 cohorts (1c+1d) of CheckMate 143 (NCT02017717) evaluated the safety/tolerability and efficacy of nivolumab plus radiotherapy (RT) ± temozolomide (TMZ) in newly diagnosed glioblastoma., Methods: In total, 136 patients were enrolled. In part A (safety lead-in), 31 patients ( n = 15, methylated/unknown MGMT promoter; n = 16, unmethylated MGMT promoter) received nivolumab and RT+TMZ (NIVO+RT+TMZ) and 30 patients with unmethylated MGMT promoter received NIVO+RT. In part B (expansion), patients with unmethylated MGMT promoter were randomized to NIVO+RT+TMZ ( n = 29) or NIVO+RT ( n = 30). Primary endpoint was safety/tolerability; secondary endpoint was overall survival (OS)., Results: NIVO+RT±TMZ was tolerable; grade 3/4 treatment-related adverse events occurred in 51.6% (NIVO+RT+TMZ) and 30.0% (NIVO+RT) of patients in part A and 46.4% (NIVO+RT+TMZ) and 28.6% (NIVO+RT) in part B. No new safety signals were detected. In part A, median OS (mOS) with NIVO+RT+TMZ was 33.38 months (95% CI, 16.2 to not estimable) in patients with methylated MGMT promoter. In patients with unmethylated MGMT promoter, mOS was 16.49 months (12.94-22.08) with NIVO+RT+TMZ and 14.41 months (12.55-17.31) with NIVO+RT. In part B, mOS was 14.75 months (10.01-18.6) with NIVO+RT+TMZ and 13.96 months (10.81-18.14) with NIVO+RT in patients with unmethylated MGMT promoter., Conclusions: CheckMate 143 was the first trial evaluating immune checkpoint inhibition with first-line treatment of glioblastoma. Results showed that NIVO can be safely combined with RT±TMZ, with no new safety signals. Toxicities, including lymphopenia, were more frequent with NIVO+RT+TMZ. OS was similar with or without TMZ in patients with unmethylated MGMT promoter, and differences by MGMT methylation status were observed., (© The Author(s) 2022. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Immunotherapy has revolutionized treatment for many hard-to-treat cancers but has yet to produce significant improvement in outcomes for patients with glioblastoma. This reflects the multiple and unique mechanisms of immune evasion and escape in this highly heterogeneous tumor. Glioblastoma engenders profound local and systemic immunosuppression and is remarkably effective at inducing T-cell dysfunction, posing a challenge to any immunotherapy-based approach. To overcome these mechanisms, multiple disparate modes of immune-oriented therapy will be required. However, designing trials that can evaluate these combinatorial approaches requires careful consideration. In this review, we explore the immunotherapy resistance mechanisms that have been encountered to date and how combinatorial approaches may address these. We also describe the unique aspects of trial design in both preclinical and clinical settings and consider endpoints and markers of response best suited for an intervention involving multiple agents., (©2021 The Authors; Published by the American Association for Cancer Research.)

Therapeutic advances for glioblastoma have been minimal over the past 2 decades. In light of the multitude of recent phase III trials that have failed to meet their primary endpoints following promising preclinical and early-phase programs, a Society for Neuro-Oncology Think Tank was held in November 2020 to prioritize areas for improvement in the conduct of glioblastoma clinical trials. Here, we review the literature, identify challenges related to clinical trial eligibility criteria and trial design in glioblastoma, and provide recommendations from the Think Tank. In addition, we provide a data-driven context with which to frame this discussion by analyzing key study design features of adult glioblastoma clinical trials listed on ClinicalTrials.gov as "recruiting" or "not yet recruiting" as of February 2021., (©2021 American Association for Cancer Research.)

Our group has employed methodologies for effective ex vivo generation of dendritic cell (DC) vaccines for patients with primary malignant brain tumors. In order to reliably produce the most potent, most representational vaccinated DC that will engender an antitumor response requires the ability to orchestrate multiple methodologies that address antigen cross-presentation, T-cell costimulation and polarization, and migratory capacity. In this chapter, we describe a novel method for augmenting the immunogenicity and migratory potential of DCs for their use as vaccines. We have elucidated methodologies to avoid the phenomenon known as immunodominance in generating cancer vaccines. We have found that culturing DC progenitors in serum-free conditions for the duration of the differentiation protocol results in a more homogeneously mature population of DCs that exhibit enhanced immunogenicity compared to DCs generated in serum-containing culture conditions. Furthermore, we demonstrate our method for generating high mobility DCs that readily migrate toward lymphoid organ chemoattractants using CCL3 protein. The combination of these two approaches represents a facile and clinically tractable methodology for generating highly mature DCs with excellent migratory capacity., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Here we present a 14-color flow cytometry panel for the evaluation of 13 myeloid and lymphoid populations within murine glioblastoma samples. Reagents, processing protocols, and downstream analyses were thoroughly validated and optimized to resolve the following populations: T cells (CD4, CD8, CD3), B cells (B220), NK cells (NK1.1), neutrophils (Ly6G), classical and non-classical monocytes (Ly6c, CD43), macrophages (F4/80, CD11b), microglia (CD45-lo, CD11b), and dendritic cells (DCs) (CD11c, MHC class II). In addition, this panel leaves Alexa Fluor 488/FITC open for the inclusion of fluorescent reporters or congenic marker staining., (Copyright © 2021. Published by Elsevier B.V.)

Glioblastoma is the the most common primary brain tumor in adults. Onset of disease is followed by a uniformly lethal prognosis and dismal overall survival. While immunotherapies have revolutionized treatment in other difficult-to-treat cancers, these have failed to demonstrate significant clinical benefit in patients with glioblastoma. Obstacles to success include the heterogeneous tumor microenvironment (TME), the immune-privileged intracranial space, the blood-brain barrier (BBB) and local and systemic immunosuppressions. Monoclonal antibody-based therapies have failed at least in part due to their inability to access the intracranial compartment. Bispecific T-cell engagers are promising antibody fragment-based therapies which can bring T cells close to their target and capture them with a high binding affinity. They can redirect the entire repertoire of T cells against tumor, independent of T-cell receptor specificity. However, the multiple challenges posed by the TME, immune privilege and the BBB suggest that a single agent approach may be insufficient to yield durable, long-lasting antitumor efficacy. In this review, we discuss the mechanism of action of T-cell engagers, their preclinical and clinical developments to date. We also draw comparisons with other classes of multispecific antibodies and potential combinations using these antibody fragment therapies., Competing Interests: Competing interests: KS, KLH, JMR, and AAM declare no conflicts of interest. JHS has an equity interest in Annias Immunotherapeutics, which has licensed intellectual property from Duke related to the use of the pepCMV vaccine in the treatment of glioblastoma multiforme. JHS has an equity interest in Istari Oncology, which has licensed intellectual property from Duke related to the use of poliovirus and D2C7 in the treatment of glioblastoma. JHS is an inventor on patents related to PEP-CMV DC vaccine with tetanus, as well as poliovirus vaccine and D2C7 in the treatment of glioblastoma. MK reports personal fees from Jackson Laboratory for Genomic Medicine and research funding from AbbVie and Bristol-Myers Squibb were paid to his institution for glioblastoma research., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Glioblastoma is an immunologically 'cold' tumor, which are characterized by absent or minimal numbers of tumor-infiltrating lymphocytes (TILs). For those tumors that have been invaded by lymphocytes, they are profoundly exhausted and ineffective. While many immunotherapy approaches seek to reinvigorate immune cells at the tumor, this requires TILs to be present. Therefore, to unleash the full potential of immunotherapy in glioblastoma, the trafficking of lymphocytes to the tumor is highly desirable. However, the process of T cell recruitment into the central nervous system (CNS) is tightly regulated. Naïve T cells may undergo an initial licensing process to enter the migratory phenotype necessary to enter the CNS. T cells then must express appropriate integrins and selectin ligands to interact with transmembrane proteins at the blood-brain barrier (BBB). Finally, they must interact with antigen-presenting cells and undergo further licensing to enter the parenchyma. These T cells must then navigate the tumor microenvironment, which is rich in immunosuppressive factors. Altered tumoral metabolism also interferes with T cell motility. In this review, we will describe these processes and their mediators, along with potential therapeutic approaches to enhance trafficking. We also discuss safety considerations for such approaches as well as potential counteragents.

Purpose: To examine the effectiveness and safety of single-isocenter multitarget stereotactic radiosurgery using a volume-adapted dosing strategy in patients with 4 to 10 brain metastases., Methods and Materials: Adult patients with 4 to 10 brain metastases were eligible for this prospective trial. The primary endpoint was overall survival. Secondary endpoints were local recurrence, distant brain failure, neurologic death, and rate of adverse events. Exploratory objectives were neurocognition, quality of life, dosimetric data, salvage rate, and radionecrosis. Dose was prescribed in a single fraction per RTOG 90-05 or as 5 Gy × 5 fractions for lesions ≥3 cm diameter, lesions involving critical structures, or single-fraction brain V 12Gy >20 mL., Results: Forty patients were treated with median age of 61 years, Karnofsky performance status 90, and 6 brain metastases. Twenty-two patients survived longer than expected from the time of protocol SRS, with 1 living patient who has not reached that milestone. Median overall survival was 8.1 months with a 1-year overall survival of 35.7%. The 1-year local recurrence rate was 5% (10 of 204 of evaluable lesions) in 12.5% (4 of 32) of the patients. Distant brain failure was observed in 19 of 32 patients with a 1-year rate of 35.8%. Grade 1-2 headache was the most common complaint, with no grade 3-5 treatment-related adverse events. Radionecrosis was observed in only 5 lesions, with a 1-year rate of 1.5%. Rate of neurologic death was 20%. Neurocognition and quality of life did not significantly change 3 months after SRS compared with pretreatment., Conclusions: These results suggest that volume-adapted dosing single-isocenter multitarget stereotactic radiosurgery is an effective and safe treatment for patients with 4 to 10 brain metastases., (© 2021 The Authors.)

We have only recently begun to understand how cancer metabolism affects antitumor responses and immunotherapy outcomes. Certain immunometabolic targets have been actively pursued in other tumor types, however, glioblastoma research has been slow to exploit the therapeutic vulnerabilities of immunometabolism. In this review, we highlight the pathways that are most relevant to glioblastoma and focus on how these immunometabolic pathways influence tumor growth and immune suppression. We discuss hypoxia, glycolysis, tryptophan metabolism, arginine metabolism, 2-Hydroxyglutarate (2HG) metabolism, adenosine metabolism, and altered phospholipid metabolism, in order to provide an analysis and overview of the field of glioblastoma immunometabolism., Competing Interests: PF reports consulting for Monteris Medical. JS has an equity interest in Istari Oncology, which has licensed intellectual property from Duke related to the use of poliovirus and D2C7 in the treatment of glioblastoma. JS is an inventor on patents related to PEP-CMV DC vaccine with tetanus, as well as poliovirus vaccine and D2C7 in the treatment of glioblastoma. JS has an equity interest in Annias Immunotherapeutics, which has licensed intellectual property from Duke related to the use of the pepCMV vaccine in the treatment of glioblastoma. MK reports advisory roles for Janssen, AbbVie, and Jackson Laboratory for Genomic Medicine, and research funding from AbbVie, Bristol-Myers Squibb, and Specialized Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mohan, Tomaszewski, Haskell-Mendoza, Hotchkiss, Singh, Reedy, Fecci, Sampson and Khasraw.)

Personalized cancer vaccines targeting neoantigens arising from somatic missense mutations are currently being evaluated for the treatment of various cancers due to their potential to elicit a multivalent, tumor-specific immune response. Several cancers express a low number of neoantigens; in these cases, ensuring the immunotherapeutic potential of each neoantigen-derived epitope (neoepitope) is crucial. In this study, we discovered that therapeutic vaccines targeting immunodominant major histocompatibility complex (MHC) I-restricted neoepitopes require a conjoined helper epitope in order to induce a cytotoxic, neoepitope-specific CD8+ T-cell response. Furthermore, we show that the universally immunogenic helper epitope P30 can fulfill this requisite helper function. Remarkably, conjoined P30 was able to unveil immune and antitumor responses to subdominant MHC I-restricted neoepitopes that were, otherwise, poorly immunogenic. Together, these data provide key insights into effective neoantigen vaccine design and demonstrate a translatable strategy using a universal helper epitope that can improve therapeutic responses to MHC I-restricted neoepitopes.

Several immunotherapy clinical trials in recurrent glioblastoma have reported long-term survival benefits in 10-20% of patients. Here we perform genomic analysis of tumor tissue from recurrent WHO grade IV glioblastoma patients acquired prior to immunotherapy intervention. We report that very low tumor mutation burden is associated with longer survival after recombinant polio virotherapy or after immune checkpoint blockade in recurrent glioblastoma patients. A relationship between tumor mutation burden and survival is not observed in cohorts of immunotherapy naïve newly diagnosed or recurrent glioblastoma patients. Transcriptomic analyses reveal an inverse relationship between tumor mutation burden and enrichment of inflammatory gene signatures in cohorts of recurrent, but not newly diagnosed glioblastoma tumors, implying that a relationship between tumor mutation burden and tumor-intrinsic inflammation evolves upon recurrence.

Introduction: Glioblastoma (GBM) has a survival rate of around 2 years with aggressive current standard of care. While other tumors have responded favorably to trials combining immunotherapy and chemotherapy, GBM remains uniformly deadly with minimal increases in overall survival. GBM differ from others due to being isolated behind the blood brain barrier, increased heterogeneity and mutational burden, and immunosuppression from the brain environment and tumor itself., Methods: We have reviewed clinical and preclinical studies investigating how different doses (dose intense (DI) and metronomic) and timing of immunotherapy following TMZ treatment can eradicate tumor cells, alter tumor mutational burden, and change immune cells., Results: Recent clinical trials with standard of care (SoC), DI and metronomic TMZ regimes are no able to completely eradicate GBM. Elevated TMZ levels in DI treatment can overcome MGMT resistance but may result in hypermutation of surviving tumor cells. Higher levels of TMZ will also generate a higher degree of lymphopenia compared to SoC and metronomic regimes in preclinical studies., Conclusion: The different levels of lymphopenia and tumor eradication discussed in this review suggest possible beneficial pairings between immunotherapy and TMZ treatment. Treatments resulting in profound lymphopenia will allow for expansion of vaccine specific T cells or of CAT T cells. Clinical and preclinical studies are currently comparing different combinations of TMZ and immunotherapy timing to treat GBM through a balance between tumor killing and immune cell expansion. More frequent immune monitoring time points in ongoing clinical trials are crucial for further development of these combinations.

Glioblastoma (WHO grade IV glioma) is the most common malignant primary brain tumor in adults. Survival has remained largely static for decades, despite significant efforts to develop new effective therapies. Immunotherapy and especially immune checkpoint inhibitors and programmed cell death (PD)-1/PD-L1 inhibitors have transformed the landscape of cancer treatment and improved patient survival in a number of different cancer types. With the exception of few select cases (e.g., patients with Lynch syndrome) the neuro-oncology community is still awaiting evidence that PD-1 blockade can lead to meaningful clinical benefit in glioblastoma. This lack of progress in the field is likely to be due to multiple reasons, including inherent challenges in brain tumor drug development, the blood-brain barrier, the unique immune environment in the brain, the impact of corticosteroids, as well as inter- and intratumoral heterogeneity. Here we critically review the clinical literature, address the unique aspects of glioma immunobiology and potential immunobiological barriers to progress, and contextualize new approaches to increase the efficacy of PD-1/PD-L1 inhibitors in glioblastoma that may identify gaps and testable relevant hypotheses for future basic and clinical research and to provide a novel perspective to further stimulate preclinical and clinical research to ultimately help patients with glioma, including glioblastoma, which is arguably one of the greatest areas of unmet need in cancer. Moving forward, we need to build on our existing knowledge by conducting further fundamental glioma immunobiology research in parallel with innovative and methodologically sound clinical trials., (©2020 American Association for Cancer Research.)

Despite standard of care for glioblastoma, including gross total resection, high-dose radiation, and dose-limited chemotherapy, this tumor remains one of the most aggressive and therapeutically challenging. The relatively small number of patients with this diagnosis compared with more common solid tumors in clinical trials commits new glioblastoma therapies to testing in small, underpowered, nonrandomized settings. Among approximately 200 registered glioblastoma trials identified between 2005 and 2015, nearly half were single-arm studies with sample sizes not exceeding 50 patients. These constraints have made demonstrating efficacy for novel therapies difficult in glioblastoma and other rare and aggressive cancers. Novel immunotherapies for glioblastoma such as vaccination with dendritic cells (DC) have yielded mixed results in clinical trials. To address limited numbers, we sequentially conducted three separate clinical trials utilizing cytomegalovirus (CMV)-specific DC vaccines in patients with newly diagnosed glioblastoma whereby each follow-up study had nearly doubled in sample size. Follow-up data from the first blinded, randomized phase II clinical trial (NCT00639639) revealed that nearly one third of this cohort is without tumor recurrence at 5 years from diagnosis. A second clinical trial (NCT00639639) resulted in a 36% survival rate at 5 years from diagnosis. Results of the first two-arm trial (NCT00639639) showed increased migration of the DC vaccine to draining lymph nodes, and this increased migration has been recapitulated in our larger confirmatory clinical study (NCT02366728). We have now observed that nearly one third of the glioblastoma study patient population receiving CMV-specific DC vaccines results in exceptional long-term survivors., (©2020 American Association for Cancer Research.)

Introduction: Despite maximal surgical resection and chemoradiation, glioblastoma (GBM) continues to be associated with significant morbidity and mortality. Novel therapeutic strategies are urgently needed. Given success in treating multiple other forms of cancer, checkpoint inhibitor immunotherapy remains foremost amongst novel therapeutic strategies that are currently under investigation., Areas Covered: Through a systematic review of both published literature and the latest preliminary data available from ongoing clinical studies, we provide an up-to-date discussion on the immune system in the CNS, a detailed mechanistic evaluation of checkpoint biology in the CNS along with evidence for disruption of these pathways in GBM, and a summary of available preclinical and clinical data for checkpoint blockade in GBM. We also include a discussion of novel, emerging targets for checkpoint blockade which may play an important role in GBM immunotherapy., Expert Opinion: Evidence indicates that while clinical success of checkpoint blockade for the treatment of GBM has been limited to date, through improved preclinical models, optimization in the context of standard of care therapies, assay standardization and harmonization, and combinatorial approaches which may include novel targets for checkpoint blockade, checkpoint inhibitor immunotherapy may yield a safe and effective therapeutic option for the treatment of GBM.

We recently reported the development of a fully-human, CD3-binding bispecific antibody for immunotherapy of malignant glioma. To translate this therapeutic (hEGFRvIII-CD3- bi-scFv) to clinical trials and to help further the translation of other similar CD3-binding therapeutics, some of which are associated with neurologic toxicities, we performed a good laboratory practice (GLP) toxicity study to assess for potential behavioral, chemical, hematologic, and pathologic toxicities including evaluation for experimental autoimmune encephalomyelitis (EAE). To perform this study, male and female C57/BL6 mice heterozygous for the human CD3 transgene (20/sex) were allocated to one of four designated groups. All animals were administered one dose level of hEGFRvIII-CD3 bi-scFv or vehicle control. Test groups were monitored for feed consumption, changes in body weight, and behavioral disturbances including signs of EAE. Urinalysis, hematologic, and clinical chemistry analysis were also performed. Vehicle and test chemical-treated groups were humanely euthanized 48 hours or 14 days following dose administration. Complete gross necropsy of all tissues was performed, and selected tissues plus all observed gross lesions were collected and evaluated for microscopic changes. This included hematoxylin-eosin histopathological evaluation and Fe-ECR staining for myelin sheath enumeration. There were no abnormal clinical observations or signs of EAE noted during the study. There were no statistical changes in food consumption, body weight gain, or final body weight among groups exposed to hEGFRvIII-CD3 bi-scFv compared to the control groups for the 2- and 14-day timepoints. There were statistical differences in some clinical chemistry, hematologic and urinalysis endpoints, primarily in the females at the 14-day timepoint (hematocrit, calcium, phosphorous, and total protein). No pathological findings related to hEGFRvIII-CD3 bi-scFv administration were observed. A number of gross and microscopic observations were noted but all were considered to be incidental background findings. The results of this study allow for further translation of this and other important CD3 modulating bispecific antibodies., Competing Interests: P.C. Gedeon and J.H. Sampson are listed as coinventors of patents regarding the use of fully human bispecific antibodies targeting EGFRvlll which belong to Duke University. This includes granted US patent #9,676,858 entitled “Human bispecific EGFRvIII antibody and CD3 engaging molecules”, granted US patent #10,053,514 entitled “Certain improved human bispecific EGFRvIII antibody engaging molecules”, and international extensions of these patents. Duke University entered a Research Support Services Agreement with Integrated Laboratory Systems, Inc. under which Integrated Laboratory Systems, Inc. functioned as a contractor for the sole purpose of assisting with the completion of the described study. Integrated Laboratory Systems, Inc. holds no stake in the described technology or study outcome. J.H. Sampson reports receiving commercial research grants from Annias and Istari; holds ownership interest (including patents) in Annias, Neuronium, Duke University, and Istari; is a consultant/advisory board member for Bristol Myers Squibb, Medicenna, Insera Health, and Annias. No potential conflicts of interest were disclosed by the other authors. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Several different self-assembling peptide systems that form nanofibers have been investigated as vaccine platforms, but design principles for adjusting the character of the immune responses they raise have yet to be well articulated. Here we compared the immune responses raised by two structurally dissimilar peptide nanofibers, one a β-sheet fibrillar system (Q11), and one an α-helical nanofiber system (Coil29), hypothesizing that integrated T-cell epitopes within the latter would promote T follicular helper (Tfh) cell engagement and lead to improved antibody titers and quality. Despite significantly different internal structures, nanofibers of the two peptides exhibited surprisingly similar nanoscale morphologies, and both were capable of raising strong antibody responses to conjugated peptide epitopes in mice without adjuvant. Both were minimally inflammatory, but as hypothesized Coil29 nanofibers elicited antibody responses with higher titers and avidities against a conjugated model epitope (OVA 323-339 ) and a candidate peptide epitope for vaccination against S. aureus. Subsequent investigation indicated that Coil29 nanofibers possessed internal CD4+ T cell epitopes: whereas Q11 nanofibers required co-assembly of additional CD4+ T cell epitopes to be immunogenic, Coil29 nanofibers did not. Coil29 nanofibers also raised stronger germinal center B cell responses and follicular helper T cell (Tfh) responses relative to Q11 nanofibers, likely facilitating the improvement of the antibody response. These findings illustrate design strategies for improving humoral responses raised by self-assembled peptide nanofibers.

Introduction : Glioblastoma (GBM) is a highly aggressive brain tumor and is one of the most lethal human cancers. Chimeric antigen receptor (CAR) T cell therapy has markedly improved survival in previously incurable disease; however, this vanguard treatment still faces challenges in GBM. Likewise, checkpoint blockade therapies have not enjoyed the same victories against GBM. As it becomes increasingly evident that a mono-therapeutic approach is unlikely to provide anti-tumor efficacy, there evolves a critical need for combined treatment strategies. Areas covered : This review highlights the clinical successes observed with CAR T cell therapy as well the current efforts to overcome its perceived limitations. The review also explores employed combinations of CAR T cell approaches with immune checkpoint blockade strategies, which aim to potentiate immunotherapeutic benefits while restricting the impact of tumor heterogeneity and T cell exhaustion. Expert opinion : Barriers such as tumor heterogeneity and T cell exhaustion have exposed the weaknesses of various mono-immunotherapeutic approaches to GBM, including CAR T cell and checkpoint blockade strategies. Combining these potentially complementary strategies, however, may proffer a rational means of mitigating these barriers and advancing therapeutic successes against GBM and other solid tumors.

Purpose: Rindopepimut is a vaccine targeting the tumor-specific EGF driver mutation, EGFRvIII. The ReACT study investigated whether the addition of rindopepimut to standard bevacizumab improved outcome for patients with relapsed, EGFRvIII-positive glioblastoma., Patients and Methods: In this double-blind, randomized, phase II study (NCT01498328) conducted at 26 hospitals in the United States, bevacizumab-naïve patients with recurrent EGFRvIII-positive glioblastoma were randomized to receive rindopepimut or a control injection of keyhole limpet hemocyanin, each concurrent with bevacizumab. The primary endpoint was 6-month progression-free survival (PFS6) by central review with a one-sided significance of 0.2., Results: Between May 2012 and 2014, 73 patients were randomized (36 rindopepimut, 37 control). Rindopepimut toxicity included transient, low-grade local reactions. As primary endpoint, PFS6 was 28% (10/36) for rindopepimut compared with 16% (6/37) for control ( P = 0.12, one-sided). Secondary and exploratory endpoints also favored the rindopepimut group including a statistically significant survival advantage [HR, 0.53; 95% confidence interval (CI), 0.32-0.88; two-sided log-rank P = 0.01], a higher ORR [30% (9/30) vs. 18% (6/34; P = 0.38)], median duration of response [7.8 months (95% CI, 3.5-22.2) vs. 5.6 (95% CI, 3.7-7.4)], and ability to discontinue steroids for ≥6 months [33% (6/18) vs. 0% (0/19)]. Eighty percent of rindopepimut-treated patients achieved robust anti-EGFRvIII titers (≥1:12,800), which were associated with prolonged survival (HR = 0.17; 95% CI, 0.07-0.45; P < 0.0001)., Conclusions: Our randomized trial supports the potential for targeted immunotherapy among patients with GBM, but the therapeutic benefit requires validation due to the small sample size and potential heterogeneity of bevacizumab response among recurrent patients with GBM. See related commentary by Wick and Wagener, p. 1535 ., (©2020 American Association for Cancer Research.)

Background: First-in-human (FIH) clinical trials require careful selection of a safe yet biologically relevant starting dose. Typically, such starting doses are selected based on toxicity studies in a pharmacologically relevant animal model. However, with the advent of target-specific and highly active immunotherapeutics, both the Food and Drug Administration and the European Medicines Agency have provided guidance that recommend determining a safe starting dose based on a minimum anticipated biological effect level (MABEL) approach., Methods: We recently developed a T cell activating bispecific antibody that effectively treats orthotopic patient-derived malignant glioma and syngeneic glioblastoma in mice (hEGFRvIII:CD3 bi-scFv). hEGFRvIII:CD3 bi-scFv is comprized of two single chain antibody fragments (bi-scFvs) that bind mutant epidermal growth factor receptor variant III (EGFRvIII), a mutation frequently seen in malignant glioma, and human CD3ε on T cells, respectively. In order to establish a FIH dose, we used a MABEL approach to select a safe starting dose for hEGFRvIII:CD3 bi-scFv, based on a combination of in vitro data, in vivo animal studies, and theoretical human receptor occupancy modeling., Results: Using the most conservative approach to the MABEL assessment, a dose of 57.4 ng hEGFRvIII:CD3 bi-scFv/kg body weight was selected as a safe starting dose for a FIH clinical study., Conclusions: The comparison of our MABEL-based starting dose to our in vivo efficacious dose and the theoretical human receptor occupancy strongly supports that our human starting dose of 57.4 ng hEGFRvIII:CD3 bi-scFv/patient kg will be safe., Competing Interests: Competing interests: JHS has an equity interest in Annias Immunotherapeutics, which has licensed intellectual property from Duke related to the use of the pepCMV vaccine in the treatment of glioblastoma multiforme. JHS has an equity interest in Istari Oncology, which has licensed intellectual property from Duke related to the use of poliovirus and D2C7 in the treatment of glioblastoma. JHS is an inventor on patents related to PEP-CMV DC vaccine with tetanus, as well as poliovirus vaccine and D2C7 in the treatment of glioblastoma., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Brain metastasis (BM), the most common adult brain tumor, develops in 20% to 40% of patients with late-stage cancer and traditionally are associated with a poor prognosis. The management of patients with BM has become increasingly complex because of new and emerging systemic therapies and advancements in radiation oncology and neurosurgery. Current therapies include stereotactic radiosurgery, whole-brain radiation therapy, surgical resection, laser-interstitial thermal therapy, systemic cytotoxic chemotherapy, targeted agents, and immune-checkpoint inhibitors. Determining the optimal treatment for a specific patient has become increasingly individualized, emphasizing the need for multidisciplinary discussions of patients with BM. Recognizing and addressing the sequelae of BMs and their treatment while maintaining quality of life and neurocognition is especially important because survival for patients with BMs has improved. The authors present current and emerging treatment options for patients with BM and suggest approaches for managing sequelae and disease recurrence., (© 2020 American Cancer Society.)

Efficacy of dendritic cell (DC) cancer vaccines is classically thought to depend on their antigen-presenting cell (APC) activity. Studies show, however, that DC vaccine priming of cytotoxic T lymphocytes (CTLs) requires the activity of endogenous DCs, suggesting that exogenous DCs stimulate antitumor immunity by transferring antigens (Ags) to endogenous DCs. Such Ag transfer functions are most commonly ascribed to monocytes, implying that undifferentiated monocytes would function equally well as a vaccine modality and need not be differentiated to DCs to be effective. Here, we used several murine cancer models to test the antitumor efficacy of undifferentiated monocytes loaded with protein or peptide Ag. Intravenously injected monocytes displayed antitumor activity superior to DC vaccines in several cancer models, including aggressive intracranial glioblastoma. Ag-loaded monocytes induced robust CTL responses via Ag transfer to splenic CD8+ DCs in a manner independent of monocyte APC activity. Ag transfer required cell-cell contact and the formation of connexin 43-containing gap junctions between monocytes and DCs. These findings demonstrate the existence of an efficient gap junction-mediated Ag transfer pathway between monocytes and CD8+ DCs and suggest that administration of tumor Ag-loaded undifferentiated monocytes may serve as a simple and efficacious immunotherapy for the treatment of human cancers.

Gliomas, the most common malignant primary brain tumours, remain universally lethal. Yet, seminal discoveries in the past 5 years have clarified the anatomy, genetics and function of the immune system within the central nervous system (CNS) and altered the paradigm for successful immunotherapy. The impact of standard therapies on the response to immunotherapy is now better understood, as well. This new knowledge has implications for a broad range of tumours that develop within the CNS. Nevertheless, the requirements for successful therapy remain effective delivery and target specificity, while the dramatic heterogeneity of malignant gliomas at the genetic and immunological levels remains a profound challenge.

The incidence of brain metastases is increasing as cancer therapies improve and patients live longer, providing new challenges to the multidisciplinary teams that care for these patients. Brain metastatic cancer cells possess unique characteristics that allow them to penetrate the blood-brain barrier, colonize the brain parenchyma, and persist in the intracranial environment. In addition, brain metastases subvert the innate and adaptive immune system, permitting evasion of the antitumor immune response. Better understanding of the above mechanisms will allow for development and delivery of more effective therapies for brain metastases. In this review, we outline the molecular mechanisms underlying development, survival, and immunosuppression of brain metastases. We also discuss current and emerging treatment strategies, including surgery, radiation, disease-specific and mutation-targeted systemic therapy, and immunotherapy., (©2019 American Association for Cancer Research.)

The last decade has seen a crescendo of FDA approvals for immunotherapies against solid tumors, yet glioblastoma remains a prominent holdout. Despite more than 4 decades of work with a wide range of immunotherapeutic modalities targeting glioblastoma, efficacy has been challenging to obtain. Earlier forms of immune-based platforms have now given way to more current approaches, including chimeric antigen receptor T-cells, personalized neoantigen vaccines, oncolytic viruses, and checkpoint blockade. The recent experiences with each, as well as the latest developments and anticipated challenges, are reviewed.

Bispecific single chain antibody fragments (bi-scFv) represent an emerging class of biotherapeutics. We recently developed a fully human bi-scFv (EGFRvIII:CD3 bi-scFv) with the goal of redirecting CD3-expressing T cells to recognize and destroy malignant, EGFRvIII-expressing glioma. In mice, we showed that EGFRvIII:CD3 bi-scFv effectively treats orthotopic patient-derived malignant glioma and syngeneic glioblastoma. Here, we developed a targeted assay for pharmacokinetic (PK) analysis of EGFRvIII:CD3 bi-scFv, a necessary step in the drug development process. Using microflow liquid chromatography coupled to a high resolution parallel reaction monitoring mass spectrometry, and data analysis in Skyline, we developed a bottom-up proteomic assay for quantification of EGFRvIII:CD3 bi-scFv in both plasma and whole blood. Importantly, a protein calibrator, along with stable isotope-labeled EGFRvIII:CD3 bi-scFv protein, were used for absolute quantification. A PK analysis in a CD3 humanized mouse revealed that EGFRvIII:CD3 bi-scFv in plasma and whole blood has an initial half-life of ∼8 min and a terminal half-life of ∼2.5 h. Our results establish a sensitive, high-throughput assay for direct quantification of EGFRvIII:CD3 bi-scFv without the need for immunoaffinity enrichment. Moreover, these pharmacokinetic parameters will guide drug optimization and dosing regimens in future IND-enabling and phase I studies of EGFRvIII:CD3 bi-scFv.