Anti-PD-1 immunotherapy is routinely used to treat advanced melanoma cancer despite producing therapeutic benefits in less than half of patients. Given that this treatment can be associated with severe adverse effects, researchers are working to develop biomarker-based tests to predict the likely response to treatment. This study aimed to qualitatively explore patients’ views on the value of such tests and how they would affect their treatment decision-making. Semi-structured interviews were conducted with melanoma patients commencing PD-1 inhibitor therapy (n = 14), who were presented with a hypothetical scenario in which a blood-based biomarker prognostic test indicated the likely or unlikely success of anti-PD-1 immunotherapy. Thematic analysis identified three major themes. (1) “More knowledge is more power”: Perceived value and benefits of the test—all participants reported that such a test would be valuable, regardless of the outcome, as it would provide them with an expectation of treatment success and assist them with their treatment decision-making. (2) “It depends”: Treatment decisions shaped by test outcome—participants described how the test outcome would affect their decision to undergo PD-1 inhibitor therapy. (3) “Faith in my doctor”: Reliance on doctor’s guidance for treatment decisions—participants stated that their treating doctor’s opinion on their test outcome and subsequent advice would ultimately guide their treatment decision. Melanoma patients see value in a test to predict anti-PD-1 immunotherapy response. An important consideration when communicating the outcome of this test is ensuring patients can discuss alternative treatment options and their recommendations for the best treatment option with their treating doctor.

We demonstrate multiple DC defects in patients with brain tumors. This includes a profound reduction in the frequency of multiple DC subsets, diminished activation marker expression, and reduced Flt3L levels in cancer patients with brain tumors compared to those without. We also demonstrate reduced intra-tumoral DCs in brain compared to lung tumors. This is the first time DC subsets have been fully characterized in a range of brain tumor patients. Importantly, corticosteroid usage was closely associated with DC defects, highlighting the adverse effects of a standard symptomatic treatment on these critical immune cells. However, tumors located within the brain also directly contribute to DC defects. Finally, we identified several mouse brain tumor models that replicate key observations in patients and may be used to further understand this endogenous DC deficiency and to develop approaches to restore DCs, ultimately leading to new combination immunotherapies for the treatment of brain cancers.

Abstract To facilitate survival, migration and evasion of immune surveillance, cancer cells tightly coordinate the synthesis and trafficking of a diverse repertoire of proteins to their cell surface and the surrounding tumor microenvironment. A key mechanism underlying this process is the intracellular membrane trafficking pathways, including vesicular transport systems. There remains a paucity of mechanistic insight into the regulatory components that mediate nascent protein trafficking and their dysregulation in cancer. Herein, we investigate Tumor Protein D54 (TPD54) as a central regulator of intracellular protein transport that is exploited by melanoma cells to promote disease progression. Integrative analyses of patient-derived tumor tissue specimens show that the expression of TPD52L2 (the gene encoding TPD54) is frequently overexpressed in melanoma and correlates with adverse clinical outcomes, including reduced responses to immune checkpoint blockade. Mechanistic investigations further revealed that TPD54 maintains Golgi integrity and orchestrates trafficking of early endosomes, anterograde vesicles and extracellular vesicles. Functionally, TPD54 augments the secretion of pro-cancerous cytokines, increases the cell surface expression of adhesion-signaling receptors (e.g. integrin-β1 and desmoglein-2), promotes melanoma cell migration and elevates their capability to undergo vasculogenic mimicry. Targeting TPD52L2 in two mouse models of melanoma (B16-F10 and HCmel12) showed significant attenuation of tumor growth, disrupted tumor vasculature, enhanced anti-tumor immunity with infiltration of CD8 + T cells and reduced metastatic disease. Collectively, these findings establish TPD54 as a critical and previously underappreciated regulator of protein trafficking in cancer cells that directly contributes to disease progression and highlights its potential as a novel therapeutic target to combat melanoma.

Abstract Objectives Glioblastoma is characterised by poor survival with few treatment advances for over 20 years. Immunotherapies, which have transformed treatment of other cancers, have been difficult to apply to glioblastoma because of its highly immune‐suppressive microenvironment, and there is a growing appreciation that the disease also induces a global suppression of the systemic immune system. However, immunotherapy research is hampered by the lack of detailed characterisation of the whole immune system in murine brain tumor models, with studies until now having focussed mainly on tumor‐infiltrating immune cells. Methods Here, we have studied common murine models of intracranial brain tumors by using high‐parameter flow cytometry and tissue immunofluorescence staining to fully characterise immune cells in the brain, draining lymph node, spleen and bone marrow. Results The highly immune‐compromised NSG mouse models were striking in their many immune perturbations, which extended beyond the known deficits in lymphocytes. Immunocompetent tumor models had significant changes in brain‐resident immune cells compared to controls, as expected; however, systemic effects were also observed with significant reductions in subsets of monocytes, macrophages and dendritic cells in the spleen and increases in bone marrow. Conclusion Our extensive and quantitative characterisation of the immune system in murine models of glioblastoma will allow for a better‐informed selection of models and advance the search for new immune‐based treatments for this deadly disease.

Abstract γδ T cells are a unique subset of T lymphocytes, exhibiting features of both innate and adaptive immune cells and are involved with cancer immunosurveillance. They present an attractive alternative to conventional T cell‐based immunotherapy due, in large part, to their lack of major histocompatibility (MHC) restriction and ability to secrete high levels of cytokines with well‐known anti‐tumour functions. To date, clinical trials using γδ T cell‐based immunotherapy for a range of haematological and solid cancers have yielded limited success compared with in vitro studies. This inability to translate the efficacy of γδ T‐cell therapies from preclinical to clinical trials is attributed to a combination of several factors, e.g. γδ T‐cell agonists that are commonly used to stimulate populations of these cells have limited cellular uptake yet rely on intracellular mechanisms; administered γδ T cells display low levels of tumour‐infiltration; and there is a gap in the understanding of γδ T‐cell inhibitory receptors. This review explores the discrepancy between γδ T‐cell clinical and preclinical performance and offers viable avenues to overcome these obstacles. Using more direct γδ T‐cell agonists, encapsulating these agonists into lipid nanocarriers to improve their pharmacokinetic and pharmacodynamic profiles and the use of combination therapies to overcome checkpoint inhibition and T‐cell exhaustion are ways to bridge the gap between preclinical and clinical success. Given the ability to overcome these limitations, the development of a more targeted γδ T‐cell agonist‐checkpoint blockade combination therapy has the potential for success in clinical trials which has to date remained elusive.

BackgroundHigh-grade gliomas including glioblastoma (GBM) and diffuse midline gliomas (DMG) represent the most lethal and aggressive brain cancers where current treatment modalities offer limited efficacy. Chimeric antigen receptor (CAR) T cell therapies have emerged as a promising strategy, boasting tumor-specific targeting and the unique ability to penetrate the blood-brain barrier. However, the effective clinical application hinges on the optimal choice of antigen, with a limited number, currently under investigation.MethodsWe employed cell surface proteomic analysis of primary human high-grade glioma samples from both adult and pediatric patients. This led to the identification of Ephrin type-A receptor 3 (EphA3) as a prevalently expressed target. We engineered a second-generation EphA3-targeted CAR T cell and assessed function using in vitro and in vivo models of GBM and DMG.ResultsEphA3-targeted CAR T cells demonstrated robust antigen-specific killing of human GBM and DMG cell lines in vitro. In an orthotopic xenograft NSG mouse model, EphA3-targeted CAR T cells not only effectively eradicated tumors but also established a functional T cell population protective on rechallenge. Remarkably, mice rechallenged with a second contralateral orthotopic tumor implantation achieved complete tumor clearance and maintained a sustained complete response 6 months following initial treatment.ConclusionBuilding on the proven safety profile of EphA3 antibodies in clinical settings, our study provides compelling preclinical evidence supporting the efficacy of EphA3-targeted CAR T cells against high-grade gliomas. These findings underscore the potential for transitioning this innovative therapy into clinical trials, aiming to revolutionize the treatment landscape for patients afflicted with these formidable brain cancers.

A new study by Yamada-Hunter et al. reveals a novel approach to promote synergy-rather than antagonism-between macrophages and engineered T cells, leading to enhanced antitumor immunity.

A new study by Yamada-Hunter et al. reveals a novel approach to promote synergy—rather than antagonism—between macrophages and engineered T cells, leading to enhanced antitumor immunity.

AbstractObjectivesCAR‐T cells are being investigated as a novel immunotherapy for glioblastoma, but clinical success has been limited. We recently described fibroblast activation protein (FAP) as an ideal target antigen for glioblastoma immunotherapy, with expression on both tumor cells and tumor blood vessels. However, CAR‐T cells targeting FAP have never been investigated as a therapy for glioblastoma.MethodsWe generated a novel FAP targeting CAR with CD3ζ and CD28 signalling domains and tested the resulting CAR‐T cells for their lytic activity and cytokine secretion function in vitro (using real‐time impedance, flow cytometry, imaging and bead‐based cytokine assays), and in vivo (using a xenograft mimicking the natural heterogeneity of human glioblastoma).ResultsFAP‐CAR‐T cells exhibited target specificity against model cell lines and potent cytotoxicity against patient‐derived glioma neural stem cells, even when only a subpopulation expressed FAP, indicating a bystander killing mechanism. Using co‐culture assays, we confirmed FAP‐CAR‐T cells mediate bystander killing of antigen‐negative tumor cells, but only after activation by FAP‐positive target cells. This bystander killing was at least partially mediated by soluble factors and amplified by IL‐2 which activated the non‐transduced fraction of the CAR‐T product. Finally, a low dose of intravenously administered FAP‐CAR‐T cells controlled, without overt toxicity, the growth of subcutaneous tumors created using a mixture of antigen‐negative and antigen‐positive glioblastoma cells.ConclusionsOur findings advance FAP as a leading candidate for clinical CAR‐T therapy of glioblastoma and highlight under‐recognised antigen nonspecific mechanisms that may contribute meaningfully to the antitumor activity of CAR‐T cells.

Background Chimeric antigen receptor (CAR) T cell therapies specific for the CD19 and B-cell maturation antigen have become an approved standard of care worldwide for relapsed and refractory B-cell malignancies. If CAR-T cell therapy for non-hematological malignancies is to achieve the same stage of clinical development, then iterative early-phase clinical testing can add value to the clinical development process for evaluating CAR-T cell products containing different CAR designs and manufactured under differing conditions.Methods We conducted a phase 1 trial of third-generation GD2-specific CAR-T cell therapy, which has previously been tested in neuroblastoma patients. In this study, the GD2-CAR-T therapy was evaluated for the first time in metastatic melanoma patients in combination with BRAF/MEK inhibitor therapy, and as a monotherapy in patients with colorectal cancer and a patient with fibromyxoid sarcoma. Feasibility and safety were determined and persistence studies, multiplex cytokine arrays on sera and detailed immune phenotyping of the original CAR-T products, the circulating CAR-T cells, and, in select patients, the tumor-infiltrating CAR-T cells were performed.Results We demonstrate the feasibility of manufacturing CAR-T products at point of care for patients with solid cancer and show that a single intravenous infusion was well tolerated with no dose-limiting toxicities or severe adverse events. In addition, we note significant improvements in CAR-T cell immune phenotype, and expansion when a modified manufacturing procedure was adopted for the latter 6 patients recruited to this 12-patient trial. We also show evidence of CAR-T cell-mediated immune activity and in some patients expanded subsets of circulating myeloid cells after CAR-T cell therapy.Conclusions This is the first report of third-generation GD2-targeting CAR-T cells in patients with metastatic melanoma and other solid cancers such as colorectal cancer, showing feasibility, safety and immune activity, but limited clinical effect.Trial registration number ACTRN12613000198729.

In this Commentary article, as part of the 100-year celebrations of the journal, we reflect on the contribution of articles published in ICB in the field of tumor immunology. A highlight is a series of interviews conducted with three Australian-based ICB authors who have contributed key papers over the years: Rajiv Khanna, Delia Nelson and Ian Frazer.

In this Commentary article, as part of the 100-year celebrations of the journal, we reflect on the contribution of articles published in ICB in the field of tumor immunology. A highlight is a series of interviews conducted with three Australian-based ICB authors who have contributed key papers over the years: Rajiv Khanna, Delia Nelson and Ian Frazer.

Cancers in the central nervous system resist therapies effective in other cancers, possibly due to the unique biochemistry of the human brain microenvironment composed of cerebrospinal fluid (CSF). However, the impact of CSF on cancer cells and therapeutic efficacy is unknown. Here, we examined the effect of human CSF on glioblastoma (GBM) tumors from 25 patients. We found that CSF induces tumor cell plasticity and resistance to standard GBM treatments (temozolomide and irradiation). We identified nuclear protein 1 (NUPR1), a transcription factor hampering ferroptosis, as a mediator of therapeutic resistance in CSF. NUPR1 inhibition with a repurposed antipsychotic, trifluoperazine, enhanced the killing of GBM cells resistant to chemoradiation in CSF. The same chemo-effective doses of trifluoperazine were safe for human neurons and astrocytes derived from pluripotent stem cells. These findings reveal that chemoradiation efficacy decreases in human CSF and suggest that combining trifluoperazine with standard care may improve the survival of patients with GBM.

The clinical success of immune-checkpoint inhibitors (ICI) in both resected and metastatic melanoma has confirmed the validity of therapeutic strategies that boost the immune system to counteract cancer. However, half of patients with metastatic disease treated with even the most aggressive regimen do not derive durable clinical benefit. Thus, there is a critical need for predictive biomarkers that can identify individuals who are unlikely to benefit with high accuracy so that these patients may be spared the toxicity of treatment without the likely benefit of response. Ideally, such an assay would have a fast turnaround time and minimal invasiveness. Here, we utilize a novel platform that combines mass spectrometry with an artificial intelligence-based data processing engine to interrogate the blood glycoproteome in melanoma patients before receiving ICI therapy. We identify 143 biomarkers that demonstrate a difference in expression between the patients who died within six months of starting ICI treatment and those who remained progression-free for three years. We then develop a glycoproteomic classifier that predicts benefit of immunotherapy (HR=2.7; p=0.026) and achieves a significant separation of patients in an independent cohort (HR=5.6; p=0.027). To understand how circulating glycoproteins may affect efficacy of treatment, we analyze the differences in glycosylation structure and discover a fucosylation signature in patients with shorter overall survival (OS). We then develop a fucosylation-based model that effectively stratifies patients (HR=3.5; p=0.0066). Together, our data demonstrate the utility of plasma glycoproteomics for biomarker discovery and prediction of ICI benefit in patients with metastatic melanoma and suggest that protein fucosylation may be a determinant of anti-tumor immunity.

Background Immune checkpoint inhibitors (ICIs) have revolutionized melanoma treatment, necessitating predictive biomarkers to identify patients likely to benefit. To that end, this study leverages a novel platform that combines liquid chromatography/mass spectrometry with a proprietary artificial-intelligence-based data processing engine, allowing for highly scalable and reproducible interrogation of glycoproteins with site-and glycan-specificity, capable of identifying blood-based predictive biomarkers using pre-treatment plasma samples from metastatic melanoma (MM) patients. Methods We interrogated 521 glycopeptide (GP) and 75 peptide biomarkers in a discovery cohort of pre-treatment plasma samples obtained from 202 patients with metastatic melanoma (MM) treated with anti-PD-1 monotherapy (pembrolizumab or nivolumab (57%), or anti-CTLA-4 (ipilimumab) with/without nivolumab (43%) (table 1). In addition to using age- and sex-adjusted regression to identify differentially abundant biomarkers where overall survival (OS) from ICI therapy start was the primary endpoint, patients were divided into those having early treatment failures (death within 6-months), intermediate controls (progression of death l between 6-months and 3-years), and sustained controls (progression-free for at least 3 years). Next, the discovery cohort was divided into a training, test, and validation set to develop and assess a repeated cross-validated LASSO-regularized Cox-based glycoproteomic classifier. To externally validate the classifier, an independent cohort of 27 MM patients were tested (table 2). Lastly, given the link between fucosylation and MM, engineered fucosylation-features were used in a second classifier. Results We identified 143 markers that significantly distinguished patients with early treatment failure from those with sustained controls (figure 1). A 14-marker classifier achieved a high degree of separation (table 3-detailed performance metrics) between those likely to benefit (i.e. those predicted to achieve long-term clinical benefit) and unlikely to benefit (Cox proportional hazard ratio/H.R. = 2.7, p-value = 0.026) (figure 2) while also yielding comparable performance in an independent cohort (H.R = 5.6, p-value = 0.027) (table 3). The secondary fucosylated-based classifier was also able to distinguish patients with and without long-term benefit (H.R = 3.5, p-value = 0.0066) (figure 4). Conclusions Using glycoproteomic profiling, our classifier predicted which MM patients treated with ICIs had nearly a 3-fold greater likelihood of durable benefit, with the finding validated in an independent cohort. Our results also suggest circulating glycoprotein fucosylation may be an important determinant of anti-tumor immunity. These data demonstrate the utility of plasma glycoproteomics for biomarker discovery and prediction of ICI benefit in patients with MM. Future directions include prospective confirmatory testing. Acknowledgements The authors thank James Richard Hartness, Jr. and Kim Vigal for their alliance management efforts and critical inputs for this abstract. References Shum B, Larkin J, Turajlic S. Predictive biomarkers for response to immune checkpoint inhibition. Semin Cancer Biol. 2022 Feb;79:4–17. doi: 10.1016/j.semcancer.2021.03.036. Epub 2021 Apr 2. PMID: 33819567. Dhar C, Ramachandran P, Xu G, Pickering C, Caval T, Rice R, Zhou B, Srinivasan A, Hundal I, Cheng R, Aiyetan P. Diagnosing and staging epithelial ovarian cancer by serum glycoproteomic profiling. medRxiv 2023.03.20.23287422 [Preprint]. March 20, 2023 [cited 2023 Jun 26]. Available from: https://doi.org/10.1101/2023.03.20.23287422 Agrawal P, Fontanals-Cirera B, Sokolova E, Jacob S, Vaiana CA, Argibay D, Davalos V, McDermott M, Nayak S, Darvishian F, Castillo M, Ueberheide B, Osman I, Fenyö D, Mahal LK, Hernando E. A Systems Biology Approach Identifies FUT8 as a Driver of Melanoma Metastasis. Cancer Cell. 2017 Jun 12;31(6):804–819.e7. doi: 10.1016/j.ccell.2017.05.007. PMID: 28609658; PMCID: PMC5649440. Ethics Approval Plasma samples were collected under MGH IRB protocols 12–488 & 11–181and Central Adelaide Local Health Network Human Research Ethics Committee protocol HREC/16/RAH/95. Written informed consent was obtained from all patients prior to inclusion in the study.

Accurate delineation of gross tumor volumes remains a barrier to radiotherapy dose escalation and boost dosing in the treatment of solid tumors, such as prostate cancer. Magnetic resonance imaging (MRI) of tumor targets has the power to enable focal dose boosting, particularly when combined with technological advances such as MRI-linear accelerator. Fibroblast activation protein (FAP) is overexpressed in stromal components of >90% of epithelial carcinomas. Herein, the authors compare targeted MRI of prostate specific membrane antigen (PSMA) with FAP in the delineation of orthotopic prostate tumors. Control, FAP, and PSMA-targeting iron oxide nanoparticles were prepared with modification of a lymphotropic MRI agent (FerroTrace, Ferronova). Mice with orthotopic LNCaP tumors underwent MRI 24 h after intravenous injection of nanoparticles. FAP and PSMA nanoparticles produced contrast enhancement on MRI when compared to control nanoparticles. FAP-targeted MRI increased the proportion of tumor contrast-enhancing black pixels by 13%, compared to PSMA. Analysis of changes in R2 values between healthy prostates and LNCaP tumors indicated an increase in contrast-enhancing pixels in the tumor border of 15% when targeting FAP, compared to PSMA. This study demonstrates the preclinical feasibility of PSMA and FAP-targeted MRI which can enable targeted image-guided focal therapy of localized prostate cancer.