Indigenous Australian genomes show deep structure and rich novel variation.

The Indigenous peoples of Australia have a rich linguistic and cultural history. How this relates to genetic diversity remains largely unknown because of their limited engagement with genomic studies. Here we analyse the genomes of 159 individuals from four remote Indigenous communities, including people who speak a language (Tiwi) not from the most widespread family (Pama-Nyungan). This large collection of Indigenous Australian genomes was made possible by careful community engagement and consultation. We observe exceptionally strong population structure across Australia, driven by divergence times between communities of 26,000-35,000 years ago and long-term low but stable effective population sizes. This demographic history, including early divergence from Papua New Guinean (47,000 years ago) and Eurasian groups1, has generated the highest proportion of previously undescribed genetic variation seen outside Africa and the most extended homozygosity compared with global samples. A substantial proportion of this variation is not observed in global reference panels or clinical datasets, and variation with predicted functional consequence is more likely to be homozygous than in other populations, with consequent implications for medical genomics2. Our results show that Indigenous Australians are not a single homogeneous genetic group and their genetic relationship with the peoples of New Guinea is not uniform. These patterns imply that the full breadth of Indigenous Australian genetic diversity remains uncharacterized, potentially limiting genomic medicine and equitable healthcare for Indigenous Australians.

The landscape of genomic structural variation in Indigenous Australians.

Indigenous Australians harbour rich and unique genomic diversity. However, Aboriginal and Torres Strait Islander ancestries are historically under-represented in genomics research and almost completely missing from reference datasets1-3. Addressing this representation gap is critical, both to advance our understanding of global human genomic diversity and as a prerequisite for ensuring equitable outcomes in genomic medicine. Here we apply population-scale whole-genome long-read sequencing4 to profile genomic structural variation across four remote Indigenous communities. We uncover an abundance of large insertion-deletion variants (20-49 bp; n = 136,797), structural variants (50 b-50 kb; n = 159,912) and regions of variable copy number (>50 kb; n = 156). The majority of variants are composed of tandem repeat or interspersed mobile element sequences (up to 90%) and have not been previously annotated (up to 62%). A large fraction of structural variants appear to be exclusive to Indigenous Australians (12% lower-bound estimate) and most of these are found in only a single community, underscoring the need for broad and deep sampling to achieve a comprehensive catalogue of genomic structural variation across the Australian continent. Finally, we explore short tandem repeats throughout the genome to characterize allelic diversity at 50 known disease loci5, uncover hundreds of novel repeat expansion sites within protein-coding genes, and identify unique patterns of diversity and constraint among short tandem repeat sequences. Our study sheds new light on the dimensions and dynamics of genomic structural variation within and beyond Australia.

Equitable Expanded Carrier Screening Needs Indigenous Clinical and Population Genomic Data.

Expanded carrier screening (ECS) for recessive monogenic diseases requires prior knowledge of genomic variation, including DNA variants that cause disease. The composition of pathogenic variants differs greatly among human populations, but historically, research about monogenic diseases has focused mainly on people with European ancestry. By comparison, less is known about pathogenic DNA variants in people from other parts of the world. Consequently, inclusion of currently underrepresented Indigenous and other minority population groups in genomic research is essential to enable equitable outcomes in ECS and other areas of genomic medicine. Here, we discuss this issue in relation to the implementation of ECS in Australia, which is currently being evaluated as part of the national Government's Genomics Health Futures Mission. We argue that significant effort is required to build an evidence base and genomic reference data so that ECS can bring significant clinical benefit for many Aboriginal and/or Torres Strait Islander Australians. These efforts are essential steps to achieving the Australian Government's objectives and its commitment "to leveraging the benefits of genomics in the health system for all Australians." They require culturally safe, community-led research and community involvement embedded within national health and medical genomics programs to ensure that new knowledge is integrated into medicine and health services in ways that address the specific and articulated cultural and health needs of Indigenous people. Until this occurs, people who do not have European ancestry are at risk of being, in relative terms, further disadvantaged.

The efficacy of combination treatment with elotuzumab and lenalidomide is dependent on crosstalk between natural killer cells, monocytes and myeloma cells.

Patients with refractory relapsed multiple myeloma respond to combination treatment with elotuzumab and lenalidomide. The mechanisms underlying this observation are not fully understood. Furthermore, biomarkers predictive of response have not been identified to date. To address these issues, we used a humanized myeloma mouse model and adoptive transfer of human natural killer (NK) cells to show that elotuzumab and lenalidomide treatment controlled myeloma growth, and this was mediated through CD16 on NK cells. In co-culture studies, we showed that peripheral blood mononuclear cells from a subset of patients with refractory relapsed multiple myeloma were effective killers of OPM2 myeloma cells when treated with elotuzumab and lenalidomide, and this was associated with significantly increased expression of CD54 on OPM2 cells. Furthermore, elotuzumab- and lenalidomide-induced OPM2 cell killing and increased OPM2 CD54 expression were dependent on both monocytes and NK cells, and these effects were not mediated by soluble factors alone. At the transcript level, elotuzumab and lenalidomide treatment significantly increased OPM2 myeloma cell expression of genes for trafficking and adhesion molecules, NK cell activation ligands and antigen presentation molecules. In conclusion, our findings suggest that multiple myeloma patients require elotuzumab- and lenalidomide-mediated upregulation of CD54 on autologous myeloma cells, in combination with NK cells and monocytes to mediate an effective anti-tumor response. Furthermore, our data suggest that increased myeloma cell CD54 expression levels could be a powerful predictive biomarker for response to elotuzumab and lenalidomide treatment.

Finding the Keys to the CAR: Identifying Novel Target Antigens for T Cell Redirection Immunotherapies.

Oncology immunotherapy has been a significant advancement in cancer treatment and involves harnessing and redirecting a patient's immune response towards their own tumour. Specific recognition and elimination of tumour cells was first proposed over a century ago with Paul Erlich's 'magic bullet' theory of therapy. In the past decades, targeting cancer antigens by redirecting T cells with antibodies using either bispecific T cell engagers (BiTEs) or chimeric antigen receptor (CAR) T cell therapy has achieved impressive clinical responses. Despite recent successes in haematological cancers, linked to a high and uniformly expressed CD19 antigen, the efficacy of T cell therapies in solid cancers has been disappointing, in part due to antigen escape. Targeting heterogeneous solid tumours with T cell therapies will require the identification of novel tumour specific targets. These targets can be found among a range of cell-surface expressed antigens, including proteins, glycolipids or carbohydrates. In this review, we will introduce the current tumour target antigen classification, outline existing approaches to discover novel tumour target antigens and discuss considerations for future design of antibodies with a focus on their use in CAR T cells.

Cellular and Molecular Mechanisms of CD8+ T Cell Differentiation, Dysfunction and Exhaustion.

T cells follow a triphasic distinct pathway of activation, proliferation and differentiation before becoming functionally and phenotypically "exhausted" in settings of chronic infection, autoimmunity and in cancer. Exhausted T cells progressively lose canonical effector functions, exhibit altered transcriptional networks and epigenetic signatures and gain constitutive expression of a broad coinhibitory receptor suite. This review outlines recent advances in our understanding of exhausted T cell biology and examines cellular and molecular mechanisms by which a state of dysfunction or exhaustion is established, and mechanisms by which exhausted T cells may still contribute to pathogen or tumour control. Further, this review describes our understanding of exhausted T cell heterogeneity and outlines the mechanisms by which checkpoint blockade differentially engages exhausted T cell subsets to overcome exhaustion and recover T cell function.

The Evolving Protein Engineering in the Design of Chimeric Antigen Receptor T Cells.

The clinical success of chimeric antigen receptor (CAR) T cell immunotherapy in the treatment of haematological cancers has encouraged the extensive development of CAR design to improve their function and increase their applicability. Advancements in protein engineering have seen modifications to both the ecto- and endo-domains of the CAR, with recent designs targeting multiple antigens and including inducible elements. These developments are likely to play an important role in inducing effective CAR T cell responses in a solid tumour context, where clinical responses have not been effective to date. This review highlights the spectrum of novel strategies being employed in CAR design, including for example variations in targeting tumour antigens by utilising different ectodomain designs such as dual chain CARs, natural receptor or ligand-based CARs, and T cell receptor fusion constructs, and also reviews some of the innovative approaches to a "universal" CAR and various multi-antigen targeting CAR strategies. We also explore how choices in the endodomain impact CAR function and how these need to be considered in the overall CAR design.

The strength of T cell receptor signal controls the polarization of cytotoxic machinery to the immunological synapse.

Killing by cytotoxic T lymphocytes (CTLs) is mediated by the secretion of lytic granules. The centrosome plays a key role in granule delivery, polarizing to the central supramolecular activation complex (cSMAC) within the immunological synapse upon T cell receptor (TCR) activation. Although stronger TCR signals lead to increased target cell death than do weaker signals, it is not known how the strength of TCR signal controls polarization of the centrosome and lytic granules. By using TCR transgenic OT-I CTLs, we showed that both high- and low-avidity interactions led to centrosome polarization to the cSMAC. However, only high-avidity interactions, which induced a higher threshold of intracellular signaling, gave rise to granule recruitment to the polarized centrosome at the synapse. By controlling centrosome and granule polarization independently, the centrosome is able to respond rapidly to weak signals so that CTLs are poised and ready for the trigger for granule delivery.

Perforin forms transient pores on the target cell plasma membrane to facilitate rapid access of granzymes during killer cell attack.

Cytotoxic lymphocytes serve a key role in immune homeostasis by eliminating virus-infected and transformed target cells through the perforin-dependent delivery of proapoptotic granzymes. However, the mechanism of granzyme entry into cells remains unresolved. Using biochemical approaches combined with time-lapse microscopy of human primary cytotoxic lymphocytes engaging their respective targets, we defined the time course of perforin pore formation in the context of the physiological immune synapse. We show that, on recognition of targets, calcium influx into the lymphocyte led to perforin exocytosis and target cell permeabilization in as little as 30 seconds. Within the synaptic cleft, target cell permeabilization by perforin resulted in the rapid diffusion of extracellular milieu-derived granzymes. Repair of these pores was initiated within 20 seconds and was completed within 80 seconds, thus limiting granzyme diffusion. Remarkably, even such a short time frame was sufficient for the delivery of lethal amounts of granzymes into the target cell. Rapid initiation of apoptosis was evident from caspase-dependent target cell rounding within 2 minutes of perforin permeabilization. This study defines the final sequence of events controlling cytotoxic lymphocyte immune defense, in which perforin pores assemble on the target cell plasma membrane, ensuring efficient delivery of lethal granzymes.

Rapid and unidirectional perforin pore delivery at the cytotoxic immune synapse.

The effective engagement of cytotoxic lymphocytes (CLs) with their target cells is essential for the removal of virus-infected and malignant cells from the body. The spatiotemporal properties that define CL engagement and killing of target cells remain largely uncharacterized due to a lack of biological reporters. We have used a novel live cell microscopy technique to visualize the engagement of primary human and mouse CL with their targets and the subsequent delivery of the lethal hit. Extensive quantitative real-time analysis of individual effector-target cell conjugates demonstrated that a single effector calcium flux event was sufficient for the degranulation of human CLs, resulting in the breach of the target cell membrane by perforin within 65-100 s. In contrast, mouse CLs demonstrated distinct calcium signaling profiles leading to degranulation: whereas mouse NKs required a single calcium flux event, CD8(+) T cells typically required several calcium flux events before perforin delivery. Irrespective of their signaling profile, every target cell that was damaged by perforin died by apoptosis. To our knowledge, we demonstrate for the first time that perforin pore delivery is unidirectional, occurring exclusively on the target cell membrane, but sparing the killer cell. Despite this, the CTL membrane was not intrinsically perforin resistant, as intact CTLs presented as targets to effector CTLs were capable of being killed by perforin-dependent mechanisms. Our results highlight the remarkable efficiency and specificity of perforin pore delivery by CLs.

EphA3-targeted chimeric antigen receptor T cells are effective in glioma and generate curative memory T cell responses.

BACKGROUND: High-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. METHODS: We 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. RESULTS: EphA3-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. CONCLUSION: Building 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.

Synthetic biology, genetic circuits and machine learning: a new age of cancer therapy.

Synthetic biology has made it possible to rewire natural cellular responses to treat disease, notably demonstrated by chimeric antigen receptor (CAR) T cells as cancer immunotherapy. Building on the success of T-cell activation using synthetic receptors, the field is now investigating how induction of noncanonical signalling pathways and sophisticated synthetic gene circuitry can enhance the antitumour phenotype of engineered T cells. This commentary explores two recently published studies that provide proof of concept for how new technologies achieve this. The first demonstrated that non-naturally occurring combinations of signalling motifs derived from various immune receptors and arranged as a CAR drove unique signal transduction pathways in T cells and improved their tumour killing ability. Here, machine learning complemented the screening process and successfully predicted CAR T-cell phenotype dependent on signalling motif choice. The second explored how synthetic zinc fingers can be engineered into controllable transcriptional regulators, where their activity was dependent on the presence or absence of FDA-approved small-molecule drugs. These studies are pivotal in expanding the design choices available for gene circuits of the future and highlight how a single cellular therapy could respond to multiple environmental cues including target cell antigen expression, the tumour microenvironment composition and small molecule drugs.

From signalling pathways to targeted therapies: unravelling glioblastoma's secrets and harnessing two decades of progress.

Glioblastoma, a rare, and highly lethal form of brain cancer, poses significant challenges in terms of therapeutic resistance, and poor survival rates for both adult and paediatric patients alike. Despite advancements in brain cancer research driven by a technological revolution, translating our understanding of glioblastoma pathogenesis into improved clinical outcomes remains a critical unmet need. This review emphasises the intricate role of receptor tyrosine kinase signalling pathways, epigenetic mechanisms, and metabolic functions in glioblastoma tumourigenesis and therapeutic resistance. We also discuss the extensive efforts over the past two decades that have explored targeted therapies against these pathways. Emerging therapeutic approaches, such as antibody-toxin conjugates or CAR T cell therapies, offer potential by specifically targeting proteins on the glioblastoma cell surface. Combination strategies incorporating protein-targeted therapy and immune-based therapies demonstrate great promise for future clinical research. Moreover, gaining insights into the role of cell-of-origin in glioblastoma treatment response holds the potential to advance precision medicine approaches. Addressing these challenges is crucial to improving outcomes for glioblastoma patients and moving towards more effective precision therapies.

Flow Cytometry Identification of Cell Compartments in the Murine Brain.

Glioma can be modelled in the murine brain through the induction of genetically engineered mouse models or intracranial transplantation. Gliomas (oligodendroglioma and astrocytoma) are thought to arise from neuronal and glial progenitor populations in the brain and are poorly infiltrated by immune cells. An improved understanding of oligodendrocytes, astrocytes, and the immune environment throughout tumor development will enhance the analysis and development of brain cancer models. Here, we describe the isolation and analysis of murine brain cell types using a combination of flow cytometry and quantitative RT-PCR strategies to analyze these individual cell populations in vivo.

Endogenous H3.3K27M derived peptide restricted to HLA-A∗02:01 is insufficient for immune-targeting in diffuse midline glioma.

Diffuse midline glioma (DMG) is a childhood brain tumor with an extremely poor prognosis. Chimeric antigen receptor (CAR) T cell therapy has recently demonstrated some success in DMG, but there may a need to target multiple tumor-specific targets to avoid antigen escape. We developed a second-generation CAR targeting an HLA-A∗02:01 restricted histone 3K27M epitope in DMG, the target of previous peptide vaccination and T cell receptor-mimics. These CAR T cells demonstrated specific, titratable, binding to cells pulsed with the H3.3K27M peptide. However, we were unable to observe scFv binding, CAR T cell activation, or cytotoxic function against H3.3K27M+ patient-derived models. Despite using sensitive immunopeptidomics, we could not detect the H3.3K27M26-35-HLA-A∗02:01 peptide on these patient-derived models. Interestingly, other non-mutated peptides from DMG were detected bound to HLA-A∗02:01 and other class I molecules, including a novel HLA-A3-restricted peptide encompassing the K27M mutation and overlapping with the H3 K27M26-35-HLA-A∗02:01 peptide. These results suggest that targeting the H3 K27M26-35 mutation in context of HLA-A∗02:01 may not be a feasible immunotherapy strategy because of its lack of presentation. These findings should inform future investigations and clinical trials in DMG.

Human EGFRvIII chimeric antigen receptor T cells demonstrate favorable safety profile and curative responses in orthotopic glioblastoma.

Objectives: Glioblastoma is a highly aggressive and fatal brain malignancy, and effective targeted therapies are required. The combination of standard treatments including surgery, chemotherapy and radiotherapy is not curative. Chimeric antigen receptor (CAR) T cells are known to cross the blood-brain barrier, mediating antitumor responses. A tumor-expressed deletion mutant of the epidermal growth factor receptor (EGFRvIII) is a robust CAR T cell target in glioblastoma. Here, we show our de novo generated, high-affinity EGFRvIII-specific CAR; GCT02, demonstrating curative efficacy in human orthotopic glioblastoma models. Methods: The GCT02 binding epitope was predicted using Deep Mutational Scanning (DMS). GCT02 CAR T cell cytotoxicity was investigated in three glioblastoma models in vitro using the IncuCyte platform, and cytokine secretion with a cytometric bead array. GCT02 in vivo functionality was demonstrated in two NSG orthotopic glioblastoma models. The specificity profile was generated by measuring T cell degranulation in response to coculture with primary human healthy cells. Results: The GCT02 binding location was predicted to be located at a shared region of EGFR and EGFRvIII; however, the in vitro functionality remained exquisitely EGFRvIII specific. A single CAR T cell infusion generated curative responses in two orthotopic models of human glioblastoma in NSG mice. The safety analysis further validated the specificity of GCT02 for mutant-expressing cells. Conclusion: This study demonstrates the preclinical functionality of a highly specific CAR targeting EGFRvIII on human cells. This CAR could be an effective treatment for glioblastoma and warrants future clinical investigation.

A combined immunopeptidomics, proteomics, and cell surface proteomics approach to identify immunotherapy targets for diffuse intrinsic pontine glioma.

Introduction: Diffuse intrinsic pontine glioma (DIPG), recently reclassified as a subtype of diffuse midline glioma, is a highly aggressive brainstem tumor affecting children and young adults, with no cure and a median survival of only 9 months. Conventional treatments are ineffective, highlighting the need for alternative therapeutic strategies such as cellular immunotherapy. However, identifying unique and tumor-specific cell surface antigens to target with chimeric antigen receptor (CAR) or T-cell receptor (TCR) therapies is challenging. Methods: In this study, a multi-omics approach was used to interrogate patient-derived DIPG cell lines and to identify potential targets for immunotherapy. Results: Through immunopeptidomics, a range of targetable peptide antigens from cancer testis and tumor-associated antigens as well as peptides derived from human endogenous retroviral elements were identified. Proteomics analysis also revealed upregulation of potential drug targets and cell surface proteins such as Cluster of differentiation 27 (CD276) B7 homolog 3 protein (B7H3), Interleukin 13 alpha receptor 2 (IL-13Rα2), Human Epidermal Growth Factor Receptor 3 (HER2), Ephrin Type-A Receptor 2 (EphA2), and Ephrin Type-A Receptor 3 (EphA3). Discussion: The results of this study provide a valuable resource for the scientific community to accelerate immunotherapeutic approaches for DIPG. Identifying potential targets for CAR and TCR therapies could open up new avenues for treating this devastating disease.

HER2 chimeric antigen receptor T cell immunotherapy is an effective treatment for diffuse intrinsic pontine glioma.

Background: Diffuse intrinsic pontine glioma (DIPG) and other diffuse midline gliomas (DMG) of the thalamus and spinal cord are rare but devastating high-grade glial tumors of childhood with no curative treatment. Despite aggressive treatment attempts the prognosis has remained poor. Chimeric antigen receptor (CAR) T cell therapy has been identified as a promising new approach in the treatment of DMG tumors; however, additional targets are urgently required given known tumor heterogeneity and the prospect of antigen escape of this cancer. Methods: Using cell surface mass spectrometry, we detected high HER2 cell surface protein across a panel of patient-derived DIPG cells, thereby identifying an existing CAR T cell therapy for use in DIPG. Primary human T cells were transduced to express a second-generation HER2 CAR and interrogated for efficacy against patient-derived DIPG cells. Results: HER2 CAR T cells demonstrated potent and antigen-specific cytotoxicity and cytokine secretion when co-cultured with patient-derived DIPG cells. Furthermore, HER2 CAR T cells provided a significant regression in intracranial DIPG xenograft tumors. Conclusions: HER2 CAR T cells are already in clinic development and are well tolerated in pediatric patients. Here we provide strong preclinical evidence for the inclusion of DIPG patients in future pediatric CNS tumor HER2 CAR T cell clinical trials.

To go or not to go? Biological logic gating engineered T cells.

Genetically engineered T cells have been successfully used in the treatment of hematological malignancies, greatly increasing both progression-free and overall survival in patients. However, the outcomes of patients treated with Chimeric Antigen Receptor (CAR) T cells targeting solid tumors have been disappointing. There is an unmet clinical need for therapies which are specifically designed to overcome the challenges associated with solid tumors such as tumor heterogeneity and antigen escape. Genetic engineering employing the use of biological logic gating in T cells is an emerging and cutting-edge field that may address these issues. The advantages of logic gating include localized secretion of anti-tumor proteins into the tumor microenvironment, multi antigen targeting of tumors and a potential increase in safety when targeting tumor antigens which may not be exclusively tumor specific. In this review, we introduce the concept of biological logic gating and how this technology addresses some of the challenges of current CAR T treatment. We outline the types of logic gating circuits and finally discuss the application of this new technology to engineered T cells, in the treatment of cancer.