The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction.

In bacteria, the protein FtsZ is the principal component of a ring that constricts the cell at division. Though all mitochondria probably arose through a single, ancient bacterial endosymbiosis, the mitochondria of only certain protists appear to have retained FtsZ, and the protein is absent from the mitochondria of fungi, animals, and higher plants. We have investigated the role that FtsZ plays in mitochondrial division in the genetically tractable protist Dictyostelium discoideum, which has two nuclearly encoded FtsZs, FszA and FszB, that are targeted to the inside of mitochondria. In most wild-type amoebae, the mitochondria are spherical or rod-shaped, but in fsz-null mutants they become elongated into tubules, indicating that a decrease in mitochondrial division has occurred. In support of this role in organelle division, antibodies to FszA and FszAgreen fluorescent protein (GFP) show belts and puncta at multiple places along the mitochondria, which may define future or recent sites of division. FszB-GFP, in contrast, locates to an electron-dense, submitochondrial body usually located at one end of the organelle, but how it functions during division is unclear. This is the first demonstration of two differentially localized FtsZs within the one organelle, and it points to a divergence in the roles of these two proteins.Mitochondria and chloroplasts divide by fission, like their bacterial ancestors. In 1995, Osteryoung and Vierling (51) discovered a chloroplast-targeted version of the bacterial cell division protein FtsZ (AtFtsZ1-1) in Arabidopsis thaliana that was most similar to the FtsZs of cyanobacteria, the ancestors of chloroplasts. The implication of this finding was that all chloroplasts, and perhaps even mitochondria, might still use FtsZ to divide. FtsZ is the most widespread and important of a dozen or so bacterial division proteins (1,13,15,36,54). FtsZ is a GTPase that bears little amino acid sequence identity but a striking structural similarity to tubulins, a family of eukaryotic cytoskeletal proteins (30, 47). Like tubulin, FtsZ monomers can self-associate and have been observed to form microtubule-like filaments in vitro (14). FtsZ has, thus, been proposed to be the prokaryotic ancestor of tubulin (12,38,47).FtsZ is associated with the invaginating inner margin of the bacterial cell membrane (7), and studies of FtsZ-green fluorescent protein (GFP) fusions showed the protein forms a ring at the division site (31). It is not known if the FtsZ cytokinetic ring, the Z-ring, generates the contractile force necessary to pull in the cell edges, or if it simply provides an assembly site for other constricting proteins.FtsZs play a critical role in the division of chloroplasts. If the expression of either of two versions of FtsZ in Arabidopsis (AtFtsZ1-1 and AtFtsZ2-1) is inhibited by antisense RNA, chloroplasts fail to divide properly, if at all (52). Gene disruption of chloroplast FtsZ in the moss Physcomitrella patens produces a similar result (59). Immunofluorescence microscopy showed that AtFtsZ1...

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Objectives The increasing success of Chimeric Antigen Receptor (CAR) T cell therapy in haematological malignancies is reinvigorating its application in many other cancer types and with renewed focus on its application to solid tumors. We present a novel CAR against glioblastoma, an aggressive, malignant glioma, with a dismal survival rate for which treatment options have remained unchanged for over a decade. Methods We use the human Retained Display (ReD) antibody platform (Myrio Therapeutics) to identify a novel single‐chain variable fragment (scFv) that recognises epidermal growth factor receptor mutant variant III (EGFRvIII), a common and tumor‐specific mutation found in glioblastoma. We use both in vitro functional assays and an in vivo orthotopic xenograft model of glioblastoma to examine the function of our novel CAR, called GCT02, targeted using murine CAR T cells. Results Our EGFRvIII‐specific scFv was found to be of much higher affinity than reported comparators reverse‐engineered from monoclonal antibodies. Despite the higher affinity, GCT02 CAR T cells kill equivalently but secrete lower amounts of cytokine. In addition, GCT02‐CAR T cells also mediate rapid and complete tumor elimination in vivo . Conclusion We present a novel EGFRvIII‐specific CAR, with effective antitumor functions both in in vitro and in a xenograft model of human glioblastoma.